Hyperglycemic Crises

ABSTRACT

Hyperglycemic emergencies, including diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS), are life-threatening complications requiring urgent treatment. DKA is more common in type 1 diabetes and results from absolute insulin deficiency, leading to hyperglycemia, dehydration, ketonemia and metabolic acidosis. Symptoms include polyuria, polydipsia, nausea, vomiting, and altered mental status. HHS is typically seen in type 2 diabetes and is defined by extreme hyperglycemia, severe dehydration, and hyperosmolarity without significant ketoacidosis. It often presents with confusion and neurological symptoms. Diagnosis of these conditions relies on blood glucose and ketone levels, blood gas, and electrolyte measurements. Fluid resuscitation, insulin therapy, and electrolyte correction are the mainstays of treatment. In HHS, intravenous insulin is used more cautiously to prevent rapid osmolar shifts and cerebral edema. Treatment protocols slightly differ in pregnancy, euglycemic ketoacidosis, and advanced kidney disease. Preventative strategies include education on sick day rules, regular clinic follow-ups in high-risk groups, and adherence to insulin therapy.

INTRODUCTION

Diabetes mellitus (DM) affects approximately 828 million people and its prevalence is expected to further grow in the coming years (1). It’s estimated that 193 million people are asymptomatic and remain undiagnosed. Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS) are acute life-threatening emergencies resulting from severe metabolic decompensation. DKA more commonly affects individuals with type 1 diabetes (T1DM) and is characterized by insulin deficiency and the triad of hyperglycemia, ketonemia and/or ketonuria, and metabolic acidosis, whereas HHS primary affects people with type 2 diabetes (T2DM) and presents with severe hyperglycemia, hyperosmolality, and severe dehydration.

Epidemiologic studies conducted in the U.S. and Europe over the past decade have revealed a concerning rise in the rate of hyperglycemic emergencies in adults with both T1DM and T2DM despite the previously noted reduction between 2000 and 2009 (2) (3) (4). The reported incidence of DKA in adults with T1DM varies across Europe, the U.S., and Israel, ranging from 0 to 56 events per 1,000 person-years. However, a study conducted in China between 2010 and 2012 reported a significantly higher rate of 263 per 1,000 person-years (5). In developing countries, DKA episodes affect 3.8%-73.4% of the diabetes population (6).

In the US, 240,000 patients had a primary diagnosis of DKA in 2020, corresponding to 10.2 cases per 1000 admissions and 27,000 had a diagnosis of HHS (1.2 cases per 1000 admissions), a significant rise in DKA cases compared to the total of 184,255 and 27,532 events for DKA and HHS respectively in 2014 (4). The cost of inpatient care for DKA in USA rose to $5.1 billion in 2014 and $6.76 billion in 2017, with the average cost per DKA admission increasing to approximately $31,000 (7) (8).

HHS is less common and is estimated to account for less than 1% of hospital admissions of patients with diabetes (9). Despite its rarity, HHS is associated with a higher mortality rate, ranging from 10% to 20%, which is about ten times higher than DKA. About 2/3 of adults admitted with DKA have T1DM, while almost 90% of the HHS patients have a known diagnosis of T2DM (3).

PATHOGENESIS

The primary distinction between DKA and HHS lies in the degree of insulin insufficiency. DKA results from severe insulin deficiency in the presence of increased counterregulatory hormones, including glucagon, cortisol, epinephrine, and growth hormone, which in turn leads to increased gluconeogenesis, accelerated glycogenolysis, and impaired peripheral glucose uptake. The combination of insulin deficiency and heightened counterregulatory hormone activity enhances lipolysis resulting in high release of free fatty acids from the adipose tissue (10). Free fatty acids are oxidized in the liver, leading to increased ketone production, ketonemia, and metabolic acidosis (11).

In HHS, there is relative insulin deficiency and residual insulin is enough to suppress lipolysis and prevent ketogenesis, but inadequate to regulate hyperglycemia. This persistent hyperglycemia is further enhanced by increased gluconeogenesis and decreased peripheral glucose uptake. Severe hyperglycemia leads to osmotic diuresis and subsequently to volume depletion. Increased thirst is often not sufficient to compensate for these losses and as a result osmolality rises, renal filtration declines, and as HHS progresses, severe dehydration ensues, frequently followed by cognitive impairment (12) (13).

Hyperglycemia in hyperglycemic crises is linked to a severe inflammatory state, marked by increased levels of proinflammatory cytokines such as tumor necrosis factor-α and interleukins-1, -6, and -8, C-reactive protein, reactive oxygen species, and lipid peroxidation biomarkers, even in the absence of apparent infection or an acute cardiovascular event (13).

PRECIPITATING FACTORS

Most admissions for DKA/HHS are precipitated by one of the following risk factors (9) (14):

Infection: The stress of the infection can exacerbate the effects of the insulin deficiency by a rise in counterregulatory hormone levels and cytokines. In T1DM cohorts, infection accounts for up to 79.4% of the cases. Similarly, infection is a trigger factor for HHS in about 40-60% of cases.
Inadequate insulin dose or poor adherence to treatment: Lack of insulin due to omission or suboptimal treatment regimens is one of the most common precipitants of DKA and HHS.
New diagnosis of diabetes: DKA can be the initial clinical presentation of T1DM. In recent years, more people with undiagnosed T2DM diabetes present with DKA or mixed DKA/HHS; part of these cohorts are eventually diagnosed with ketosis prone diabetes (discussed in the special cases section).
Medical or surgical emergencies, such as myocardial infarction, stroke, or trauma, can be complicated by diabetes related emergencies.
Medications: Certain medications can impair glucose metabolism, increase gluconeogenesis and insulin resistance. Glucocorticoids can lead to significant hyperglycemia and subsequently DKA or HHS. A rare complication of SGLT-2 inhibitors is euglycemic ketoacidosis that is further discussed below.

DIAGNOSTIC CRITERIA AND CLINICAL MANIFESTATIONS

Based on the recent global consensus, the following three criteria are used to establish the diagnosis of DKA (13):

Hyperglycemia ≥200 mg/dL (11.1 mmol/L) or a prior history of diabetes irrespective of the presenting glucose reading.
Elevated ketone body concentration: venous or capillary β-hydroxybutyrate ≥3.0 mmol/L or in the early stages of DKA urine ketone 2+ or greater.
Metabolic acidosis: pH<7.3 and/or bicarbonate concentration <18 mmol/L.

It’s worth noting that urine ketone testing, which measures acetoacetate, may underestimate the level of ketonemia due to a lag in the formation of acetoacetate and overestimate it in the advanced stages of DKA due to the increased clearance of β-hydroxybutyrate and conversion to acetoacetate (10). Moreover, ketone tests based on the nitroprusside reaction can give false-positive results in the presence of drugs containing sulfhydryl groups (15, 16). Hence, measurement of venous or capillary β-hydroxybutyrate is recommended for the diagnosis. The use of bedside capillary ketone monitors is now advocated as the best standard of care.

The most common symptoms are nausea and vomiting and abdominal pain, which can mimic an acute abdomen. General symptoms include fatigue and malaise. Osmotic symptoms (polydipsia, polyuria) and unintentional weight loss often precede the diagnosis. Individuals can exhibit neurological symptoms including confusion, headache, lethargy, and coma. Kussmaul breathing (deep, labored breathing) occurs in 28% of cases (17). Fruity breath odor due to ketone production, another distinct sign of DKA, might be present. Tachycardia and hypotension due to dehydration are often present along with dry mucous membranes and decreased skin turgor.

Contrary to DKA, HHS is characterized by absence of severe ketonemia and metabolic acidosis. There are four diagnostic criteria as per global consensus (13).

Plasma glucose >600 mg/dL (33.3 mmol/L).
Hyperosmolality defined as effective osmolality >300 mOsm/kg or total serum osmolality > 320 mOsm/kg.
Absence of significant ketonemia: β-hydroxybutyrate <3.0 mmol/L or urine ketone < 2+.
Absence of acidosis: pH≥7.3 and bicarbonate concentration ≥ 15 mmol/L.

General symptoms include significant fatigue and weakness. Osmotic symptoms and weight loss often precede the acute presentation. Neurological symptoms, including confusion, focal neurological deficits, lethargy, seizures, and coma are more frequent in HHS. Signs of severe dehydration and volume depletion include tachycardia and severe hypotension as well as general signs of dehydration. When left untreated, it can lead to multiorgan failure and death (9).

The differences between DKA and HHS are shown in Table 1. Patients may manifest symptoms and laboratory studies of both DKA and HHS as DKA and HHS represent a spectrum of insulin deficiency disorders.

Table 1. DIFFERENCES BETWEEN DKA AND HHS
Clinical / Laboratory feature	DKA	HHS
Onset	Rapid (hours to 1-2 days)	Gradual (several days to weeks)
Blood glucose	200-600 mg/dL (11.1-13.3 mmol/L)	>600 mg/dl (13.3 mmol/L)
Ketones	Ketonemia >3 mmol/L or ketonuria 2+ or higher	Absent or <3 mmol/L
pH (acidosis)	<7.3	≥7.3
Bicarbonate	<18 mmol/L	≥ 15 mmol/L
Osmolality	Moderately elevated	Severely elevated (>320 mOsm/kg)
Neurological symptoms	Mild to moderate confusion	Severe confusion, seizures, coma

Other Laboratory Findings

Leukocytosis is a common finding in patients with DKA or HHS and might be resulting from acute stress, but a white blood cell count > 25,000/μL may indicate an underlying infection and warrants further investigations (18). Hypertriglyceridemia is frequently seen in HHS and is nearly always present in DKA (19). Additionally, elevated amylase and lipase levels can be found in DKA (20).

In DKA, the anion gap, calculated as [Na⁺] - ([Cl⁻] + [HCO₃⁻]), is typically elevated (>12 mEq/L), reflecting the progression of ketoacidosis. However, other factors such as nausea, vomiting, and renal losses can attenuate this rise by contributing to bicarbonate loss and deranged electrolytes. Moreover, Kussmaul breathing induces respiratory alkalosis, often resulting in a mixed metabolic and respiratory acid-base disturbance.

DIFFERENTIAL DIAGNOSIS

DKA must be differentiated from the following conditions:

Alcoholic Ketoacidosis: Like DKA, it’s characterized by high anion gap metabolic acidosis but with normal or low glucose levels (21).
Starvation Ketoacidosis: Malnutrition or prolonged fasting can lead to ketosis and mild acidosis; however blood glucose levels are normal or low (22).
Lactic Acidosis: Often seen in sepsis and dehydration, it leads to metabolic acidosis in the absence of ketonemia. It’s sometimes seen in individuals with impaired renal function and diabetes treated with metformin (10).
Toxins: Substances such as methanol and ethylene glycol can lead to high anion gap metabolic acidosis.
Renal Failure: Acute or chronic kidney disease is associated with metabolic acidosis and uremia, which can present with symptoms similar to those of DKA as discussed later (23).

Besides distinguishing HHS from DKA, several conditions can mimic or coexist with HHS (24).

Sepsis: Characterized by dehydration and altered mental status, sepsis can also act as a precipitating factor for HHS.
Stroke: When focal neurological deficits are present, HHS may be mistaken for stroke. Glucose testing is essential for an accurate diagnosis.
Uremia: Acute or chronic kidney disease can cause altered mental status, but in the absence of diabetes, blood glucose levels tend to remain normal.
Electrolyte disorders: Hyponatremia or hypernatremia can lead to confusion and neurological symptoms including seizures, lethargy, and coma. Presence of hyperosmolality and hyperglycemia along with electrolyte disorders can unmask an underlying hyperglycemic crisis.

COMPLICATIONS

Hypoglycemia: One of the most common complications of treatment especially in patients with DKA (25).
Hypokalemia: Due to intracellular shift of potassium following insulin treatment, hypokalemia is often seen during treatment of hyperglycemic emergencies. It’s estimated to affect about 55% of DKA and 51% of HHS patients (25).
Hyperchloremic Non–Anion Gap Acidosis: This transient and typically self-resolving condition can result from excessive normal saline infusion or the metabolism of ketoanions to bicarbonate during DKA resolution (26) (27).
Cerebral Edema: This complication is more common in DKA, particularly in younger patients, but has also been reported in HHS. A declining level of consciousness, lethargy, and headache should raise suspicion. If untreated, it can progress to seizures, pupillary abnormalities, bradycardia, and respiratory arrest, with a high risk of mortality and permanent neurological damage. The exact cause remains unclear but may involve osmotic shifts, hypoperfusion, and inflammatory processes (10) (28).
Hypoxemia and rarely non-cardiogenic pulmonary edema: It may be associated with a decrease in the colloid osmotic pressure leading to pulmonary edema.
Thrombosis: Both DKA and HHS are prothrombotic states (29) (30). The risk is higher in HHS potentially due to the concurrent hypernatremia or raised vasopressin concentrations, which are considered thrombogenic. Hyperglycemia is also linked to a pro-inflammatory effect on the endothelium. An individual risk assessment for venous thromboembolism (VTE) should be performed for all patients presenting with DKA or HHS to decide if prophylactic or therapeutic dose of anticoagulation should be prescribed (31) (9). Given limited clinical trial data, therapeutic anticoagulation dose in all patients presenting with HHS is not recommended.
Acute kidney failure: A common complication of DKA and HHS, usually resulting from severe dehydration due to osmotic diuresis.

TREATMENT

Fluids

Administration of intravenous (IV) fluids restores circulating intravascular volume and thus organ perfusion, facilitating the excretion of glucose and ketone bodies. Additionally, it improves insulin sensitivity by reducing the effect of counterregulatory hormones. Isotonic fluids have been the preferred choice for over 50 years. Normal saline 0.9% has been the standard fluid; however, concerns have been raised about its potential to cause hyperchloremic metabolic acidosis, particularly when administered in large volumes. Recent prospective and observational studies, as well as meta-analyses, have shown that using balanced crystalloid solutions such as Ringer’s lactate leads to faster resolution of DKA, shorter hospital stays, and lower incidence of hyperchloremic metabolic acidosis (32) (33, 34).

In DKA, an infusion of 15-20 ml per Kg body weight within the first hour is usually appropriate in adults without renal or cardiac compromise (35). As a general rule, administration of isotonic saline or crystalloid solutions at a rate of 500–1,000 mL/h during the first 2–4 hours is recommended (13). Once intravascular volume is repleted, fluid replacement rate and choice of fluid are guided by vital signs, fluid balance, and serum electrolyte levels (8) .In hypernatremia, isotonic solutions are still the preferred choice due to lower risk of rapid correction of sodium levels and cerebral edema (36). Fluid replacement should restore estimated deficits within the first 24–48 h. Rapid replacement might not be appropriate in individuals with chronic cardiac failure, chronic kidney disease (CKD), frailty, and older age. If there are concerns about hyperchloremic metabolic acidosis, Ringer’s lactate solution can be used instead. When plasma glucose falls below 250 mg/dL (13.9 mmol/L), 5–10% dextrose in addition to the 0.9% sodium chloride is suggested to prevent hypoglycemia while insulin is used to correct ketonemia.

In HHS, the goal of treatment is to replace approximately 50% of the fluid deficit within the first 12 h and the remainder in the following 12 h. Similarly to DKA, initial administration rate of isotonic saline is 500–1,000 mL/h during the first 2–4 h. Fluid replacement alone leads to reduction in glucose levels which in turn decreases serum osmolality due to the water shift into the intracellular space. This results in increasing sodium levels, but this is not necessarily an indication for hypotonic solutions unless the osmolality is not adequately decreasing. If the rise in serum sodium is much greater than 2.4 mmol/L for every 5.5 mmol/L fall in blood glucose, this suggests inadequate fluid replacement and requires a higher infusion rate (37). If fluid replacement is adequate but glucose and osmolality are not falling at the desired rate, then 0.45% sodium chloride solution should be considered. Overall, the goals of treatment are a decrease in osmolality between 3 and 8 mOsmol/kg per hour, a sodium reduction by no more than 10 mmol/L in 24 hours, and hourly glucose fall by up to 5mmol/L (37).

Insulin

Insulin therapy is the mainstay of DKA treatment and should be started immediately after the diagnosis using a fixed-rate intravenous insulin infusion started at 0.1 units/kg/h. Short-acting insulin is the preferred choice. An insulin bolus (0.1 units/kg/hour) given intravenously or intramuscularly is suggested in some treatment protocols if a delay in obtaining venous access is expected, followed by the fixed rate infusion (13) (38). When blood glucose falls below 250 mg/dL (13.9 mmol/L), the rate should be halved to 0.05 units/kg/h. Subsequently, the infusion continues until the ketoacidosis is resolved and rate adjustments are made depending on the glucose levels with a target glucose of 200 mg/dl (11.1 mmol/l) (13).

For patients on long-acting insulin before admission, basal insulin can be continued during the administration of the IV insulin infusion, which will later enable the transition to a subcutaneous basal bolus regimen (39). In the newly diagnosed patients, basal insulin is initiated at 0.15–0.3 units/kg. Once DKA has resolved and oral intake is adequate, IV insulin can be discontinued, and rapid acting insulin is resumed with meals or initiated in the newly diagnosed. For patients who hadn’t been on simultaneous IV insulin and SC basal insulin during treatment, the infusion should be stopped at least 1-2 hours after the administration of SC insulin. If oral intake is poor, transition to variable rate insulin infusion along with glucose solutions is recommended. Criteria for resolution of DKA include (40):

Blood glucose< 200 mg/dl (11.1 mmol/l)
Venous pH > 7.3 and / or bicarbonate ≥18 mmol/L
Plasma ketone <0.6 mmol/L

The anion gap is no longer used as a criterion, as it may also be elevated in hyperchloremic metabolic acidosis.

In HHS, mild or moderate ketonemia (blood β-hydroxybutyrate ≥1.0 to <3.0 mmol/L or urine ketones <2+) in the absence of acidosis (pH ≥7.3 and bicarbonate ≥18 mmol/L) is treated with IV fluids and a fixed-rate IV insulin infusion is only started once the glucose stops falling; this is to prevent large osmotic shifts and subsequently neurological complications (37). If insulin is required, the recommended initial rate is also more conservative at 0.05 units/kg/h.

Mixed DKA/HHS is defined as hyperosmolality (>320 mOsm/kg), β-hydroxybutyrate ≥3.0 mmol/L or ketonuria ≥2+ and presence of acidosis (pH <7.30, or bicarbonate <18 mmol/L) and has been reported in more than one-third of people with hyperglycemic crises (41). DKA and HHS share some common mechanisms in the underlying pathophysiology. In both conditions, counterregulatory hormones are elevated, while the release of cytokines leads to reduced response to insulin. It’s possible that glucotoxicity, inflammation, and oxidative stress all lead to a relative insulin deficiency due to beta cell exhaustion resulting in this overlap (37, 42). Similarly to DKA, it requires higher doses of insulin (starting rate for fixed rate insulin infusion: 0.1 units/kg/h) and IV fluids with the goal to achieve a positive balance of 3–6 L during the first 12 h and the remaining replacement in the following 12 h, although complete resolution may take up to 72 h (37). Transition to SC insulin follows the same principles as DKA.

The criteria for the resolution of HHS has recently been agreed such that overall serum osmolality (total and effective) should fall below 300 mOsm/kg, blood glucose below 250 mg/dL (13.9 mmol/L), urine output is above 0.5 mL/kg/h, and cognitive status has improved (43).

Bicarbonate

Bicarbonate is not routinely administered since IV fluids and insulin usually suffice to correct the metabolic acidosis of DKA (34). Higher risk of hypokalemia, cerebral edema, and development of paradoxical central nervous system acidosis have been reported with bicarbonate treatment (10, 40) and therefore their use is limited in severe metabolic acidosis (i.e., pH <7.0). In this case, 100 mmol of sodium bicarbonate (8.4% solution) in 400 mL of an isotonic solution can be administered every 2 h to achieve a pH >7.0 (43).

Potassium

Total body potassium is reduced in DKA/HHS due to renal losses resulting from osmotic diuresis, extrarenal losses due to vomiting, and concurrent hyperaldosteronism, however this reduction is often masked by the potassium movement from the intracellular to the extracellular space due to the lack of insulin and presence of acidosis. A further reduction results from treatment with insulin that increases uptake of potassium by cells and fluids that restore the intravascular volume and metabolic balance leading to increased potassium excretion in the urine (44). Potassium replacement should be started when serum levels are below 5.5 mmol/L with a target range of 4–5 mmol/L. This is usually achieved with 20–30 mmol of potassium in each liter of intravenous solution. Potassium levels below 3.5 mmol/L require a higher rate of 10 mmol/h and insulin therapy should be deferred until the potassium level is above 3.5 mmol/L to reduce risk of lethal arrhythmias and respiratory muscle weakness (45).

Phosphate

Hypophosphatemia can result from the osmotic shift into the extracellular fluid and the renal losses due to osmotic diuresis. Replacement is indicated in the presence of muscle weakness or respiratory / cardiac distress and phosphate levels below 1.0 mmol/L. In this case 20–30 mmol of potassium phosphate is added to the replacement fluids.

Figure 1. Key points for the management of DKA.

Figure 2. Key points for the management of HHS.

SPECIAL SITUATIONS

Pregnancy

DKA is rare in pregnancy and has an estimated incidence of 0.5–3% with up to 30% being euglycemic DKA (glucose less than 13.9 mmol/l) (46). Fetal demise rates remain as high as 35% for a single episode of DKA despite substantial improvements in perinatal and neonatal care (47). DKA most commonly presents in the third trimester and in women with T1DM followed by gestational diabetes (48). Since euglycemic DKA can present in one third of the cases, increased awareness and low threshold for ketone testing is required when women present with nausea, vomiting, abdominal pain, or recent osmotic symptoms.

During pregnancy, insulin resistance progressively increases to ensure adequate fetal nutrition, while insulin secretion rises to meet these higher demands. Additionally, pregnancy is a ketogenic state characterized by increased lipolysis and ketogenesis, driven by relative maternal starvation and hypoglycemia (49). This explains why DKA can develop in response to triggers such as insulin omission, steroid use, or infection.

Hyperosmolar hyperglycemic state (HHS) is rare in pregnancy due to physiological adaptations, including an 8–10 mmol/kg reduction in maternal plasma osmolality and a lower threshold for vasopressin secretion, both of which reduce the risk of hyperosmolality. However, as insulin resistance increases throughout gestation, HHS is more likely to occur in women with type 2 diabetes mellitus (T2DM) (50).

Diabetic emergencies in pregnancy should be managed in level 2 critical care units such as HDU or ICU. Differences in the management of DKA in pregnancy include the need for fetal monitoring with frequent cardiotocography (CTGs) and in some cases ultrasounds and potentially a more conservative approach in the fluid resuscitation that should be guided by frequent assessment of the hemodynamic status (51). Isotonic saline (0.9%) remains the fluid of choice and should be administered in boluses of 500 ml over 10-15 min if the SBP is less than 90 mmHg and repeated if SBP remains below target. Following initial resuscitation, maintenance fluids can be prescribed as per adult guideline for management of DKA with a recommended rate of 5-15 ml/kg/hour. When it comes to insulin administration, current weight should be considered to determine the infusion rate. Once DKA is resolved, stricter glycemic control should be promptly achieved based on current recommended targets (fasting glucose <5.3 mmol/l, 1 hour post meals< 7.8 mmol/l).

Diabetic ketoacidosis (DKA) can lead to fetal cardiac arrhythmias and, in up to one-third of cases, fetal demise (52). Fetal heart rhythm often normalizes after correction of maternal metabolic acidosis, though this may take 4–8 hours (51).The decision for urgent delivery should be individualized, taking into account gestational age and response to therapy (46).

As noted, euglycemic DKA (euDKA) is common in pregnancy. This is attributed to increased fetal glucose utilization via enhanced placental glucose transporter expression, increased renal glucose excretion due to higher glomerular filtration, and hemodilution from expanded plasma volume. The treatment of euDKA differs from standard DKA management, requiring early initiation of 5% dextrose alongside normal saline via a separate line.

In HHS, weight measurement can assist with fluid replacement. A loss of weight more than 10% of the last clinic weight measurement indicates severe volume depletion and fetal risk (50). Despite stricter glucose targets during gestation, osmolality reduction should not exceed the recommended rate due to risk of cerebral edema.

Recurrent DKA

Recurrent episodes of DKA occur more frequently in females, young age groups, ethnic minorities, and individuals with suboptimal glycemic control (HbA1c > 86 mmol/mol). The most frequent trigger is insulin omission (53). This is often driven by psychosocial factors, including difficulty accepting a chronic condition, depression, eating disorders, fear of weight gain, and severe anxiety about hypoglycemia (54-56). Low socioeconomic status and substance abuse have also been identified as risk factors.

Family conflict and a lack of parental involvement in diabetes management also contribute, particularly in adolescents (57). An observational retrospective case control study showed that 75% of all patients with recurrent DKA did not attend at least one appointment within the previous 12 months; patients had a mean age of 31 years reinforcing the younger age distribution of recurrent DKA cases (58). Data from 6 institutions in Chicago showed a prevalence of recurrent DKA at 21.6% with 5.8% of the cohort presenting with 4 or more episodes. Individuals with fragmented care in different healthcare institutions were at higher risk of recurrence and were more commonly of African American/Black ethnicity (59).

Preventing further episodes of DKA is the primary goal in this high-risk population. Strategies include insulin administration by family members or community nurses, structured insulin regimens, and hybrid closed-loop systems when there are no concerns about technology use. Ongoing support from a diabetes specialist psychologist and programs for complex cases, such as fear of hypoglycemia or eating disorders, are also important. Additionally, virtual appointments can provide flexibility and reduce missed appointments, while technology-based communication, such as messaging, can facilitate frequent contact with the diabetes care team.

Ketosis Prone Diabetes

Ketosis prone diabetes is a distinct variant form that has been described in recent decades, in which patients present with DKA without the underlying autoimmunity of T1DM (GAD and anti-islet cell antibody negative). The genetic aspect remains under investigation with contradictory results so far. It most frequently presents in African, Asian and Indian and Hispanic populations. It’s estimated that it accounts for 25% to 50% of African Americans and Hispanics with a new diagnosis of DKA (60). However, the overall prevalence remains unknown. It’s also more common in middle aged individuals, males, and overweight or obese people (61).

One of the unique aspects of this entity is the reversibility with high chance of insulin cessation and diabetes remission. This usually occurs after several months of insulin therapy and lifestyle interventions with only one fourth of patients remaining insulin dependent (62). Recurrent DKA is rare, and the clinical course generally resembles that of T2DM with oral agents such as metformin often being an effective treatment.

Ketoacidosis stems from the lack of beta cell insulin secretion on the background of severe hyperglycemia. There is often no preceding trigger factor. Patients may report osmotic symptoms prior to DKA presentation. The mechanism for the acute onset of severe hyperglycemia is currently unknown. Once normoglycemia is restored, glucose-stimulated insulin secretion starts to recover and maximizes approximately 12 weeks after the resolution of DKA and initiation of insulin treatment (63) (64). Improvement in c-peptide levels suggests temporary functional abnormalities of beta cells as opposed to permanent damage. Insulin sensitivity also remarkably improves following treatment.

Biochemical parameters during the acute presentation are consistent with the classic form of DKA. Management of DKA doesn’t differ, however once patients are established on subcutaneous insulin, close monitoring is advised since requirements are expected to decrease in the following months (60).

Hyperglycemic Emergencies In Chronic Kidney Disease

DKA IN CHRONIC KIDNEY DISEASE

Chronic kidney disease (CKD) remains a major complication of diabetes, with end-stage renal disease (ESRD) affecting up to 31.3% of cases (65). Declining kidney function leads to impaired gluconeogenesis and reduced insulin degradation, resulting in lower insulin requirements. However, despite these lower needs, individuals with advanced CKD can still develop DKA and, more rarely, HHS.

Water excretion is impaired and osmotic diuresis decreases, which is less pronounced in people with impaired renal function, and it is absent in those with anuria. On the contrary, polydipsia is still present due to hypertonicity leading to increased water intake, and subsequent expansion of interstitial and intravascular volume.

Chronic metabolic acidosis is common in advanced CKD due to decreased acid excretion and high endogenous and exogenous acid loads (66). Acidosis deteriorates rapidly with the accumulation of ketone bodies. Distinguishing between DKA and acidosis of uremia remains challenging as both share similar symptoms (67). Ketone measurements are therefore crucial for prompt diagnosis especially in the presence of markedly elevated anion gap (>20). The recommended diagnostic threshold remains a ß-hydroxybutyrate level of more than 3 mmol/L (68). The diagnostic criteria for DKA do not differ in individuals with CKD.

Clinical findings vary significantly depending on the stage of renal impairment. In individuals with adequate diuresis, volume status is often reduced, further exacerbated by extrarenal losses such as vomiting and diarrhea. In those with decreased diuresis or anuria, volume status may be expanded, leading to signs of fluid overload such as shortness of breath, raised jugular venous pressure, peripheral edema, hypertension, and pulmonary edema.

In patients on dialysis hyperglycemia can be corrected with hemodialysis, with studies reporting a 36% decrease in blood glucose levels two hours post-dialysis (69); hemodialysis also helps correct hyperkalemia and acidosis; however, ketogenesis may persist due to insufficient insulin. The timing of the latest dialysis session, the volume of fluid removed, the presence of extra renal or excessive insensible losses affects the above parameters making diagnosis even more challenging. For this reason, some authors suggest measuring capillary ketone and lactate levels in all people with ESRD and metabolic acidosis (63).

The management of DKA depends on the stage of CKD, which directly influences fluid balance and insulin requirements. In ESRD, insulin requirements are lower, so an initial infusion rate of 0.05 units/kg/hour is recommended instead of 1 unit/kg/hour. The insulin dose should be adjusted based on treatment response, aiming for blood glucose reduction of 3–5 mmol/L per hour, plasma ketone reduction of 0.5 mmol/L per hour, and serum bicarbonate increase of 3 mmol/L per hour. The reduction of effective osmolality should not exceed 8 mOsm⁄kg⁄hour to minimize the risk of cerebral osmotic demyelination.

If the initial assessment shows normal or increased volume status, intravenous fluids are not required unless there is hypotension or other signs of volume depletion. If fluid overload is present, insulin infusion alone may be sufficient. However, severe overload with pulmonary edema requires dialysis, and renal consultation is recommended. If volume status is unclear, central pressure monitoring can help guide management. If the patient is hypovolemic (GI losses, excessive insensible losses, dry mucous membranes, reduced skin turgor, hypotension, weight at or below their post-dialysis weight), fluid resuscitation with boluses of 250 ml of sodium chloride 0.9% with frequent monitoring of clinical and laboratory parameters is recommended. In individuals on dialysis, pre-dialysis weight should be targeted (67).

Serum electrolytes should be closely monitored with potassium supplementation reserved for patients with hypokalemia. Serum potassium levels are often elevated, and hyperkalemia tends to be more severe in people on dialysis for the same level of hyperglycemia. In either case, it is expected to improve with the administration of intravenous insulin (70). Continuous cardiac monitoring is recommended when potassium exceeds 5.5 mmol/L. Correction with 40 mmol/L of potassium chloride is recommended when serum potassium is below 3.5 mmol/l (71).

Emergency hemodialysis should be considered in major hyperglycemia, severe metabolic acidosis in anuric patients, significant hypertonicity with often co-existent severe hyponatremia, and persistent hyperkalemia that does not respond to insulin administration. Rapid correction of blood glucose levels and plasma osmolality should be avoided to reduce risk of cerebral edema (72). Use of bicarbonates is generally limited for severe metabolic acidosis (pH<6.9) (34). Since bicarbonate regeneration is insufficient in advanced CKD, their use can be considered when pH<7.2.

EUGLYCEMIC DKA IN ADVANCED CKD

Euglycemic ketosis can present during continuous renal replacement (CRRT) with glucose free solutions and low caloric intake. During CRRT, glucose is removed from the blood (from 30 to 160 g per day depending on glycemia and hemofiltration rate) and glycogen stores are depleted within 2-3 days when glucose free solutions are used (73). This leads to increased glucagon levels and enhances gluconeogenesis and ketogenesis, resulting in euglycemic ketoacidosis. It can present even in the absence of diabetes (74). A high anion gap metabolic acidosis in people on CRRT should raise suspicion and prompt ketone testing. Ketosis is managed by raising the daily caloric intake (some authors suggest 25 kcal/kg/day) with a supplemental dextrose infusion along with insulin administration. Glucose-containing CRRT solutions should also be considered (73).

HHS IN ADVANCED CKD

HHS is rare in people with advanced CKD, due to lack of significant diuresis, but a mixed HHS/DKA picture can be seen considering that glucose accumulates in the extracellular space leading to hypertonicity and significant hyperglycemia. Diagnostic criteria remain the same in advanced CKD, however since urea is often increased, effective osmolality is a more reliable marker for both diagnosis and assessing response to treatment (23).

Mixed DKA/HHS should be managed as DKA. There are no evidence-based recommendations on the management of HHS in people with advanced CKD. Aggressive fluid resuscitation (30 mL/kg crystalloid) is considered safe for dialysis-dependent patients in other settings, such as sepsis-induced hypotension, based on retrospective data (75) (76). Since people with HHS are usually significantly dehydrated, a similar initial approach with frequent reassessment of fluid responsiveness and volume status could be considered.

PREVENTION

Approximately one in five patients admitted for DKA will be readmitted within 30 days (77). Insulin omission and underlying infections are the most common precipitating factors. As mentioned before, psychological factors, low socioeconomic status, younger age, and substance abuse are often present in people with recurrent episodes of DKA. Identifying patients at risk is crucial to prevent diabetic emergencies; this can be achieved with more frequent clinic visits, structured education programs for example those designed for type 1 diabetes and disordered eating, additional support from the psychiatry service, access to continuous glucose monitoring, and when appropriate, use of hybrid closed loop systems. Funding of community programs targeting people who struggle to access medical care due to socioeconomic reasons should be encouraged especially if we consider the financial implications of diabetic emergencies and complications (78) (79).

Patient education on sick day rules is important for DKA prevention and should be discussed periodically. Temporary discontinuation of SGLT-2 inhibitors in acute illness or planned surgery, when oral intake of food and water is restricted, is recommended to reduce the risk of euglycemic ketoacidosis (80). Education of family members and school staff can help prevent or at least recognize promptly the symptoms of DKA in children and adolescents with type 1 diabetes. Likewise, close observation and early detection of symptoms can help prevent HHS in older adults (36).

SUMMARY

Hyperglycemic emergencies, including DKA and HHS, are serious, life-threatening complications and require increased prompt recognition and urgent medical intervention. DKA is more common in type 1 diabetes and results from absolute insulin deficiency, while HHS is typically seen in type 2 diabetes and is defined by extreme hyperglycemia, severe dehydration, and hyperosmolarity without significant ketoacidosis. The diagnosis of these conditions relies on blood glucose and ketone levels, blood gas, and electrolyte measurements. Treatment focuses on fluid resuscitation, insulin therapy, and electrolyte correction. In HHS, intravenous insulin is used more cautiously to prevent rapid osmolar shifts and cerebral edema. Treatment protocols may need to be adjusted in special populations, including pregnant women, people presenting with euglycemic ketoacidosis, or advanced kidney disease. Preventative strategies including education on sick day rules, regular clinic follow-ups in high-risk groups, and adherence to insulin therapy, are essential in reducing the incidence of these emergencies.

REFERENCES

Collaboration NCDRF. Worldwide trends in diabetes prevalence and treatment from 1990 to 2022: a pooled analysis of 1108 population-representative studies with 141 million participants. Lancet. 2024;404(10467):2077-93.
Zhong VW, Juhaeri J, Mayer-Davis EJ. Trends in Hospital Admission for Diabetic Ketoacidosis in Adults With Type 1 and Type 2 Diabetes in England, 1998-2013: A Retrospective Cohort Study. Diabetes Care. 2018;41(9):1870-7.
Benoit SR, Hora I, Pasquel FJ, Gregg EW, Albright AL, Imperatore G. Trends in Emergency Department Visits and Inpatient Admissions for Hyperglycemic Crises in Adults With Diabetes in the U.S., 2006-2015. Diabetes Care. 2020;43(5):1057-64.
Leiva-Gea I, Fernandez CA, Cardona-Hernandez R, Lozano MF, Bahillo-Curieses P, Arroyo-Diez J, et al. Increased Presentation of Diabetic Ketoacidosis and Changes in Age and Month of Type 1 Diabetes at Onset during the COVID-19 Pandemic in Spain. J Clin Med. 2022;11(15).
Fazeli Farsani S, Brodovicz K, Soleymanlou N, Marquard J, Wissinger E, Maiese BA. Incidence and prevalence of diabetic ketoacidosis (DKA) among adults with type 1 diabetes mellitus (T1D): a systematic literature review. BMJ Open. 2017;7(7):e016587.
Haile HK, Fenta TG. Magnitude, risk factors and economic impacts of diabetic emergencies in developing countries: A systematic review. PLoS One. 2025;20(2):e0317653.
Ramphul K, Joynauth J. An Update on the Incidence and Burden of Diabetic Ketoacidosis in the U.S. Diabetes Care. 2020;43(12):e196-e7.
Desai D, Mehta D, Mathias P, Menon G, Schubart UK. Health Care Utilization and Burden of Diabetic Ketoacidosis in the U.S. Over the Past Decade: A Nationwide Analysis. Diabetes Care. 2018;41(8):1631-8.
Pasquel FJ, Umpierrez GE. Hyperosmolar hyperglycemic state: a historic review of the clinical presentation, diagnosis, and treatment. Diabetes Care. 2014;37(11):3124-31.
Dhatariya KK, Glaser NS, Codner E, Umpierrez GE. Diabetic ketoacidosis. Nat Rev Dis Primers. 2020;6(1):40.
Kitabchi AE, Umpierrez GE, Murphy MB, Barrett EJ, Kreisberg RA, Malone JI, et al. Management of hyperglycemic crises in patients with diabetes. Diabetes Care. 2001;24(1):131-53.
Delaney MF, Zisman A, Kettyle WM. Diabetic ketoacidosis and hyperglycemic hyperosmolar nonketotic syndrome. Endocrinol Metab Clin North Am. 2000;29(4):683-705, V.
Umpierrez GE, Davis GM, ElSayed NA, Fadini GP, Galindo RJ, Hirsch IB, et al. Hyperglycemic Crises in Adults With Diabetes: A Consensus Report. Diabetes Care. 2024;47(8):1257-75.
Ahuja W, Kumar N, Kumar S, Rizwan A. Precipitating Risk Factors, Clinical Presentation, and Outcome of Diabetic Ketoacidosis in Patients with Type 1 Diabetes. Cureus. 2019;11(5):e4789.
Laffel L. Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes. Diabetes Metab Res Rev. 1999;15(6):412-26.
Dhatariya K. Blood Ketones: Measurement, Interpretation, Limitations, and Utility in the Management of Diabetic Ketoacidosis. Rev Diabet Stud. 2016;13(4):217-25.
Adhikari PM, Mohammed N, Pereira P. Changing profile of diabetic ketosis. J Indian Med Assoc. 1997;95(10):540-2.
Slovis CM, Mork VG, Slovis RJ, Bain RP. Diabetic ketoacidosis and infection: leukocyte count and differential as early predictors of serious infection. Am J Emerg Med. 1987;5(1):1-5.
Umpierrez G, Freire AX. Abdominal pain in patients with hyperglycemic crises. J Crit Care. 2002;17(1):63-7.
Yadav D, Nair S, Norkus EP, Pitchumoni CS. Nonspecific hyperamylasemia and hyperlipasemia in diabetic ketoacidosis: incidence and correlation with biochemical abnormalities. Am J Gastroenterol. 2000;95(11):3123-8.
Garg SK, Garg P. Differential Diagnosis of Ketoacidosis in Hyperglycemic Alcoholic Diabetic Patient: Role of Insulin. Indian J Crit Care Med. 2021;25(10):1203-4.
Gall AJ, Duncan R, Badshah A. Starvation ketoacidosis on the acute medical take. Clin Med (Lond). 2020;20(3):298-300.
Stathi D, Dhatariya KK, Mustafa OG. Management of diabetes-related hyperglycaemic emergencies in advanced chronic kidney disease: Review of the literature and recommendations. Diabet Med. 2025;42(2):e15405.
Adeyinka A, Kondamudi NP. Hyperosmolar Hyperglycemic Syndrome.StatPearls. Treasure Island (FL)2025.
Dhatariya KK, Nunney I, Higgins K, Sampson MJ, Iceton G. National survey of the management of Diabetic Ketoacidosis (DKA) in the UK in 2014. Diabet Med. 2016;33(2):252-60.
Rewers A, Kuppermann N, Stoner MJ, Garro A, Bennett JE, Quayle KS, et al. Effects of Fluid Rehydration Strategy on Correction of Acidosis and Electrolyte Abnormalities in Children With Diabetic Ketoacidosis. Diabetes Care. 2021;44(9):2061-8.
Mahler SA, Conrad SA, Wang H, Arnold TC. Resuscitation with balanced electrolyte solution prevents hyperchloremic metabolic acidosis in patients with diabetic ketoacidosis. Am J Emerg Med. 2011;29(6):670-4.
Siwakoti K, Giri S, Kadaria D. Cerebral edema among adults with diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome: Incidence, characteristics, and outcomes. J Diabetes. 2017;9(2):208-9.
Pillai S, Davies G, Lawrence M, Whitley J, Stephens J, Williams PR, et al. The effect of diabetic ketoacidosis (DKA) and its treatment on clot microstructure: Are they thrombogenic? Clin Hemorheol Microcirc. 2021;77(2):183-94.
Wei WT, Lin SM, Hsu JY, Wu YY, Loh CH, Huang HK, et al. Association between Hyperosmolar Hyperglycemic State and Venous Thromboembolism in Diabetes Patients: A Nationwide Analysis in Taiwan. J Pers Med. 2022;12(2).
Burzynski J. DKA and thrombosis. CMAJ. 2005;173(2):132; author reply -3.
Li S, Mikhael B, van Zyl DG. Choice of Intravenous Fluid for Resuscitation in Diabetic Ketoacidosis. N Engl J Med. 2025;392(9):923-6.
Jahangir A, Jahangir A, Siddiqui FS, Niazi MRK, Yousaf F, Muhammad M, et al. Normal Saline Versus Low Chloride Solutions in Treatment of Diabetic Ketoacidosis: A Systematic Review of Clinical Trials. Cureus. 2022;14(1):e21324.
Self WH, Evans CS, Jenkins CA, Brown RM, Casey JD, Collins SP, et al. Clinical Effects of Balanced Crystalloids vs Saline in Adults With Diabetic Ketoacidosis: A Subgroup Analysis of Cluster Randomized Clinical Trials. JAMA Netw Open. 2020;3(11):e2024596.
Lizzo JM, Goyal A, Gupta V. Adult Diabetic Ketoacidosis.StatPearls. Treasure Island (FL)2025.
Gosmanov AR, Gosmanova EO, Kitabchi AE. Hyperglycemic Crises: Diabetic Ketoacidosis and Hyperglycemic Hyperosmolar State. In: Feingold KR, Anawalt B, Blackman MR, Boyce A, Chrousos G, Corpas E, et al., editors. Endotext. South Dartmouth (MA)2000.
Mustafa OG, Haq M, Dashora U, Castro E, Dhatariya KK, Joint British Diabetes Societies for Inpatient Care G. Management of Hyperosmolar Hyperglycaemic State (HHS) in Adults: An updated guideline from the Joint British Diabetes Societies (JBDS) for Inpatient Care Group. Diabet Med. 2023;40(3):e15005.
Kitabchi AE, Murphy MB, Spencer J, Matteri R, Karas J. Is a priming dose of insulin necessary in a low-dose insulin protocol for the treatment of diabetic ketoacidosis? Diabetes Care. 2008;31(11):2081-5.
Thammakosol K, Sriphrapradang C. Effectiveness and safety of early insulin glargine administration in combination with continuous intravenous insulin infusion in the management of diabetic ketoacidosis: A randomized controlled trial. Diabetes Obes Metab. 2023;25(3):815-22.
Dhatariya KK, Joint British Diabetes Societies for Inpatient C. The management of diabetic ketoacidosis in adults-An updated guideline from the Joint British Diabetes Society for Inpatient Care. Diabet Med. 2022;39(6):e14788.
Jiang DH, Herrin J, Van Houten HK, McCoy RG. Evaluation of High-Deductible Health Plans and Acute Glycemic Complications Among Adults With Diabetes. JAMA Netw Open. 2023;6(1):e2250602.
Hassan EM, Mushtaq H, Mahmoud EE, Chhibber S, Saleem S, Issa A, et al. Overlap of diabetic ketoacidosis and hyperosmolar hyperglycemic state. World J Clin Cases. 2022;10(32):11702-11.
Kitabchi AE, Umpierrez GE, Miles JM, Fisher JN. Hyperglycemic crises in adult patients with diabetes. Diabetes Care. 2009;32(7):1335-43.
Arora S, Cheng D, Wyler B, Menchine M. Prevalence of hypokalemia in ED patients with diabetic ketoacidosis. Am J Emerg Med. 2012;30(3):481-4.
Murthy K, Harrington JT, Siegel RD. Profound hypokalemia in diabetic ketoacidosis: a therapeutic challenge. Endocr Pract. 2005;11(5):331-4.
Sibai BM, Viteri OA. Diabetic ketoacidosis in pregnancy. Obstet Gynecol. 2014;123(1):167-78.
Dhanasekaran M, Mohan S, Erickson D, Shah P, Szymanski L, Adrian V, et al. Diabetic Ketoacidosis in Pregnancy: Clinical Risk Factors, Presentation, and Outcomes. J Clin Endocrinol Metab. 2022;107(11):3137-43.
Stathi D, Lee FN, Dhar M, Bobotis S, Arsenaki E, Agrawal T, et al. Diabetic Ketoacidosis in Pregnancy: A Systematic Review of the Reported Cases. Clin Med Insights Endocrinol Diabetes. 2025;18:11795514241312849.
Metzger BE, Ravnikar V, Vileisis RA, Freinkel N. "Accelerated starvation" and the skipped breakfast in late normal pregnancy. Lancet. 1982;1(8272):588-92.
Nayak S, Lippes HA, Lee RV. Hyperglycemic hyperosmolar syndrome (HHS) during pregnancy. J Obstet Gynaecol. 2005;25(6):599-601.
Mohan M BK, Lindow S. Management of diabetic ketoacidosis in pregnancy. . The Obstetrician & Gynaecologist. 2017;19: 55–62.
de Veciana M. Diabetes ketoacidosis in pregnancy. Semin Perinatol. 2013;37(4):267-73.
Dabelea D, Rewers A, Stafford JM, Standiford DA, Lawrence JM, Saydah S, et al. Trends in the prevalence of ketoacidosis at diabetes diagnosis: the SEARCH for diabetes in youth study. Pediatrics. 2014;133(4):e938-45.
Weinstock RS, Xing D, Maahs DM, Michels A, Rickels MR, Peters AL, et al. Severe hypoglycemia and diabetic ketoacidosis in adults with type 1 diabetes: results from the T1D Exchange clinic registry. J Clin Endocrinol Metab. 2013;98(8):3411-9.
Talbot F, Nouwen A. A review of the relationship between depression and diabetes in adults: is there a link? Diabetes Care. 2000;23(10):1556-62.
Gavard JA, Lustman PJ, Clouse RE. Prevalence of depression in adults with diabetes. An epidemiological evaluation. Diabetes Care. 1993;16(8):1167-78.
Skinner TC. Recurrent diabetic ketoacidosis: causes, prevention and management. Horm Res. 2002;57 Suppl 1:78-80.
Cooper H, Tekiteki A, Khanolkar M, Braatvedt G. Risk factors for recurrent admissions with diabetic ketoacidosis: a case-control observational study. Diabet Med. 2016;33(4):523-8.
Mays JA, Jackson KL, Derby TA, Behrens JJ, Goel S, Molitch ME, et al. An Evaluation of Recurrent Diabetic Ketoacidosis, Fragmentation of Care, and Mortality Across Chicago, Illinois. Diabetes Care. 2016;39(10):1671-6.
Umpierrez GE, Smiley D, Kitabchi AE. Narrative review: ketosis-prone type 2 diabetes mellitus. Ann Intern Med. 2006;144(5):350-7.
Lebovitz HE, Banerji MA. Ketosis-Prone Diabetes (Flatbush Diabetes): an Emerging Worldwide Clinically Important Entity. Curr Diab Rep. 2018;18(11):120.
Mauvais-Jarvis F, Sobngwi E, Porcher R, Riveline JP, Kevorkian JP, Vaisse C, et al. Ketosis-prone type 2 diabetes in patients of sub-Saharan African origin: clinical pathophysiology and natural history of beta-cell dysfunction and insulin resistance. Diabetes. 2004;53(3):645-53.
Banerji MA, Chaiken RL, Lebovitz HE. Long-term normoglycemic remission in black newly diagnosed NIDDM subjects. Diabetes. 1996;45(3):337-41.
Maldonado M, Hampe CS, Gaur LK, D'Amico S, Iyer D, Hammerle LP, et al. Ketosis-prone diabetes: dissection of a heterogeneous syndrome using an immunogenetic and beta-cell functional classification, prospective analysis, and clinical outcomes. J Clin Endocrinol Metab. 2003;88(11):5090-8.
Koye DN, Magliano DJ, Nelson RG, Pavkov ME. The Global Epidemiology of Diabetes and Kidney Disease. Adv Chronic Kidney Dis. 2018;25(2):121-32.
Kim HJ. Metabolic Acidosis in Chronic Kidney Disease: Pathogenesis, Clinical Consequences, and Treatment. Electrolyte Blood Press. 2021;19(2):29-37.
Al Sadhan A, ElHassan E, Altheaby A, Al Saleh Y, Farooqui M. Diabetic Ketoacidosis in Patients with End-stage Kidney Disease: A Review. Oman Med J. 2021;36(2):e241.
Galindo RJ, Pasquel FJ, Vellanki P, Zambrano C, Albury B, Perez-Guzman C, et al. Biochemical Parameters of Diabetes Ketoacidosis in Patients with End-stage Kidney Disease and Preserved Renal Function. J Clin Endocrinol Metab. 2021;106(7):e2673-e9.
Sudha MJ, Salam HS, Viveka S, Udupa AL. Assessment of changes in insulin requirement in patients of type 2 diabetes mellitus on maintenance hemodialysis. J Nat Sci Biol Med. 2017;8(1):64-8.
Galindo RJ, Pasquel FJ, Fayfman M, Tsegka K, Dhruv N, Cardona S, et al. Clinical characteristics and outcomes of patients with end-stage renal disease hospitalized with diabetes ketoacidosis. BMJ Open Diabetes Res Care. 2020;8(1).
Seddik AA, Bashier A, Alhadari AK, AlAlawi F, Alnour HH, Bin Hussain AA, et al. Challenges in management of diabetic ketoacidosis in hemodialysis patients, case presentation and review of literature. Diabetes Metab Syndr. 2019;13(4):2481-7.
Gupta A, Rohrscheib M, Tzamaloukas AH. Extreme hyperglycemia with ketoacidosis and hyperkalemia in a patient on chronic hemodialysis. Hemodial Int. 2008;12 Suppl 2:S43-7.
Ting S, Chua HR, Cove ME. Euglycemic Ketosis During Continuous Kidney Replacement Therapy With Glucose-Free Solution: A Report of 8 Cases. Am J Kidney Dis. 2021;78(2):305-8.
Coutrot M, Hekimian G, Moulin T, Brechot N, Schmidt M, Besset S, et al. Euglycemic ketoacidosis, a common and underecognized complication of continuous renal replacement therapy using glucose-free solutions. Intensive Care Med. 2018;44(7):1185-6.
Rajdev K, Leifer L, Sandhu G, Mann B, Pervaiz S, Habib S, et al. Fluid resuscitation in patients with end-stage renal disease on hemodialysis presenting with severe sepsis or septic shock: A case control study. J Crit Care. 2020;55:157-62.
Kanbay M, Copur S, Mizrak B, Ortiz A, Soler MJ. Intravenous fluid therapy in accordance with kidney injury risk: when to prescribe what volume of which solution. Clin Kidney J. 2023;16(4):684-92.
Hurtado CR, Lemor A, Vallejo F, Lopez K, Garcia R, Mathew J, et al. Causes and Predictors for 30-Day Re-Admissions in Adult Patients with Diabetic Ketoacidosis in the United States: A Nationwide Analysis, 2010-2014. Endocr Pract. 2019;25(3):242-53.
Culica D, Walton JW, Prezio EA. CoDE: Community Diabetes Education for uninsured Mexican Americans. Proc (Bayl Univ Med Cent). 2007;20(2):111-7.
Prezio EA, Pagan JA, Shuval K, Culica D. The Community Diabetes Education (CoDE) program: cost-effectiveness and health outcomes. Am J Prev Med. 2014;47(6):771-9.
Lam D, Shaikh A. Real-Life Prescribing of SGLT2 Inhibitors: How to Handle the Other Medications, Including Glucose-Lowering Drugs and Diuretics. Kidney360. 2021;2(4):742-6.

Risk of Fasting and Non-Fasting Hypertriglyceridemia in Coronary Vascular Disease and Pancreatitis

ABSTRACT

Cardiovascular disease (CVD) remains a major cause of mortality in the Western world and in spite of the reduction of CVD risk by the use of lipid lowering agents per current treatment goals there remains substantial residual and absolute risk of CVD in high-risk populations. Focus on elevated triglyceride (TG) levels deserves renewed attention, particularly as one-third of all adults in the United States suffer from elevated TG and a growing number of people are diagnosed with metabolic syndrome or type 2 diabetes mellitus (T2DM). The dyslipidemia of metabolic syndrome and T2DM is characterized by low high-density lipoprotein cholesterol (HDL-c) concentrations and marked elevations in triglyceride rich lipoproteins (TRL). There has been growing data that points towards an association of fasting and non-fasting TGs with CVD, including a number of genetic studies suggesting causality. However, the association of TG as an independent risk faster in CVD is confounded by its inverse metabolic relationship with HDL-c and the heterogeneity of TG lipoproteins. Current guidelines suggest diagnosis of hypertriglyceridemia based on fasting levels where the length of fasting is recommended to be 9-12 hours. Although non-fasting TG levels may be a better indicator of risk, the lack of standardization of non-fasting TG measurements, lack of specific reference ranges, and the variability of postprandial lipid measurements have hampered their routine clinical use. Current guidelines focus mainly on LDL-c levels; however, lowering TG may provide additional benefit for CVD prevention. Lifestyle changes including dietary changes and exercise play an important role in the treatment of hyperlipidemia. Pharmacological agents used in the treatment of hypertriglyceridemia include niacin, fibrates, fish oil, and statins. Most guidelines recommend treating elevated TG for prevention of pancreatitis. This chapter will discuss the role of elevated TG in pancreatitis and CVD risk.

INTRODUCTION

Cardiovascular disease (CVD) remains a major cause of mortality in the Western world, especially in individuals with obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM). With increasing incidence of the metabolic syndrome and T2DM worldwide, the global burden of CVD will also increase (1). In spite of the reduction of CVD risk by 25-35% with the use of lipid lowering agents, especially statins, there remains substantial residual and absolute risk of CVD in high-risk populations such as T2DM (2). Elevated low-density lipoprotein (LDL-c) is a well-established CVD risk factor and has been the primary target for lipid lowering treatment. However, growing evidence suggests that an elevated triglyceride (TG) level is also an independent risk factor (3,4). Borderline high TGs or high TGs defined by National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) as TG concentration (150 – 199 mg/dL) and (200 – 499 mg/dL) respectively are present in 30% of the US adult population (5) and these levels have been associated with increased risk of CVD. The dyslipidemia of metabolic syndrome and T2DM is characterized by raised TG concentrations, low high-density lipoprotein cholesterol (HDL-c) concentrations and marked elevations in triglyceride rich lipoproteins (TRL). This triad is termed mixed or atherogenic dyslipidemia (6). Impaired metabolism of TRLs in the postprandial state have been observed in insulin resistant states such as visceral obesity, metabolic syndrome, and T2DM and this has been linked to the development of atherosclerosis (7).

Severe hypertriglyceridemia and very severe hyperlipidemia defined by Endocrine Society Clinical Practice Guideline on Evaluation and Treatment of Hypertriglyceridemia as TG concentration (1000 – 1999 mg/dL) and (>2000 mg/dL) carries an increased risk for pancreatitis (8). Case series and uncontrolled studies have shown that severely elevated TG levels are associated with the chylomicronemia syndrome and an increased risk of pancreatitis. Serum TG levels of 1000 mg/dL and higher have been observed in 12% to 38% of patients with acute pancreatitis (9). Hence, understanding the role of hypertriglyceridemia in CVD, chylomicronemia syndrome and risk of pancreatitis is important.

METABOLISM OF TRIGLYCERIDE RICH LIPOPROTEINS AND TRIGLYCERIDEMIA

Triglyceride Rich Lipoprotein Metabolism

Triglyceride rich lipoproteins (TRL) consist of chylomicrons carrying triglycerides from the diet, VLDLs synthesized in the liver, and their respective remnant particles. After a fatty meal, dietary triglycerides are hydrolyzed in the intestine to free fatty acids and monoglycerides. Fatty acids and monoglycerides are then absorbed by enterocytes and resynthesized to form triglycerides. Triglycerides within the intestinal enterocytes are assembled with apolipoprotein (apo) B-48 into large chylomicrons which are released from the cells into the lymphatic system. They access the plasma via the thoracic duct and are rapidly metabolized by lipoprotein lipase (LPL) to yield chylomicron remnants. These are taken up by remnant receptors and by LDL receptors in the liver. Free fatty acids liberated by the action of LPL are available to adipose tissue for storage and to other tissues (e.g., skeletal muscle, heart) for use as energy substrates. Lipids derived from chylomicron remnants, from de novo lipid synthesis, and from lipolysis of adipose tissue are reassembled in the liver as very-low-density lipoprotein (VLDL) particles, which are secreted into the plasma. VLDL particles are metabolized by LPL to yield intermediate density lipoprotein (IDL) particles, which are metabolized by LPL and hepatic lipase to yield low density lipoprotein (LDL) particles. IDL can be taken up by the liver through an apo E-dependent process, and LDL is taken up by the liver through the binding of apoB100 to LDL receptors. Small VLDL particles, IDL particles, and LDL particles may be taken up by peripheral tissues to deliver nutrients, cholesterol, and fat-soluble vitamins (10-12).

Hypertriglyceridemia

Hypertriglyceridemia is a normal physiological state that occurs post ingestion of a meal where lipids undergo the above-mentioned metabolism. In insulin- resistant states there is an exaggerated lipid response leading to pathological hypertriglyceridemia which is thought to be atherogenic. In insulin-resistant states there is an increase in the production of VLDL by the liver and decreased hepatic uptake of VLDL, IDL and LDL. There is a reduction in LPL activity resulting in high triglyceride concentrations, especially in the postprandial state. The over secretion of VLDL, which competes with chylomicron remnants for clearance through the common pathway, can exacerbate the post prandial response. The large amount of TRLs and their prolonged residence time in the circulation leads to increased exchange of cholesteryl ester for triglycerides between TRL and LDL or HDL particles mediated by cholesteryl ester transfer protein (CETP). This process enriches LDL and HDL with triglyceride, and these particles are subsequently more readily hydrolyzed by hepatic lipase resulting in smaller, denser LDL particles and lower concentrations of HDL (13). These abnormalities result in a characteristic dyslipidemia in insulin resistant states, which is now recognized to be atherogenic.

Apo C-III

Apolipoprotein C-III (APOC3) has a key role in lipoprotein metabolism and regulation of triglyceride levels. APOC3 is synthesized in the liver and transported on triglyceride-rich lipoproteins. It inhibits LPL mediated hydrolysis of triglycerides, and at high concentrations can also inhibit hepatic lipase activity. In addition, APOC3 impairs the hepatic uptake of triglyceride rich lipoproteins by remnant receptors. Thus, increased APOC3 levels are an independent risk factor for CVD (14-16); genetic variants of APOC3 leading to lower levels are associated with a reduced risk for CVD (17-19).

ANGPTL

Angiopoietin-like proteins (ANGPTL) are regulators of lipoprotein metabolism. ANGPT3 and ANGPTL4 are natural inhibitors of LPL. Loss of function variants in these proteins have been associated with decreased triglyceride levels and decreased CVD. Murine studies have found that the suppression of ANGPTL3 decreases atherosclerosis (20).

Severe Hypertriglyceridemia

In severe or very severe TG levels (> 1000 mg/dL), which occur as a result of defective lipolysis or excessive production of endogenous triglyceride, the LPL removal system is saturated. There is decreased degradation of dietary TGs incorporated into chylomicrons and a rapid increase of TG levels post fat-rich meals (worsened by dietary simple sugars, fructose, and alcohol) in susceptible individuals causing pancreatitis. The mechanism by which hypertriglyceridemia causes pancreatitis is not understood, but could include local accumulation of free fatty acids and serum hyperviscosity (8).

HYPERTRIGLYCERIDEMIA AND RISK OF PANCREATITIS AND CHYLOMICRONEMIA SYNDROME

Severe hypertriglyceridemia (> 1000 mg/dL) is an infrequent laboratory finding and is generally associated with genetic disorders of lipid metabolism or TG levels exacerbated by secondary causes. Familial chylomicronemia is a rare monogenic disorder that can cause severe hypertriglyceridemia. It is defined as the presence of chylomicronemia (TG > 1000 mg/dL) along with one or more of the following: eruptive xanthomas, lipemia retinalis, or abdominal findings of pain, acute pancreatitis and/or hepatosplenomegaly (21). Multifactorial chylomicronemia syndrome (MFCS) which is a much more common cause of severe hypertriglyceridemia is caused by the accumulation of genetic, non-genetic, and environmental factors. Individuals with severe hypertriglyceridemia may present with these classic findings and pancreatitis or may be asymptomatic. The mechanisms by which hypertriglyceridemia may lead to acute pancreatitis are not known. Possible mechanisms include intra-pancreatic hydrolysis of high triglycerides by pancreatic lipase leading to accumulation of fatty acids in the pancreas which may be toxic and lead to inflammation and ischemia. Another proposed mechanism is increased viscosity by high chylomicron levels leading to ischemia (22). A study looking at the frequency of signs and symptoms of hypertriglyceridemia including pancreatitis found that the incidence of pancreatitis and eruptive xanthomas was low unless TG levels were significantly elevated, e.g. > 1700 mg/dL (20 mmol/l); patients with extreme hypertriglyceridemia had a combination of primary and secondary factors (T2DM, obesity, alcohol intake, pregnancy) contributing to their high TG levels (23). Murphy et al. in their cohort study estimated the risk of pancreatitis with differing degrees of TG elevations and showed that the crude incidence of pancreatitis was 0.91 per 1000 person years (95% CI, 0.76 – 1.09) in individuals with TG levels <150 mg/dL, 1.24 (95% CI, 1.07 – 1.44) with TG levels 150 – 499 mg/dL and 2.48 (95% CI, 1.79 – 3.42) with TG levels >500 mg/dL (24). Increased incidence is seen with increased TG levels. The level of TG at which pancreatitis can be attributed to hypertriglyceridemia is not well defined nor is the level of TG reduction that is associated with reduced risk known. A study by Lindkist et al. (22) looked at the association of moderately elevated serum TG levels and acute pancreatitis. In this study, 33,260 individuals were followed for a median 25.7 years where overall incidence of acute pancreatitis in the cohort was 35.5/100,000-person years. There was a statistically significant association between TG levels and risk of pancreatitis with adjusted HR for pancreatitis of 1.21 (95% CI, 1.07 – 1.36) per 1 mmol/l (~88.5 mg/dL) increment in TG and a significant increased risk for acute pancreatitis in individuals with TG levels > 1.64 mmol/l (145 mg/dL). The analysis in this study was restricted to individuals with TG levels < 6 mmol/l (530 mg/dL) producing statistically significant results and showing that TG levels much lower than previously believed can be associated with an increased risk of acute pancreatitis. Another study evaluated the association between lower follow-up TG levels and the incidence of recurrent clinical events for patients with severe hypertriglyceridemia (>500 mg/dL). This study included 41,210 individuals with < 1% having a history of pancreatitis. Individuals with severe hypertriglyceridemia with follow up TG levels <200 mg/dL experienced a lower rate of recurrent pancreatitis episodes, with an adjusted rate ratio of 0.45 (95% CI, 0.34 – 0.60) compared to those with TG levels >500 mg/dL (25). There is an increased risk of pancreatitis with severe hypertriglyceridemia and in individuals with elevated dietary TG levels and in some cases pharmacological intervention is necessary to prevent severe complications such as pancreatitis.

HYPERTRIGLYCERIDEMIA AS A CARDIOVASCULAR RISK FACTOR

Epidemiological Data Supporting TG as a CVD Risk Factor

The association of elevated TG values with CVD remains controversial. Establishing TG level as an independent risk factor for CVD is confounded by its inverse metabolic relationship with HDL-c and the heterogeneity of TG risk lipoproteins. Growing evidence suggests that an elevated TG level is an independent risk factor for CVD and represents an important biomarker of CVD risk because of their association with atherogenic remnant particles. A meta-analysis by Hokanson and Austin (4), showed increased plasma TG levels are associated with a significant increase in the risk of CVD independent of HDL-c level. An overall relative risk (RR) for CVD of 1.32 for men and 1.76 for women per 1 mmol/L (~88.5 mg/dL) increase in TGs was noted. However, this analysis was limited to Caucasian study subjects. A non-overlapping meta-analysis involving data from 26 prospective studies in Asian and Pacific populations reported a RR for CVD of 1.8 (95% CI, 1.49 – 2.19), comparing subjects in the top fifth with the bottom fifth of TG levels (26). Sarwar et al. (27) reported data from two prospective cohort studies: the Reykjavik study and the European Prospective Investigational of Cancer (EPIC) - Norfolk study, which together comprised 44,237 Western middle-aged men and women and a total of 3582 incident cases of CVD. Comparing individuals with TGs in the top tertile with the bottom tertile, the adjusted odds ratio for CVD was 1.76 (95% CI, 1.39 – 2.21) in the Reykjavik study and 1.57 (95% CI, 1.10 – 2.24) in the EPIC-Norfolk study. However, adjustment for HDL-c substantially attenuated the magnitude of association of TG level with CVD. They also performed an updated meta-analysis of the Western population studies adding information to include a total of >10,000 CVD cases from 29 Western prospective studies involving a total of > 260,000 participants, and report an adjusted odds ratio of 1.72 (95% CI, 1.56 – 1.90) comparing top and the bottom tertiles of TG values (27). A more recent meta-analysis by Murad, et al. (9), included 35 studies with a total of 927,218 subjects who suffered 132,460 deaths and 72,654 cardiac events, myocardial infarctions or pancreatitis; with odds ratio of 1.80 (95% CI, 1.31 – 2.49) for cardiac events, 1.31 (95% CI, 1.15 – 1.49) for myocardial infarctions, and 3.96 (95%, CI 1.27 – 12.34) for pancreatitis.

Genetic Data Linking TG to CVD

Recent human genetic studies show that elevated TGs and TRLs are causal risk factors for CVD. A Mendelian randomization study based on several genetic variants affecting remnant cholesterol and/or HDL showed that a 1 mmol/l (39 mg/dL) increase in non-fasting remnant cholesterol is associated with a 2.8-fold causal risk for ischemic heart disease, independent of reduced HDL cholesterol (28). A meta-analysis of 46 lipid genome-wide-association studies (GWAS) together comprising >100,000 individuals of European descent identified four novel loci associated with CVD that were related to HDL-c and TG levels suggesting elevated TG metabolism may also be associated with CVD risk (29). Another large Mendelian randomization study based on a single APOA5 variant (-1131T>C) that regulates TG showed an association with CVD risk. The odds ratio for coronary heart disease was 1·18 (95% CI 1·11–1·26; p=2·6×10⁻⁷) per C allele, which was concordant with the hazard ratio of 1·10 (95% CI 1·08–1·12) per 16% higher TG concentration recorded in prospective studies (30). This finding is similar to that seen in the study by Jorgensen et al. (31), where doubling of genetically raised remnant cholesterol and TG levels due to APOA5 genetic variants was associated with an increased risk of myocardial infarctions. In addition, a study using individuals from the Copenhagen City Heart Study with genetic variants in lipoprotein lipase (LPL), tested whether low concentrations of non-fasting TG were associated with reduced all-cause mortality in observational analyses (n = 13,957). The results showed that each genetically-derived 1 mmol/l (~88.5 mg/dL) reduction in TG levels was associated with a halved risk of all-cause mortality (32). Two large studies examining the relationship between the gene encoding apolipoprotein C3 (APOC3) found that loss of function mutations in APOC3 were associated with low levels of TG and reduced risk of CVD (17,19). ANGPTLs have also been linked to CVD. A large human study found that individuals with loss of function variants in ANGPTL3 had lower TG levels, as well as lower levels of HDL-c and LDL-c compared to control subjects, and decreased odds of CVD (20). Similarly, individuals with loss of function variants of ANGPTL4 also had lower TG levels and decreased risk for CVD (33). A study by Ference et al did a randomization analysis of a total of 654,783 participants to determine if there was any association with risk of CVD per unit of change in ApoB from either LDL-C lowering variants in the LDL receptor gene (LDLR) or TG-lowering variants in the lipoprotein lipase gene. It showed that there was a similar decrease in risk of CVD per unit difference in ApoB from either TG-lowering LPL variants or LDL-C lowering LDLR variants. Due to differences in the composition of LDL and VLDL, TG levels must be decreased to a much greater extent than LDL-c to achieve a comparable decrease in ApoB levels. This has important implications in the interpretation of TG lowering trials, as studies may not sufficiently lower TG to effectively decrease ASCVD risk. To be noted, the results from this study are from genetic variants and not lipid lowering therapies (34). A genetic cause of lower LDL-c confers a lifetime reduction with associated decline of ASCVD risk up to three to four times greater than with statin therapy, which has a shorter period of lower LDL-c. This finding is likely similar for TG. Collectively, these studies strongly point to a causal effect of elevated TG and TRLs with CVD.

Clinical Trial Evidence Supporting Lowering TG Reduces CVD

Results with clinical outcomes trials of fibrate therapy have been variable but primarily indicate a reduction in CV events. Post hoc analysis of several of these trials provides consistent evidence showing a clinical benefit in subgroups with elevated TG levels. A meta-analysis of the effect of TG lowering in 18 trials providing data for 45,058 participants showed that fibrate therapy produced a 10% RR reduction (95% CI 0-18) for major cardiovascular events (p=0.048) and a 13% RR reduction (95% CI 7-19) for coronary events (p<0.0001) (35). The Helsinki Heart Study (HHS) , a primary prevention trial showed that in an average follow up of 5 years, there was a 34% (95% CI 8.2-52.6, P =0.02) RR reduction in CVD in those treated with gemfibrozil compared to placebo (36). In an 18 year follow up of this study, individuals randomized to gemfibrozil had a 24% adjusted RR reduction (p=0.05) in CVD, and individuals with elevated TG and body mass index (BMI) showed significant benefit from treatment with gemfibrozil. Those with TG level in the highest tertiles had a 71% lower RR of CVD mortality (p <0.001) (37). The Bezafibrate Infarction Prevention (BIP) study assessed the role of fibrates in secondary prevention. The initial reports showed no significant RR reductions in CVD outcomes in bezafibrate treated vs. placebo-treated subjects. However, a post-hoc analysis of individuals with TG >200 mg/dL demonstrated significant RR reduction by 39.5% (p=0.02); there was no significant RR in those with TG values < 200 mg/dL (38,39). In a subgroup analysis of patients in the BIP study by Tenenbaum at al. (40), patients with CVD, metabolic syndrome and TG > 150 mg/dL experienced significant benefits from the treatment with bezafibrate. Bezafibrate was associated with a reduced risk of any MI and nonfatal MI with HRs of 0.71 (95% CI, 0.54-0.95) and 0.67 (95% CI, 0.49-0.91), respectively. The cardiac mortality risk tended to be lower in patients taking bezafibrate (HR, 0.74; 95% CI, 0.54-1.03). The Veterans Affairs High-Density Lipoprotein Intervention (VA-HIT) involved 2531 males with CVD with low HDL-c and relatively low LDL-c who were treated with gemfibrozil or placebo and monitored for 5.1 years. Gemfibrozil safely reduced the risk of death from CVD or nonfatal myocardial infarction by 22 percent (41). For every 100 mg/dL increment in baseline TG there was a 14% increase in coronary risk (p=0.045). Further, those with highest tertile of TG values (>180 mg/dL) exhibited a more marked decrease in coronary risk with gemfibrozil compared with those in lower tertiles (42). In the Action to Control Cardiovascular Risk in Diabetes (ACCORD) lipid study using statin + fibrate combination therapy, fenofibrate + simvastatin had no effect on the primary outcome vs. simvastatin alone for all patients. However, in the fenofibrate + simvastatin group, there was a 31% reduction in CV risk in the subgroup with baseline TG levels in the upper tertile vs. simvastatin monotherapy (43).

Pemafibrate, a new selective PPAR-α activator, approved in Japan in 2017 for hyperlipidemia, is metabolized by cytochromes CYP2C8, CYP2C9, and CYP3A4, indicating potential drug interactions (44). The Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT) trial evaluated its cardiovascular benefits in patients with high TGs, including those with diabetes. While pemafibrate significantly reduced TGs, remnant cholesterol, and apo C-III after 4 months, it also raised LDL cholesterol and apo B and did not improve HDL cholesterol. Adverse effects included renal issues and a risk of venous thromboembolism (45). This trial found no significant difference in major cardiovascular events among patients treated with pemafibrate compared to placebo. Despite initial promise, the trial's findings diminished support for combining fibrates like pemafibrate with statins for cardiovascular risk reduction.

PPAR alpha activators may decrease atherosclerosis by various pathways, not limited to affecting circulating lipid levels. Plutzky and Zandbergen discuss the reduction of acute phase reactants produced in the liver, including C-reactive protein, serum amyloid A, and fibrinogen, by PPAR alpha agonists. These agents may also limit endothelial dysfunction through inhibition of the vasoconstrictor endothelin-1 and by promoting vasodilation by enhanced expression of endothelial nitric oxide synthase. PPARα agonists may also limit early atherogenesis by inhibiting cytokine-induced expression of a key molecule, vascular adhesion molecule-1, required for adhesion of leukocytes to injured vasculature (46). Additionally, PPAR alpha activators may activate PPARs differently, resulting in variable effects.

The Reduction of Cardiovascular Events with Icosapent Ethyl–Intervention (REDUCE-IT) and Statin Residual Risk with Epanova in High Cardiovascular Risk Patients with Hypertriglyceridemia (STRENGTH) trials present examples of conflicting cardiovascular outcomes in patients receiving omega-3 fatty acid therapy. The REDUCE-IT trail with 8,179 patients showed a decrease in major cardiac events with high dose icosapent ethyl (4g/d EPA), while the STRENGTH trial with 13,086 patients showed no effects on cardiac events with high dose EPA/DHA carboxylic acid (4g/d) (47,48). A difference in the two studies could be due to the EPA levels achieved in each study. REDUCE-IT achieved an EPA level of 144 (mg/mL) while the STRENGTH trial achieved an EPA level of 89.6 (mg/mL) (49). Another issue is that in the REDUCE-IT trial, mineral oil was used as the placebo in the control group which resulted in higher atherogenic lipoproteins in that arm. This raised concerns that both the negative effects from the mineral oil in the control group and the positive effects of EPA in the treatment group underlies the observed reduction in major cardiac events seen in the trial. However, Olshansky et al reviewed eight studies that used mineral oil as a placebo which showed no evidence that mineral oil in the dosage used in the REDUCE-IT trial had any effect on clinical outcomes (50). Given the opposite results of the two trials, further investigation is needed in regards to EPA levels and drug formulations. A secondary analysis of the STRENGTH trial showed no cardiovascular benefits at the highest levels of DHA or EPA (51). A meta-analysis which includes the REDUCE-IT and STRENGTH trails show that there was a higher risk of atrial fibrillation in groups treated with omega-3 fatty acids than placebo (52). These studies cast doubt on the benefits of lowering TG levels to reduce ASCVD and necessitate further evaluation in the potential adverse effects of omega 3 fatty acids.

Collectively, studies suggest that fibrate monotherapy leading to a reduction in TG levels prevents coronary events. Many of these studies also show improvements in HDL-c levels which may contribute to the improvements in CVD seen; however, recent HDL-c raising studies (using CETP inhibitors) have not found improved cardiovascular benefits suggesting that the decrease in TG levels contributed to the reduction of CVD seen in the fibrate studies. It has been difficult to demonstrate an ASCVD benefit with TG lowering in patients taking statins. At present, guidelines have not been recommending TG lowering in patients with elevated TG levels to reduce CVD. The lack of evidence to support an ASCVD benefit for TG lowering in patients already using statins may be due to insufficient lowering of TG levels, effects that counter the beneficial effects of lowering TG levels (e.g. in the PROMINENT trial non-HDL-c increased), or perhaps inclusion of inappropriate study populations.

Post-Prandial TG as a CVD Risk Factor

There is also growing evidence that postprandial hypertriglyceridemia may be a better indicator of the presence or development of CVD than fasting hypertriglyceridemia. In the Women’s Health Study, a prospective cohort of 26,330 initially healthy women with over 11 years of follow up, it was observed that higher non-fasting TG levels were strongly associated with an increased risk of future cardiovascular events independent of baseline cardiac risk factors, levels of other lipids, and markers of insulin resistance. The concentrations of lipids and apolipoproteins differed minimally when measurements were performed on non-fasting compared to fasting blood samples, except for TG, which were higher when non-fasting. There was a > 4-fold increased risk of a cardiovascular event among individuals with postprandial TG concentrations peaking at 2-4 hours following a meal. This study showed that HDL-c, TG, total cholesterol/HDL-c ratio, and apolipoprotein B predict CVD when measured in non-fasting samples. By contrast, total cholesterol, LDL, and non-HDL cholesterol, in addition to apolipoprotein B-100 and B-100/A-I ratio, may provide less useful CVD risk information when measured non-fasting (53,54). In a Norwegian study which included 42,600 women and 43,641 men ages 20 – 50 years at inclusion, with a mean follow-up of 27 years, non-fasting TG were positively associated with CVD death in both genders, with hazard ratios being higher in women than in men. However, after adjustment for cholesterol, systolic blood pressure, and smoking, and in a sub-sample also HDL-c, the associations were distinctly attenuated (55). In another study, the Copenhagen City Heart Study, a prospective cardiovascular study of the Danish general population initiated in 1976, 7581 women and 6391 men who had lipids measured at baseline in 1976-1978, were followed for up to 31 years without losses to follow-up, and most were not taking lipid-lowering therapy. The study found that the cumulative incidence of myocardial infarction, ischemic heart disease, and death increased with increasing levels of non-fasting TG levels. Non-fasting TG level were a better predictor of coronary heart disease in women whereas non-fasting cholesterol level was a better predictor in men. However, non-fasting cholesterol levels were not found to be associated with total mortality (56,57). A Japanese study which included 4,988 participants with diabetes already on statin therapy, evaluated the relationship between fasting and non-fasting TGs and cardiovascular events. It showed that cardiac events were associated with elevated fasting and non-fasting TG. However, the study found that non-fasting TG was more helpful for risk assessment for future CVD instead of fasting TG (58). Data from these studies provide evidence for a link between non-fasting TG and cardiovascular disease and support the concept that non-fasting TG levels may strongly predict the risk of cardiovascular events.

Mechanisms by Which TG are a CVD Risk Factor

The exact mechanism by which TG may promote vascular disease remains to be elucidated. A possible explanation for TG being associated with increased CVD is that elevated levels of postprandial TG may indicate a high content of TRLs derived from chylomicrons and VLDL. Given their relatively small size, these TRLs can enter the arterial wall, and contribute to the formation of foam cells and thus cause atherosclerosis. The remnant particles under normal conditions are rapidly taken up by the liver. However, in people with the metabolic syndrome or T2DM, hepatic clearance of remnant particles can be delayed and thus there is a predisposition towards increased production of remnant particles and small dense LDL and HDL particles. Thus, increased production along with prolonged exposure of circulating remnant particles enhances the possibility for the particles to be trapped in the arterial wall. Accordingly, remnant lipoproteins have been shown to increase the risk of atherosclerotic heart disease. This suggests a need to direct attention towards diagnosis and treatment of high TG levels in conjunction with treating high cholesterol levels (59). These studies also draw importance to further investigate independent association of fasting and non-fasting hypertriglyceridemia in CVD.

PREVALENCE AND ASSESMENT OF HYPERTRIGLYCERIDEMIA

Prevalence of Hypertriglyceridemia

There is high prevalence of hypertriglyceridemia in the US which necessitates periodic assessment of TG levels, especially in individuals with increased risk. A study looking at 5680 subjects, greater than or equal to 20 years of age who participated in the National Health and Nutrition Examination Survey from 2001 and 2006 evaluated the epidemiology of adults with hypertriglyceridemia. This study reports about 67.8% of the study participants had a normal TG level (<150 mg/dL), 14.2% had borderline high TG levels (150 – 200 mg/dL) and 16.3% had high TG levels (200 - 500 mg/dL). The prevalence of severe high TG (500 – 2000 mg/dL) was noted to be 1.7% equating to about 2.4 million Americans. Three participants were noted to have TG levels > 2000 mg/dL. The participants with severe high TG tended to be men (75.3%), non-Hispanic whites (70.1%), and aged 40 to 59 years (58.5%), and more than 14% of those reported having diabetes mellitus, and 31.3% reported having hypertension (60). A study published in 2018 surveyed 9593 American adults between 2007-2014 to determine the TG levels in patients taking and not taking a statin. It showed almost one-third of people taking statins have unsatisfactory TG levels, as well as one-fourth of overall US adults (61).

Assessment of Hypertriglyceridemia

Plasma lipids and lipoproteins are generally measured in the fasting state and guidelines for therapy for CVD prevention are based on these measurements. The Endocrine Society clinical practice guidelines on evaluation and treatment of hyperlipidemia suggest diagnosis of hypertriglyceridemia based on fasting levels where length of fast is recommended to be 12 hours (8). In insulin resistant states postprandial TG may be more relevant to CVD risk. To assess postprandial TG there is a need to identify an accurate and standardized methodology to measure postprandial triglycerides and TRLs. Currently, the lack of standardization of non-fasting TG measurements, lack of specific reference ranges, and the variability of postprandial lipid measurements have hampered their routine clinical use (62). A Fat Tolerance Test (FTT) has been used to assess post prandial lipoproteins. An expert panel suggests that individuals with fasting TG concentrations between 1-2 mmol/l (89-180 mg/dL) would have better risk assessment by being tested with a FTT than with just fasting TG. Individuals with fasting TG concentration of less than 1 mmol/l (88.5 mg/dL) commonly do not have exaggerated and delayed response of TGs to a FTT, whereas individuals with elevated fasting TG values above 2 mmol/l (180 mg/dL) are expected to have an exaggerated and delayed response of TG to a FTT. These two patient populations would not benefit from a FTT for better risk assessment(63).

Fat Tolerance Testing

Given that humans spend most of their awake time in a post prandial state, various factors including fasting concentrations of serum TGs, time of the day when test is undertaken, the fat content and quality of FTT need to be considered. An expert panel statement recommends measuring total TGs to evaluate the post prandial lipemia response 4 hours after a standardized FTT performed after an 8 hour fast. There has been significant variability in the fat- rich meals used for FTT ranging from dairy products, eggs, oils, to liquid formulations. An expert panel suggests a FTT meal consisting of 75 g fat including both saturated and unsaturated fatty acids (63). ApoB-48 is an alternative marker for the assessment of post prandial hypertriglyceridemia as it measures the number of circulating chylomicrons and their remnants after a meal (there is one ApoB-48 per chylomicron particle). The level of ApoB-48 is very low compared to ApoB-100 in the fasting state but it increases after a FTT. However, the lack of internationally recognized standardized assays and reference ranges, limited availability of the ApoB-48 assay, and high costs limit the utilization of ApoB-48 in clinical settings (62,64).

Secondary Causes of Hypertriglyceridemia

Individuals found to have any elevation of fasting TG should be evaluated for secondary causes including endocrine conditions and medications (Table 1) (65,66). Patients with untreated diabetes, obesity, and insulin resistant states commonly have elevated TG levels (67,68). Other endocrine disorders such as hypothyroidism, Cushing’s disease, and growth hormone deficiency can also be associated with elevated TG levels (8). TG levels can also significantly increase during pregnancy owing to estrogen-induced stimulation of the secretion of hepatic TRLs (69). In women with underlying disorders of TG metabolism, this increase in TG levels during pregnancy can be associated with pancreatitis and fetal loss. Alcohol intake increases hepatic fatty acid synthesis and decreases breakdown resulting in increased hepatic VLDL secretion and hypertriglyceridemia. Lipodystrophies, either primary or as seen in HIV treated patients or with other diseases is also associated with hypertriglyceridemia (8). There are several monogenic autosomal recessive disorders that lead to hypertriglyceridemia (table 1). LPL deficiency, apo CII deficiency, and GPIHBP1 loss of function mutations (or antibodies to LPL or GPIHBP1) are associated with impaired LPL activity and present in young patients with an increased risk of chylomicronemia and pancreatitis. Additional genetic syndromes in the differential diagnosis of hypertriglyceridemia include mixed or familial combined hyperlipidemia (FCHL), type III dysbetalipoproteinemia, and familial hypertriglyceridemia (FHTG) (70). Many patients have multiple genetic variants and combined with environmental factors, can cause hypertriglyceridemia. However, a study with 563 patients with severe hypertriglyceridemia showed that 14.4% had heterozygous rare variants known to cause hypertriglyceridemia while 3.8% of the control group had these variants as well indicating that these variants are incompletely or partly penetrant. Polygenic risk can now be assessed by a polygenic score. An elevated score increases the probability of developing hypertriglyceridemia, but it is not an absolute predictor of developing hypertriglyceridemia (71). Many drugs also raise triglyceride levels (table 1). Oral estrogens increase the hepatic secretion of VLDL causing an increase in serum TG levels (72). Other medications include Tamoxifen/Raloxifene, retinoids, beta blockers, thiazide inhibitors, corticosteroids, immunosuppressants, antipsychotics, and antiretroviral protease inhibitors (8). Clomiphene which has been successfully used to aid fertility in women with certain anovulatory disorders, is a synthetic estrogen analog whose biochemical structure is similar to that of tamoxifen. It has been noted to induce severe hypertriglyceridemia and pancreatitis in patients with baseline hypertriglyceridemia (73). If possible, individuals with secondary hypertriglyceridemia should have the secondary cause addressed, and such individuals may then not need primary, TG-lowering therapy. However, secondary causes of hypertriglyceridemia cannot always be addressed, in which case providers should consider TG-lowering therapy.

Table 1. Causes of Hypertriglyceridemia
Disorders	Drugs	Monogenic*
Hypothyroidism Uncontrolled Diabetes Obesity Chronic renal failure Nephrotic syndrome Pregnancy HIV Cushing’s syndrome Lipodystrophy Inflammatory disease – rheumatoid arthritis, lupus, psoriasis, etc.	Alcohol Estrogens Beta blockers Tamoxifen/Raloxifene Glucocorticoids Atypical anti-psychotics Cyclosporine Protease inhibitors Clomiphene	Lipoprotein lipase deficiency Apolipoprotein CII deficiency Apolipoprotein AV deficiency GPIHBP1 deficiency Lipase Maturation factor 1 (LMF1)

Table 1. Causes of Hypertriglyceridemia

Disorders

Drugs

Monogenic*

Hypothyroidism

Uncontrolled Diabetes

Obesity

Chronic renal failure

Nephrotic syndrome

Pregnancy

HIV

Cushing’s syndrome

Lipodystrophy

Inflammatory disease – rheumatoid arthritis, lupus, psoriasis, etc.

Alcohol

Estrogens

Beta blockers

Tamoxifen/Raloxifene

Glucocorticoids

Atypical anti-psychotics

Cyclosporine

Protease inhibitors

Clomiphene

Lipoprotein lipase deficiency

Apolipoprotein CII deficiency

Apolipoprotein AV deficiency

GPIHBP1 deficiency

Lipase Maturation factor 1 (LMF1)

*autosomal recessive disorders

GUIDELINES FOR TRIGLYCERIDE EVALUTION AND MANAGEMENT

The Endocrine Society Clinical Guidelines

The Endocrine Society Guidelines recommend that the diagnosis of hypertriglyceridemia be based on fasting serum triglyceride levels and defines TG levels of 150 to 199 mg/dL as mild hypertriglyceridemia; 200 to 999 mg/dL as moderate; 1,000 to 1,999 mg/dL as severe; and 2,000 mg/dL or greater as very severe hypertriglyceridemia. The screening for elevated TG levels for all adults is recommended as part of a lipid panel at least every five years. These guidelines recommend against the routine measurement of lipoprotein particle heterogeneity. The guidelines also recommend screening patients with hypertriglyceridemia for secondary causes (medications, alcohol use, endocrine diseases, renal disease, liver disease) and that patients with primary hyperlipidemia be evaluated for family history of dyslipidemia and CVD. The guidelines recommend the use of non-HDL-c (goal 30 mg/dL higher than the LDL-c goal) for both risk stratification and as a target for therapy in patients with moderate hypertriglyceridemia. Initial treatment of patients with mild to moderate hypertriglyceridemia should include lifestyle therapy. For patients with severe to very severe hypertriglyceridemia, dietary modifications in combination with drug treatment should be considered. A fibrate is recommended as a first-line agent in patients with severe or very severe hyperlipidemia (8).

American Association Of Clinical Endocrinologists (AACE) Guidelines

Similar to other guidelines, the AACE clinical practice guidelines recommend evaluating all adults >20 years of age for dyslipidemia every 5 years for risk assessment with a fasting (9 to 12-hour fast) lipid profile. In addition, it recommends more frequent assessments for patients with a family history of premature CVD. However, unlike other guidelines, AACE also recommends Apo B measurements to assess for residual risk in patients with increased TG levels (>150 mg/dL) or low HDL-c levels (< 40 mg/dL). TG levels less than 150 mg/dL are defined as normal, 150 – 199 mg/dL as borderline high, 200 – 499 mg/dL as high, and levels >500 mg/dL or greater as very high, and AACE recommends maintaining TG levels less than 150 mg/dL. Fibrates are recommended for the treatment of severe hypertriglyceridemia (>500 mg/dL) and lifestyle changes including physical activity, weight loss, and smoking cessation are recommended as first line therapy in moderate hypertriglyceridemia (74). For adults with hypertriglyceridemia and ASCVD or at increased risk of ASCVD, the use of eicosapentaenoic acid (EPA) is recommended in addition to statins, but not combinations of EPA and docosahexaenoic acid (DHA), and niacin use is strongly discouraged. There is insufficient evidence to support pharmacologic treatment recommendations for adults with severe hypertriglyceridemia (≥500 mg/dL) (75).

American College of Cardiology (ACC) Statement on Triglycerides and Cardiovascular Disease

In 2021 the ACC published an Expert Consensus Decision Pathway on the management of ASCVD risk reduction in patients with persistent hypertriglyceridemia. In this statement, persistent hypertriglyceridemia is defined as fasting triglycerides ≥150 mg/dL despite 4–12 weeks of lifestyle changes, stable use of maximally tolerated statins (if indicated), and management of secondary causes. Before starting non-statin therapies, at least two fasting lipid panels taken at least two weeks apart should guide clinical decisions. Both fasting and non-fasting lipid profiles are acceptable for ASCVD risk assessment in adults not on lipid-lowering therapy. However, fasting lipid testing is preferred when: a) Diagnosing metabolic syndrome (requires fasting triglycerides ≥150 mg/dL); b) Evaluating lipid disorders in those with a family history of premature ASCVD or genetic lipid disorders; c) Monitoring response to lifestyle or medication therapy in patients on lipid-lowering treatment; and d) Identifying and managing triglycerides ≥500 mg/dL to assess pancreatitis risk. In most people, postprandial triglyceride increases are modest. For non-fasting triglycerides ≥400 mg/dL, a repeat fasting test is advised. The expert panel supported the 2018 AHA/ACC guidelines on lifestyle changes, statins, and LDL-C–lowering therapies, and assessed the added benefit of triglyceride-lowering non-statin treatments across four patient groups: a) Secondary prevention in patients with ASCVD and triglycerides ≥150 mg/dL (fasting) or ≥175 mg/dL (non-fasting), but <500 mg/dL.
b) Diabetes (≥40 years) with no ASCVD, with similar triglyceride thresholds c) No ASCVD or diabetes (≥20 years) with similar triglyceride thresholds. d) Severe hypertriglyceridemia (≥20 years) with triglycerides ≥500 mg/dL, especially ≥1,000 mg/dL. Treatment options for these patient groups includes a combination of intensive lifestyle modifications, optimization of statin therapy, and, when appropriate, the addition of fibrates or omega-3 fatty acids to help manage triglyceride levels and reduce ASCVD risk (76).

National Lipid Association (NLA)

The National Lipid Association guidelines recommend obtaining a fasting or a non-fasting lipoprotein profile in all adults (>20 years) every 5 years. It defines TG level of <150 mg/dL as normal, 150 – 199 mg/dL borderline high, 200 – 499 mg/dL as high and levels of >500 mg/dL as very high. The NLA Expert Panel views non-HDL-c as a better primary target for medication than LDL-c and recommends levels of non-HDL-c < 130 mg/dL as the desirable level of atherogenic cholesterol for primary prevention of CVD and non-HDL-c <100 mg/dL for high-risk patients or patients with ASCVD. An elevated TG level is not a target of therapy, except when very high (>500 mg/dL). NLA recommends that when TG levels are between 200 – 499 mg/dL, the targets of therapy are non-HDL-c and LDL-c to reduce risk of CVD events and when TG levels are very high (>500 mg/dL, and especially if >1000 mg/dL), reducing the concentration to <500 mg/dL to prevent pancreatitis becomes the primary goal. The NLA recommends lifestyle interventions as first step in efforts to reduce triglycerides. If drug therapy is indicated NLA guidelines recommend using fibric acids, omega-3 fatty acids, or nicotinic acid as first line agents if fasting TG level is >1000 mg/dL. For patients with TG levels of 500 – 999 mg/dL a triglyceride-lowering agent or a statin is considered reasonable and for TG level between 200 – 499 mg/dL a statin generally is considered fist-line drug therapy with addition of a triglyceride-lowering agent if non-HDL-c is not at goal post initiation of statin (77).

European Society of Cardiology (ESC)/European Atherosclerosis Society (EAS) Guidelines

The ESC/EAS guidelines also recommend checking lipid levels in the fasting state. Triglyceride levels are classified as optimal (<1.2 mmol/L or <100 mg/dL), borderline (1.2–1.7 mmol/L or 100–150 mg/dL), moderately elevated (1.7–5.7 mmol/L or 150–500 mg/dL), severe (5.7–10.0 mmol/L or 500–880 mg/dL), and extreme (>10 mmol/L or >880 mg/dL). First-line management of hypertriglyceridemia includes dietary changes and weight loss. The 2019 ESC/EAS guidelines note increased ASCVD risk at triglycerides >1.7 mmol/L (150 mg/dL) but recommend starting pharmacotherapy only in high-risk patients with levels >2.3 mmol/L (200 mg/dL), after ruling out secondary causes. There are no specific triglyceride treatment targets, as evidence linking triglyceride lowering to ASCVD risk reduction is limited (78). Statins are recommended as first choice for high-risk individuals with hypertriglyceridemia (triglycerides >2.3 mmol/L or 200 mg/dL). In patients already at LDL-C goal on statins but with persistent triglycerides >2.3 mmol/L (200 mg/dL), fenofibrate or bezafibrate may be considered. For high-risk (or higher) patients with triglycerides >1.5 mmol/L (135 mg/dL) despite statins and lifestyle changes, omega-3 fatty acids (icosapent ethyl 2 g/day) may be considered in combination with statin therapy (79).

TG Assessment Strategies

The different guidelines in general recommend screening for lipids using a fasting lipid profile, screening for secondary causes of dyslipidemia, and focusing on lifestyle interventions as the first approach to lower elevated TG. A practical approach is to request fasting lipid panels on patients, but to obtain non-fasting (random) panels if fasting samples cannot be provided. For any patient with a TG level that is elevated (for example, > 200 mg/dL), screen for secondary causes and address these if possible. For significantly elevated TG (e.g., > 500 mg/dL) consider the addition of TG-lowering therapy, with the goal of preventing any further elevations increasing the risk for pancreatitis. For individuals with moderately elevated TG (e.g., 150-500mg/dL) then we recommend the consideration of TG-lowering therapy on an individual basis. For example, individuals with existing CVD or at high CVD risk, or those with very low HDL levels might be candidates for therapy, whereas in those with low CVD risk, or desirable HDL levels, additional focus on lifestyle interventions to lower TG may be the appropriate first line of therapy. The use of non-HDL cholesterol levels can help guide decisions.

MANANGEMENT OF HYPERTRIGLYCERIDEMIA

We recommend different therapeutic interventions for hypertriglyceridemia dependent on the underlying etiology and triglyceride level(s).

In patients with mild-to-moderate hypertriglyceridemia (150-499 mg/dL), statin therapy should be incorporated based on ASCVD risk. Eicosapentaenoic acid (EPA) ethyl ester can be added in adults on statins who have moderately elevated TG levels >150 mg/dL with ASCVD or diabetes plus 2 additional risk factors to reduce risk of cardiovascular disease.
For patients with multifactorial chylomicronemia syndrome, the primary goal of treatment focuses on achieving TG level below 500 mg/dL to prevent pancreatitis. This is achieved through fibrate therapy, with addition of omega-3 fatty acid. Niacin therapy can be added for further TG lowering, although this can worsen diabetes control.

Individuals with familial chylomicronemia syndrome are typically nonresponsive to fibrate and omega-3 fatty acid therapy due to complete absence of lipolytic activity. Historically these individuals were treated with a low-fat diet, restricted to 10-30 g/day of fat or 10-15% of calories as fat, with further limitation of long-chain fatty acids; however, we now suggest consideration of new drugs to suppress apoCIII to reduce TG levels in patients living with familial chylomicronemia syndrome.

A more detailed discussion of the above-mentioned therapeutic agents follows below.

Lifestyle Intervention

Studies have shown that the consumption of a Western diet which includes highly processed, calorie-dense and nutrient poor foods leads to an exaggerated lipemia. In addition, factors such as physical inactivity, cigarette smoking, excessive alcohol intake, and obesity worsen lipemia (63). Hence, the control of secondary factors and lifestyle changes are considered to be the first line approach of the clinical management of both fasting hypertriglyceridemia and post prandial hyperlipidemia. Appropriate dietary changes include limiting fat and simple sugar content, caloric restriction resulting in weight loss, restriction of alcohol intake, and increased exercise are fundamental for management of hypertriglyceridemia (62,63). The type of carbohydrate consumed may affect serum triglycerides and a diet rich in simple carbohydrates and sugar-sweetened beverages is associated with hypertriglyceridemia. As compared with starches, sugars, particularly sucrose and fructose, tend to increase serum triacylglycerol concentrations by about 60%. Because fructose bypasses a major rate-determining step in glycolysis, a high influx of fructose to the liver promotes triacylglycerol synthesis and VLDL production (80). The effects of sucrose or fructose on fasting TG may be more pronounced in men, sedentary overweight individuals, or those with the metabolic syndrome. Sucrose and fructose also increase postprandial TG levels and may augment the lipemia associated with fat-containing meals (81). There is mounting evidence that physical activity lowers risk for CVD (82). Mestek et al. (83) reported that aerobic exercise lowered the postprandial TG response to a high fat meal in subjects with the metabolic syndrome. The effects of exercise in reducing postprandial lipemia are seen both acutely right after exercise as well as delayed effects through the next day. Additionally, exercise does not need to be a single continuous bout but instead could be spread out throughout the day. Accumulated physical activity appears to be as effective in lowering postprandial TGs concentrations as a single bout (84). The mechanisms leading to decreased TG levels post meals are not completely understood and need further investigation.

Statins

Statins are the most widely used lipid lowering agents and have beneficial effects on cardiovascular morbidity and mortality. Statins are effective in lowering non-HDL-c, mainly because of their LDL lowering action and to a certain extent lowering TG levels. The higher the baseline TG levels, the greater the TG lowering effect. Available data also indicate that statins can reduce postprandial TG values (66). Statins inhibit HMG-CoA reductase, hence up-regulate the LDL receptor due to the intracellular depletion of cholesterol in the liver. Increased numbers of LDL receptors may improve the removal of TRL remnants in postprandial state. It is also postulated that statins inhibit VLDL synthesis (85). Parhofer et al showed that 10 mg of atorvastatin per day for 4 weeks improves, but does not normalize, post prandial lipoprotein metabolism in hypertriglyceridemic patients (86). Other studies have also shown that atorvastatin improved fasting as well as postprandial lipemia (87,88).

Fibrates

Fibrates have the most pronounced effect on lowering plasma TG levels of the currently available lipid lowering therapies. Through activation of peroxisomal proliferator activated receptor (PPAR) alpha, fibrates decrease TGs by increasing LPL activity and decreasing apolipoprotein CIII production leading to increased lipolysis. Fibrates also increase fatty acid oxidation in the liver leading to a decrease in VLDL secretion (63). The Endocrine Society Clinical Practice Guidelines on Evaluation and Treatment of Hypertriglyceridemia recommend that a fibrate be used as a first line agent for reduction of TGs in patients at risk for triglyceride- induced pancreatitis (8). The ACCORD trial evaluated the benefit of adding fenofibrate to simvastatin therapy concluded that the addition of fenofibrate in patients with diabetes did not reduce the rate of CVD events. However, in the fenofibrate + simvastatin group there was a significant reduction in cardiovascular risk in the subgroup with clinically significant dyslipidemia marked by elevated TG levels and low HDL levels (43). Rosenson et al. reported that fenofibrate treatment for 6 weeks significantly decreased both postprandial hypertriglyceridemia and the inflammatory response after the ingestion of a test meal consisting of a milkshake including standardized fat content (68% of energy) that was adjusted to body surface area (50 g/m²) in patients with hypertriglyceridemia and the metabolic syndrome (89). In a small study (n = 10), bezafibrate was shown to significantly decrease postprandial endothelial dysfunction and elevations of both exogenous and endogenous triglycerides in patients with metabolic syndrome (90). The effects of fibrates in decreasing postprandial TRLs may play a role in their vascular protective effects.

Niacin

Niacin decreases TG levels and has pronounced effects on increasing HDL concentration. The mechanism of action of niacin remains unclear, but it is proposed that niacin decreases TG synthesis and hepatic secretion of VLDL. The Coronary Drug Project was a randomized controlled trial that looked at the role of immediate-release niacin as a solo agent for coronary prevention. The Coronary Drug Project showed that niacin was associated with a significant reduction in cardiovascular events (91,92). Studies have shown that both immediate-release and extended-release niacin suppress postprandial hypertriglyceridemia (93,94). The Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) trial showed that the addition of niacin to statin therapy in patients with CVD and LDL cholesterol levels of less than 70 mg per deciliter had no incremental clinical benefit during a 36-month follow-up period, despite significant improvements in HDL cholesterol and TG levels (2). However, a trend towards benefit (hazard ratio 0.74; p=0.073) was found for the subset of patients with both the highest TG levels and lowest HDL levels (>198 and <33mg/dL respectively) (95). Lipids in this study were measured in fasting state. Similarly, the Heart Protection Study 2-Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2 –THRIVE) study, which compared niacin + laropiprant (a prostaglandin D2 receptor antagonist used as an anti-flushing agent) + statin vs statin alone did not find added benefit of niacin. However, this lack of additional benefit may be related to the patient population studied which did not have elevated TG levels (96) and a possible benefit may be seen for subjects with both elevated TG and low HDL. Further studies are needed to access the effects of niacin on hypertriglyceridemia in metabolic syndrome and patients with T2DM.

Ezetimibe

Ezetimibe is a cholesterol lowering agent that inhibits the intestinal absorption of cholesterol (97). Recent studies show that ezetimibe alone or in conjunction with statins also reduces postprandial hypertriglyceridemia. Masuda, et al. showed that ezetimibe significantly decreased TGs in the fasting state along with a decrease in postprandial elevations of cholesterol and TG levels in the chylomicrons (CM) size range, suggesting that the postprandial production of CM particles was suppressed by ezetimibe (98). In a study by Olijhoek et al, combination therapy with low dose simvastatin and ezetimibe was shown to preserve post-fat load endothelial function when compared to treatment with high-dose simvastatin monotherapy in male metabolic syndrome patients (99). The Improved Reduction of Outcomes: Vytorin Efficacy International trial (IMPROVE-IT), a multicenter, randomized, double-blind trial of 18,144 moderate-high risk patients stabilized following ACS, was conducted to investigate if the addition of ezetimibe to a statin improves cardiovascular outcomes relative to statin monotherapy in these patients. The results from this study suggest that the addition of ezetimibe to statin therapy improves cardiovascular outcomes, but likely via further LDL-c lowering (100).

Fish Oil

Omega 3 polyunsaturated fatty acids (PUFAs) have dose dependent TG lowering effects resulting from variety of mechanisms including decreased VLDL secretion and improved VLDL TG clearance (101). In the Japan EPA Lipid Intervention Study (JELIS) trial, 18,645 patients in Japan were recruited between 1996 and 1999 and assigned to receive either 1800 mg of eicosapentaenoic acid (EPA) daily with statin or statin only. A 19% relative reduction in major coronary events (p = 0.011) was seen in patients in the EPA group. Unstable angina and non-fatal coronary events were significantly reduced; however, sudden cardiac death and coronary death did not differ between the groups (102). As discussed above, REDUCE-IT and STRENGTH studied the effects of high dose omega-3 fatty acids and had differing results. The REDUCE-IT trail with 8,179 patients showed a decrease in major cardiac events with high dose icosapent ethyl (4g/d EPA), while the STRENGTH trial with 13,086 patients showed no effects on cardiac events with high dose EPA/DHA carboxylic acid (4g/d) (47,48).

A few studies have examined the effects of fish oil supplementation on postprandial lipemia and found that fish oil use decreases fasting and postprandial triglyceride levels (103,104). A study looking at the effect of fish oil, exercise and the combined treatments on fasting and postprandial chylomicron metabolism showed that combining fish oil with chronic exercise, reduced the plasma concentration of pro-atherogenic chylomicron remnants; in addition it reduced the fasting and postprandial TG response in viscerally obese insulin resistant subjects (105).

For additional information on drugs to treat hyperlipidemia see the chapters on triglyceride lowering drugs and cholesterol lowering drugs in Endotext (106,107).

Therapies Targeting APOC3

APOC3 is a CVD risk factor due to its association with increased triglyceride levels. Antisense oligonucleotides (ASOs) are novel therapeutic agents that bind mRNA leading to its degradation. An ASO to APOC3 was found to lower APOC3 and triglyceride levels. A RCT evaluating this ASO in patients with hypertriglyceridemia (fasting triglyceride levels between 350-2000 mg/dL if not on triglyceride-lowering therapy, or 225-2000 mg/dL if on a fibrate) found that it led to reductions in triglyceride levels of 30-71% over the 13-week trial period. After the ASO was discontinued triglyceride levels returned towards baseline levels over the next 13 weeks. There were no safety concerns in this trial (108).

Volanesorsen is an antisense oligonucleotide inhibitor of apolipoprotein CIII (apoCIII) mRNA, designed to decrease triglycerides by reducing hepatic apoCIII production (109). In Europe, Volanesorsen was approved in 2019 for the treatment of adults with genetically confirmed familial chylomicronemia syndrome (FCS) at high risk of pancreatitis and for which diet and triglyceride lowering therapy was insufficient. In the US, Volanesorsen received orphan drug status in 2015. Two multicenter international phase three randomized, placebo-controlled, double-blind trials, COMPASS and APPROACH, evaluated the efficacy and safety of Volanesorsen in patients with multifactorial severe hypertriglyceridemia or FCS (110,111). Injection-site reactions and thrombocytopenia were observed in both studies. In the APPROACH trial 77% of individuals who received 300 mg volanesorsen subcutaneously once weekly, had fasting triglyceride levels less than 750 mg/dL compared to 10% in the placebo group at 3 months (OR, 186.16; 95% CI, 12.86 to could not be estimated; P<0.001) (111). A common side effect with volanesorsen is thrombocytopenia and it is currently only approved for use in Europe. Volanesorsen at 300 mg once weekly, decreased mean fasting plasma triglyceride concentration by 71.2% (95% CI-79.3 to -63.2) after 3 months of therapy compared to 0.9% (-13.9 to 12.2) in participants in the placebo group (p<0.0001) in the COMPASS trial (110). Inhibition of APOC3 may be a therapeutic option in individuals with LPL deficiency (112); at present there are no effective therapies except for extreme dietary restrictions for these individuals.

Olezarsen (Tryngolza™) is another antisense oligonucleotide inhibitor of apoCIII manufactured by Ionis Pharmaceuticals, Inc. and FDA approved for treatment of FCS as an adjunct to diet on December 19, 2024. The composition of olezarsen incorporates the nucleotide sequence and backbone chemical composition found in volanesorsen, differing only by inclusion of Triantennary N-acetylgalactosamine (GalNAc3) (113). GalNAc3 is a carbohydrate ligand for asialoglycoprotein receptors found on the surface of hepatocytes which promotes uptake of the drug into hepatocyte nuclei where olezarsen binds APOC3 mRNA prompting degradation by ribonuclease H1-mediated cleavage of the sense strand. The BALANCE trial was a multicenter international Phase III, randomized, double-blinded, placebo-controlled study examining the safety and efficacy of olezarsen (113). Participants were assigned to receive 50 or 80 mg olezarsen, or a placebo every 4 weeks for 49 weeks. Fasting triglycerides at 6-months were significantly decreased in the 80 mg cohort as compared with placebo (−43.5 percentage points; 95% confidence interval [CI], −69.1 to −17.9; P<0.001) but not with the 50-mg dose (−22.4 percentage points;95% CI, −47.2 to 2.5; P = 0.08).

Plozasiran (ARO-APOC3) is a small interfering RNA (siRNA) therapy targeting cytoplasmic APOC3 mRNA, differing from nucleus-acting drugs like volanesorsen and olezarsen (114). In the phase 3 PALISADE trial involving patients with FCS with severe hypertriglyceridemia, plozasiran significantly reduced triglyceride levels by up to 80%, independent of sex or genetic background. At 10 months, reductions in median triglyceride levels of 2044 mg/dL (23.0 mmol/L) reached −80 and −78% in the 25 and 50 mg plozasiran groups, respectively (p < 0.001) (115). It also lowered non-HDL cholesterol and increased HDL and LDL cholesterol (with LDL remaining below 55 mg/dL). The treatment reduced the risk of acute pancreatitis and showed a similar safety profile to placebo, though it caused a transient rise in liver enzymes and a possible increase in HbA1c in diabetic or prediabetic patients. Plozasiran has been designated an orphan drug by the EMA and received FDA breakthrough therapy status in 2024. A phase 3 cardiovascular outcomes trial is planned.

Inhibition of APOC3 may be a therapeutic option in individuals with LPL deficiency (112); at present there are no effective therapies except for extreme dietary restrictions for these individuals.

Therapies Targeting ANGPTL3

ANGPTL3 is another potential target for triglyceride lowering. Genetic causes of decreased activity are associated with lower TG, HDL, and LDL levels as well as a decreased risk for CVD. A small trial using a human monoclonal antibody to target ANGPTL3 reported decreases in triglycerides up to 76% (20). A recent study by Harada-Shiba et al, a phase 1 study took a total of 96 Caucasian and Japanese patients and randomized them to receive varying doses and routes of evinacumab or placebo. In the evinacumab cohorts, reduced TGs were rapidly seen in a dose-dependent manner. The study showed no serious or severe treatment emergent adverse events (116). Further clinical trials using either monoclonal antibody or antisense technology are ongoing. Zodasiran, a GalNAc-conjugated siRNA targeting ANGPTL3 mRNA, was evaluated in the phase 2 ARCHES-2 trial involving 204 patients with mixed hyperlipidemia. It produced dose-dependent triglyceride reductions of up to 63%, with 88% of patients on the highest dose (200 mg) achieving target TG levels. The treatment also lowered LDL-C, non-HDL-C, HDL-C, remnant cholesterol, lipoprotein(a), and apoB, though LDL-C reductions were less in patients with higher baseline TG levels. Zodasiran was generally well tolerated, with no major safety concerns. A transient HbA1c rise and increased urinary tract infections were observed in diabetic patients at the highest dose (117).

New Agents on the Horizon

FGF21 is a key metabolic regulator that enhances insulin sensitivity, promotes fatty acid oxidation, and facilitates triglyceride-rich lipoprotein (TRL) clearance, ultimately lowering non-HDL cholesterol and improving atherosclerosis in animal models (118-120). Its analog, pegozafermin, developed to extend FGF21’s half-life and reduce off-target effects, significantly reduced triglycerides (up to 54%) and increased HDL-C in clinical trials involving patients with elevated triglycerides (121) . In the phase 2 ENTRIGUE trial, nearly 80% of treated patients achieved TG levels below 500 mg/dL, with additional improvements in apoB, apoC-III, and HDL-C (122). Pegozafermin was well tolerated, with only mild-to-moderate GI side effects. A phase 3 trial (ENTRUST) is ongoing to further evaluate its long-term efficacy and safety.

Weight Management Therapies and Hypertriglyceridemia

Please see other chapters for further discussion of the role of weight management (68).

CONCLUSION

Recent data strongly indicate that fasting as well as non-fasting hypertriglyceridemia is a risk factor for atherosclerosis and CVD. Current treatment goals aimed at lowering LDL-c still do not eliminate residual risk of CVD. Current guidelines focus mainly on LDL-c levels and correction of hypertriglyceridemia is not the focus of current treatment. However, focus on elevated hypertriglyceridemia deserves renewed attention, particularly as one-third of all adults in the United States suffer from elevated TG and growing number of people are diagnosed with metabolic syndrome or T2DM. There is a need for more studies specifically testing the benefits of lowering hypertriglyceridemia. Additionally, the usefulness of “fat tolerance test” using a standardized meal, analogous to a glucose tolerance test, warrants further evaluation as potential indicator of a metabolic state identifying individuals at higher risk for cardiovascular events. Given the association with CVD, elevated postprandial TGs levels may represent a particularly attractive therapeutic target and further studies particularly looking at the effect of various lipid lowering agents on postprandial along with fasting TGs are necessary.

Table 2. Targeted Treatment of Hypertriglyceridemia
	Primary Treatment	Secondary Treatment
ASCVD (150-499 mg/dL)	Statin	Omega-3 Fatty Acid
MFCS	Fibrate	Omega-3 Fatty Acid, Niacin
FCS	Olezarsen

REFERENCES

Chapman MJ, Ginsberg HN, Amarenco P, Andreotti F, Boren J, Catapano AL, Descamps OS, Fisher E, Kovanen PT, Kuivenhoven JA, Lesnik P, Masana L, Nordestgaard BG, Ray KK, Reiner Z, Taskinen MR, Tokgozoglu L, Tybjaerg-Hansen A, Watts GF, European Atherosclerosis Society Consensus P. Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management. Eur Heart J. 2011;32(11):1345-1361.
Boden WE, Probstfield JL, Anderson T, Chaitman BR, Desvignes-Nickens P, Koprowicz K, McBride R, Teo K, Weintraub W. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N Engl J Med. 2011;365(24):2255-2267.
Talayero BG, Sacks FM. The role of triglycerides in atherosclerosis. Curr Cardiol Rep. 2011;13(6):544-552.
Hokanson JE, Austin MA. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta- analysis of population-based prospective studies. J Cardiovasc Risk. 1996;3(2):213-219.
Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). Jama. 2001;285(19):2486-2497.
Abdel-Maksoud M, Sazonov V, Gutkin SW, Hokanson JE. Effects of modifying triglycerides and triglyceride-rich lipoproteins on cardiovascular outcomes. J Cardiovasc Pharmacol. 2008;51(4):331-351.
Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation. 1979;60(3):473-485.
Berglund L, Brunzell JD, Goldberg AC, Goldberg IJ, Sacks F, Murad MH, Stalenhoef AF, Endocrine s. Evaluation and treatment of hypertriglyceridemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2012;97(9):2969-2989.
Murad MH, Hazem A, Coto-Yglesias F, Dzyubak S, Gupta S, Bancos I, Lane MA, Erwin PJ, Berglund L, Elraiyah T, Montori VM. The association of hypertriglyceridemia with cardiovascular events and pancreatitis: a systematic review and meta-analysis. BMC Endocr Disord. 2012;12:2.
Champe P. Cholesterol and Steroid Metabolism. Vol 3rd Edition. Philadelphia: Lippincott Williams & Wilkins.
Melmed S. Disorders of Lipid Metabolism. Williams Textbook of Endocrinology. Philadelphia: Elsevier/Saunders; 2011.
Feingold KR, Grunfeld C. Introduction to Lipids and Lipoproteins. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Adiels M, Olofsson SO, Taskinen MR, Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipidemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol. 2008;28(7):1225-1236.
Mendivil CO, Rimm EB, Furtado J, Chiuve SE, Sacks FM. Low-density lipoproteins containing apolipoprotein C-III and the risk of coronary heart disease. Circulation. 2011;124(19):2065-2072.
Ooi EM, Barrett PH, Chan DC, Watts GF. Apolipoprotein C-III: understanding an emerging cardiovascular risk factor. Clinical science. 2008;114(10):611-624.
Sacks FM, Alaupovic P, Moye LA, Cole TG, Sussex B, Stampfer MJ, Pfeffer MA, Braunwald E. VLDL, apolipoproteins B, CIII, and E, and risk of recurrent coronary events in the Cholesterol and Recurrent Events (CARE) trial. Circulation. 2000;102(16):1886-1892.
Jorgensen AB, Frikke-Schmidt R, Nordestgaard BG, Tybjaerg-Hansen A. Loss-of-function mutations in APOC3 and risk of ischemic vascular disease. N Engl J Med. 2014;371(1):32-41.
Pollin TI, Damcott CM, Shen H, Ott SH, Shelton J, Horenstein RB, Post W, McLenithan JC, Bielak LF, Peyser PA, Mitchell BD, Miller M, O'Connell JR, Shuldiner AR. A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science. 2008;322(5908):1702-1705.
Tg, Hdl Working Group of the Exome Sequencing Project NHL, Blood I, Crosby J, Peloso GM, Auer PL, Crosslin DR, Stitziel NO, Lange LA, Lu Y, Tang ZZ, Zhang H, Hindy G, Masca N, Stirrups K, Kanoni S, Do R, Jun G, Hu Y, Kang HM, Xue C, Goel A, Farrall M, Duga S, Merlini PA, Asselta R, Girelli D, Olivieri O, Martinelli N, Yin W, Reilly D, Speliotes E, Fox CS, Hveem K, Holmen OL, Nikpay M, Farlow DN, Assimes TL, Franceschini N, Robinson J, North KE, Martin LW, DePristo M, Gupta N, Escher SA, Jansson JH, Van Zuydam N, Palmer CN, Wareham N, Koch W, Meitinger T, Peters A, Lieb W, Erbel R, Konig IR, Kruppa J, Degenhardt F, Gottesman O, Bottinger EP, O'Donnell CJ, Psaty BM, Ballantyne CM, Abecasis G, Ordovas JM, Melander O, Watkins H, Orho-Melander M, Ardissino D, Loos RJ, McPherson R, Willer CJ, Erdmann J, Hall AS, Samani NJ, Deloukas P, Schunkert H, Wilson JG, Kooperberg C, Rich SS, Tracy RP, Lin DY, Altshuler D, Gabriel S, Nickerson DA, Jarvik GP, Cupples LA, Reiner AP, Boerwinkle E, Kathiresan S. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med. 2014;371(1):22-31.
Dewey FE, Gusarova V, Dunbar RL, O'Dushlaine C, Schurmann C, Gottesman O, McCarthy S, Van Hout CV, Bruse S, Dansky HM, Leader JB, Murray MF, Ritchie MD, Kirchner HL, Habegger L, Lopez A, Penn J, Zhao A, Shao W, Stahl N, Murphy AJ, Hamon S, Bouzelmat A, Zhang R, Shumel B, Pordy R, Gipe D, Herman GA, Sheu WHH, Lee IT, Liang KW, Guo X, Rotter JI, Chen YI, Kraus WE, Shah SH, Damrauer S, Small A, Rader DJ, Wulff AB, Nordestgaard BG, Tybjaerg-Hansen A, van den Hoek AM, Princen HMG, Ledbetter DH, Carey DJ, Overton JD, Reid JG, Sasiela WJ, Banerjee P, Shuldiner AR, Borecki IB, Teslovich TM, Yancopoulos GD, Mellis SJ, Gromada J, Baras A. Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease. N Engl J Med. 2017;377(3):211-221.
Leaf DA. Chylomicronemia and the chylomicronemia syndrome: a practical approach to management. Am J Med. 2008;121(1):10-12.
Lindkvist B, Appelros S, Regner S, Manjer J. A prospective cohort study on risk of acute pancreatitis related to serum triglycerides, cholesterol and fasting glucose. Pancreatology. 2012;12(4):317-324.
Sandhu S, Al-Sarraf A, Taraboanta C, Frohlich J, Francis GA. Incidence of pancreatitis, secondary causes, and treatment of patients referred to a specialty lipid clinic with severe hypertriglyceridemia: a retrospective cohort study. Lipids Health Dis. 2011;10:157.
Murphy MJ, Sheng X, MacDonald TM, Wei L. Hypertriglyceridemia and acute pancreatitis. JAMA Intern Med. 2013;173(2):162-164.
Christian JB, Arondekar B, Buysman EK, Jacobson TA, Snipes RG, Horwitz RI. Determining triglyceride reductions needed for clinical impact in severe hypertriglyceridemia. Am J Med. 2014;127(1):36-44 e31.
Patel A, Barzi F, Jamrozik K, Lam TH, Ueshima H, Whitlock G, Woodward M, Asia Pacific Cohort Studies C. Serum triglycerides as a risk factor for cardiovascular diseases in the Asia-Pacific region. Circulation. 2004;110(17):2678-2686.
Sarwar N, Danesh J, Eiriksdottir G, Sigurdsson G, Wareham N, Bingham S, Boekholdt SM, Khaw KT, Gudnason V. Triglycerides and the risk of coronary heart disease: 10,158 incident cases among 262,525 participants in 29 Western prospective studies. Circulation. 2007;115(4):450-458.
Varbo A, Benn M, Tybjaerg-Hansen A, Jorgensen AB, Frikke-Schmidt R, Nordestgaard BG. Remnant cholesterol as a causal risk factor for ischemic heart disease. J Am Coll Cardiol. 2013;61(4):427-436.
Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, Pirruccello JP, Ripatti S, Chasman DI, Willer CJ, Johansen CT, Fouchier SW, Isaacs A, Peloso GM, Barbalic M, Ricketts SL, Bis JC, Aulchenko YS, Thorleifsson G, Feitosa MF, Chambers J, Orho-Melander M, Melander O, Johnson T, Li X, Guo X, Li M, Shin Cho Y, Jin Go M, Jin Kim Y, Lee JY, Park T, Kim K, Sim X, Twee-Hee Ong R, Croteau-Chonka DC, Lange LA, Smith JD, Song K, Hua Zhao J, Yuan X, Luan J, Lamina C, Ziegler A, Zhang W, Zee RY, Wright AF, Witteman JC, Wilson JF, Willemsen G, Wichmann HE, Whitfield JB, Waterworth DM, Wareham NJ, Waeber G, Vollenweider P, Voight BF, Vitart V, Uitterlinden AG, Uda M, Tuomilehto J, Thompson JR, Tanaka T, Surakka I, Stringham HM, Spector TD, Soranzo N, Smit JH, Sinisalo J, Silander K, Sijbrands EJ, Scuteri A, Scott J, Schlessinger D, Sanna S, Salomaa V, Saharinen J, Sabatti C, Ruokonen A, Rudan I, Rose LM, Roberts R, Rieder M, Psaty BM, Pramstaller PP, Pichler I, Perola M, Penninx BW, Pedersen NL, Pattaro C, Parker AN, Pare G, Oostra BA, O'Donnell CJ, Nieminen MS, Nickerson DA, Montgomery GW, Meitinger T, McPherson R, McCarthy MI, McArdle W, Masson D, Martin NG, Marroni F, Mangino M, Magnusson PK, Lucas G, Luben R, Loos RJ, Lokki ML, Lettre G, Langenberg C, Launer LJ, Lakatta EG, Laaksonen R, Kyvik KO, Kronenberg F, Konig IR, Khaw KT, Kaprio J, Kaplan LM, Johansson A, Jarvelin MR, Janssens AC, Ingelsson E, Igl W, Kees Hovingh G, Hottenga JJ, Hofman A, Hicks AA, Hengstenberg C, Heid IM, Hayward C, Havulinna AS, Hastie ND, Harris TB, Haritunians T, Hall AS, Gyllensten U, Guiducci C, Groop LC, Gonzalez E, Gieger C, Freimer NB, Ferrucci L, Erdmann J, Elliott P, Ejebe KG, Doring A, Dominiczak AF, Demissie S, Deloukas P, de Geus EJ, de Faire U, Crawford G, Collins FS, Chen YD, Caulfield MJ, Campbell H, Burtt NP, Bonnycastle LL, Boomsma DI, Boekholdt SM, Bergman RN, Barroso I, Bandinelli S, Ballantyne CM, Assimes TL, Quertermous T, Altshuler D, Seielstad M, Wong TY, Tai ES, Feranil AB, Kuzawa CW, Adair LS, Taylor HA, Jr., Borecki IB, Gabriel SB, Wilson JG, Holm H, Thorsteinsdottir U, Gudnason V, Krauss RM, Mohlke KL, Ordovas JM, Munroe PB, Kooner JS, Tall AR, Hegele RA, Kastelein JJ, Schadt EE, Rotter JI, Boerwinkle E, Strachan DP, Mooser V, Stefansson K, Reilly MP, Samani NJ, Schunkert H, Cupples LA, Sandhu MS, Ridker PM, Rader DJ, van Duijn CM, Peltonen L, Abecasis GR, Boehnke M, Kathiresan S. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466(7307):707-713.
Triglyceride Coronary Disease Genetics C, Emerging Risk Factors C, Sarwar N, Sandhu MS, Ricketts SL, Butterworth AS, Di Angelantonio E, Boekholdt SM, Ouwehand W, Watkins H, Samani NJ, Saleheen D, Lawlor D, Reilly MP, Hingorani AD, Talmud PJ, Danesh J. Triglyceride-mediated pathways and coronary disease: collaborative analysis of 101 studies. Lancet. 2010;375(9726):1634-1639.
Jorgensen AB, Frikke-Schmidt R, West AS, Grande P, Nordestgaard BG, Tybjaerg-Hansen A. Genetically elevated non-fasting triglycerides and calculated remnant cholesterol as causal risk factors for myocardial infarction. Eur Heart J. 2013;34(24):1826-1833.
Thomsen M, Varbo A, Tybjaerg-Hansen A, Nordestgaard BG. Low nonfasting triglycerides and reduced all-cause mortality: a mendelian randomization study. Clin Chem. 2014;60(5):737-746.
Myocardial Infarction G, Investigators CAEC, Stitziel NO, Stirrups KE, Masca NG, Erdmann J, Ferrario PG, Konig IR, Weeke PE, Webb TR, Auer PL, Schick UM, Lu Y, Zhang H, Dube MP, Goel A, Farrall M, Peloso GM, Won HH, Do R, van Iperen E, Kanoni S, Kruppa J, Mahajan A, Scott RA, Willenberg C, Braund PS, van Capelleveen JC, Doney AS, Donnelly LA, Asselta R, Merlini PA, Duga S, Marziliano N, Denny JC, Shaffer CM, El-Mokhtari NE, Franke A, Gottesman O, Heilmann S, Hengstenberg C, Hoffman P, Holmen OL, Hveem K, Jansson JH, Jockel KH, Kessler T, Kriebel J, Laugwitz KL, Marouli E, Martinelli N, McCarthy MI, Van Zuydam NR, Meisinger C, Esko T, Mihailov E, Escher SA, Alver M, Moebus S, Morris AD, Muller-Nurasyid M, Nikpay M, Olivieri O, Lemieux Perreault LP, AlQarawi A, Robertson NR, Akinsanya KO, Reilly DF, Vogt TF, Yin W, Asselbergs FW, Kooperberg C, Jackson RD, Stahl E, Strauch K, Varga TV, Waldenberger M, Zeng L, Kraja AT, Liu C, Ehret GB, Newton-Cheh C, Chasman DI, Chowdhury R, Ferrario M, Ford I, Jukema JW, Kee F, Kuulasmaa K, Nordestgaard BG, Perola M, Saleheen D, Sattar N, Surendran P, Tregouet D, Young R, Howson JM, Butterworth AS, Danesh J, Ardissino D, Bottinger EP, Erbel R, Franks PW, Girelli D, Hall AS, Hovingh GK, Kastrati A, Lieb W, Meitinger T, Kraus WE, Shah SH, McPherson R, Orho-Melander M, Melander O, Metspalu A, Palmer CN, Peters A, Rader D, Reilly MP, Loos RJ, Reiner AP, Roden DM, Tardif JC, Thompson JR, Wareham NJ, Watkins H, Willer CJ, Kathiresan S, Deloukas P, Samani NJ, Schunkert H. Coding Variation in ANGPTL4, LPL, and SVEP1 and the Risk of Coronary Disease. N Engl J Med. 2016;374(12):1134-1144.
Ference BA, Kastelein JJP, Ray KK, Ginsberg HN, Chapman MJ, Packard CJ, Laufs U, Oliver-Williams C, Wood AM, Butterworth AS, Di Angelantonio E, Danesh J, Nicholls SJ, Bhatt DL, Sabatine MS, Catapano AL. Association of Triglyceride-Lowering LPL Variants and LDL-C-Lowering LDLR Variants With Risk of Coronary Heart Disease. JAMA. 2019;321(4):364-373.
Jun M, Zhu B, Tonelli M, Jardine MJ, Patel A, Neal B, Liyanage T, Keech A, Cass A, Perkovic V. Effects of fibrates in kidney disease: a systematic review and meta-analysis. J Am Coll Cardiol. 2012;60(20):2061-2071.
Frick MH, Elo O, Haapa K, Heinonen OP, Heinsalmi P, Helo P, Huttunen JK, Kaitaniemi P, Koskinen P, Manninen V, et al. Helsinki Heart Study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987;317(20):1237-1245.
Tenkanen L, Manttari M, Kovanen PT, Virkkunen H, Manninen V. Gemfibrozil in the treatment of dyslipidemia: an 18-year mortality follow-up of the Helsinki Heart Study. Arch Intern Med. 2006;166(7):743-748.
Haim M, Benderly M, Brunner D, Behar S, Graff E, Reicher-Reiss H, Goldbourt U. Elevated serum triglyceride levels and long-term mortality in patients with coronary heart disease: the Bezafibrate Infarction Prevention (BIP) Registry. Circulation. 1999;100(5):475-482.
Bezafibrate Infarction Prevention s. Secondary prevention by raising HDL cholesterol and reducing triglycerides in patients with coronary artery disease. Circulation. 2000;102(1):21-27.
Tenenbaum A, Motro M, Fisman EZ, Tanne D, Boyko V, Behar S. Bezafibrate for the secondary prevention of myocardial infarction in patients with metabolic syndrome. Arch Intern Med. 2005;165(10):1154-1160.
Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med. 1999;341(6):410-418.
Miller M, Cosgrove B, Havas S. Update on the role of triglycerides as a risk factor for coronary heart disease. Curr Atheroscler Rep. 2002;4(6):414-418.
Ginsberg HN, Elam MB, Lovato LC, Crouse JR, 3rd, Leiter LA, Linz P, Friedewald WT, Buse JB, Gerstein HC, Probstfield J, Grimm RH, Ismail-Beigi F, Bigger JT, Goff DC, Jr., Cushman WC, Simons-Morton DG, Byington RP. Effects of combination lipid therapy in type 2 diabetes mellitus. N Engl J Med. 2010;362(17):1563-1574.
Xu J, Ashjian E. Treatment of Hypertriglyceridemia: A Review of Therapies in the Pipeline. J Pharm Pract. 2023;36(3):650-661.
Pradhan AD, Paynter NP, Everett BM, Glynn RJ, Amarenco P, Elam M, Ginsberg H, Hiatt WR, Ishibashi S, Koenig W, Nordestgaard BG, Fruchart JC, Libby P, Ridker PM. Rationale and design of the Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT) study. Am Heart J. 2018;206:80-93.
Zandbergen F, Plutzky J. PPARalpha in atherosclerosis and inflammation. Biochim Biophys Acta. 2007;1771(8):972-982.
Nicholls SJ, Lincoff AM, Garcia M, Bash D, Ballantyne CM, Barter PJ, Davidson MH, Kastelein JJP, Koenig W, McGuire DK, Mozaffarian D, Ridker PM, Ray KK, Katona BG, Himmelmann A, Loss LE, Rensfeldt M, Lundstrom T, Agrawal R, Menon V, Wolski K, Nissen SE. Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk: The STRENGTH Randomized Clinical Trial. JAMA. 2020;324(22):2268-2280.
Bhatt DL, Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, Doyle RT, Jr., Juliano RA, Jiao L, Granowitz C, Tardif JC, Ballantyne CM, Investigators R-I. Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia. N Engl J Med. 2019;380(1):11-22.
Mason RP, Eckel RH. Mechanistic Insights from REDUCE-IT STRENGTHen the Case Against Triglyceride Lowering as a Strategy for Cardiovascular Disease Risk Reduction. Am J Med. 2021;134(9):1085-1090.
Olshansky B, Chung MK, Budoff MJ, Philip S, Jiao L, Doyle RT, Jr., Copland C, Giaquinto A, Juliano RA, Bhatt DL. Mineral oil: safety and use as placebo in REDUCE-IT and other clinical studies. Eur Heart J Suppl. 2020;22(Suppl J):J34-J48.
Nissen SE, Lincoff AM, Wolski K, Ballantyne CM, Kastelein JJP, Ridker PM, Ray KK, McGuire DK, Mozaffarian D, Koenig W, Davidson MH, Garcia M, Katona BG, Himmelmann A, Loss LE, Poole M, Menon V, Nicholls SJ. Association Between Achieved omega-3 Fatty Acid Levels and Major Adverse Cardiovascular Outcomes in Patients With High Cardiovascular Risk: A Secondary Analysis of the STRENGTH Trial. JAMA Cardiol. 2021;6(8):910-917.
Lombardi M, Carbone S, Del Buono MG, Chiabrando JG, Vescovo GM, Camilli M, Montone RA, Vergallo R, Abbate A, Biondi-Zoccai G, Dixon DL, Crea F. Omega-3 fatty acids supplementation and risk of atrial fibrillation: an updated meta-analysis of randomized controlled trials. Eur Heart J Cardiovasc Pharmacother. 2021;7(4):e69-e70.
Mora S, Rifai N, Buring JE, Ridker PM. Fasting compared with nonfasting lipids and apolipoproteins for predicting incident cardiovascular events. Circulation. 2008;118(10):993-1001.
Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA. 2007;298(3):309-316.
Lindman AS, Veierod MB, Tverdal A, Pedersen JI, Selmer R. Nonfasting triglycerides and risk of cardiovascular death in men and women from the Norwegian Counties Study. European journal of epidemiology. 2010;25(11):789-798.
Langsted A, Freiberg JJ, Tybjaerg-Hansen A, Schnohr P, Jensen GB, Nordestgaard BG. Nonfasting cholesterol and triglycerides and association with risk of myocardial infarction and total mortality: the Copenhagen City Heart Study with 31 years of follow-up. Journal of internal medicine. 2011;270(1):65-75.
Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. Jama. 2007;298(3):299-308.
Tada H, Nomura A, Yoshimura K, Itoh H, Komuro I, Yamagishi M, Takamura M, Kawashiri MA. Fasting and Non-Fasting Triglycerides and Risk of Cardiovascular Events in Diabetic Patients Under Statin Therapy. Circ J. 2020;84(3):509-515.
Nordestgaard BG, Langsted A, Freiberg JJ. Nonfasting hyperlipidemia and cardiovascular disease. Current drug targets. 2009;10(4):328-335.
Christian JB, Bourgeois N, Snipes R, Lowe KA. Prevalence of severe (500 to 2,000 mg/dl) hypertriglyceridemia in United States adults. Am J Cardiol. 2011;107(6):891-897.
Fan W, Philip S, Granowitz C, Toth PP, Wong ND. Hypertriglyceridemia in statin-treated US adults: the National Health and Nutrition Examination Survey. J Clin Lipidol. 2019;13(1):100-108.
Boren J, Matikainen N, Adiels M, Taskinen MR. Postprandial hypertriglyceridemia as a coronary risk factor. Clin Chim Acta. 2014;431:131-142.
Kolovou GD, Mikhailidis DP, Kovar J, Lairon D, Nordestgaard BG, Ooi TC, Perez-Martinez P, Bilianou H, Anagnostopoulou K, Panotopoulos G. Assessment and clinical relevance of non-fasting and postprandial triglycerides: an expert panel statement. Curr Vasc Pharmacol. 2011;9(3):258-270.
Smith D, Watts GF, Dane-Stewart C, Mamo JC. Post-prandial chylomicron response may be predicted by a single measurement of plasma apolipoprotein B48 in the fasting state. Eur J Clin Invest. 1999;29(3):204-209.
Feingold KR, Brinton EA, Grunfeld C. The Effect of Endocrine Disorders on Lipids and Lipoproteins. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, Dungan K, Grossman A, Hershman JM, Hofland J, Kalra S, Kaltsas G, Koch C, Kopp P, Korbonits M, Kovacs CS, Kuohung W, Laferrere B, McGee EA, McLachlan R, Morley JE, New M, Purnell J, Sahay R, Singer F, Stratakis CA, Trence DL, Wilson DP, eds. Endotext. South Dartmouth (MA)2000.
Herink M, Ito MK. Medication Induced Changes in Lipid and Lipoproteins. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, Dungan K, Grossman A, Hershman JM, Hofland J, Kalra S, Kaltsas G, Koch C, Kopp P, Korbonits M, Kovacs CS, Kuohung W, Laferrere B, McGee EA, McLachlan R, Morley JE, New M, Purnell J, Sahay R, Singer F, Stratakis CA, Trence DL, Wilson DP, eds. Endotext. South Dartmouth (MA)2000.
Feingold KR, Grunfeld C. Diabetes and Dyslipidemia. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Feingold KR, Grunfeld C. Obesity and Dyslipidemia. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Grimes SB, Wild R. Effect of Pregnancy on Lipid Metabolism and Lipoprotein Levels. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Miller M, Stone NJ, Ballantyne C, Bittner V, Criqui MH, Ginsberg HN, Goldberg AC, Howard WJ, Jacobson MS, Kris-Etherton PM, Lennie TA, Levi M, Mazzone T, Pennathur S, American Heart Association Clinical Lipidology T, Prevention Committee of the Council on Nutrition PA, Metabolism, Council on Arteriosclerosis T, Vascular B, Council on Cardiovascular N, Council on the Kidney in Cardiovascular D. Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association. Circulation. 2011;123(20):2292-2333.
Dron JS, Hegele RA. Genetics of Hypertriglyceridemia. Front Endocrinol (Lausanne). 2020;11:455.
Kissebah AH, Harrigan P, Wynn V. Mechanism of hypertriglyceridaemia associated with contraceptive steroids. Horm Metab Res. 1973;5(3):184-190.
Castro MR, Nguyen TT, O'Brien T. Clomiphene-induced severe hypertriglyceridemia and pancreatitis. Mayo Clin Proc. 1999;74(11):1125-1128.
Jellinger PS, Smith DA, Mehta AE, Ganda O, Handelsman Y, Rodbard HW, Shepherd MD, Seibel JA, Dyslipidemia ATFfMo, Prevention of A. American Association of Clinical Endocrinologists' Guidelines for Management of Dyslipidemia and Prevention of Atherosclerosis. Endocr Pract. 2012;18 Suppl 1:1-78.
Patel SB, Wyne KL, Afreen S, Belalcazar LM, Bird MD, Coles S, Marrs JC, Peng CC, Pulipati VP, Sultan S, Zilbermint M. American Association of Clinical Endocrinology Clinical Practice Guideline on Pharmacologic Management of Adults With Dyslipidemia. Endocr Pract. 2025;31(2):236-262.
Virani SS, Morris PB, Agarwala A, Ballantyne CM, Birtcher KK, Kris-Etherton PM, Ladden-Stirling AB, Miller M, Orringer CE, Stone NJ. 2021 ACC Expert Consensus Decision Pathway on the Management of ASCVD Risk Reduction in Patients With Persistent Hypertriglyceridemia: A Report of the American College of Cardiology Solution Set Oversight Committee. J Am Coll Cardiol. 2021;78(9):960-993.
Jacobson TA, Ito MK, Maki KC, Orringer CE, Bays HE, Jones PH, McKenney JM, Grundy SM, Gill EA, Wild RA, Wilson DP, Brown WV. National lipid association recommendations for patient-centered management of dyslipidemia: part 1--full report. J Clin Lipidol. 2015;9(2):129-169.
Ginsberg HN, Packard CJ, Chapman MJ, Boren J, Aguilar-Salinas CA, Averna M, Ference BA, Gaudet D, Hegele RA, Kersten S, Lewis GF, Lichtenstein AH, Moulin P, Nordestgaard BG, Remaley AT, Staels B, Stroes ESG, Taskinen MR, Tokgozoglu LS, Tybjaerg-Hansen A, Stock JK, Catapano AL. Triglyceride-rich lipoproteins and their remnants: metabolic insights, role in atherosclerotic cardiovascular disease, and emerging therapeutic strategies-a consensus statement from the European Atherosclerosis Society. Eur Heart J. 2021;42(47):4791-4806.
Visseren FLJ, Mach F, Smulders YM, Carballo D, Koskinas KC, Back M, Benetos A, Biffi A, Boavida JM, Capodanno D, Cosyns B, Crawford C, Davos CH, Desormais I, Di Angelantonio E, Franco OH, Halvorsen S, Hobbs FDR, Hollander M, Jankowska EA, Michal M, Sacco S, Sattar N, Tokgozoglu L, Tonstad S, Tsioufis KP, van Dis I, van Gelder IC, Wanner C, Williams B, Societies ESCNC, Group ESCSD. 2021 ESC Guidelines on cardiovascular disease prevention in clinical practice. Eur Heart J. 2021;42(34):3227-3337.
Fried SK, Rao SP. Sugars, hypertriglyceridemia, and cardiovascular disease. Am J Clin Nutr. 2003;78(4):873S-880S.
Johnson RK, Appel LJ, Brands M, Howard BV, Lefevre M, Lustig RH, Sacks F, Steffen LM, Wylie-Rosett J, American Heart Association Nutrition Committee of the Council on Nutrition PA, Metabolism, the Council on E, Prevention. Dietary sugars intake and cardiovascular health: a scientific statement from the American Heart Association. Circulation. 2009;120(11):1011-1020.
Li J, Siegrist J. Physical activity and risk of cardiovascular disease--a meta-analysis of prospective cohort studies. Int J Environ Res Public Health. 2012;9(2):391-407.
Mestek ML, Plaisance EP, Ratcliff LA, Taylor JK, Wee SO, Grandjean PW. Aerobic exercise and postprandial lipemia in men with the metabolic syndrome. Medicine and science in sports and exercise. 2008;40(12):2105-2111.
Maraki MI, Sidossis LS. The latest on the effect of prior exercise on postprandial lipaemia. Sports Med. 2013;43(6):463-481.
Karpe F. Postprandial lipemia--effect of lipid-lowering drugs. Atheroscler Suppl. 2002;3(1):41-46.
Parhofer KG, Laubach E, Barrett PH. Effect of atorvastatin on postprandial lipoprotein metabolism in hypertriglyceridemic patients. J Lipid Res. 2003;44(6):1192-1198.
Parhofer KG, Barrett PH, Schwandt P. Atorvastatin improves postprandial lipoprotein metabolism in normolipidemlic subjects. J Clin Endocrinol Metab. 2000;85(11):4224-4230.
Schaefer EJ, McNamara JR, Tayler T, Daly JA, Gleason JA, Seman LJ, Ferrari A, Rubenstein JJ. Effects of atorvastatin on fasting and postprandial lipoprotein subclasses in coronary heart disease patients versus control subjects. Am J Cardiol. 2002;90(7):689-696.
Rosenson RS, Wolff DA, Huskin AL, Helenowski IB, Rademaker AW. Fenofibrate therapy ameliorates fasting and postprandial lipoproteinemia, oxidative stress, and the inflammatory response in subjects with hypertriglyceridemia and the metabolic syndrome. Diabetes Care. 2007;30(8):1945-1951.
Ohno Y, Miyoshi T, Noda Y, Oe H, Toh N, Nakamura K, Kohno K, Morita H, Ito H. Bezafibrate improves postprandial hypertriglyceridemia and associated endothelial dysfunction in patients with metabolic syndrome: a randomized crossover study. Cardiovasc Diabetol. 2014;13:71.
Clofibrate and niacin in coronary heart disease. Jama. 1975;231(4):360-381.
Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, Friedewald W. Fifteen year mortality in Coronary Drug Project patients: long-term benefit with niacin. J Am Coll Cardiol. 1986;8(6):1245-1255.
King JM, Crouse JR, Terry JG, Morgan TM, Spray BJ, Miller NE. Evaluation of effects of unmodified niacin on fasting and postprandial plasma lipids in normolipidemic men with hypoalphalipoproteinemia. Am J Med. 1994;97(4):323-331.
Usman MH, Qamar A, Gadi R, Lilly S, Goel H, Hampson J, Mucksavage ML, Nathanson GA, Rader DJ, Dunbar RL. Extended-release niacin acutely suppresses postprandial triglyceridemia. Am J Med. 2012;125(10):1026-1035.
Guyton JR, Slee AE, Anderson T, Fleg JL, Goldberg RB, Kashyap ML, Marcovina SM, Nash SD, O'Brien KD, Weintraub WS, Xu P, Zhao XQ, Boden WE. Relationship of lipoproteins to cardiovascular events: the AIM-HIGH Trial (Atherothrombosis Intervention in Metabolic Syndrome With Low HDL/High Triglycerides and Impact on Global Health Outcomes). J Am Coll Cardiol. 2013;62(17):1580-1584.
Group HTC, Landray MJ, Haynes R, Hopewell JC, Parish S, Aung T, Tomson J, Wallendszus K, Craig M, Jiang L, Collins R, Armitage J. Effects of extended-release niacin with laropiprant in high-risk patients. N Engl J Med. 2014;371(3):203-212.
Nutescu EA, Shapiro NL. Ezetimibe: a selective cholesterol absorption inhibitor. Pharmacotherapy. 2003;23(11):1463-1474.
Masuda D, Nakagawa-Toyama Y, Nakatani K, Inagaki M, Tsubakio-Yamamoto K, Sandoval JC, Ohama T, Nishida M, Ishigami M, Yamashita S. Ezetimibe improves postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia. Eur J Clin Invest. 2009;39(8):689-698.
Olijhoek JK, Hajer GR, van der Graaf Y, Dallinga-Thie GM, Visseren FL. The effects of low-dose simvastatin and ezetimibe compared to high-dose simvastatin alone on post-fat load endothelial function in patients with metabolic syndrome: a randomized double-blind crossover trial. J Cardiovasc Pharmacol. 2008;52(2):145-150.
Cannon CP, Blazing MA, Giugliano RP, McCagg A, White JA, Theroux P, Darius H, Lewis BS, Ophuis TO, Jukema JW, De Ferrari GM, Ruzyllo W, De Lucca P, Im K, Bohula EA, Reist C, Wiviott SD, Tershakovec AM, Musliner TA, Braunwald E, Califf RM, Investigators I-I. Ezetimibe Added to Statin Therapy after Acute Coronary Syndromes. N Engl J Med. 2015;372(25):2387-2397.
Harris WS, Miller M, Tighe AP, Davidson MH, Schaefer EJ. Omega-3 fatty acids and coronary heart disease risk: clinical and mechanistic perspectives. Atherosclerosis. 2008;197(1):12-24.
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet. 2007;369(9567):1090-1098.
Tinker LF, Parks EJ, Behr SR, Schneeman BO, Davis PA. (n-3) fatty acid supplementation in moderately hypertriglyceridemic adults changes postprandial lipid and apolipoprotein B responses to a standardized test meal. J Nutr. 1999;129(6):1126-1134.
Park Y, Harris WS. Omega-3 fatty acid supplementation accelerates chylomicron triglyceride clearance. J Lipid Res. 2003;44(3):455-463.
Slivkoff-Clark KM, James AP, Mamo JC. The chronic effects of fish oil with exercise on postprandial lipaemia and chylomicron homeostasis in insulin resistant viscerally obese men. Nutr Metab (Lond). 2012;9:9.
Feingold KR, Grunfeld C. Cholesterol Lowering Drugs. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Feingold K, Grunfeld C. Triglyceride Lowering Drugs. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, eds. Endotext. South Dartmouth (MA)2000.
Gaudet D, Alexander VJ, Baker BF, Brisson D, Tremblay K, Singleton W, Geary RS, Hughes SG, Viney NJ, Graham MJ, Crooke RM, Witztum JL, Brunzell JD, Kastelein JJ. Antisense Inhibition of Apolipoprotein C-III in Patients with Hypertriglyceridemia. N Engl J Med. 2015;373(5):438-447.
Paik J, Duggan S. Volanesorsen: First Global Approval. Drugs. 2019;79(12):1349-1354.
Gouni-Berthold I, Alexander VJ, Yang Q, Hurh E, Steinhagen-Thiessen E, Moriarty PM, Hughes SG, Gaudet D, Hegele RA, O'Dea LSL, Stroes ESG, Tsimikas S, Witztum JL, group Cs. Efficacy and safety of volanesorsen in patients with multifactorial chylomicronaemia (COMPASS): a multicentre, double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Diabetes Endocrinol. 2021;9(5):264-275.
Witztum JL, Gaudet D, Freedman SD, Alexander VJ, Digenio A, Williams KR, Yang Q, Hughes SG, Geary RS, Arca M, Stroes ESG, Bergeron J, Soran H, Civeira F, Hemphill L, Tsimikas S, Blom DJ, O'Dea L, Bruckert E. Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome. N Engl J Med. 2019;381(6):531-542.
Gaudet D, Brisson D, Tremblay K, Alexander VJ, Singleton W, Hughes SG, Geary RS, Baker BF, Graham MJ, Crooke RM, Witztum JL. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med. 2014;371(23):2200-2206.
Stroes ESG, Alexander VJ, Karwatowska-Prokopczuk E, Hegele RA, Arca M, Ballantyne CM, Soran H, Prohaska TA, Xia S, Ginsberg HN, Witztum JL, Tsimikas S, Balance I. Olezarsen, Acute Pancreatitis, and Familial Chylomicronemia Syndrome. N Engl J Med. 2024;390(19):1781-1792.
Chebli J, Larouche M, Gaudet D. APOC3 siRNA and ASO therapy for dyslipidemia. Curr Opin Endocrinol Diabetes Obes. 2024;31(2):70-77.
Watts GF, Rosenson RS, Hegele RA, Goldberg IJ, Gallo A, Mertens A, Baass A, Zhou R, Muhsin M, Hellawell J, Leeper NJ, Gaudet D, Group PS. Plozasiran for Managing Persistent Chylomicronemia and Pancreatitis Risk. N Engl J Med. 2025;392(2):127-137.
Harada-Shiba M, Ali S, Gipe DA, Gasparino E, Son V, Zhang Y, Pordy R, Catapano AL. A randomized study investigating the safety, tolerability, and pharmacokinetics of evinacumab, an ANGPTL3 inhibitor, in healthy Japanese and Caucasian subjects. Atherosclerosis. 2020;314:33-40.
Rosenson RS, Gaudet D, Hegele RA, Ballantyne CM, Nicholls SJ, Lucas KJ, San Martin J, Zhou R, Muhsin M, Chang T, Hellawell J, Watts GF, Team A-T. Zodasiran, an RNAi Therapeutic Targeting ANGPTL3, for Mixed Hyperlipidemia. N Engl J Med. 2024;391(10):913-925.
Szczepanska E, Gietka-Czernel M. FGF21: A Novel Regulator of Glucose and Lipid Metabolism and Whole-Body Energy Balance. Horm Metab Res. 2022;54(4):203-211.
Geng L, Lam KSL, Xu A. The therapeutic potential of FGF21 in metabolic diseases: from bench to clinic. Nat Rev Endocrinol. 2020;16(11):654-667.
Schlein C, Talukdar S, Heine M, Fischer AW, Krott LM, Nilsson SK, Brenner MB, Heeren J, Scheja L. FGF21 Lowers Plasma Triglycerides by Accelerating Lipoprotein Catabolism in White and Brown Adipose Tissues. Cell Metab. 2016;23(3):441-453.
Rader DJ, Maratos-Flier E, Nguyen A, Hom D, Ferriere M, Li Y, Kompa J, Martic M, Hinder M, Basson CT, Yowe D, Diener J, Goldfine AB, Team CXS. LLF580, an FGF21 Analog, Reduces Triglycerides and Hepatic Fat in Obese Adults With Modest Hypertriglyceridemia. J Clin Endocrinol Metab. 2022;107(1):e57-e70.
Bhatt DL, Bays HE, Miller M, Cain JE, 3rd, Wasilewska K, Andrawis NS, Parli T, Feng S, Sterling L, Tseng L, Hartsfield CL, Agollah GD, Mansbach H, Kastelein JJP, Investigators EP. The FGF21 analog pegozafermin in severe hypertriglyceridemia: a randomized phase 2 trial. Nat Med. 2023;29(7):1782-1792.

Assay of Thyroid Hormone and Related Substances

ABSTRACT

This chapter reviews how improvements in the sensitivity and specificity of thyroid tests [total and free thyroid hormones (T4 and T3), TSH, thyroid autoantibodies (TRAb, TPOAb, and TgAb) and thyroglobulin (Tg)] have advanced the detection and treatment of thyroid disorders. The strengths and limitations of current methodologies [Radioimmunoassay (RIA), Immunometric assay (IMA) and Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)] are discussed, together with their propensity for analyte-specific and non-specific interferences relating to analyte heterogeneity (TSH, TgAb and Tg), analyte-specific autoantibodies (T4Ab, T3Ab, TSHAb and TgAb) and interferences from heterophile antibodies (HAb) or assay reagents such as Biotin and Rhuthenium. Currently, between-method differences preclude establishing universal thyroid test reference ranges. However, collaborations between the International Federation of Clinical Chemistry (IFCC), the committee for the standardization of thyroid function tests (C-STFT), and the in-vitro diagnostic (IVD) industry are now focused on eliminating these between-method differences.

INTRODUCTION

Figure 1 shows the timeline for improvements in the sensitivity and specificity of thyroid test methodologies made over the last 60 years (1). In the 1950s the only thyroid test available was an indirect estimate of the serum total (free + protein-bound) thyroxine (T4) concentration, using the protein bound iodine (PBI) technique (2). Early technological advances in radioimmunoassay (RIA) (3-6), immunometric assay (IMA) (7-11), and most recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies (12-14) have progressively improved the sensitivity and specificity of thyroid tests. Currently, most thyroid testing is made on serum specimens using automated IMA methodology to measure total thyroid hormones (TT4 and TT3), estimate free thyroid hormones (FT4 and FT3) (13,15,16), and measure TSH (13) and thyroglobulin (Tg) (14,17). Automated IMA methodology is also used to detect autoantibodies that target the TSH receptor (TRAb) (18-20), the thyroid peroxidase enzyme (TPOAb) (21), and the thyroglobulin protein (TgAb) (22-24). When indicated, the thyroid hormone binding proteins thyroxine binding globulin (TBG), transthyretin (TTR)/prealbumin (TBPA), and albumin can also be measured (25-27). The IFCC and CDC continue their efforts to encourage test manufacturers to identify the causes of, and reduce the magnitude of, between-method variability in thyroid hormone and TSH measurements (12,28-33). Isotope-dilution liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS) has become the reference measurement procedure (RMP) for total thyroid hormone measurements (28) and free hormone (FT4 and FT3) measurement in equilibrium dialysates (15,28,34,35). TSH methods are now being re-standardized to the new International Reference Preparation (81/615) and harmonized to the all-method mean (13,33,36). Although serum Tg can now be detected by LC-MS/MS as tryptic peptides (37-42), the clinical value relative to the expense of this technique is still debated (14,41,43). Thus, despite technical improvements in sensitivity, specificity and standardization, the problem of substantial between-method variabilities remains for all tests (13,14,28,30,32,33,35,44-46). Establishing universal thyroid test reference ranges that would apply to all methods, by removing current between-method biases, would greatly benefit healthcare systems worldwide. Current guidelines for managing pregnant (47,48) and non-pregnant patients with hypothyroidism (49-52), hyperthyroidism (53,54), thyroid nodules (55), or differentiated thyroid cancers (DTC) (17,23,56-60) are also referenced.

Figure 1. Timeline for the Major Technical Advances in Thyroid Testing. The figure shows the development of increasingly more sensitive TSH tests: first generation, (1G), second generation (2G), and third generation (3G), and advances in the methodologies used to measure total thyroid hormones (TT4 and TT3), indirectly estimate free thyroid hormones (FT4 and FT3), directly measure FT4, and measure the thyroid autoantibodies TPOAb, TgAb, and TRAb and Thyroglobulin (Tg). From reference 1.

TOTAL THYROID HORMONE MEASUREMENTS (TT4 and TT3)

Thyroxine (T4) circulates 99.97 percent bound to the plasma proteins, primarily TBG (60-75 %) but also transthyretin TTR/TBPA (15-30 %) and albumin (~10 %) (25,26,61). In contrast 99.7 % of Triiodothyronine (T3) is bound to TBG (26,61). The total (free + protein-bound) thyroid hormones (TT4 and TT3) circulate at nanomolar concentrations that are considerably easier to measure than the free hormone moieties (FT4 and FT3) that circulate in the picomolar range (62). Serum TT4 methods have evolved over the past five decades from protein-bound iodine and competitive protein binding tests (2,63) to non-isotopic immunometric assays and most recently, isotope dilution tandem mass spectrometry (ID-LC-MS/MS) methods (13,64,65) (66). Since total thyroid hormone concentrations are influenced by conditions that change the binding protein concentrations (Figure 2), the measurement of the free thyroid hormone is considered more clinically reliable (13).

Figure 2. Conditions that Influence Thyroid Hormone Binding Proteins. From references 25, 27 and 61.

Total thyroid hormone methods typically require the inclusion of inhibitors, such as 8-anilino-1-napthalene-sulphonic acid to block hormone binding to serum proteins and facilitate the binding of thyroid hormone to the antibody reagent(s) (67). T3 concentrations are ten-fold lower than T4, so measuring T3 has always presented a greater sensitivity and precision challenge than measuring T4. Currently both TT4 and TT3 are measured by immunometric assays performed on automated platforms using enzymes, fluorescence, or chemiluminescent molecules as signals (11,13,62).

Between-method variability among eleven TT4 and twelve TT3 immunoassays are shown in Figure 3 (28) from sera from healthy individuals and compared with values reported by isotope dilution tandem mass spectrometry (ID-LC-MS/MS) - the reference measurement procedure (RMP) that uses primary T4 and T3 standards for calibration (13,28). Although most methods fell short of the optimal 5 percent goal established by the C-STFT, 4/11 TT4 assays agreed within 10 percent of the reference, whereas most TT3 assays exhibited a positive bias that would necessitate re-standardization (28,68,69). Thus, as would be expected, TT4 assays are more reliable than TT3 assays. However, variability persists likely resulting from matrix differences between calibrators and patient sera, the efficiency of the blocking agent employed, and reagent lot-to-lot variability (13,69-72).

Figure 3. (a) TT4 and (b) TT3 Between-Method Variability. The figure shows the variability among 11 TT4 (A-P) and 12 TT3 (A-M) methods (shown as assay means 2 sd) relative to the RMP for the method. For the assays differing >10% from the RMP mean, the numerical value of the mean is listed (28).

TT4 and TT3 Reference Ranges

The problem of between-method differences in TT4 and TT3 measurements (Figure 3) is compounded by the continued use of non-SI units by some countries. TT4 reference ranges have approximated 58 to 160 nmol/L (4.5-12.5 µg/dL) for more than four decades. However, in euthyroid pregnant women there is an approximate 2-fold rise in TBG concentrations by mid-gestation that produce a steady TT4 increase beginning in the first trimester and plateauing at approximately 1.5-fold pre-pregnancy levels by mid-gestation (73-75). As a result, some have suggested that the non-pregnant TT4 reference range be adjusted by a factor of 1.5 when assessing thyroid status in the latter half of gestation (47,73,74,76). TT3 reference ranges generally approximate 1.2 - 2.7 nmol/L (80 –180 ng/dL) (77), but as shown in Figure 3, TT3 displays more between-method variability than TT4 (69,78).

FREE THYROID HORMONE TESTS (FT4 and FT3)

In accordance with the free hormone hypothesis, it is the free thyroid hormone fractions (0.02 % of TT4 and 0.2 % of TT3) that exert biologic activity at the cellular level (79) and protein-bound hormone is considered biologically inert. Since binding-protein abnormalities are highly prevalent (Figure 2) (25,27,61), free hormone measurements (FT4 and FT3) are preferable to total hormone (TT4 and TT3) (13,15). However, the measurement of free hormone concentrations independent of protein-bound hormone remains technically challenging (13,15,80). This is especially the case for FT3, because FT3 immunoassays are more susceptible to interference by free fatty acids and drugs present in the circulation, prompting many laboratories to prefer a TT3 over a FT3 assay (13). FT4 and FT3 fall into two categories – direct methods that employ a physical separation of free from protein-bound hormone and indirect free hormone estimate tests (16).

Direct FT4 and FT3 Methods

Direct free hormone methods have employed equilibrium dialysis (ED) (13,81,82), ultrafiltration (83-85), or gel filtration (86) to separate free hormone from the dominant protein-bound moiety. The IFCC has now established equilibrium dialysis, isotope dilution, liquid chromatography, tandem mass spectrometry (ED ID-LC-MS/MS) using primary calibrators as the RMP for FT4 measurements (13,32,87-89). Specifically, equilibrium dialysis of serum is performed under defined conditions before measuring FT4 in the dialysate by ID-LC-MS/MS (12,34,35). Manufacturers are recommended to use this RMP to recalibrate their FT4 immunoassay tests (13). However, even direct methods that employ equilibrium dialysis or ultrafiltration to separate free from protein-bound hormone are not immune from technical problems relating to dilution, adsorption, membrane defects, temperature, the influence of endogenous binding protein inhibitors, fatty acid formation, and sample-related effects (13,80,82,90). Because direct free hormone methods are technically demanding, inconvenient, and expensive, they are typically only readily available in reference laboratories and most clinical laboratories use FT4 and FT3 estimate tests - immunoassay “sequestration” methods (see below). However, a direct free hormone test can be especially useful for evaluating thyroid status when immunoassay values appear discordant with the clinical presentation and/or the TSH measurement (15,91). All current FT4 and FT3 estimate tests remain binding-protein dependent to some extent (69).

EQUILIBRIUM DIALYSIS (ED)

Early equilibrium dialysis methods used I¹³¹ and later I¹²⁵ labeled T4 tracers to measure the free T4 fraction, that when multiplied by a total hormone measurement gave an estimate of the free hormone concentration (81). Subsequently, symmetric dialysis in which serum was dialyzed without dilution (or employing a near-physiological medium) was used to overcome dilution effects (82). By the early 1970s higher affinity T4 antibodies (>1x10¹¹ L/mol) and high specific activity T4-I¹²⁵ tracers were used to develop sensitive RIA methods that could directly measure FT4 and FT3 in dialysates and ultrafiltrates (83,92). Subsequent improvements have involved employing more physiological buffer diluents and improving the dialysis cell design (82,92). More recently, isotope-dilution liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS) (93) has been used to measure FT4 in ultrafiltrates (94) and dialysates (13,32,35,36,87,95,96).

ULTRAFILTRATION METHODS

Ultrafiltration has also been used to remove protein-bound T4 prior to LC-MS/MS measurement of FT4 in the ultrafiltrate (97). Direct FT4 measurements employing ultrafiltration are sometimes higher than those made by equilibrium dialysis, because ultrafiltration avoids dilution effects (98). Moreover, ultrafiltration is not influenced by dialyzable inhibitors of T4-protein binding that can be present in conditions such as non-thyroidal illness (NTI) (90). However, ultrafiltration can be prone to errors when there is a failure to completely exclude protein-bound hormone and/or adsorption of hormone onto the filters, glassware, and tubing (99). In addition, ultrafiltration is temperature dependent such that ultrafiltration performed at ambient temperature (25°C) will report FT4 results that are 67 percent lower than ultrafiltration performed at 37°C (97). However, FT4 concentrations measured by ID-LC-MS/MS following either ultrafiltration at 37°C or equilibrium dialysis usually correlate (100).

GEL ABSORPTION METHODS

Some early direct FT4 methods used Sephadex LH-20 columns to separate free from bound hormone before eluting the free T4 from the column for measurement by a sensitive RIA. However, because of a variety of technical issues, assays based on this methodologic approach are not currently used (62).

Indirect Free T4 and Free T3 Estimate Tests

The first free hormone estimate tests were free hormone “indexes” (FT4I and FT3I) – a correction of the total hormone concentration for the influence of binding proteins assessed either using a direct TBG measurement or a binding-protein estimate (uptake) test (101,102). Current free hormone estimate tests are typically automated immunoassays that employ an antibody to sequester a small amount of the total hormone that is purportedly proportional to the free hormone concentration (13,15). Both index tests (FT4I and FT3I) and FT4 and FT3 immunoassays are typically protein-dependent to some extent and may under- or overestimate free hormone when binding proteins are grossly abnormal (80,103-105). As with TT4 methods, current FT4 immunoassays have significant between-method variability and biases (relative to the RMP) that far exceed the biological FT4 variation (Figure 4) (13,28,69). Recalibrating methods against the RMP has been shown to significantly reduce biases (32). It is hoped that manufacturers will continue to work to eliminate between-method biases and establish reference intervals that would apply to all methods (106).

Figure 4. FT4 Between-Method Variability in FT4 Immunoassays. This figure shows deviations in FT4 measurements made by 13 different immunoassays relative to the reference measurement procedure (RMP = ED-ID-LC-MS/MS) (89).

TWO TEST INDEX METHODS (FT4I AND FT3I)

Free hormone indices (FT4I and FT3I) are unitless mathematical calculations made by correcting the total hormone test result for the influence of binding proteins, primarily TBG (107). These indexes that have been used for more than 50 years require two separate tests to estimate free hormone (80). The first test involves the measurement of total hormone (TT4 or TT3) whereas the second test assesses the binding protein concentration by either a direct TBG immunoassay (103), a Thyroid Hormone Binding Ratio (THBR) or “Uptake” test (102), or an isotopic determination of the free hormone fraction (80,108).

TBG Immunoassays

Data has been conflicting concerning whether indexes that employ THBR in preference to a direct TBG are diagnostically superior (109). Free hormone indexes calculated using TBG measurement (TT4/TBG) may offer improved diagnostic accuracy over THBR when the total hormone concentration is abnormally high (i.e. hyperthyroidism), or when drug therapies interfere with THBR tests (110). Regardless, the TT4/TBG index is not totally independent of the TBG concentration, nor does it correct for albumin or transthyretin binding protein abnormalities (figure 2) (104).

Thyroid Hormone Binding Ratio (THBR) / "Uptake" Tests

The first "T3 uptake" tests developed in the 1950s employed the partitioning of T3-I¹³¹ tracer between the plasma proteins in the specimen and an inert scavenger (red cell membranes, talc, charcoal, ion-exchange resin, or antibody) (111-113). The "uptake" of T3 tracer onto the scavenger provided an indirect, reciprocal estimate of the TBG concentration in the specimen. Initially, T3 uptake tests were reported as percent uptakes (free/total tracer). Sera with normal TBG concentrations typically had approximately 30 percent of the T3 tracer taken up by the scavenger. During the 1970s methods were refined by replacing I¹³¹-T3 tracers by I¹²⁵-T3 with a calculation of the hormone uptake based on the ratio of isotopic counts between the absorbent, and total minus absorbent counts. Results were expressed as a ratio with normal sera having an assigned value of 1.00 (108). Historically, the use of T3 as opposed to T4 tracer was made for practical reasons relating to the ten-fold lower affinity of TBG for T3 versus T4, facilitating a higher percentage of T3 tracer binding to the scavenger, thereby allowing shorter isotopic counting times. Because current methods use non-isotopic proprietary T4 or T3 "analogs", counting time is no longer an issue and current tests may use a "T4 uptake" approach - which may be more appropriate for correcting for T4-binding protein effects. Differences between T3 and T4 "uptakes" have not been extensively studied (114). Although all THBR tests are to some degree TBG dependent, the calculated FT4I and FT3I usually provides an adequate correction for mild TBG abnormalities (i.e. pregnancy and estrogen therapy) (73,102,103,115) but may fail to correct for grossly abnormal binding proteins (26) seen in euthyroid patients with congenital TBG extremes (103,104,116), familial dysalbuminemias (62,105,117-119), thyroid hormone autoantibodies (120-122), or medications that directly or indirectly influence thyroid hormone binding to plasma proteins (13,62,104,123).

Isotopic Index Methods

The first free hormone tests developed in the 1960s were indexes calculated from the product of the free hormone fraction, measured isotopically by dialysis, and TT4 measured by PBI and later RIA (81). These early isotopic detection systems were technically demanding and included paper chromatography, electrophoresis, magnesium chloride precipitation, and column chromatography (81,124-126). The free fraction index approach was later extended to ultrafiltration (83,85) and symmetric dialysis (127), the latter measuring the rate of transfer of isotopically labeled hormone across a membrane separating two chambers containing the same undiluted specimen. Ultrafiltration and symmetric dialysis had the advantage of eliminating dilution effects that influenced tracer dialysis values (82,128). However, free hormone indexes calculated using an isotopic free fraction were not completely independent of the TBG concentration and were influenced by tracer purity and the buffer matrix employed (92,129).

Clinical Utility of Two-Test Index Methods (FT4I and FT3I)

In the past some have favored the two-test FT4I approach for evaluating the thyroid status of patients with abnormal binding protein states like pregnancy or NTI (73,82). However, the continued use of these FT4I tests remains controversial (130). Until FT4 immunoassays are re-standardized to remove biases (13,69), FT4I remains a useful confirmatory test when binding proteins are abnormal or for diagnosing central hypothyroidism (69).

Free Thyroid Hormone Immunoassay Methods (FT4 and FT3)

Currently, most free hormone testing is made using automated FT4 and FT3 immunoassays (62,131). These immunoassays are based on "one-step", "labeled antibody" or "two-step" principles (80). For more than twenty years controversy has surrounded the standardization and diagnostic accuracy of these methods, especially in pathophysiologic conditions associated with the binding protein abnormalities such as pregnancy (15,73,131). These assays are subject to variability due to polymorphisms, drug interactions, high free fatty acid (FFA) levels, or thyroid binding inhibitors such as those present in non-thyroidal illness (NTI) (11,30, 62, 69, 90, 99,104,105,121,132). Studies of the inverse FT4/TSH log/linear relationship have emphasized the need to evaluate each method with clinical specimens containing abnormal binding proteins (94,133,134). Currently, most FT4 and FT3 immunoassays display significant negative or positive biases that exceed the intra-individual biological variability (12,13). As shown in Figure 4, all but one of the FT4 immunoassays tested had a negative bias relative to the FT4 RMP. Although the IVD industry is being encouraged to recalibrate their free hormone immunoassays against the RMP to reduce between-method biases (13, 28, 69, 87,135), implementation of a global re-calibration effort has been delayed by cost as well as practical, educational, and regulatory complexity.

ONE-STEP FT4 AND FT3 METHODS

The “one-step” approach uses a proprietary labeled hormone analog, designed for minimal interaction with thyroid hormone binding proteins, that competes with hormone in the specimen for a solid-phase anti-hormone antibody in a classic competitive immunoassay format (15,62,80). After washing away unbound constituents, the free hormone concentration should be inversely proportional to the labeled analog bound to the solid support. Although conceptually attractive, the diagnostic utility of the one-step approach has been shown to be dependent on the degree that the analog is "inert" with respect to binding proteins (80,94,133,134).

LABELED ANTIBODY FT4 AND FT3 METHODS

Labeled antibody methods are "one-step" methods that use a labeled antibody in preference to a labeled hormone analog. The free hormone in the specimen competes with solid-phase hormone for the labeled antibody and is quantified as a function of the fractional occupancy of hormone-antibody binding sites in the reaction mixture (15,62,80,136). The labeled antibody approach is used as the basis for several automated immunoassay platforms because it is easy to automate and considered less binding-protein dependent than the labeled analog approach, since the solid phase hormone does not compete with endogenous free hormone for hormone binding proteins (15,80,137-139).

TWO-STEP, BACK TITRATION FT4 AND FT3 METHODS

The two-step approach was first developed by Ekins and colleagues in the late 1970s (79,113). Two-step methods typically employ immobilized T4 or T3 antibody (for FT4 and FT3 immunoassays, respectively) to sequester a small proportion of total hormone from a diluted serum specimen without disturbing the original free to protein-bound equilibrium (62,80). After removing unbound serum constituents by washing, a labeled probe (originally^125-I T4, or more recently a macromolecular T4 conjugate) is added to quantify unoccupied antibody-binding sites that are inversely related to the free hormone concentration - a procedure that has been referred to as "back-titration (80).

CLINICAL UTILITY OF FT4 AND T3 IMMUNOASSAY MEASURMENTS

Current reference ranges for FT4 and FT3 immunoassays are method-dependent because of calibration biases that preclude establishing a universal reference range that would apply across methods (13,68,86). These biases are evident for FT4 immunoassay methods shown in Figure 4. Most FT4 methods give diagnostically reliable results when binding proteins are near-normal, provided that a method-specific reference range is employed (69). However, both TT3 and FT3 immunoassays tend to be inaccurate in the low range (78,140) and have no value for diagnosing or monitoring treatment for hypothyroidism (52,141), although FT3 measurements can be useful for diagnosing or confirming unusual cases of hyperthyroidism.

Ambulatory Patients

FT4 and FT3 tests are used in preference to TT4 or TT3 measurements because they have better diagnostic accuracy for detecting hypo- and hyperthyroidism in patients with abnormal thyroid hormone binding proteins (figure 2). FT4 typically serves as a second-line test for confirming primary thyroid dysfunction detected by an abnormal TSH, but is the first-line test when thyroid status is unstable (early phase of treating hypo- or hyperthyroidism); in the presence of pituitary/hypothalamic disease (when TSH is unreliable); or when patients are taking drugs such as dopamine or glucocorticoids that are known to affect TSH secretion (10,104,110,142). Mild "subclinical" thyroid dysfunction is characterized by a TSH/FT4 discordance (abnormal TSH/normal FT4) reflecting the intrinsic complex nature of the inverse log/linear TSH/FT4 relationship (8,10,143) - a relationship that is modified by age and sex (144,145). Thus, small changes in FT4, even within normal limits, are expected to produce a mild degree of TSH abnormality - between 0.05 and 0.3 mIU/L (with subclinical hyperthyroidism) and 5 and 10 mIU/L (with subclinical hypothyroidism). An unexpected TSH/FT4 discordance if confirmed, should prompt an investigation for interference with FT4, TSH or both tests (91,146,147). FT4 interference can result from severe binding protein abnormalities such as congenital TBG excess or deficiency (26,62,103,148,149), dysalbuminemias (105,150-152), thyroid hormone autoantibodies (147,153-155), or drug interferences (62,104,123).

Pregnant Patients

Current reference ranges for FT4 immunoassays are method-dependent because of calibration biases that precludes establishing a universal reference range that would apply across methods (Figure 4) (156,157). This between-method variability has profound effects on the setting of the FT4 reference range for pregnancy (Figure 5). As with non-pregnant patients, TSH is the first-line test to use for assessing thyroid status during pregnancy (48,158). However, FT4 measurement is needed for monitoring anti-thyroid drug treatment of hyperthyroid pregnant patients who have an undetectable TSH. The question whether an isolated low FT4 during pregnancy is a maternal or fetal risk factor, remains controversial (159,160), although some studies suggest that low FT4 may be a risk factor for gestational diabetes and fetal complications (161-163). Non-pregnant FT4 reference ranges do not apply to pregnancy since FT4 progressively declines as gestation progresses, necessitating the use of a trimester-specific reference ranges (73,158,164,165). Setting universal trimester-specific FT4 reference ranges is currently hampered by the between-method differences shown in Figure 4 and 5 (69,156,165), compounded by the differences related to ethnicity (166-170), iodine intake (171-173), smoking (174), and BMI (145,166). Establishing institution-specific trimester-specific reference ranges from the 2.5 to 97.5 percentiles by recruiting at least 400 pregnant patients (170) is not practical for most institutions. After the proposed re-standardization of FT4 methods against the RMP the feasibility of establishing universal trimester-specific reference ranges will improve (13,69,135). However, binding protein effects will remain, and population-specific factors will still have to be considered.

Figure 5. Between-Method FT4 Variability Impacts Thyroid Testing in Pregnancy. The figure shows the upper and lower FT4 reference limits (2.5–97.5%) from 43 published studies of FT4 measurements made in each trimester of pregnancy by four different methods: Abbott (1), Beckman (2), Roche (3) and Siemens (4). The data shows the expected trend for higher FT4 in the first trimester, resulting from thyroidal human chorionic gonadotropin (HCG) stimulation which is maximal in early pregnancy. The data is re-drawn with permission from reference 156.

Hospitalized Patients with Nonthyroidal Illnesses (NTI)

The diagnostic performance of current FT4 methods has not been evaluated in hospitalized patients with NTI where the severity of illness, binding protein inhibitors, and drug therapies can negatively impact the reliability of both thyroid hormone and TSH testing (10,30,62,90,122,132,181-183). Three categories of hospitalized patients deserve special attention: a) patients with NTI without known thyroid dysfunction who have a high or low T4 status; b) patients with primary hypothyroidism and concurrent NTI and, c) patients with hyperthyroidism and concurrent NTI (13). Because the diagnostic reliability of FT4 testing is still questionable in sick hospitalized patients, a combination of both T4 (FT4 or TT4) and TSH may be needed to assess thyroid status in this setting (10,13).

In most clinical situations where FT4 and TSH results are discordant, the TSH test is the most diagnostically reliable, provided that the patient does not have pituitary failure or receiving medications such as glucocorticoids or dopamine that directly inhibit TSH secretion (110,142,181). Repetitive TSH testing may be helpful in resolving the cause of an abnormal FT4, because the TSH abnormalities of NTI are typically transient (Figure 6b) whereas the TSH abnormality will persist if due to underlying thyroid dysfunction (184-187). In some cases, it may be useful to test for TPOAb as a marker for underlying thyroid autoimmunity.

Figure 6. Effects of Nonthyroidal Illness (NTI) on Thyroid Tests. Figure 6a shows the magnitude and direction of changes in total (TT4 and TT3) and free (FT4 and FT3) thyroid hormone IMA tests versus FT4 measured by the RMP (ED-ID-LC-MS/MS), as the severity of illness increases, followed by recovery. Figure 6b shows the magnitude and direction of TSH changes as the severity of illness increases, followed by recovery. Data redrawn from reference 188 with permission.

Pediatric Patients

The determination of normal reference limits for pediatric age groups is especially challenging, given the limited number of studies involving large numbers of healthy children (175-177). Most studies report that serum TSH peaks after birth and steadily declines throughout childhood to reach adult levels at puberty. Likewise, FT3 declines across the pediatric age groups during childhood and approaches the adult range at puberty, whereas FT4 levels for infants less than a year old are higher than for children 1 to 18 years old who have FT4 comparable to adults (175-180).

Interferences with Thyroid Hormone Tests

Only the ordering physician can suspect interference with a test result and request that the laboratory perform interference checks. This is because the hallmark of interference is discordance between the test result and the clinical presentation of the patient, and most specimens are sent to the laboratory with no clinical information. Failure to recognize interferences can have adverse clinical consequences (91,146,189-197).

Laboratory checks for interferences include, a) showing a discordance between different manufacturers methods (196,198-200), b) re-measurement of the analyte after adding a blocker of Heterophile antibodies (HAb) (196,200,201), c) performing linearity studies or d) precipitating interfering immunoglobulins with polyethylene glycol (PEG) (196,198). A change in the analyte concentration in response to any one of these maneuvers suggests interference, but a lack of an effect does not rule out interference.

Interferences can be classified as either (a) non-analyte-specific, or (b) analyte-specific (191,195,199).

NON-ANALYTE SPECIFIC INTERFERENCES

Protein Interferences

Either paraproteins or abnormal immunoglobulins can interfere with immunoassays (90,202-205).

Congenital TBG excess or deficiency: Free hormone immunoassays and free T4 index tests may be susceptible to interference from grossly abnormal TBG concentrations, such as seen in congenital TBG excess or deficiency states (26,62,103,148,149).

Pregnancy: Estrogen stimulation increases TBG, and consequently both TT4 and TT3, concentrations progressively rise to plateau at 2.5-fold pre-pregnancy values by mid-gestation (73). Despite the rise in total hormone, both FT4 and FT3 decline during gestation, in accordance with the law of mass action (73,157,158,206,207). However, the degree of FT4 decline during pregnancy is variable and method-dependent (Figure 5). The declining albumin concentrations typical of late gestation also affect some methods (208).

Familial Dysalbuminemias and Transthyretin Hyperthyroxinemias: Autosomal dominant mutations in the albumin or transthyretin (prealbumin) gene (209) can result in altered protein structures with enhanced affinity for thyroxine and/or triiodothyronine. These abnormal proteins can interfere with FT4 and/or FT3 measurements and result in inappropriately high FT4 and/or FT3 immunoassay values (105,151,210-212). Familial Dysalbuminemic Hyperthyroxinemia (FDH) is a rare condition with a prevalence of ~1.8 percent in the Hispanic population (119,213). It arises from a few genetic variants in the albumin gene, with the R218H being the most common. Some variants result in extremely high TT4, whereas other mutations (i.e. L66P) affect mainly TT3 (150). Affected individuals are euthyroid and have normal TSH and FT4 when measured by direct techniques such as equilibrium dialysis (105). Unfortunately, most FT4 estimate tests (immunoassays and indexes) report falsely high values for FDH patients that may prompt inappropriate treatment for presumed hyperthyroidism if the condition is not recognized (105,119).

Heterophile Antibodies (HAb)

It is well recognized that heterophile antibodies (HAb) - human poly-specific antibodies targeting animal antigens, can interfere with immunometric assays causing falsely high/positive or falsely low/negative test results (214,215). The most common interferant is human anti-mouse antibodies (HAMA) (199,215-220). Rheumatoid factor (RF), an immunoglobulin commonly associated with autoimmune conditions, is also considered a heterophile antibody that can interfere by targeting human antigens (199,217,221,222). Although HAb usually causes false positive tests, false-negative tests have also been reported (214). HAb has been shown to interfere with multiple endocrine tests that use IMA principles, including free and total thyroid hormones, TSH, Tg, and TgAb (138,193,200,214,221,223-225). The prevalence of HAbs is variable but has been reported as high as eleven percent (223,226,227). In recent years assay manufacturers have increased the immunoglobulin blocker reagents added to their tests and this has reduced HAb interference somewhat (223,225-227). However, interference is still seen in some patients with a high enough HAb to overcome the assay blocker (198,223,228). HAb interference mostly affects non-competitive immunometric assays (IMA) that employ monoclonal antibodies of murine origin (216). Assays based on the competitive format that employ high affinity polyclonal antibody reagents, are rarely affected (216). The test marketed by one manufacturer can be severely affected, whereas the test from a different manufacturer may appear unaffected (200). This is why the first step for investigating interference is re-measurement of the analyte by a different method. It should be noted that patients receiving recent vaccines, blood transfusions, or monoclonal antibodies (given for treatment or scintigraphy), as well as veterinarians and those in contact with animals, are especially prone to test interferences caused by induced HAb and human anti-mouse antibodies (HAMA) (198,229).

Anti-Reagent Antibodies

Interference can be caused by antibodies targeting assay reagents. For example, a number of reports have found that anti-rhuthenium antibodies can interfere with TSH, FT4, and FT3 tests (200,230). In addition, antibodies targeting either streptavidin (231,232) or Biotin (233,234) can interfere with assays employing streptavidin or biotin reagents.

High Dose Dietary Biotin

Some IMA tests have employed a biotin-streptavidin separation system (232). Patients who take a high dose of dietary biotin risk having test interferences with such methods (232). Depending on the specific test formulation, biotin interference can cause falsely high- or low- test results (234-236). Manufacturers are now prioritizing replacing their biotin-streptavidin separation systems to eliminate this problem (237).

ANALYTE-SPECIFIC INTERENCES

Analyte-specific interferences typically result from autoantibodies targeting the analyte (238). Autoantibodies targeting both TSH (macro-TSH) (238-241) and both thyroid hormones (T4 and/or T3) (154,155) have been reported. Autoantibody interferences may be more prevalent in patients with non-thyroid autoimmune conditions (242,243). Depending on the analyte and test formulation, thyroid hormone and TSH autoantibodies typically cause falsely high tests (239,244). It should be noted that transplacental passage of either HAb or anti-analyte autoantibodies (i.e. TSHAb or T4Ab) have the potential to interfere with neonatal screening tests (245-247). Specifically, maternal TSH autoantibodies can cross the placenta and cause a falsely high TSH screening test in the newborn mimicking congenital hypothyroidism (247), whereas maternal T4 autoantibodies could cause a falsely high neonatal T4 test and mask the presence of congenital hypothyroidism (246).

Thyroid Hormone Autoantibodies (T4Ab/T3Ab)

T4 and T3 autoantibodies can falsely elevate total hormone, free hormone, or THBR measurements depending on the method employed (153,155,210). The prevalence of thyroid hormone autoantibodies occurs in approximately 2 percent of the general population but may be present in over 30 percent of patients with autoimmune thyroid disease or other autoimmune conditions (242,243,248). However, despite their high prevalence, significant interference caused by thyroid autoantibodies is not common and depends on the qualitative characteristics of the autoantibody present (i.e. its affinity for the test reagents). Furthermore, different methods exhibit such interferences to a greater or lesser extent (120,154). Because autoantibody interference is difficult for the laboratory to detect proactively, it is the physician who should first suspect interference from an unexpected discordance between the clinical presentation of the patient and the test result(s) (249,250).

TSH (THYROID STIMULATING HORMONE) MEASUREMENT

Over the last five decades the dramatic improvements in TSH assay sensitivity and specificity have revolutionized thyroid testing and firmly established TSH as the first-line test for ambulatory patients who are not receiving drugs known to alter TSH secretion (10,13,251). Serum TSH has become the therapeutic target for levothyroxine (L-T4) replacement therapy for hypothyroidism (52) and suppression therapy for differentiated thyroid cancer (DTC) (57,252,253). The diagnostic superiority of TSH versus FT4 measurement arises from the inverse, predominantly log/linear, TSH/FT4 relationship, that is modified to some extent by factors such as age, sex, active smoking, and TPOAb status (8,10,13,143,144,254,255).

TSH Assays

TSH assay "quality" has historically been defined by clinical sensitivity – the ability to discriminate between hyperthyroid and euthyroid TSH values (8,10,13,256). The first generation of RIA methods had a detection limit approximating 1.0 mIU/L (1,3,257) that limited their clinical utility to diagnosing primary hyperthyroidism (258) and necessitated the use of TRH stimulation to diagnose hyperthyroidism, characterized by an absent TRH-stimulated TSH response (259-261). With the advent of immunometric assay (IMA) methodology that uses a combination of poly- and/or monoclonal antibodies targeting different TSH epitopes in a "sandwich" format (262-264), a ten-fold improvement in TSH assay sensitivity (~ 0.1 mIU/L) was achieved when using isotopic (I¹²⁵) signals (265). This level of sensitivity facilitated the determination of the lower TSH reference limit (0.3-0.4 mIU/L) and the detection of overt hyperthyroidism without the need for TRH stimulation (266,267) but was still insufficient for distinguishing between differing degrees of hyperthyroidism (i.e. subclinical versus overt) (268). Assay sensitization continued until a third generation of TSH IMAs was developed by employing non-isotopic signals that could achieve a sensitivity of 0.01 mIU/L (8,251,267). Initially different non-isotopic signals were used that gave rise to a lexicon of terminology to distinguish between assays: immunoenzymometric assays (IEMA) used enzyme signals; immunofluorometric assays (IFMA) used fluorophors as signals, immunochemiluminometric assays (ICMA) used chemiluminescent molecules as signals, and immunobioluminometric assays (IBMA) used bioluminescent signal molecules (112,267). Current TSH methods are mostly automated ICMAs that achieve third generation functional sensitivity (FS = ≤0.01 mIU/L) - a FS level that has now become the standard of care (10,13,269).

FUNCTIONAL SENSITIVITY (FS) = THE LOWEST REPORTABLE ASSAY LIMIT

During the period of active TSH assay sensitization, different non-isotopic IMAs made competing claims for sensitivity. Methods were described as: "sensitive", "highly sensitive", "ultrasensitive", or "supersensitive" - marketing terms that had no scientific definition. This confusion led to a debate concerning what was the most clinically relevant parameter to use to determine the lowest reliable reportable TSH value for clinical practice (10,251,267). Functional sensitivity (FS) became defined as the lowest analyte concentration measured with 20 percent coefficient of variation (10) established over a clinically relevant timespan (6-8 weeks for TSH). FS is now recognized as the parameter that best represents the between-run precision for measuring low analyte concentrations in clinical practice (10,270,271). FS is used to define the lower reportable limit for not only TSH but also Tg and TgAb, as well as other non-thyroid assays for which analytic sensitivity is critical (10). FS protocols recognize that immunoassays tend to be matrix-sensitive and specify that precision be determined in human sera rather than a quality control material that uses an artificial protein matrix (71,72,272). The timespan used for determining precision is also analyte-specific and should reflect the frequency of testing employed in clinical practice - 6 to 8 weeks for TSH, but 6 to 12 months for the Tg and TgAb - assays that are used as tumor markers for monitoring DTC. An optimal timescale is important, because low-end precision erodes over time due to a myriad of variables including reagent lot-to-lot variability (71). Note that the FS parameter is more stringent than other biochemical sensitivity parameters such as limit of detection (LOD - a within-run parameter) and limit of quantitation (LOQ - a between-run parameter without stipulations regarding the matrix and the timespan used for determining precision (72,271,273). A ten-fold difference in FS has been used to define each generation of increasingly more sensitive methods (17,251,271,274). Thus, TSH RIA methods with FS approximating 1.0 mIU/L were designated "first generation", the TSH immunoradiometric (IRMA) methods that had a functional sensitivity approximating 0.1 mIU/L were designated " second generation", and current TSH ICMAs with FS approximating 0.01 mIU/L are now designated "third generation" assays (267,270,275).

TSH BIOLOGIC VARIABILITY

TSH is a heterogeneous glycoprotein (276-278), and TRH-mediated changes in TSH glycosylation and thus detection by IMA methodology (279,280) have the potential to influence immunoactivity (277,281). Alterations in TSH glycosylation can occur in a number of pathophysiologic circumstances (278,282). Seasonal variability in TSH has been shown with 10% higher TSH levels in the winter than in the summer months (283). However, FT4 and FT3 levels show no such seasonal variability (283). The demonstration that harmonization of TSH methods successfully minimizes between-method differences (69), suggesting that under normal conditions current TSH IMAs appear to be "glycosylation blind" and detect different TSH glycoforms in an equimolar fashion (277,278). However, future studies need to include sera from conditions where TRH dysregulation may lead to abnormal TSH glycosylation and bioactivity, such as pituitary dysfunction, NTI, and aging (278,280,281,284-286).

TSH intra-individual variability is relatively narrow (20-25 percent) in both non-pregnant and pregnant subjects, as compared with between-person variability (13,287,288). In fact, the serum TSH of euthyroid volunteers was found to vary only ~0.5 mIU/L when tested every month over a span of one year (287). Twin studies suggest that there are genetic factors that determine hypothalamic-pituitary-thyroid setpoints (289-291). These studies report that the inheritable contribution to the serum TSH level approximates 65 percent (290,291). This genetic influence appears in part to involve single nucleotide polymorphisms in thyroid hormone pathway genes such as the phosphodiesterase gene (PDE8B) (292), polymorphisms causing gain (293) or loss of function TSH receptors (294), and the type II deiodinase enzyme polymorphisms (293). Undoubtedly, such polymorphisms account for some of the euthyroid outliers that skew TSH reference range calculations (295). The narrow TSH within-person variability and low (< 0.6) index of individuality (IoI) (287,288) limits the clinical utility of using the TSH population-based reference range to detect thyroid dysfunction in an individual patient (288,296-298). When evaluating patients with marginally (confirmed) low (0.1–0.4 mIU/L) or high (4–10 mIU/L) TSH abnormalities, it is more important to consider the degree of TSH abnormality relative to patient-specific risk factors for cardiovascular disease rather than the degree of the abnormality relative to the TSH reference range (13,52,299).

TSH REFERENCE RANGES

As with the thyroid hormone tests, the significant biases between different TSH methods (Figure 7) prevent establishing universal population or trimester-specific reference ranges that would apply across methods (13,170). These method biases also impact the detection of subclinical hypothyroidism (299,300). Since TSH is a complex glycoprotein, no reference measurement procedure (RMP) is available, or will likely be feasible in the future (13), given the current lack of commutability between the pituitary TSH reference preparations and patient specimens (33). A harmonization approach (31,301), whereby methods are recalibrated to the "all method mean", has been shown to have the potential to effectively eliminate current between-method TSH differences that are most pronounced at pathophysiologic levels (29,302). Better harmonization may also be possible using a reference panel of serum specimens (33). The IFCC is actively working with the IVD industry to encourage manufacturers to harmonize their methods. A reduction of between-method variability could eliminate the need to establish method-specific TSH reference ranges - a practice that is costly and inconvenient given the large numbers of rigorously screened participants that are necessary to establish reliable 2.5th to 97.5th percentiles for a population (87,303). However, even after harmonization minimizes inter-method differences, it remains to be determined to what extent universal ranges would be impacted by other factors such as age (254,304), ethnicity (254), and iodine intake (305). It may be that a reference range established in one geographic location may not be representative of a different locale or population. The harmonization of TSH methods would be advantageous for consolidating data from different studies and establishing universal reference limits (13).

Figure 7. TSH Between-Method Variability. Figure shows deviations in TSH measurements made in the low (<0.5), medium (0.5-5.0), and high (>5) mIU/L range using 14 different immunoassays. Data is expressed as deviations from the trimmed all method mean (88).

TSH POPULATION REFERENCE RANGE

The log/linear TSH/FT4 relationship (8,10,143,144,255) dictates that TSH will be the first abnormality to appear as mild (subclinical) as hypo- or hyperthyroidism develops. It follows that the setting of the TSH reference limits critically influences the frequency of diagnosing subclinical thyroid disease (50,53). It is recommended that “TSH reference intervals should be established from the 95 percent confidence limits of the log-transformed values of at least 120 rigorously screened normal euthyroid volunteers who have: (a) no detectable thyroid autoantibodies, TPOAb or TgAb; (b) no personal or family history of thyroid dysfunction; (c) no visible or palpable goiter and, (d) who are taking no medications (except estrogen)” (10,303).

Multiple factors influence population TSH reference limits, especially the upper (97.5th percentile) limit. Different methods report different ranges for the same population resulting from between-methods biases (Figure 7) (13). A key factor affecting the upper limit is the stringency used for eliminating individuals with thyroid autoimmunity from the population (306-308). Other factors relate to population demographics such as sex (254), ethnicity (254,309,310), iodine intake (311,312), BMI (313,314), and smoking status (311,315). The relationship between TSH and age is complex with most studies in iodine sufficient populations reporting an increase in the TSH upper limit with age (143,254,308,309,316). This has led to the suggestion that age-and sex-specific TSH reference limits be used (50,316). Conflicting data on this issue could merely represent population differences with an increasing prevalence of thyroid autoimmunity in iodine-sufficient populations (254,317). Whereas in iodine deficient populations, increasing autonomy of nodular goiter can result in decreased TSH with aging (318). Some studies have reported that a mild TSH elevation in elderly individuals may convey a survival benefit (319), whereas other studies dispute this (320). However, TSH is a labile hormone, and studies cannot assume that a TSH abnormality found in a single determination is representative of thyroid status in the long-term (321).

PEDIATRIC TSH REFERENCE RANGES

The adult TSH population reference range does not apply to neonates or children. Serum TSH values are generally higher in neonates and then gradually decline until the adult range is reached after puberty (178,179,322,323). This necessitates using age-specific TSH reference ranges for diagnosing thyroid dysfunction in different pediatric age groups.

SUBCLINICAL THYROID DYSFUNCTION

Subclinical Hyperthyroidism (SCHY) is defined as a low (<2.5th percentile) but detectable TSH (0.01 - 0.3 mIU/L range) without a FT4 abnormality. SCHY seems relatively independent of the method used (324-326). Endogenous SCHY prevalence is low (0.7 %) in iodine-sufficient populations (254) but may increase as an iatrogenic consequence of L-T4 replacement therapy (327-330). SCHY is a risk factor for osteoporosis and increased fracture risk (331) as well as atrial fibrillation and cardiovascular disease (325,332-334), especially in older patients.

Subclinical Hypothyroidism (SCHO) is defined as a TSH above the upper (>97.5th percentile) TSH reference limit without a FT4 abnormality (50,300,308,335). However, the setting of the TSH upper limit remains controversial, thus the prevalence of SCHO is highly variable - 4 to 8.5 percent rising to 15 percent in older populations (254,299,307,335). In most cases, SCHO is associated with TPOAb positivity, indicative of an autoimmune etiology (307). The clinical consequences of SCHO relate to the degree of TSH elevation (336,337). Most guidelines recommend L-T4 treatment of SCHO when TSH is above 10 mIU/L (49,50), but below 10 mIU/L L-T4 treatment is usually based on patient-specific risk factors (50). There is active debate concerning the efficacy of treating SCHO to prevent progression (338-340), or improve renal (341), cardiovascular (333,336,342-346), or lipid (347,348) abnormalities that can be associated with SCHO.

THYROID DYSFUNCTION IN PREGNANCY

Overt hypo- or hyperthyroidism is associated with both maternal and fetal complications (349-352). However, the impact of maternal subclinical thyroid dysfunction remains controversial (51), although no maternal or fetal complications appear associated with subclinical hyperthyroidism during pregnancy (349,353). First trimester "gestational hyperthyroidism" is typically transient and hCG-related (354). In contrast, short-term and long-term outcome studies of maternal subclinical hypothyroidism (51,355) are complicated by heterogeneity among studies arising from a myriad of factors influencing TSH cutoffs, such as gestational stage, TSH method used, maternal TPOAb status, and current and pre-pregnancy iodine intake (160,172). Using gestational age-specific reference intervals the frequency of SCHO in first trimester pregnancy approximates 2-5 percent (355,356). Studies have found that subclinical hypothyroidism is associated with increased frequency of maternal and fetal complications, especially when TPOAb is positive (51,160,349,357-362). Maternal complications have included miscarriage (358), preeclampsia (363,364), placental abruption (350), preterm delivery (349,358,365,366), and post-partum thyroiditis (359). Fetal complications have included intrauterine growth retardation and low birth weight (350,353) and possible impaired neuropsychological development (367,368). It remains controversial whether L-T4 treatment of SCHO in early gestation decreases the risk of complications (358,362,369).

Trimester-Specific TSH Reference Ranges. As with non-pregnant patients, TSH is the first-line test used for assessing thyroid status during pregnancy when gestation-related TSH changes occur (47,51,76,158,355). Currently, method specific TSH reference ranges are needed for each trimester because of between-method variability (Figure 8). In the first trimester, there is a transient rise in FT4 caused by high hCG concentrations stimulating the TSH receptor - because hCG shares some homology with TSH (370-372). The degree of TSH suppression is inversely related to the hCG concentration and can be quite profound in patients with hyperemesis who have an especially high hCG (165,370,372-374). As gestation progresses, TSH tends to return towards pre-pregnancy levels (165). Recent studies from different geographic areas with diverse iodine intakes using different TSH methods have reported higher trimester-specific TSH upper limits than recommended by previous guidelines (51,159,164,165, 355, 375). In response, the American Thyroid Association have revised their pregnancy guidelines (47,48) to replace trimester-specific reference limits by a universal upper TSH limit of 4.0 mIU/L, when TPOAb is negative and no local reference range data is available (376). However, at this time between-method biases (Figure 7) clearly preclude proposing universal TSH cut offs that would apply to all methods and all populations including pregnant patients (69,87,164,165). IVD manufacturers are being encouraged to harmonize their TSH methods so that universal reference limits can be established for pregnancy (69,87). Requiring each institution to establish their own trimester-specific reference ranges is impractical, given the costs, logistics and ethical considerations involved in recruiting the more than 400 disease-free pregnant women that would be needed to represent each trimester (158). Even after methods are re-standardized (FT4) or harmonized (TSH), trimester-specific reference ranges would still be influenced by differences in ethnicity and iodine intake, especially the pre-pregnancy iodine intake that influences thyroidal iodine stores (172). In addition, since the TSH upper limit is skewed by the inclusion of individuals with thyroid autoimmunity, reliable method-specific TPOAb cutoffs need to be established (165,372,377).

Figure 8. Between-Method TSH Variability Impacts Thyroid Testing in Pregnancy. The figure is a summary of 43 published studies showing the upper and lower TSH reference limits (2.5–97.5 %) measured in each trimester of pregnancy by four different methods – Abbott (1), Beckman (2), Roche (3), and Siemens (4). The data shows the expected trend for a lower TSH in the first trimester, resulting from thyroidal human chorionic gonadotropin (HCG) stimulation of thyroxine, which is maximal in the first trimester. The data is re- drawn with permission from reference 156.

Clinical Utility of TSH Measurement

AMBULATORY PATIENTS

In the outpatient setting the reliability of TSH testing is not usually influenced by the time of day of the blood draw, because the diurnal TSH peak occurs between midnight and 0400 (378-381). However, seasonal changes in TSH have been shown, with TSH approximately 10 % higher in winter than in summer (283). Third generation TSH assays (FS ~0.01 mIU/L) have now become the standard of care because they can reliably detect the full spectrum of thyroid dysfunction from overt hyperthyroidism to overt hypothyroidism, provided that hypothalamic-pituitary function is intact, and thyroid status stable (10,49,382,383). TSH is also used for optimizing L-T4 therapy - a drug with a narrow therapeutic range (49,384). Because TSH secretion is slow to respond to changes in thyroxine status there is no need to withhold the L-T4 dose on the day of the blood test (10,384). In addition, in differentiated thyroid cancer (DTC) patients, targeting the degree of TSH suppression relative to recurrence risk plays a critical role in management (57,385,386).

HOSPITALIZED PATIENTS WITH NONTHYROIDAL ILLNESSES (NTI)

Non-thyroidal illness, sometimes called the "sick euthyroid syndrome" is associated with alterations in hypothalamic/pituitary function and thyroid hormone peripheral metabolism, often exacerbated by drug influences (104,181,186,387-389). Routine thyroid testing in the hospital setting is not recommended because thyroid test abnormalities are frequently seen in sick euthyroid patients (Figure 6) (388-391). TSH also usually remains within normal limits or may become somewhat depressed in the early phase, especially in response to drug therapies such as dopamine or glucocorticoid (104,110,181). During the recovery phase, TSH frequently rebounds above the reference range (184). High TSH may also be seen associated with psychiatric illness (392). It is important to distinguish the generally mild, transient TSH alterations typical of NTI from the more profound and persistent TSH changes associated with hyper- or hypothyroidism (10,183,185,390).

Causes of Misleading TSH Measurements

A diagnostically misleading TSH can result from biological factors or interferants in the serum such as drugs, heterophile antibodies (i.e. HAMA), or endogenous TSH autoantibodies (91,195,197,378,393). In most cases such interferences cause a falsely high TSH.

BIOLOGIC FACTORS CAUSING MISLEADING TSH

Unstable Thyroid Function

TSH can be misleading when there is unstable thyroid status - such as in the early phase of treating hypo- or hyperthyroidism or non-compliance with L-T4 therapy - when there is a lag in the resetting of pituitary TSH to reflect a new thyroid status (394). During such periods of instability TSH will be misleading and FT4 will be the more diagnostically reliable test.

Pituitary/Hypothalamic Dysfunction

Pituitary dysfunction is rare in ambulatory patients (395). TSH measurement is unreliable in cases of both central hypothyroidism and central hyperthyroidism (285,395-397).

Central hypothyroidism (CH) is rare, 1/1000 less prevalent than primary hypothyroidism, 1/160,000 detected by neonatal screening) (395). CH can arise from disease at either the pituitary or hypothalamic level, or both (395). A major limitation of using a TSH-centered screening strategy is that current TSH tests will miss a diagnosis of CH because TSH IMAs are “glycosylation blind” and detect the abnormally glycosylated biologically inactive TSH as “normal” TSH, despite clinical hypothyroidism (276,285,396). This limitation necessitates that the clinical diagnosis of CH be confirmed biochemically as a low FT4/normal-low TSH discordance. and that L-T4 replacement therapy for CH be optimized using the serum FT4 not TSH. It should be noted that in the absence of clinical suspicion, investigations for pituitary dysfunction should only be initiated after ruling-out technical interference.

TSH secreting pituitary adenomas are characterized by a non-suppressed TSH associated with high thyroid hormone levels and clinical hyperthyroidism (397,398). Since this is a rare type of pituitary adenoma (0.7 %), technical interferences such as heterophile antibody (HAb) or TSH autoantibodies (macro TSH) should be excluded before initiating inconvenient and unnecessary pituitary imaging or dynamic diagnostic testing such as T3 suppression or TRH stimulation. This clinical/biochemical discordance reflects the TSH isoforms with enhanced biologic activity secreted by the adenoma. As with CH, current TSH IMA methods cannot distinguish these abnormal isoforms from normal TSH. Failure to diagnose the pituitary as the cause of the hyperthyroidism can lead to inappropriate thyroid ablation. The treatment of choice is surgery but in cases of surgical failure somatostatin analog treatment has been found effective (398). Note that the biochemical profile (high thyroid hormones and non-suppressed TSH) resembles that seen with thyroid hormone resistance syndromes (399,400) or interference from thyroid autoantibodies (120).

Resistance to Thyroid Hormone (RTH)

Resistance to thyroid hormone is caused by mutations in the THRB gene encoding the thyroid hormone receptor Band is biochemically characterized by high thyroid hormone (FT4 +/- T3) levels and a non-suppressed, sometimes slightly elevated TSH (402,403). Tissues expressing primarily the thyroid hormone receptor B are hypothyroid (e.g. the liver), whereas organs with a predominant expression of thyroid receptor A (e.g. the heart) display alterations consistent with thyroid hormone excess (400,401). Early cases of thyroid hormone resistance were shown to result from mutations in the thyroid hormone receptor B (400). More recently the syndromes with decreased sensitivity to thyroid hormones have been broadened to include mutations in thyroid hormone transporters (e.g. MCT8), the metabolism of thyroid hormone (e.g. SBP2), and resistance mediated by mutations in thyroid receptor A (401) (for detailed discussion see the Endotext chapter entitled “Impaired Sensitivity to Thyroid Hormone: Defects of Transport, Metabolism and Action”). These insensitivity and resistance syndromes display a spectrum of clinical and biochemical profiles and can now be identified by genetic testing.

Activating or Inactivating TSH Receptor Mutations

Non-autoimmune hyperthyroidism resulting from an activating mutation of the TSH receptor (TSHR) is rare (293,402). A spectrum of loss-of-function TSHR mutations (TSH resistance) causing clinical and subclinical hypothyroidism despite high thyroid hormone levels, have also been described (295,400,403,404). Because TSHR mutations are a rare cause of TSH/FT4 discordances, technical interferences should first be excluded before considering a TSHR mutation as the cause of these discordant biochemical profiles.

TECHNICAL FACTORS CAUSING MISLEADING TSH

Non-Analyte Specific Interferences

Heterophile Antibodies (HAbs) such as Human Anti Mouse Antibody (HAMA) can cause falsely high TSH IMA tests (200,220,241,405,406) and interfere with neonatal TSH screening (407). Since the HAb in some patient's sera interfere strongly with some manufacturers tests but appear inert in others (200), re-measurement using a different manufacturers assay should be the first test to identify interference. A fall in TSH in response to a blocker-tube treatment (43) is typically used to confirm HAb interference.

Anti-Reagent Antibody Interferences. As discussed for free hormone tests, some patients have antibodies that target test reagents such as rhuthenium and cause interference with TSH tests. (408). It should be noted that the anti-rhuthenium antibodies of different patients may affect different analytes to differing degrees (230,409,410).

Biotin Interferences. Tests employing streptavidin or biotin reagents are prone to interferences from antibodies targeting either streptavidin (231) or biotin (233). Alternatively, high dose biotin ingestion has been known to produce interference in an analyte-specific, platform-specific manner (241,411). The popularity of biotin therapy is now prompting assay manufacturers to reformulate their tests to remove biotin interference (237,412).

Analyte-Specific Interferences

Analyte-specific interferences typically result from autoantibodies targeting the analyte. Depending on the analyte and test formulation, autoantibody interferences most commonly cause falsely high test results. It should be noted that transplacental passage of both heterophile antibodies or anti-analyte autoantibodies (i.e. TSHAb or T4Ab) have the potential to interfere with neonatal screening tests (245-247,413). Patients with autoantibodies targeting both TSH and prolactin (PRL) have been described (414).

TSH Autoantibodies (Macro TSH). Analytically suspicious TSH measurements are not uncommon (205,238,239,244,415) and have been reported in up to five percent of specimens subjected to rigorous screening (405). There have been many reports of TSHAb, often referred to as "macro TSH” causing spuriously high TSH results in a range of different methods used for both adult (238,416) and neonatal screening (244,415). The prevalence of TSHAb approximates 0.8 percent but can be as high as 1.6 percent in patients with subclinical hypothyroidism (238). The most convenient test for TSHAb is to show a lowering of TSH in response to a polyethylene glycol (PEG) precipitation of immunoglobulins (415-417). Alternatively, column chromatography can show TSH immunoactivity in a high molecular weight peak representing a bioinactive TSH-immunglobulin complex (415,416).

TSH Variants. TSH variants are a rare cause of interference (403). Nine different TSH beta variants have been identified to date (286). These mutant TSH molecules may have altered immunoactivity and be detected by some TSH IMA methods but not others (403). The bioactivity of these TSH mutants is variable and can range from normal to bio-inert (286,403), resulting in discordances between the TSH concentration and clinical status (403) and/or a discordant TSH/FT4 relationship (286). These TSH genetic variants are one of the causes of central congenital hypothyroidism (418,419).

THYROID SPECIFIC AUTOANTIBODIES (TRAb, TPOAb and TgAb)

Tests for antibodies targeting thyroid-specific antigens such as thyroid peroxidase (TPO), thyroglobulin (Tg) and TSH receptors (TSHR) are used as markers for autoimmune thyroid conditions (420-422). Over the last four decades, thyroid antibody test methodologies have evolved from semi-quantitative agglutination, complement fixation techniques and whole animal bioassays to specific ligand assays using recombinant antigens or cell culture systems transfected with the human TSH receptor (20,420). Unfortunately, the diagnostic and prognostic value of these tests has been hampered by methodologic differences as well as difficulties with assay standardization (423,424). Although most thyroid autoantibody testing is currently made on automated immunoassay platforms, methods vary in sensitivity, specificity, and the numeric values they report because of standardization issues (45,377,425). Thyroid autoantibody testing can be useful for diagnosing or monitoring treatment for several clinical conditions, although these tests should be selectively employed as adjunctive tests to other diagnostic testing procedures.

TSH Receptor Autoantibodies (TRAb)

The TSH receptor (TSHR) serves as a major autoantigen (19,422,426-428). Thyroid gland stimulation occurs when TSH binds to the TSHR on thyrocyte plasma membranes and activates the cAMP and phospholipase C signaling pathways (427). The TSH receptor belongs to the G protein-coupled class of transmembrane receptors. It undergoes complex posttranslational processing in which the ectodomain of the receptor is cleaved to release a subunit into the circulation (426). The TSH-like thyroid stimulator found uniquely in the serum of Graves’ disease patients was first described using a guinea pig bioassay system in 1956 (429). Later, a mouse thyroid bioassay system was used to show this serum factor displayed a prolonged stimulatory effect as compared to TSH and hence was termed to be a “long-acting thyroid stimulator” or LATS (430,431). Much later, the LATS factor was recognized not to be a TSH-like protein but an antibody capable of stimulating the TSH receptor that was the cause of Graves’ hyperthyroidism (432). TSH receptor antibodies (TRAb) have also become implicated in the pathogenesis of Graves’ ophthalmopathy (432-436). TRAbs are heterogeneous (polyclonal) and fall into two general classes both of which can be associated with autoimmune thyroid disorders – (a) thyroid stimulating autoantibodies (TSAb) that mimic the actions of TSH and cause Graves’ hyperthyroidism and (b), blocking antibodies (TBAb) that block TSH binding to its receptor and can cause hypothyroidism (19,20,420,427,432,437-440). TSH, TSAb and TBAb appear to bind to different sites on the TSH receptor ectoderm with similar affinities and often overlapping epitope specificities (441). In some cases of Graves’ hyperthyroidism, TBAb have been detected in association with TSAb (442,443) and the dominance of one over the other can change over time in response to treatment (444,445). Because both TSAb and TBAb can be present in the same patient, the relative concentrations and receptor binding characteristics of these two classes of TRAb can influence the severity of Graves’ hyperthyroidism and the response to antithyroid drug therapy or pregnancy (426,442,446-448). For completeness, it should also be mentioned that a third class of “neutral” TRAb has also been described, of which the functional significance has yet to be determined (432,438,448,449).

Two different methodologic approaches have been used to quantify TSH receptor antibodies (425,437,450,451): (a) TSH receptor antibody tests (TRAb assays) also called TSH Binding Inhibition Immunoglobulin (TBII) assays, and (b) Bioassays that use whole cells transfected with human or chimeric TSH receptors that produce a biologic response (cAMP or bioreporter gene) when TSAb or TBAb are present in a serum specimen. In recent years automated immunometric assays using recombinant human TSHR constructs have been shown to have high sensitivity for reporting positive results in Graves' disease sera (18,425). However, assay sensitivity varies among current receptor versus bioassay methods (452).

TSH RECEPTOR (TRAb)/TSH BINDING INHIBITORY IMMUNOGLOBULIN (TBII)

TRAb methods detect serum immunoglobulins that bind TSHR but do not functionally discriminate stimulating from blocking antibodies (453). TRAb methods are based on standard competitive or noncompetitive principles. The first generation of methods were liquid-based whereby immunoglobulins in the serum inhibited the binding of ¹²⁵I-labeled TSH or enzyme-labeled TSH to a TSH receptor preparation (451). These methods used TSH receptors of human, guinea pig, or porcine origin (454). After 1990, a second generation of both isotopic and non-isotopic methods were developed that used and immobilized porcine or recombinant human TSH receptors (451,455). These second-generation methods were shown to have significantly more sensitivity for detecting Graves' thyroid stimulating immunoglobulins than first generation tests (425). In 2003 a third generation of non-isotopic methods were developed that were based on serum immunoglobulins competing for immobilized TSHR preparation (recombinant human or porcine TSHR) with a monoclonal antibody (M22) (420,425,451,455-457). Third generation assays have also shown a good correlation and comparable overall diagnostic sensitivity with bioassay methods (425,442,458). Current third generation TRAb tests have now been automated on several immunoassay platforms (425). However, between-method variability remains high and between assay precision is often suboptimal (CVs > 10 %) despite calibration using the same International Reference Preparation (08/204) (423,459). This fact makes it difficult to compare values using different methods and indicates that further efforts focused on additional assay improvements are needed (420,423,455).

Over the last ten years automated IMA methods have dramatically lowered the cost and increased the availability of TRAb testing (18,428,452). Automated TRAb IMAs are not functional tests and do not distinguish between stimulating and blocking TRAbs (455), however, this distinction is usually unnecessary, since it is evident from clinical evidence of hyper- or hypothyroid features. Also, both TSHR stimulating and blocking antibodies may be detected simultaneously in the same patient and cause diagnostic confusion (460). Because the sensitivity and specificity of current third generation TRAb tests is over 98 percent, TRAb testing can be useful for determining the etiology of hyperthyroidism (425,428), as an independent risk factor for Graves’ ophthalmopathy (435,436,440), and may be useful for monitoring responses to therapy (76,425). TRAb measured prior to radioiodine therapy for Graves' hyperthyroidism can also help predict the risk for exacerbating ophthalmopathy (433,436,461). There is conflicting data concerning the value of using TRAb to predict the response to antithyroid drug treatment or the risk of relapse (443,458,462,463). An important application of TRAb testing is to detect high TRAb concentrations in pregnant patients with a history of autoimmune thyroid disease or active or previously treated Graves’ hyperthyroidism, in whom transplacental passage of stimulating or blocking TRAb can cause neonatal hyper- or hypothyroidism, respectively (76,352,425,437,451,464-466). Because the expression of thyroid dysfunction may be different in the mother and infant, automated IMA methods have the advantage of being able to detect both stimulating and blocking antibodies (467). It is currently recommended that TRAb be measured in the first trimester in all pregnant patients with active Graves’ hyperthyroidism or who have received prior ablative (radioiodine or surgery) therapy for Graves’ disease in whom TRAb can remain high even after patients have been rendered hypothyroid and are being maintained on L-T4 replacement therapy (47,48). When TRAb is high in the first trimester additional TRAb testing is recommended at 18-22 and 30-34 weeks (47,48,76,420,442,468).

BIOASSAY METHODS (TSAb/TBAb)

The first TSH receptor assays used surgical human thyroid specimens, mouse, or guinea pig thyroid cells, or rat FRTL-5 cell lines to detect TSH receptor antibodies. These methods typically required pre-extraction of immunoglobulins from the serum specimen (429,437,439,469,470). Later, TRAb bioassays used cells with endogenously expressed or stably transfected human TSH receptors and unextracted serum specimens (471-473). Current TRAb bioassays are functional assays that use intact (typically CHO) cells transfected with human or chimeric TSH receptors, which when exposed to serum containing TSH receptor antibodies use cAMP or a reporter gene (luciferase) as a biological marker for any stimulating or blocking activity in a serum (425,451,463). Bioassays are more technically demanding than the more commonly used receptor assays because they use viable cells. However, these functional assays can be modified to detect TBAb that may coexist with TSAb in the same sera and make interpretation difficult (451). The most recent development is for second generation assays to use a chimeric human/rat LH TSHR to effectively eliminate the influence of blocking antibodies. This new approach has shown excellent sensitivity and specificity for diagnosing Graves' hyperthyroidism and clinical utility for monitoring the effects of anti-thyroid drug therapy (463).

Thyroid Peroxidase Autoantibodies (TPOAb)

TPO is a large, dimeric, membrane-associated, globular glycoprotein that is expressed on the apical surface of thyrocytes. TPO autoantibodies (TPOAb) found in sera typically have high affinities for an immunodominant region of the intact TPO molecule. When present, these autoantibodies vary in titer and IgG subclass and display complement-fixing properties (474). Studies have shown that epitope fingerprints are genetically conserved suggesting a possible functional importance (475). However, it is still unclear whether the TPOAb epitope profile correlates with the presence of, or potential for, the development of thyroid dysfunction (474-477). TPOAb antibodies were initially detected as antibodies against thyroid microsomes (antimicrosomal antibodies, AMA) using semi-quantitative complement fixation and tanned erythrocyte hemaagglutination techniques (478). Studies have identified the principal antigen in AMA tests as the thyroid peroxidase (TPO) enzyme, a 100 kD glycosylated protein present in thyroid microsomes. Manual agglutination tests have now been replaced by more specific, automated TPOAb immunoassay or immunometric assay methods that use purified or recombinant TPO (10,420,479-482). There is considerable inter-method variability of current TPOAb assays (correlation coefficients 0.65 and 0.87), despite calibration against the same International Reference Preparation (MRC 66/387) (420,479,480,482). It appears that both the methodologic principles of the test and the purity of the TPO reagent used may influence the sensitivity, specificity, and reference range of the method (420,479). The variability in sensitivity limits and the reference ranges of different methods has led to different interpretations regarding the normalcy of having a detectable TPOAb (377,420,424,482).

TPOAb CLINICAL SIGNIFICANCE

Estimates of TPOAb prevalence depend on the sensitivity and specificity of the method employed (377,424,482). In addition, ethnic and/or geographic factors (such as iodine intake) influence the TPOAb prevalence in population studies (317). For example, TPOAb prevalence is significantly higher (~11 percent) in countries like the United States and Japan where dietary iodine is sufficient, as compared with iodine deficient areas in Europe (~ 6 percent) (254,483). The prevalence of TPOAb is higher in women of all age groups and ethnicities, presumably reflecting the higher propensity for autoimmunity as compared with men (254,483). Approximately 70-80 percent of patients with Graves' disease and virtually all patients with Hashimoto’s or post-partum thyroiditis have detected TPOAb (479,484). TPOAb has, in fact, been implicated as a cytotoxic agent in the destructive thyroiditic process (477,485,486).

TPOAb prevalence is also significantly higher in various non-thyroidal autoimmune disorders in which no apparent thyroid dysfunction is evident (487). Aging is associated with an increasing prevalence of TPOAb that parallels the increasing prevalence of both subclinical and clinical hypothyroidism (254). In fact, the NHANES III survey reported that TPOAb prevalence increases with age and approaches 15-20 percent in elderly females even in the iodine-sufficient United States (254). This same study found that the odds ratio for hypothyroidism was strongly associated with the presence of TPOAb but not TgAb, suggesting that primarily TPOAb contribute to the autoimmune etiology of hypothyroidism (254). Although the presence of TgAb alone did not appear to be associated with hypothyroidism or TSH elevations, the combination of TPOAb and TgAb versus TPOAb alone may be more pathologically significant (Figure 9), however further studies would be needed to confirm this (254,307,477). It is now apparent that the presence of TPOAb in apparently euthyroid individuals (TSH within reference range) appears to be a risk factor for future development of overt hypothyroidism that subsequently becomes evident at the rate of approximately two percent per year in such populations (474,488,489). Furthermore, TPO-positivity in pregnant women is a risk factor for preterm birth (490).

Figure 9. Prevalence of thyroid antibodies in women (A) and men (B). Abscissa TSH values correspond to the upper and lower limits of the intervals spanning each set of bars. Asterisks denote a significant difference in prevalence from the TSH range with lowest antibody prevalence, 0.1 and 1.5 mIU/liter for women and 0.1 and 2.0 mIU/liter for men from reference 307.

TPOAb measurement can serve as a useful prognostic indicator for future thyroid dysfunction (489,491). However, a hypoechoic ultrasound pattern can often be seen before the biochemical TPOAb abnormality appears (492). Further, some individuals with unequivocal TSH elevations, presumably resulting from autoimmune destructive disease of the thyroid, do not have TPOAb detected (307). Presumably, this paradoxical absence of TPOAb in some patients with elevated TSH likely reflects the suboptimal sensitivity and/or specificity of current TPOAb tests or a non-autoimmune cause of thyroid failure (i.e. atrophic thyroiditis) (254,307,482,493).

Although changes in autoantibody concentrations often occur with treatment, or reflect a change in disease activity, serial TPOAb measurements are not recommended for monitoring treatment for autoimmune thyroid diseases (49,479,494). This is not surprising since treatment of these disorders addresses the consequence (thyroid dysfunction) and not the cause (autoimmunity) of the disease. However, where it may have an important clinical application is to use the presence of serum TPOAb as a risk factor for developing thyroid dysfunction in patients receiving amiodarone, interferon-alpha, interleukin-2, or lithium therapies which all appear to act as triggers for initiating autoimmune thyroid dysfunction in susceptible (especially TPOAb-positive) individuals (10,110,495-500).

During pregnancy the presence of TPOAb has been linked to reproductive complications such as miscarriage, infertility, IVF failure, fetal death, pre-eclampsia, pre-term delivery, post-partum thyroiditis, and depression (47,76,484,501-508). However, whether this association represents cause or effect remains unresolved.

Thyroglobulin Autoantibodies (TgAb)

Thyroglobulin autoantibodies belong predominantly to the immunoglobulin G (IgG) class, are not complement fixing and are generally conformational (509). Tg autoantibodies were the first thyroid antibody to be detected in the serum of patients with autoimmune thyroid disorders using tanned red cell hemagglutination techniques (478). Subsequently, methodologies for detecting TgAb have evolved in parallel with those for TPOAb measurement, from semi-quantitative techniques to more sensitive ELISA and RIA methods and now to non-isotopic competitive or non-competitive immunoassays (45,420,482,510,511). Unfortunately, the between-method variability of TgAb assays is even greater than that of the TPOAb tests (Figure 10) (45,420,510-512). Additionally, high levels of thyroglobulin in the serum have the potential to influence TgAb measurements (511). Between-method variability is influenced by the purity and the epitope specificity of the Tg reagent, as well as the patient-specific epitope specificity of the TgAb secreted (513). As with TPOAb methods, TgAb tests have highly variable sensitivity limits and manufacturer-recommended cut-off values for "positivity", despite the use of the same International Reference Preparation (MRC 65/93) (Figure 10) (45,510-512,514). Whereas the FS limit is the recommended cutoff to define TgAb-positivity for DTC monitoring, the FS is typically much lower than the manufacturer-recommended cut-off for “positivity” (Figure 10) (10,45). This is because manufacturer-recommended cutoffs (MCO) are set for diagnosing thyroid autoimmunity and are too high to detect the low TgAb levels that can interfere with Tg measurements (515,516). Although there are reports that low levels of TgAb may be present in normal euthyroid individuals, it is unclear whether this represents assay noise due to matrix effects or "natural" antibodies (21). Further complicating this question are studies suggesting that there may be qualitative differences in TgAb epitope specificities expressed by normal individuals versus patients with either differentiated thyroid cancers (DTC) or autoimmune thyroid disorders (517). These differences in test sensitivity and specificity negatively impact the reliability of determining the TgAb status (positive versus negative) of specimens prior to Tg testing of DTC patients.

Figure 10. TgAb Measurements Made by Different Methods. Figure shows the relative TgAb concentrations reported for 143 DTC patient sera with evidence of TgAb interference with serum Tg measurements. Each serum was measured by four different methods each with a different manufacturer-recommended cutoff value for “TgAb positivity” (open bars) and with different experimentally determined (10) functional sensitivity limits (closed bars). From reference 45.

CLINICAL UTILITY OF TgAb TESTING

Tg autoantibodies (TgAb) are encountered in autoimmune thyroid conditions, usually in association with TPOAb (254,489,490). However, the NHANES III survey found that only three percent of subjects with no risk factors for thyroid disease had serum TgAb present without detectable TPOAb (Figure 9) (254,307). Furthermore, there was no association between the isolated presence of TgAb and TSH abnormalities in these subjects (254,307). This suggests that it is unnecessary to measure both TPOAb and TgAb for a routine evaluation for thyroid autoimmunity (307,420,489). However, when autoimmune thyroid disease is present, there is some evidence that assessing the combination of TPOAb and TgAb has greater diagnostic utility than the TPOAb measurement alone (Figure 9) (307,489,490,518). In pregnant women, both TPOAb and TgAb-positivity have been shown to be risk factors for preterm birth (490).

The role of TgAb for monitoring patients with DTC is two-fold: 1) to authenticate that a Tg measurement is not compromised by TgAb interference, and 2) as an independent surrogate tumor-marker (519,520). Immunoassay methods detect TgAb in approximately 25 percent of patients presenting with DTC, double the TgAb prevalence of the general population (45,254,521,522). In patients with thyroid nodules the presence of TgAb is a risk factor for lymph node metastases (520,523,524) and may be a useful marker for papillary thyroid cancer in cases of indeterminate cytology (523,525,526). The prevalence of TgAb is typically higher in patients with papillary versus follicular tumors (22,510,519,527-529). After TgAb-positive patients are rendered disease-free by surgery, TgAb concentrations typically progressively decline during the first few post-operative years and typically become undetectable after a median of three years of follow-up (22,530,531). In contrast, a rise in, or de novo appearance of, TgAb is often the first indication of tumor recurrence (14,22,531,532). In patients with persistent disease, serially determined TgAb concentrations may serve as an independent surrogate tumor marker for changes in tumor mass (Figure 11) (14,17,22,520,530,531,533-536). However, the use of the TgAb trend as a surrogate tumor marker necessitates that TgAb be measured by the same method in preferably the same laboratory, because of the large differences in the sensitivities and cut off values for “positivity” between different methods (Figure 10) (9,45,511,512,514,519,521).

THYROGLOBULIN (Tg)

Thyroglobulin plays a central role in a variety of pathophysiologic thyroid conditions, including acting as an autoantigen for thyroid autoimmunity (421,509,537). Serum Tg levels can serve as a marker for iodine status of a population (538-540) and genetic defects in Tg biosynthesis causing dyshormonogenesis can result in congenital hypothyroidism (10,541,542). Because Tg has a thyroid-tissue specific origin, a serum Tg measurement can be used to investigate the etiology of congenital hypothyroidism (athyreosis versus dyshormonogenesis) (543,544). Likewise, a paradoxically low serum Tg can be used to distinguish factitious hyperthyroidism from the high Tg expected with endogenous hyperthyroidism (14,545-547). However, the primary clinical use of Tg measurement is as a post-operative tumor-marker test used to monitor patients with follicular-derived (differentiated) thyroid cancer (DTC) (14,17,57,271,274,548-550).

Most Tg testing is currently by rapid, automated immunometric assays (IMA), most of which now have second generation functional sensitivity (FS≤ 0.1 µg/L) - a sensitivity level that obviates the need for recombinant human TSH (rhTSH) stimulation (57,274,551-554). TgAb interference, causes falsely low/undetectable serum Tg IMA tests and this is the major limitation of using IMA methodology since this direction of interference can mask disease (14,17,23,512,521,555,556). Currently, most laboratories first establish the TgAb status of the specimen (negative or positive) and restrict Tg-IMA testing to TgAb-negative sera, while reflexing TgAb-positive specimens to other methodologies believed less prone to TgAb interference from TgAb - RIA (14,274,512,521) or LC-MS/MS (14,24,43,555,557,558).

Technical Limitations of Tg Methods

Thyroglobulin measurement remains technically challenging. Five methodologic problems impair the clinical utility of this test: (a) suboptimal functional sensitivity; (b) between-method biases; (c) "hook" problems (some IMA methods) and interferences caused by (e) Heterophile antibodies (HAb) and/or (f) Tg autoantibodies (TgAb).

Tg ASSAY SENSITIVITY

As with TSH, assay functional sensitivity (FS) represents the lowest analyte concentration that can be measured in human serum with 20 percent CV, calculated from runs made over a clinically relevant timespan (6 -12 months for Tg) and using at least two different lots of reagents (10). These stipulations are necessary because assay precision erodes over time, especially during the long clinical interval (6-12 month) typically used when monitoring Tg as a tumor marker for DTC, during which time assay reagents and conditions can change (9,71,559). The use of FS as the assay sensitivity limit is more relevant than either a limit of detection (LOD) or limit of quantitation (LOQ) calculation - parameters that do not stipulate using a clinically relevant time span for assessing precision (10,560). The FS protocol (10) also stipulates that precision be determined in human sera rather than a commercial QC preparation, because instruments and methods are matrix-sensitive (72,560). Tg IMA methods should have precision determined in TgAb-negative human sera (560) and TgAb-positive human serum pools should be used to determine the precision of Tg methodologies used to measure TgAb-positive specimens - most commonly RIA or LC-MS/MS.

In accord with TSH a generational approach to Tg assay nomenclature has been adopted (1,17). Early Tg RIAs (5) had FS approximating 1 μg/L and were designated "first generation" assays. Currently, some RIAs, IMAs and LC-MS/MS methods still only have first generation functional sensitivity (FS = 0.5-1.0 µg/L) (17,271,274,512,561,562). However, in recent years 2^nd generation assays (FS 0.05-0.10 µg/L) have become the standard of care (57,271,274,549,550,561,563). These second-generation tests obviate the need for recombinant human TSH (rhTSH) stimulation, because basal Tg correlates with rhTSH-stimulated Tg (17,57,561). However, the use of a second-generation assay does not eliminate the need for periodic ultrasound examinations, because many histologically confirmed lymph nodes metastases may not secrete enough Tg to be detected (14,563,564).

SERUM Tg REFERENCE RANGES

The adult serum Tg reference range approximates 2-40 µg/L (10,565). Newborn infants have a higher serum Tg that falls to the adult range after two years of age (566). However, most Tg testing is made following surgery (thyroidectomy or lobectomy) for DTC, the Tg reference range is only relevant in the preoperative period (567-570). Different Tg methods may report two-fold differences in numeric values for the same serum specimen (14,274,571). This between-method variability reflects differences in assay standardization as well as the assay specificity for detecting different Tg isoforms in the serum (512,572-576). When evaluating a thyroidectomized patient, the assay reference range should be adjusted for thyroid mass (thyroidectomy versus lobectomy) as well as the TSH status of the patient (10,570).

BETWEEN METHOD Tg BIASES

Thyroglobulin in frozen sera is remarkably stable. The between-run precision for repetitive serum Tg measurements made over 6-12 months (the typical DTC monitoring interval), approximates 10 percent. In contrast, between-method variability can exceed 30 percent (14,274,516,571) despite CRM-457 standardization (584,585). In fact, in some cases different methods can report more than a two-fold difference in Tg for the same serum specimen (14,274,571). This between-method variability significantly exceeds the biologic variability of Tg in normal euthyroid subjects (~16 %) (559,577). This between-method variability reflects matrix differences between methods as well as specificity differences for detecting different Tg isoforms in the serum (45,512,572-574,576).

Some Tg should be detected in all TgAb-negative normal euthyroid subjects when using a second-generation IMA method standardized against the International Reference Preparation CRM-457. Although the intra-individual serum Tg variability is relatively narrow (CV ~15 %) (577), the Tg population reference range is quite broad (2-40 µg/L) (512,565,575,578). It follows that 1 gram of normal thyroid tissue gives rise to ~1.0 µg/L Tg in the circulation, unless TSH is elevated (10,579). Following a lobectomy, euthyroid patients should be evaluated using a mass-adjusted reference range (1.5 - 20 µg/L). The range should be lowered a further 50 percent (0.75 - 10 µg/L) during TSH-suppression (10,570). After thyroidectomy, the typical 1-to-2-gram thyroid remnant (580) would be expected to produce a serum Tg below 2 µg/L (at low-normal TSH) (581,582). By this same reasoning, truly athyreotic patients would be expected to have no Tg detected irrespective of their TSH status (10). However, a rising Tg trend after lobectomy in the absence of recurrent disease is not unexpected due to a compensatory increase in normal remnant tissue (583).

Since TgAb interferes with different methods to differing extents (14,45,586), a false negative TgAb test could also lead to significant between-method differences with the potential to disrupt serial Tg monitoring and negatively impact clinical management (516). Between method variability is the reason current guidelines stress the necessity of using the same Tg method (and preferably the same laboratory) for monitoring Tg trends and the need to re-baseline the Tg level if a change in method becomes necessary (57,587).

Figure 11. Between-Method Serum Tg Variability in DTC Patients +/- TgAb. Serum Tg measured by different methodologies in patients with distant metastatic DTC who were either TgAb-negative (panel A) or TgAb-positive (panel B). Three Tg methodologies were compared: IMA, LC-MS/MS (MS-M = Mayo; MS-Q = Quest), and RIA. Tg measurements below the assay FS limit are indicated in the shaded areas and expressed as a percentage relative to the total number of tests performed with that method. Patients who died of DTC-related complications are shown by solid symbols. From reference 14.

HIGH-DOSE HOOK EFFECT

Tumor marker tests employing IMA methodology can be prone to so-called "high-dose hook effects", whereby very high antigen concentrations can overwhelm the binding capacity of the monoclonal antibody reagents leading to a falsely normal/low value (9,588-591). Manufacturers have largely overcome hook problems by adopting a two-step procedure, whereby a wash step is used to remove unbound antigen after the first incubation of specimen with the capture monoclonal antibody before introducing the labeled monoclonal during which the signal binds the captured antigen during a second incubation (580). When using IMA methodology, it is the laboratory’s responsibility to determine whether a hook effect is likely to generate falsely normal or low values.

There are two approaches for detecting and overcoming hook effects with Tg IMA methods when an unexpectedly low serum Tg value is encountered for a patient with known metastatic disease: 1) Measure the Tg in the specimen at two dilutions. For example, a hook effect is likely present when the value of the test serum measured at a 1/5 or 1/10 dilution is higher than that obtained with the undiluted specimen. 2) Assess the recovery of added Tg antigen. If a hook effect is present, the Tg result will be inappropriately low.

INTERFERENCES WITH Tg MEASUREMENT

Heterophile Antibody (HAb) Interferences

HAb interferes with Tg IMAs, but not RIA or Tg-LC-MS/MS methodologies (43, 214, 221, 223, 228, 592-595). HAb interferences are thought to reflect the binding of HAb to the monoclonal antibody IMA reagents (murine origin). RIA methods are not prone to HAb interference because their polyclonal antibody reagents (rabbit origin) do not bind human IgG. In most cases HAb interferences are characterized by a false-positive Tg-IMA result (223,228,592), although falsely low Tg IMA results have also been reported (214,596). Recent reports find that Tg LC-MS/MS methodology appears free from HAb interferences (43,595). A presumptive test for HAb interference is a lowering of the analyte value in the presence of a blocking agent (43,201,597). The laboratory cannot proactively test for HAb because specimens are typically sent to the laboratory without clinical information. Physicians should request the laboratory test for HAb interference when an apparently disease-free patient has an unexpectedly high Tg result.

Tg Autoantibody (TgAb) Interference

TgAb interference with Tg measurement remains the major limitation for using Tg as a DTC tumor marker. TgAb has the potential to interfere with Tg measured by each of the current methodologies: IMA, RIA and LC-MS/MS. The prevalence of TgAb in DTC patients approximates 25 percent - twice that of the general population (9,254,522). There appears to be no threshold TgAb concentration that precludes TgAb interference (45,57,386,510,512,521). TgAb is thought to interfere by both in vitro (epitope masking) (45,512,521,598) and/or in vivo mechanisms such as enhanced TgAb-mediated Tg clearance (599-603). High TgAb concentrations do not necessarily interfere, whereas low TgAb may profoundly interfere (9,22,45,521,555,598,604,605). Unfortunately, the recovery approach appears to be unreliable for detecting TgAb interference (512,521,598).

TgAb Interference - In-Vivo Mechanisms. Studies over past decades have suggested that the presence of TgAb enhances Tg metabolic clearance. In 1967 Weigle showed enhanced clearance of endogenously ^I25-labeled Tg in rabbits, after inducing TgAb by immunizing the animals with an immunogenic Tg preparation (599,603). In humans, Tg and TgAb acute responses to sub-total thyroidectomy have also suggested that TgAb may increase Tg metabolic clearance (603,606). Changes (a rise or fall) in TgAb versus Tg-RIA concentrations have typically been concordant and appropriate for clinical status, whereas the direction of change in Tg-IMA is typically discordant with Tg-RIA and clinical status (45,274,521,556). In general, the change in TgAb concentrations tends to be steeper than for Tg-RIA (521), as would be consistent with TgAb-mediated Tg clearance, perhaps because some TgAbs act as "sweeper" antibodies that facilitate clearance of antigen (602,603,607).

TgAb Interference - In-Vitro Mechanisms. TgAb interferes with Tg measurement in a qualitative, quantitative, and method-dependent manner (22,45,521,608,609). The potential for in vitro interference is multifactorial and depends not only on the assay methodology (IMA, RIA or LC-MS/MS) (39), but also the concentration and epitope specificity of the TgAb secreted by the patient (22,512,610). RIA methodology appears to quantify total Tg (free Tg + TgAb-bound Tg) whereas IMA primarily detects only the free Tg moiety, i.e. Tg molecules with epitopes not masked by TgAb complexing. Steric masking of Tg epitopes is the reason why TgAb interference with IMA methodology is always unidirectional (underestimation) and why a low Tg-IMA/Tg-RIA ratio has been used to indicate TgAb interference (45,521,555,611,612). The recently developed Tg-LC-MS/MS methodology uses trypsin digestion of Tg-TgAb complexes to liberate a proteotypic Tg peptide. This conceptually attractive approach was primarily developed to overcome TgAb interference with IMA methods thereby eliminating falsely low/undetectable Tg-IMA results that can mask disease. However, recent studies report that a high percentage (>40 %) of TgAb-positive DTC patients with structural disease have paradoxically undetectable Tg-LC-MS/MS tests (14,24,43,555,557,558). More studies are needed to determine why LC-MS/MS fails to detect Tg in TgAb-positive DTC patients with disease. Possibilities to investigate include tumor Tg polymorphisms that prevent the production of the Tg-specific tryptic peptide (38), suboptimal trypsinization of Tg-TgAb complexes, or Tg levels that are truly below detection because of increased clearance of Tg-TgAb complexes by the hepatic asialoglycoprotein receptor (599-602).

TgAb interference with Tg-RIA Methodology. Radioimmunoassay (RIA) was the earliest methodology used to measure Tg (5). Thyroglobulin antigen (from serum or added ¹²⁵I-Tg tracer) competes for a low concentration of polyclonal (PAb) (usually rabbit) Tg antibody. After incubation, the Tg-PAb complex is precipitated by an anti-rabbit second antibody and the serum Tg concentration is quantified from the ¹²⁵I-Tg in the precipitate. The first Tg-RIAs developed in the 1970s were insensitive (~2 µg/L) (5,613). Over subsequent decades some Tg-RIAs have achieved first generation functional sensitivity (FS = 0.5 µg/L) by using a long (48-hour) pre-incubation before adding a high specific activity ¹²⁵I-Tg tracer (614,615). The use of a high affinity polyclonal antibody (616) coupled with a species-specific second antibody appears to minimize TgAb interference. Resistance to TgAb interference is evidenced by appropriately normal Tg-RIA values for TgAb-positive euthyroid controls (512) and detectable Tg-RIA in TgAb-positive DTC patients with structural disease (14,555). The clinical performance of this Tg-RIA contrasts with IMA methods that fail to detect Tg in some TgAb-positive normal euthyroid subjects (512), some TgAb-positive Graves' hyperthyroid patients (14,617), or TgAb-positive patients with structural disease (14,512,618). It should be noted that the propensity of TgAb to interfere with Tg-RIA determinations and cause under- or overestimation (546,608) depends on the patient-specific interactions between Tg and TgAb in the specimen and the RIA reagents (609).

TgAb interference with Tg-IMA Methodology. Most Tg testing is currently made by automated IMAs, whereby antigen is captured by two monoclonal antibodies (MAb) that target different epitopes on the Tg protein (619). TgAb interferes with IMA methodology by steric inhibition – i.e. by blocking the epitope(s) necessary for Tg to bind the MAb(s), so that the MAb-Tg-MAb reaction cannot take place and Tg is reported as falsely low or undetectable. This mechanism of epitope masking is supported by timed recovery studies. Clinically, TgAb interference is evident from the paradoxically low/undetectable Tg-IMA seen for TgAb-positive normal controls (512), patients with Graves' hyperthyroidism (14,617), and DTC patients with active disease (Figures 10 and 11) (14,43,555). High Tg concentrations can overwhelm the TgAb binding capacity rendering Tg-IMA concentrations detectable and lessening the degree of interference (45,555). It follows that as Tg concentrations rise, more Tg is free, the influence of TgAb lessens and the discordance between Tg-IMA and Tg-RIA lessens (Figure 11B) (45,555). Although some IMA methods have claimed to overcome TgAb interference by using monoclonal antibodies directed against specific epitopes not involved in thyroid autoimmunity (580), this approach has not overcome TgAb interferences in clinical practice, possibly because less restricted TgAb epitopes are associated with thyroid carcinomas than with autoimmune thyroid conditions (510,517,620).

TgAb Interference with Tg LC-MS/MS. Liquid Chromatography, Tandem Mass Spectrometry (LC-MS/MS) is the newest methodology used to measure Tg. This methodology measures Tg by trypsinizing the Tg-TgAb complexes in the serum to generate a Tg-specific peptide(s) that can be measured by LC-MS/MS (37-39,41,580,621). Most Tg LC-MS/MS methods only have first generation functional sensitivity (FS ~ 0.5 µg/L) (24,39,40) although more sensitive methods are being developed (621). Tg-LC-MS/MS methodology has been shown free from HAb interferences (43,595) and has been promoted as being free from TgAb interference (24,39,40). However, these claims are not supported by clinical studies in which paradoxically undetectable LC-MS/MS Tg tests are seen for many TgAb-positive DTC patients with structural disease (14,24,43,555,557,558). The higher the TgAb, the more likely that no Tg would be detected by LC-MS/MS in patients with disease (558). It currently appears that when TgAb is present LC-MS/MS methodology offers no diagnostic advantage over IMA.

Clinical Utility of TGAb Used as a Surrogate DTC Tumor Marker

The serum TgAb trend has become recognized as a postoperative surrogate DTC tumor-marker. A declining TgAb trend is a good prognostic sign, whereas a stable or rising TgAb may indicate persistent/recurrent disease (23,57,509,519,521,530,531,533,536,612,622-624). The TgAb half-life in blood approximates 10 weeks (522). Following successful surgery (± radioiodine treatment), TgAb typically falls more than 50 percent in first post-operative year and often decreases to <10 percent after 3-4 years eventually becoming undetectable with reduced stimulation of the immune system by lower Tg antigen levels (45,57,274,522,524,530,531,625). The time needed for a TgAb-positive patient to become TgAb-negative in response to successful treatment is inversely related the initial TgAb concentration, perhaps representing the long-lived memory of plasma cells (274,626). Patients exhibiting a TgAb decline of more than 50 percent by the end of the first post-operative year have been shown to have a low recurrence risk (515,531,534,612,627). However, a significant percentage (~5 %) of TgAb-negative patients may develop transient de novo TgAb-positivity in the early post-operative period, presumably in response to Tg antigen released by surgical trauma (532,628,629). A rise in TgAb can also be seen soon after fine needle aspiration (FNA) biopsy (630-632) or more chronically (months) in response to radiolytic damage following radioiodine treatment (22,633,634). However, the 5 percent of DTC patients that display a sustained de novo TgAb appearance are likely to have recurrent disease (Figure 11B) (532,635). These TgAb-negative to TgAb-positive conversions are the reason why guidelines mandate that TgAb be measured with every Tg test (23,57,635). Patients with persistent disease may exhibit only a marginal TgAb decline or have stable, rising or a de novo TgAb appearance (511,521,531-533,612,622). If serum Tg remains detectable after TgAb becomes negative (~3 % of cases), the risk for disease remains (Figure 11A). Since TgAb tests differ in sensitivity and specificity (Figure 10) (23,45,513,514,636) it is essential to measure the serum TgAb trend by the same method, preferably in the same laboratory (23,45,57,482,511,512,514,535,636).

Serum Tg Monitoring of Patients with DTC

Over the past decade, the incidence of DTC has substantially risen with the detection of small thyroid nodules and micropapillary cancers by ultrasound and other anatomic imaging modalities (57,637-640). Although most DTC patients are rendered disease-free by their initial surgery, approximately 15 percent of patients experience recurrences and approximately 5 percent die from disease-related complications (580,641-644). A risk-stratified approach to diagnosis and treatment is now recommended by current guidelines (57).

In most cases, persistent/recurrent disease is detected within the first five post-operative years, although recurrences can occur decades after initial surgery necessitating life-long monitoring for recurrence (642,643). Since most patients have a low pre-test probability for disease, protocols for follow-up need a high negative predictive value (NPV) to eliminate unnecessary testing, as well as a high positive predictive value (PPV) for identifying patients with persistent/recurrent disease. Because Tg testing is generally recognized as being more sensitive for detecting disease than diagnostic ¹³¹I whole body scanning (645), biochemical testing (serum Tg. + TgAb) is used in conjunction with periodic ultrasound (57,645). The persistent technical limitations of Tg and TgAb measurements necessitate close physician-laboratory cooperation.

The majority (~75 %) of DTC patients have no Tg antibodies detected (521). In the absence of TgAb, four factors influence the interpretation of serum Tg concentrations: (1) the mass of thyroid tissue present (normal tissue + tumor); (2) The intrinsic ability of the tumor to secrete Tg; (3) the presence of any inflammation of, or injury to, thyroid tissue following fine needle aspiration biopsy, surgery, RAI therapy, or thyroiditis; and (4) the degree of TSH receptor stimulation by TSH, hCG, or TSAb (10). The presence of TgAb necessitates a shift in focus from monitoring serum Tg as the primary tumor-marker, to monitoring the serum TgAb trend as a surrogate tumor-marker (519).

Figure 12. TgAb Effects on Serial Tg IMA and Tg RIA Measurements. Serial TgAb, Tg-RIA and Tg-IMA measurements made in two DTC patients who underwent a change in TgAb status (panel A, positive to negative) or (panel B negative to positive) before death from structural DTC. These cases illustrate why a Tg measurement cannot be interpreted without knowing the TgAb status of the patient (57). The de novo appearance of TgAb (Patient B) either reflects a change in tumor-derived Tg heterogeneity (secretion of a more immunogenic Tg molecule), or recognition of tumor-derived Tg by the immune system. In contrast, TgAb can become undetectable despite the exacerbation of disease (Patient A).

Figure 13. TgAb Trends in Response to Treatment. Typical trends in TgAb following thyroidectomy in patients rendered disease-free by thyroidectomy (pattern A) versus patents with persistent/recurrent disease (pattern B). TgAb levels may rise or become detectable de novo in response to an increase in Tg antigen following surgical injury, lymph node recurrence(s), lymph node resection(s), FNA biopsy of metastatic lymph nodes or radioiodine therapy.

PRE-OPERATIVE Tg MEASUREMENT

An elevated Tg is merely a non-specific indicator of thyroid pathology and cannot be used to diagnose malignancy (568). However, studies have reported that a Tg elevation detected decades before a DTC diagnosis, is a risk factor for thyroid malignancy (567-569,646-648). This suggests that most thyroid cancers secrete Tg protein to an equal or greater degree than normal thyroid tissue, underscoring the importance of using Tg as a DTC tumor marker. Approximately 50 percent of DTC patients have an elevated preoperative serum Tg the highest being seen in follicular > oncocytic (formerly “Hurthle cell cancer”) > papillary thyroid carcinoma (567-569). Up to one-third of tumors may be poor Tg secretors relative to tumor mass, especially BRAF-positive tumors that are associated with reduced expression of Tg protein (649). Although current guidelines do not recommend routine pre-operative serum Tg measurement (57,549,650), some believe that a preoperative serum Tg (drawn before or more than two weeks after FNA) can provide information regarding the tumor’s intrinsic ability to secrete Tg and thus aid with the interpretation of postoperative Tg changes (567-569,648,650). For example, knowing that a tumor is an inefficient Tg secretor could prompt a physician to focus more on anatomic imaging and less on postoperative Tg monitoring (649,651,652).

POST-OPERATIVE Tg MEASUREMENT

Because TSH exerts such a strong influence on serum Tg concentrations it is important to promptly initiate thyroid hormone therapy after surgery to establish a stable post-operative Tg baseline to begin biochemical monitoring. When surgery is followed by RAI treatment it may take time (months) to establish a stable Tg baseline because the Tg rises in response to TSH-stimulation and may be augmented by Tg release from radiolytic damage of the thyroid remnant. Short-term rhTSH stimulation is expected to produce an approximate 10-fold serum Tg elevation (561), whereas chronic endogenous TSH stimulation following thyroid hormone withdrawal results in an approximate 20-fold serum Tg rise (653). Serum Tg measurements performed as early as 6 to 8 weeks after thyroidectomy have been shown to have prognostic value - the higher the serum Tg the greater the risk of persistent/recurrent disease (526, 654, 655). Since the half-life of Tg in the circulation approximates 3 days (656), the acute Tg release resulting from the surgical trauma and healing of surgical margins should largely resolve within the first six months, provided that post-operative thyroid hormone therapy prevents TSH from rising. Patients who receive RAI for remnant ablation may exhibit a slow Tg decline over subsequent years, presumably reflecting the long-term radiolytic destruction of remnant tissue (657,658).

The Tg secretion expected from the ~1 gram of normal remnant tissue left after thyroidectomy (580) is expected to produce a serum Tg concentration ~1.0 µg/L, provided TSH is not elevated (10). A recent study found that in the first six months following thyroidectomy (without RAI treatment) disease-free PTC patients had a serum Tg nadir < 0.5 µg/L when TSH was maintained below 0.5 mIU/L (274,581,582). This is consistent with earlier studies using receiver operator curve (ROC) analysis that found a 6-week serum Tg of <1.0 µg/L, when measured during TSH suppression, had a 98 percent negative predictive value (NPV) for disease (although positive predictive value (PPV) was only 43 percent) (654).

LONG-TERM Tg MONITORING (WITHOUT TSH STIMULATION)

The higher the post-operative serum Tg measured without TSH stimulation, the greater the risk for persistent/recurrent disease (654). If a stable TSH is maintained (≤0.5 mIU/L) (274,582) changes in serum Tg will reflect changes in tumor mass. Under these conditions a rising Tg would be suspicious for tumor recurrence whereas declining Tg levels suggests the absence or regression of disease. When using a sensitive Tg-IMA method, the trend in serum Tg (measured without TSH stimulation) is a more reliable indicator of disease status than using a fixed Tg cutoff value for disease (57,274,548,562,587,654,659-661). It is the degree of Tg elevation, not merely a "detectable" Tg that is the risk factor for disease, since Tg “detectability” varies according to the method used (563,575,578,582). As with other tumor-markers, such as calcitonin, the Tg doubling time (measured without TSH stimulation) is a useful prognostic marker that has an inverse relationship to mortality (252,581,660,662-666). However, between-method variability necessitates that the serum Tg trend be established using the same method, and preferably the same laboratory (Figure 11) (57,587). One approach used to mitigate between-run imprecision and improve the reliability of assessing the Tg trend has been to measure the current specimen concurrently (in the same run) with the patient’s previous archived specimen, thereby eliminating run-to-run variability and increasing the confidence to detect small Tg changes (9,10,587).

SERUM Tg RESPONSES TO TSH STIMULATION

The degree of tumor differentiation determines the presence and density of TSH receptors that in large part determines the magnitude of the serum Tg response to TSH stimulation (667,668). The serum Tg rise in response to endogenous TSH (thyroid hormone withdrawal) is twice that seen with short-term rhTSH stimulation (~20-fold versus ~10-fold, respectively) (386,653,669). Recombinant human TSH (rhTSH) administration was adopted as a standardized approach for stimulating serum Tg into the measurable range of the insensitive first-generation tests (386,549,561,653,669,670). A rhTSH-stimulated serum Tg cut-off of ≥2.0 µg/L, measured 72 hours after the second dose of rhTSH, was found to be a risk factor for disease (653,669). A "positive" rhTSH response had a higher NPV (>95 percent) than the basal Tg measured by an insensitive first-generation test, (553,564,654,671). However, a negative rhTSH test did not guarantee the absence of tumor (653,671). Furthermore, the reliability of adopting a fixed numeric rhTSH-Tg cut-off value for a positive response is problematic, given that different methods can report different numeric Tg values for the same specimen (Figure 11) (14, 512, 575). Other variables include differences in the dose of rhTSH delivered relative to absorption from the injection site as well as the surface area and age of the patient (672,673). One critical variable is the TSH sensitivity of tumor tissues, with poorly differentiated tumors having blunted TSH-mediated Tg responses (649,651,652,668). When using a sensitive second-generation Tg-IMA, an undetectable basal Tg (<0.10 µg/L) had a comparable NPV to rhTSH stimulation and was rarely associated with a "positive" rhTSH-stimulated response (>2.0 µg/L) (561, 563, 575, 674, 675). This would be expected given the strong relationship between basal Tg and rhTSH-stimulated Tg values (553,561,578,676). Once sensitive Tg-IMA methods had become the standard of care, it became apparent that rhTSH-stimulation provided no additional information over and above a basal Tg measured by second generation assay (57, 553, 561, 563, 575, 578, 674-676).

One potential use of rhTSH-stimulated Tg would be to test for HAb interferences. Specifically, when a Tg-IMA value appears clinically inappropriate (usually high), an absent rhTSH-stimulated Tg response would suggest interference that could be confirmed by a blocker tube test (561). An alternative reason for an absent/blunted rhTSH-stimulated response would be the presence of TgAb (578), with TgAb-enhanced clearance of Tg-TgAb complexes (599,602,606).

Tg MEASUREMENT IN FNA NEEDLE WASHOUTS (FNA-Tg)

Because the Tg protein is tissue-specific, the detection of Tg in non-thyroidal tissues or fluids (such as pleural fluid) indicates the presence of metastatic thyroid cancer (677). Struma ovarii is the only (rare) condition in which the Tg in the circulation does not originate from the thyroid (678,679). Cystic thyroid nodules are commonly encountered in clinical practice, the large majority arising from follicular epithelium and the minority from parathyroid epithelium. A high concentration of Tg or parathyroid hormone (PTH) measured in the cyst fluid provides a reliable indicator of the tissue origin of the cyst (thyroid versus parathyroid, respectively), information critical for surgical decision-making (677,680). Lymph node metastases are found in up to 50 percent of patients with papillary cancers but only 20 percent of follicular cancers (681,682). High-resolution ultrasound has now become an important component of postoperative surveillance for recurrence (57,386,669). Although ultrasound characteristics are helpful for distinguishing benign reactive lymph nodes from those suspicious for malignancy, the finding of Tg in the needle washout of a lymph node biopsy has higher diagnostic accuracy than the ultrasound appearance (632,683-691). An FNA needle washout is now widely accepted as a useful adjunctive test that improves the diagnostic sensitivity of a cytological evaluation of a suspicious lymph node or thyroid mass, even in the presence of TgAb (683-687). The current protocol for obtaining FNA-Tg samples recommends rinsing the biopsy needle in 1.0 mL of saline and sending this specimen to the laboratory for Tg analysis. In thyroidectomized patients a common cutoff value for a "positive" FNA-Tg result is 1.0 µg/L, however this cutoff can vary by method and institution (685,686,690-692). For investigations of suspicious lymph nodes in patients with an intact thyroid, a higher FNA-Tg cutoff value (~35-40 µg/L) is recommended (683). There is still controversy whether TgAb interferes with FNA-Tg analyses (528,684). It should be noted that when the serum TgAb concentration is high there can be TgAb contamination of the FNA wash fluid. Although a ~40-fold dilution of TgAb in the wash fluid would be expected, this could still be insufficient to lower TgAb below detection and eliminate the possibility of TgAb interference with the FNA-Tg IMA test producing a falsely low result. The FNA needle wash-out procedure can also be used to detect calcitonin in neck masses of patients with primary and metastatic medullary thyroid cancer (680,693,694). In addition, FNA-PTH determinations may be useful for identifying lymph nodes arising from parathyroid tissue (680).

THYROID SPECIFIC mRNAs USED AS THYROID TUMOR MARKERS

Reverse transcription-polymerase chain reaction (RT-PCR) has been used to detect thyroid-specific mRNAs (Tg, TSHR, TPO and NIS) in the peripheral blood of patients with DTC (579,695-697). Initial studies suggested that circulating Tg mRNA might be employed as a useful tumor marker for thyroid cancer, especially in TgAb-positive patients in whom Tg measurements were subject to TgAb interference (695,698,699). More recently, this approach has been applied to the detection of NIS, TPO, and TSH receptor (TSHR) mRNAs (699,700). Although some studies have suggested that thyroid specific mRNA measurements could be useful for cancer diagnosis and detecting recurrent disease, most studies have concluded that they offer no advantages over sensitive serum Tg measurements (579,699,701). Further, the recent report of false positive Tg mRNA results in patients with congenital athyreosis (702) suggests that Tg mRNA can arise as an assay artifact originating from non-thyroid tissues, or illegitimate transcription (703,704). Conversely, false negative Tg mRNA results have also been observed in patients with documented metastatic disease (705,706). Although Tg, TSHR, NIS and TPO are generally considered “thyroid specific” proteins, mRNAs for these antigens have been detected in non-thyroidal tissues such as lymphocytes, leukocytes, kidney, hepatocytes, brown fat and skin (427,707,708)). Additional sources of variability in mRNA analyses relate to the use of primers that detect splice variants, sample-handling techniques that introduce variability, and difficulties in quantifying the mRNA detected (701,705). The general consensus is that thyroid specific mRNA measurements lack the optimal specificity and practicality to be useful tumor markers (579,699,701). MicroRNA (miRNA) has recently been proposed as an alternate candidate biomarker when Tg measurement is unreliable (709). The growing number of reports of functional TSH receptors and Tg mRNA present in non-thyroidal tissues further suggests that these mRNA measurements will have limited clinical utility in the management of DTC in the future (427,707,708). Further studies in thyroid cancer genomics may yield additional DTC tumor markers with optimal sensitivity and specificity to monitor DTC (710).

REFERENCES

Spencer CA. Laboratory Thyroid Tests: A Historical Perspective. Thyroid. 2023;33(4):407-419.
Chaney A. Protein-bound iodine. Advances in clinical chemistry. 1958;Vol. 1(2):81-109.
Utiger RD. RADIOIMMUNOASSAY OF HUMAN PLASMA THYROTROPIN. J Clin Invest. 1965;44(8):1277-1286.
Chopra IJ. A radioimmunoassay for measurement of thyroxine in unextracted serum. J Clin Endocrinol Metab.1972;34(6):938-947.
Van Herle AJ, Uller RP, Matthews NL, Brown J. Radioimmunoassay for measurement of thyroglobulin in human serum. J Clin Invest. 1973;52:1320-1327.
Chopra IJ. A radioimmunoassay for measurement of 3,3',5'-triiodothyronine (reverse T3). J Clin Invest.1974;54(3):583-592.
Woodhead J, Addison G, Hales C. The immunoradiometric assay and related techniques. Br Med Bull.1974;30:44-49.
Spencer CA, LoPresti JS, Patel A, Guttler RB, Eigen A, Shen D, Gray D, Nicoloff JT. Applications of a new chemiluminometric thyrotropin assay to subnormal measurement. J Clin Endocrinol Metab. 1990;70:453-460.
Spencer CA TM, Kazarosyan M,. Current Status and Performance Goals for Serum Thyroglobulin Assays. Clin Chem. 1996;42(In Press 1/96).
Baloch Z, Carayon P, Conte-Devolx B, Demers LM, Feldt-Rasmussen U, Henry JF, LiVosli VA, Niccoli-Sire P, John R, Ruf J, Smyth PP, Spencer CA, Stockigt JR. Laboratory medicine practice guidelines. Laboratory support for the diagnosis and monitoring of thyroid disease. Thyroid. 2003;13(1):3-126.
Dufour DR. Laboratory tests of thyroid function: uses and limitations. Endocrinol Metab Clin North Am.2007;36(3):579-594, v.
Thienpont LM, Van Uytfanghe K, Beastall G, Faix JD, Ieiri T, Miller WG, Nelson JC, Ronin C, Ross HA, Thijssen JH, Toussaint B. Report of the IFCC Working Group for Standardization of Thyroid Function Tests; part 2: free thyroxine and free triiodothyronine. Clin Chem. 2010;56(6):912-920.
Van Uytfanghe K, Ehrenkranz J, Halsall D, Hoff K, Loh TP, Spencer CA, Köhrle J. Thyroid Stimulating Hormone and Thyroid Hormones (Triiodothyronine and Thyroxine): An American Thyroid Association-Commissioned Review of Current Clinical and Laboratory Status. Thyroid. 2023;33(9):1013-1028.
Petrovic I, LoPresti J, Fatemi S, Gianoukakis A, Burman K, Gomez-Lima CJ, Nguyen CT, Spencer CA. Influence of Thyroglobulin Autoantibodies on Thyroglobulin Levels Measured by Different Methodologies: IMA, LC-MS/MS, and RIA. J Clin Endocrinol Metab. 2024;109(12):3254-3263.
Thienpont LM, Van Uytfanghe K, Poppe K, Velkeniers B. Determination of free thyroid hormones. Best practice & research Clinical endocrinology & metabolism. 2013;27(5):689-700.
Westbye AB, Aas FE, Kelp O, Dahll LK, Thorsby PM. Analysis of free, unbound thyroid hormones by liquid chromatography-tandem mass spectrometry: A mini-review of the medical rationale and analytical methods. Anal Sci Adv. 2023;4(7-8):244-254.
Giovanella L, D'Aurizio F, Algeciras-Schimnich A, Görges R, Petranovic Ovcaricek P, Tuttle RM, Visser WE, Verburg FA. Thyroglobulin and thyroglobulin antibody: an updated clinical and laboratory expert consensus. Eur J Endocrinol. 2023;189(2):R11-r27.
Tozzoli R, D'Aurizio F, Villalta D, Giovanella L. Evaluation of the first fully automated immunoassay method for the measurement of stimulating TSH receptor autoantibodies in Graves' disease. Clin Chem Lab Med. 2017;55(1):58-64.
Kahaly GJ, Diana T, Olivo PD. TSH Receptor Antibodies: Relevance & Utility. Endocr Pract. 2020;26(1):97-106.
Lupo MA, Olivo PD, Luffy M, Wolf J, Kahaly GJ. US-based, Prospective, Blinded Study of Thyrotropin Receptor Antibody in Autoimmune Thyroid Disease. J Clin Endocrinol Metab. 2024.
Jensen EA, Petersen PH, Blaabjerg O, Hansen PS, Brix TH, Hegedüs L. Establishment of reference distributions and decision values for thyroid antibodies against thyroid peroxidase (TPOAb), thyroglobulin (TgAb) and the thyrotropin receptor (TRAb). Clin Chem Lab Med. 2006;44(8):991-998.
Spencer CA. Clinical review: Clinical utility of thyroglobulin antibody (TgAb) measurements for patients with differentiated thyroid cancers (DTC). J Clin Endocrinol Metab. 2011;96(12):3615-3627.
Verburg FA, Luster M, Cupini C, Chiovato L, Duntas L, Elisei R, Feldt-Rasmussen U, Rimmele H, Seregni E, Smit JW, Theimer C, Giovanella L. Implications of thyroglobulin antibody positivity in patients with differentiated thyroid cancer: a clinical position statement. Thyroid. 2013;23(10):1211-1225.
Netzel BC, Grebe SK, Carranza Leon BG, Castro MR, Clark PM, Hoofnagle AN, Spencer CA, Turcu AF, Algeciras-Schimnich A. Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes. J Clin Endocrinol Metab. 2015;100(8):E1074-1083.
Schussler GC. The thyroxine-binding proteins. Thyroid. 2000;10(2):141-149.
Pappa T, Ferrara AM, Refetoff S. Inherited defects of thyroxine-binding proteins. Best practice & research Clinical endocrinology & metabolism. 2015;29(5):735-747.
L Bartalena, D Gallo, E Piantanida. Serum thyroid hormone-binding proteins. In: Encyclopedia of Endocrine Diseases. 2024:442-447.
Thienpont LM, Van Uytfanghe K, Van Houcke S. Standardization activities in the field of thyroid function tests: a status report. Clin Chem Lab Med. 2010;48(11):1577-1583.
Thienpont LM, Van Uytfanghe K, Beastall G, Faix JD, Ieiri T, Miller WG, Nelson JC, Ronin C, Ross HA, Thijssen JH, Toussaint B. Report of the IFCC Working Group for Standardization of Thyroid Function Tests; part 1: thyroid-stimulating hormone. Clin Chem. 2010;56(6):902-911.
Jonklaas J, Sathasivam A, Wang H, Gu J, Burman KD, Soldin SJ. Total and free thyroxine and triiodothyronine: measurement discrepancies, particularly in inpatients. Clin Biochem. 2014;47(13-14):1272-1278.
Vesper HW, Myers GL, Miller WG. Current practices and challenges in the standardization and harmonization of clinical laboratory tests. Am J Clin Nutr. 2016;104 Suppl 3(Suppl 3):907s-912s.
Ribera A, Zhang L, Ribeiro C, Vazquez N, Thonkulpitak J, Botelho JC, Danilenko U, van Uytfanghe K, Vesper HW. Practical considerations for accurate determination of free thyroxine by equilibrium dialysis. J Mass Spectrom Adv Clin Lab. 2023;29:9-15.
Cowper B, Lyle AN, Vesper HW, Van Uytfanghe K, Burns C. Standardisation and harmonisation of thyroid-stimulating hormone measurements: historical, current, and future perspectives. Clin Chem Lab Med.2024;62(5):824-829.
Thienpont LM, Beastall G, Christofides ND, Faix JD, Ieiri T, Jarrige V, Miller WG, Miller R, Nelson JC, Ronin C, Ross HA, Rottmann M, Thijssen JH, Toussaint B. Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum. Clin Chem Lab Med. 2007;45(7):934-936.
Van Houcke SK, Van Uytfanghe K, Shimizu E, Tani W, Umemoto M, Thienpont LM. IFCC international conventional reference procedure for the measurement of free thyroxine in serum: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Working Group for Standardization of Thyroid Function Tests (WG-STFT)(1). Clin Chem Lab Med. 2011;49(8):1275-1281.
Jansen HI, van der Steen R, Brandt A, Olthaar AJ, Vesper HW, Shimizu E, Heijboer AC, Van Uytfanghe K, van Herwaarden AE. Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum. Clin Chem Lab Med. 2023;61(9):1605-1611.
Hoofnagle AN, Becker JO, Wener MH, Heinecke JW. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin Chem. 2008;54(11):1796-1804.
Hoofnagle AN, Roth MY. Clinical review: improving the measurement of serum thyroglobulin with mass spectrometry. J Clin Endocrinol Metab. 2013;98(4):1343-1352.
Clarke NJ, Zhang Y, Reitz RE. A novel mass spectrometry-based assay for the accurate measurement of thyroglobulin from patient samples containing antithyroglobulin autoantibodies. Journal of Investigative Medicine: the official publication of the American Federation for Clinical Research. 2012;60(8):1157-1163.
Kushnir MM, Rockwood AL, Roberts WL, Abraham D, Hoofnagle AN, Meikle AW. Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem. 2013;59(6):982-990.
Netzel BC, Grant RP, Hoofnagle AN, Rockwood AL, Shuford CM, Grebe SK. First Steps toward Harmonization of LC-MS/MS Thyroglobulin Assays. Clin Chem. 2016;62(1):297-299.
Shuford CM, Walters JJ, Holland PM, Sreenivasan U, Askari N, Ray K, Grant RP. Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised. Anal Chem. 2017;89(14):7406-7415.
Barbesino G, Algeciras-Schimnich A, Bornhorst J. Thyroglobulin Assay Interferences: Clinical Usefulness of Mass-Spectrometry Methods. J Endocr Soc. 2022;7(1):bvac169.
Thienpont LM, De Brabandere VI, Stöckl D, De Leenheer AP. Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry. Biol Mass Spectrom.1994;23(8):475-482.
Spencer C, Petrovic I, Fatemi S. Current thyroglobulin autoantibody (TgAb) assays often fail to detect interfering TgAb that can result in the reporting of falsely low/undetectable serum Tg IMA values for patients with differentiated thyroid cancer. J Clin Endocrinol Metab. 2011;96(5):1283-1291.
Westbye AB, Aas FE, Dahl SR, Zykova SN, Kelp O, Dahll LK, Thorsby PM. Large method differences for free thyroid hormone assays in the hyperthyroid range can affect assessment of hyperthyroid status: Comparison of Abbott Alinity to Roche Cobas, Siemens Centaur and equilibrium dialysis LC-MS/MS. Clin Biochem. 2023;121-122:110676.
Stagnaro-Green A, Abalovich M, Alexander E, Azizi F, Mestman J, Negro R, Nixon A, Pearce EN, Soldin OP, Sullivan S, Wiersinga W. Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and postpartum. Thyroid. 2011;21(10):1081-1125.
Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Laurberg P, Lazarus JH, Mandel SJ, Peeters RP, Sullivan S. 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017;27(3):315-389.
Garber JR, Cobin RH, Gharib H, Hennessey JV, Klein I, Mechanick JI, Pessah-Pollack R, Singer PA, Woeber KA. Clinical practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyroid Association. Thyroid. 2012;22(12):1200-1235.
Pearce SH, Brabant G, Duntas LH, Monzani F, Peeters RP, Razvi S, Wemeau JL. 2013 ETA Guideline: Management of Subclinical Hypothyroidism. Eur Thyroid J. 2013;2(4):215-228.
Lazarus J, Brown RS, Daumerie C, Hubalewska-Dydejczyk A, Negro R, Vaidya B. 2014 European thyroid association guidelines for the management of subclinical hypothyroidism in pregnancy and in children. Eur Thyroid J. 2014;3(2):76-94.
Jonklaas J, Bianco AC, Bauer AJ, Burman KD, Cappola AR, Celi FS, Cooper DS, Kim BW, Peeters RP, Rosenthal MS, Sawka AM. Guidelines for the treatment of hypothyroidism: prepared by the american thyroid association task force on thyroid hormone replacement. Thyroid. 2014;24(12):1670-1751.
Biondi B, Bartalena L, Cooper DS, Hegedüs L, Laurberg P, Kahaly GJ. The 2015 European Thyroid Association Guidelines on Diagnosis and Treatment of Endogenous Subclinical Hyperthyroidism. Eur Thyroid J. 2015;4(3):149-163.
Ross DS, Burch HB, Cooper DS, Greenlee MC, Laurberg P, Maia AL, Rivkees SA, Samuels M, Sosa JA, Stan MN, Walter MA. 2016 American Thyroid Association Guidelines for Diagnosis and Management of Hyperthyroidism and Other Causes of Thyrotoxicosis. Thyroid. 2016;26(10):1343-1421.
Gharib H, Papini E, Garber JR, Duick DS, Harrell RM, Hegedüs L, Paschke R, Valcavi R, Vitti P. Medical Guidelines for Clinical Practice for the Diagnosis and Management of Thyroid Nodules--2016 Update. Endocr Pract. 2016;22(5):622-639.
Francis GL, Waguespack SG, Bauer AJ, Angelos P, Benvenga S, Cerutti JM, Dinauer CA, Hamilton J, Hay ID, Luster M, Parisi MT, Rachmiel M, Thompson GB, Yamashita S. Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2015;25(7):716-759.
Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, Pacini F, Randolph GW, Sawka AM, Schlumberger M, Schuff KG, Sherman SI, Sosa JA, Steward DL, Tuttle RM, Wartofsky L. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016;26(1):1-133.
Filetti S, Durante C, Hartl D, Leboulleux S, Locati LD, Newbold K, Papotti MG, Berruti A. Thyroid cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Ann Oncol. 2019;30(12):1856-1883.
Bible KC, Kebebew E, Brierley J, Brito JP, Cabanillas ME, Clark TJ, Jr., Di Cristofano A, Foote R, Giordano T, Kasperbauer J, Newbold K, Nikiforov YE, Randolph G, Rosenthal MS, Sawka AM, Shah M, Shaha A, Smallridge R, Wong-Clark CK. 2021 American Thyroid Association Guidelines for Management of Patients with Anaplastic Thyroid Cancer. Thyroid. 2021;31(3):337-386.
Giannoula E, Exadaktylou P, Melidis C, Koutsouki G, Katsadouros I, Tsangaridi A, Charalambous P, Papadopoulou K, Frangos S, Iakovou I. Real-world applicability of differentiated thyroid cancer guidelines. Hell J Nucl Med.2024;27(2):121-130.
Bartalena L, Robbins J. Thyroid hormone transport proteins. Clin Lab Med. 1993;13(3):583-598.
Stockigt JR. Free thyroid hormone measurement. A critical appraisal. Endocrinol Metab Clin North Am.2001;30(2):265-289.
Murphy BE, Pattee CJ. Determination of Thyroxine Utilizing the Property of Protein-Binding. J Clin Endocrinol Metab. 1964;24:187-196.
Thienpont LM, Fierens C, De Leenheer AP, Przywara L. Isotope dilution-gas chromatography/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of triiodo-L-thyronine in serum. Rapid Commun Mass Spectrom. 1999;13(19):1924-1931.
Soldin SJ, Soukhova N, Janicic N, Jonklaas J, Soldin OP. The measurement of free thyroxine by isotope dilution tandem mass spectrometry. Clin Chim Acta. 2005;358(1-2):113-118.
Li W, Gao B, Yang T, Tang L, Song D, Li H, Sun K, Xiao P. Development and validation of an isotope dilution-liquid chromatography (ID-LC-MS/MS)-based candidate reference measurement procedure for total 3,3',5-triiodothyronine in human serum. Anal Bioanal Chem. 2025.
Matsuda M, Sakata S, Komaki T, Nakamura S, Kojima N, Takuno H, Miura K. Effect of 8-anilino-1-naphthalene sulfonic acid (ANS) on the interaction between thyroid hormone and anti-thyroid hormone antibodies. Clin Chim Acta. 1989;185(2):139-146.
Thienpont LM, Van Uytfanghe K, Marriott J, Stokes P, Siekmann L, Kessler A, Bunk D, Tai S. Feasibility study of the use of frozen human sera in split-sample comparison of immunoassays with candidate reference measurement procedures for total thyroxine and total triiodothyronine measurements. Clin Chem. 2005;51(12):2303-2311.
Faix JD, Miller WG. Progress in standardizing and harmonizing thyroid function tests. Am J Clin Nutr. 2016;104 Suppl 3(Suppl 3):913s-917s.
Zhou Q, Li S, Li X, Wang W, Wang Z. Comparability of five analytical systems for the determination of triiodothyronine, thyroxine and thyroid-stimulating hormone. Clin Chem Lab Med. 2006;44(11):1363-1366.
Algeciras-Schimnich A, Bruns DE, Boyd JC, Bryant SC, La Fortune KA, Grebe SK. Failure of current laboratory protocols to detect lot-to-lot reagent differences: findings and possible solutions. Clin Chem. 2013;59(8):1187-1194.
Van Houcke SK, Thienpont LM. "Good samples make good assays" – the problem of sourcing clinical samples for a standardization project. Clin Chem Lab Med. 2013;51(5):967-972.
Lee RH, Spencer CA, Mestman JH, Miller EA, Petrovic I, Braverman LE, Goodwin TM. Free T4 immunoassays are flawed during pregnancy. Am J Obstet Gynecol. 2009;200(3):260.e261-266.
Wilson KL, Casey BM, McIntire DD, Cunningham FG. Is total thyroxine better than free thyroxine during pregnancy? Am J Obstet Gynecol. 2014;211(2):132.e131-136.
Korevaar TI, Chaker L, Medici M, de Rijke YB, Jaddoe VW, Steegers EA, Tiemeier H, Visser TJ, Peeters RP. Maternal total T4 during the first half of pregnancy: physiologic aspects and the risk of adverse outcomes in comparison with free T4. Clin Endocrinol (Oxf). 2016;85(5):757-763.
De Groot L, Abalovich M, Alexander EK, Amino N, Barbour L, Cobin RH, Eastman CJ, Lazarus JH, Luton D, Mandel SJ, Mestman J, Rovet J, Sullivan S. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2012;97(8):2543-2565.
Klee GG. Clinical usage recommendations and analytic performance goals for total and free triiodothyronine measurements. Clin Chem. 1996;42(1):155-159.
Welsh KJ, Soldin SJ. Diagnosis of Endocrine Disease: How reliable are free thyroid and total T3 hormone assays? Eur J Endocrinol. 2016;175(6):R255-r263.
Ekins R. Measurement of free hormones in blood. Endocr Rev. 1990;11(1):5-46.
Faix JD. Principles and pitfalls of free hormone measurements. Best practice & research Clinical endocrinology & metabolism. 2013;27(5):631-645.
Sterling K, Brenner MA. Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation. J Clin Invest. 1966;45(1):153-163.
Nelson JC, Weiss RM. The effect of serum dilution on free thyroxine (T4) concentration in the low T4 syndrome of nonthyroidal illness. J Clin Endocrinol Metab. 1985;61(2):239-246.
Sophianopoulos J, Jerkunica I, Lee CN, Sgoutas D. An improved ultrafiltration method for free thyroxine and triiodothyronine in serum. Clin Chem. 1980;26(1):159-162.
Wang YS, Hershman JM, Pekary AE. Improved ultrafiltration method for simultaneous measurement of free thyroxin and free triiodothyronine in serum. Clin Chem. 1985;31(4):517-522.
Weeke J, Boye N, Orskov H. Ultrafiltration method for direct radioimmunoassay measurement of free thyroxine and free tri-iodothyronine in serum. Scand J Clin Lab Invest. 1986;46(4):381-389.
Romelli PB, Pennisi F, Vancheri L. Measurement of free thyroid hormones in serum by column adsorption chromatography and radioimmunoassay. J Endocrinol Invest. 1979;2(1):25-40.
Thienpont LM, Van Uytfanghe K, Van Houcke S, Das B, Faix JD, MacKenzie F, Quinn FA, Rottmann M, Van den Bruel A. A Progress Report of the IFCC Committee for Standardization of Thyroid Function Tests. Eur Thyroid J.2014;3(2):109-116.
Thienpont L, Uytfanghe KV, Grande LD, Reynders D, Das B, Faix J, MacKenzie F, Decallonne B, Hishinuma A, Lapauw B, Taelman P, Crombrugge PV, Bruel AVd, Velkeniers B, Williams P. Harmonization of Serum Thyroid-Stimulating Hormone Measurements Paves the Way for the Adoption of a More Uniform Reference Interval. Clin Chem. 2017;63:1248-1260.
De Grande LAC, Van Uytfanghe K, Reynders D, Das B, Faix JD, MacKenzie F, Decallonne B, Hishinuma A, Lapauw B, Taelman P, Van Crombrugge P, Van den Bruel A, Velkeniers B, Williams P, Thienpont LM. Standardization of Free Thyroxine Measurements Allows the Adoption of a More Uniform Reference Interval. Clin Chem. 2017;63(10):1642-1652.
Iitaka M, Kawasaki S, Sakurai S, Hara Y, Kuriyama R, Yamanaka K, Kitahama S, Miura S, Kawakami Y, Katayama S. Serum substances that interfere with thyroid hormone assays in patients with chronic renal failure. Clin Endocrinol (Oxf). 1998;48(6):739-746.
Moran C, Schoenmakers N, Halsall D, Oddy S, Lyons G, van den Berg S, Gurnell M, Chatterjee K. Approach to the Patient With Raised Thyroid Hormones and Nonsuppressed TSH. J Clin Endocrinol Metab. 2024;109(4):1094-1108.
Nelson JC, Tomei RT. Direct determination of free thyroxin in undiluted serum by equilibrium dialysis/radioimmunoassay. Clin Chem. 1988;34(9):1737-1744.
Hopley CJ, Stokes P, Webb KS, Baynham M. The analysis of thyroxine in human serum by an 'exact matching' isotope dilution method with liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom.2004;18(10):1033-1038.
Jonklaas J, Kahric-Janicic N, Soldin OP, Soldin SJ. Correlations of free thyroid hormones measured by tandem mass spectrometry and immunoassay with thyroid-stimulating hormone across 4 patient populations. Clin Chem.2009;55(7):1380-1388.
Van Uytfanghe K, Stöckl D, Ross HA, Thienpont LM. Use of frozen sera for FT4 standardization: investigation by equilibrium dialysis combined with isotope dilution-mass spectrometry and immunoassay. Clin Chem.2006;52(9):1817-1821.
Yue B, Rockwood AL, Sandrock T, La'ulu SL, Kushnir MM, Meikle AW. Free thyroid hormones in serum by direct equilibrium dialysis and online solid-phase extraction--liquid chromatography/tandem mass spectrometry. Clin Chem. 2008;54(4):642-651.
Christofides ND, Midgley JE. Inaccuracies in free thyroid hormone measurement by ultrafiltration and tandem mass spectrometry. Clin Chem. 2009;55(12):2228-2229; author reply 2229-2230.
Tikanoja S. Ultrafiltration devices tested for use in a free thyroxine assay validated by comparison with equilibrium dialysis. Scand J Clin Lab Invest. 1990;50(6):663-669.
Fritz KS, Wilcox RB, Nelson JC. Quantifying spurious free T4 results attributable to thyroxine-binding proteins in serum dialysates and ultrafiltrates. Clin Chem. 2007;53(5):985-988.
Tractenberg RE, Jonklaas J, Soldin SJ. Agreement of immunoassay and tandem mass spectrometry in the analysis of cortisol and free t4: interpretation and implications for clinicians. Int J Anal Chem. 2010;2010.
Tuttlebee J, R B. A comparison of free thyroxine concentration and the free thyroxine index as diagnostic tests of thyroid function. Ann Clin Biochem. 1981;18:88-92.
Roberts RF, La'ulu SL, Roberts WL. Performance characteristics of seven automated thyroxine and T-uptake methods. Clin Chim Acta. 2007;377(1-2):248-255.
Nelson JC, Tomei RT. Dependence of the thyroxin/thyroxin-binding globulin (TBG) ratio and the free thyroxin index on TBG concentrations. Clin Chem. 1989;35(4):541-544.
Stockigt JR, Lim CF. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best practice & research Clinical endocrinology & metabolism. 2009;23(6):753-767.
Cartwright D, O'Shea P, Rajanayagam O, Agostini M, Barker P, Moran C, Macchia E, Pinchera A, John R, Agha A, Ross HA, Chatterjee VK, Halsall DJ. Familial dysalbuminemic hyperthyroxinemia: a persistent diagnostic challenge. Clin Chem. 2009;55(5):1044-1046.
D'Aurizio F, Kratzsch J, Gruson D, Petranović Ovčariček P, Giovanella L. Free thyroxine measurement in clinical practice: how to optimize indications, analytical procedures, and interpretation criteria while waiting for global standardization. Crit Rev Clin Lab Sci. 2023;60(2):101-140.
Felicetta JV, Green WL. Value of free thyroxine index. N Engl J Med. 1980;302(26):1480-1481.
Larsen PR, Alexander NM, Chopra IJ, Hay ID, Hershman JM, Kaplan MM, Mariash CN, Nicoloff JT, Oppenheimer JH, Solomon DH, et al. Revised nomenclature for tests of thyroid hormones and thyroid-related proteins in serum. J Clin Endocrinol Metab. 1987;64(5):1089-1094.
Litherland PG, Bromage NR, Hall RA. Thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA) measurement, compared with the conventional T3 uptake in the diagnosis of thyroid disease. Clin Chim Acta.1982;122(3):345-352.
Kundra P, Burman KD. The effect of medications on thyroid function tests. Med Clin North Am. 2012;96(2):283-295.
Witherspoon LR, Shuler SE, Garcia MM. The triiodothyronine uptake test: an assessment of methods Clin Chem.1981;27:1272-1276.
Hay ID, Bayer MF, Kaplan MM, Klee GG, Larsen PR, Spencer CA. American Thyroid Association assessment of current free thyroid hormone and thyrotropin measurements and guidelines for future clinical assays. The Committee on Nomenclature of the American Thyroid Association. Clin Chem. 1991;37(11):2002-2008.
Midgley JE. Direct and indirect free thyroxine assay methods: theory and practice. Clin Chem. 2001;47(8):1353-1363.
Harpen MD, Lee WN, Siegel JA, Greenfield MA. Serum binding of triiodothyronine: theoretical and practical implications for in vitro triiodothyronine uptake. Endocrinology. 1982;110(5):1732-1739.
Faix JD, Rosen HN, Velazquez FR. Indirect estimation of thyroid hormone-binding proteins to calculate free thyroxine index: comparison of nonisotopic methods that use labeled thyroxine ("T-uptake"). Clin Chem.1995;41(1):41-47.
Burr WA, Ramsden DB, Hoffenberg R. Hereditary abnormalities of thyroxine-binding globulin concentration. A study of 19 kindreds with inherited increase or decrease of thyroxine-binding globulin. Q J Med. 1980;49(195):295-313.
Jensen IW, Faber J. Familial dysalbuminemic hyperthyroxinemia. Acta Med Scand. 1987;221(5):469-473.
Hoshikawa S, Mori K, Kaise N, Nakagawa Y, Ito S, Yoshida K. Artifactually elevated serum-free thyroxine levels measured by equilibrium dialysis in a pregnant woman with familial dysalbuminemic hyperthyroxinemia. Thyroid.2004;14(2):155-160.
Ting MJM, Zhang R, Lim EM, Ward BK, Wilson SG, Walsh JP. Familial Dysalbuminemic Hyperthyroxinemia as a Cause for Discordant Thyroid Function Tests. J Endocr Soc. 2021;5(4):bvab012.
Zouwail SA, O'Toole AM, Clark PM, Begley JP. Influence of thyroid hormone autoantibodies on 7 thyroid hormone assays. Clin Chem. 2008;54(5):927-928.
Fillée C, Cumps J, Ketelslegers JM. Comparison of three free T4 (FT4) and free T3 (FT3) immunoassays in healthy subjects and patients with thyroid diseases and severe non-thyroidal illnesses. Clin Lab. 2012;58(7-8):725-736.
Sapin R, Schlienger JL, Gasser F, Noel E, Lioure B, Grunenberger F, Goichot B, Grucker D. Intermethod discordant free thyroxine measurements in bone marrow-transplanted patients. Clin Chem. 2000;46(3):418-422.
Surks MI, Sievert R. Drugs and thyroid function. N Engl J Med. 1995;333(25):1688-1694.
Levinson SS, Rieder SV. Parameters affecting a rapid method in which Sephadex is used to determine the percentage of free thyroxine in serum. Clin Chem. 1974;20(12):1568-1572.
Oppenheimer JH, Squef R, Surks MI, Hauer H. BINDING OF THYROXINE BY SERUM PROTEINS EVALUATED BY EQUILIBRUM DIALYSIS AND ELECTROPHORETIC TECHNIQUES. ALTERATIONS IN NONTHYROIDAL ILLNESS. J Clin Invest. 1963;42(11):1769-1782.
Snyder SM, Cavalieri RR, Ingbar SH. Simultaneous measurement of percentage free thyroxine and triiodothyronine: comparison of equilibrium dialysis and Sephadex chromatography. J Nucl Med. 1976;17(7):660-664.
Ross HA, Benraad TJ. Is free thyroxine accurately measurable at room temperature? Clin Chem. 1992;38(6):880-886.
Uchimura H, Nagataki S, Tabuchi T, MMizuno, Ingbar S. Measurements of free thyroxine: comparison of per cent of free thyroxine in diluted and undiluted sera. J Clin Endocrinol Metab. 1976;42:561-566.
Witherspoon LR, Shuler SE, Garcia MM, Zollinger LA. Effects of contaminant radioactivity on results of 125I-radioligand assay. Clin Chem. 1979;25(11):1975-1977.
Midgley JE, Hoermann R. Measurement of total rather than free thyroxine in pregnancy: the diagnostic implications. Thyroid. 2013;23(3):259-261.
Ma Z, Liu Z, Deng Y, Bai X, Zhou W, Zhang C. Free thyroid hormone: Methods and standardization. Clin Chim Acta. 2025;565:119944.
Toldy E, Locsei Z, Szabolcs I, Bezzegh A, Kovács GL. Protein interference in thyroid assays: an in vitro study with in vivo consequences. Clin Chim Acta. 2005;352(1-2):93-104.
van Deventer HE, Mendu DR, Remaley AT, Soldin SJ. Inverse log-linear relationship between thyroid-stimulating hormone and free thyroxine measured by direct analog immunoassay and tandem mass spectrometry. Clin Chem.2011;57(1):122-127.
Gounden V, Jonklaas J, Soldin SJ. A pilot study: subclinical hypothyroidism and free thyroid hormone measurement by immunoassay and mass spectrometry. Clin Chim Acta. 2014;430:121-124.
Kratzsch J, Baumann NA, Ceriotti F, Lu ZX, Schott M, van Herwaarden AE, Henriques Vieira JG, Kasapic D, Giovanella L. Global FT4 immunoassay standardization: an expert opinion review. Clin Chem Lab Med.2021;59(6):1013-1023.
Piketty ML, Bounaud MP, Bounaud JY, Lebtahi R, Valat C, Askienazy S, Begon F, Besnard JC. Multicentre evaluation of a two-step automated enzyme immunoassay of free thyroxine. Eur J Clin Chem Clin Biochem.1992;30(8):485-492.
Christofides ND, Sheehan CP. Multicenter evaluation of enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine. Clin Chem. 1995;41(1):24-31.
Martel J, Després N, Ahnadi CE, Lachance JF, Monticello JE, Fink G, Ardemagni A, Banfi G, Tovey J, Dykes P, John R, Jeffery J, Grant AM. Comparative multicentre study of a panel of thyroid tests using different automated immunoassay platforms and specimens at high risk of antibody interference. Clin Chem Lab Med. 2000;38(8):785-793.
Sapin R, d'Herbomez M. Free thyroxine measured by equilibrium dialysis and nine immunoassays in sera with various serum thyroxine-binding capacities. Clin Chem. 2003;49(9):1531-1535.
Masika LS, Zhao Z, Soldin SJ. Is measurement of TT3 by immunoassay reliable at low concentrations? A comparison of the Roche Cobas 6000 vs. LC-MSMS. Clin Biochem. 2016;49(12):846-849.
Livingston M, Birch K, Guy M, Kane J, Heald AH. No role for tri-iodothyronine (T3) testing in the assessment of levothyroxine (T4) over-replacement in hypothyroid patients. Br J Biomed Sci. 2015;72(4):160-163.
Berberoğlu M. Drugs and thyroid interaction. Pediatr Endocrinol Rev. 2003;1 Suppl 2:251-256.
Brown SJ, Bremner AP, Hadlow NC, Feddema P, Leedman PJ, O'Leary PC, Walsh JP. The log TSH-free T4 relationship in a community-based cohort is nonlinear and is influenced by age, smoking and thyroid peroxidase antibody status. Clin Endocrinol (Oxf). 2016;85(5):789-796.
Hadlow NC, Rothacker KM, Wardrop R, Brown SJ, Lim EM, Walsh JP. The relationship between TSH and free T₄in a large population is complex and nonlinear and differs by age and sex. J Clin Endocrinol Metab.2013;98(7):2936-2943.
Chaker L, Korevaar TI, Medici M, Uitterlinden AG, Hofman A, Dehghan A, Franco OH, Peeters RP. Thyroid Function Characteristics and Determinants: The Rotterdam Study. Thyroid. 2016;26(9):1195-1204.
Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol (Oxf). 2006;64(3):234-244.
Teti C, Nazzari E, Galletti MR, Mandolfino MG, Pupo F, Pesce G, Lillo F, Bagnasco M, Benvenga S. Unexpected Elevated Free Thyroid Hormones in Pregnancy. Thyroid. 2016;26(11):1640-1644.
Soheilipour F, Fazilaty H, Jesmi F, Gahl WA, Behnam B. First report of inherited thyroxine-binding globulin deficiency in Iran caused by a known de novo mutation in SERPINA7. Mol Genet Metab Rep. 2016;8:13-16.
Jin HY. Thyroxine binding globulin excess detected by neonatal screening. Ann Pediatr Endocrinol Metab.2016;21(2):105-108.
Greenberg SM, Ferrara AM, Nicholas ES, Dumitrescu AM, Cody V, Weiss RE, Refetoff S. A novel mutation in the Albumin gene (R218S) causing familial dysalbuminemic hyperthyroxinemia in a family of Bangladeshi extraction. Thyroid. 2014;24(6):945-950.
Osaki Y, Hayashi Y, Nakagawa Y, Yoshida K, Ozaki H, Fukazawa H. Familial Dysalbuminemic Hyperthyroxinemia in a Japanese Man Caused by a Point Albumin Gene Mutation (R218P). Jpn Clin Med. 2016;7:9-13.
Cho YY, Song JS, Park HD, Kim YN, Kim HI, Kim TH, Chung JH, Ki CS, Kim SW. First Report of Familial Dysalbuminemic Hyperthyroxinemia With an ALB Variant. Ann Lab Med. 2017;37(1):63-65.
Pietras SM, Safer JD. Diagnostic confusion attributable to spurious elevation of both total thyroid hormone and thyroid hormone uptake measurements in the setting of autoantibodies: case report and review of related literature. Endocr Pract. 2008;14(6):738-742.
Massart C, Elbadii S, Gibassier J, Coignard V, Rasandratana A. Anti-thyroxine and anti-triiodothyronine antibody interferences in one-step free triiodothyronine and free thyroxine immunoassays. Clin Chim Acta. 2009;401(1-2):175-176.
Loh TP, Leong SM, Loke KY, Deepak DS. Spuriously elevated free thyroxine associated with autoantibodies, a result of laboratory methodology: case report and literature review. Endocr Pract. 2014;20(8):e134-139.
Okosieme OE, Agrawal M, Usman D, Evans C. Method-dependent variation in TSH and FT4 reference intervals in pregnancy: A systematic review. Ann Clin Biochem. 2021;58(5):537-546.
Osinga JAJ, Nelson SM, Walsh JP, Ashoor G, Palomaki GE, López-Bermejo A, Bassols J, Aminorroaya A, Broeren MAC, Chen L, Lu X, Brown SJ, Veltri F, Huang K, Männistö T, Vafeiadi M, Taylor PN, Tao FB, Chatzi L, Kianpour M, Suvanto E, Grineva EN, Nicolaides KH, D'Alton ME, Poppe KG, Alexander E, Feldt-Rasmussen U, Bliddal S, Popova PV, Chaker L, Visser WE, Peeters RP, Derakhshan A, Vrijkotte TGM, Pop VJM, Korevaar TIM. Defining gestational thyroid dysfunction through modified non-pregnancy reference intervals: an individual participant meta-analysis. J Clin Endocrinol Metab. 2024.
Osinga JAJ, Derakhshan A, Palomaki GE, Ashoor G, Männistö T, Maraka S, Chen L, Bliddal S, Lu X, Taylor PN, Vrijkotte TGM, Tao FB, Brown SJ, Ghafoor F, Poppe K, Veltri F, Chatzi L, Vaidya B, Broeren MAC, Shields BM, Itoh S, Mosso L, Popova PV, Anopova AD, Kishi R, Aminorroaya A, Kianpour M, López-Bermejo A, Oken E, Pirzada A, Vafeiadi M, Bramer WM, Suvanto E, Yoshinaga J, Huang K, Bassols J, Boucai L, Feldt-Rasmussen U, Grineva EN, Pearce EN, Alexander EK, Pop VJM, Nelson SM, Walsh JP, Peeters RP, Chaker L, Nicolaides KH, D'Alton ME, Korevaar TIM. TSH and FT4 Reference Intervals in Pregnancy: A Systematic Review and Individual Participant Data Meta-Analysis. J Clin Endocrinol Metab. 2022;107(10):2925-2933.
Chan S, Boelaert K. Optimal management of hypothyroidism, hypothyroxinaemia and euthyroid TPO antibody positivity preconception and in pregnancy. Clin Endocrinol (Oxf). 2015;82(3):313-326.
Varner MW, Mele L, Casey BM, Peaceman AM, Reddy UM, Wapner RJ, Thorp JM, Saade GR, Tita ATN, Rouse DJ, Sibai BM, Costantine MM, Mercer BM, Caritis SN. Progression of Gestational Subclinical Hypothyroidism and Hypothyroxinemia to Overt Hypothyroidism After Pregnancy: Pooled Analysis of Data from Two Randomized Controlled Trials. Thyroid. 2024;34(9):1171-1176.
Oguz A, Tuzun D, Sahin M, Usluogullari AC, Usluogullari B, Celik A, Gul K. Frequency of isolated maternal hypothyroxinemia in women with gestational diabetes mellitus in a moderately iodine-deficient area. Gynecol Endocrinol. 2015;31(10):792-795.
Haddow JE, Craig WY, Neveux LM, Palomaki GE, Lambert-Messerlian G, Malone FD, D'Alton ME. Free Thyroxine During Early Pregnancy and Risk for Gestational Diabetes. PloS one. 2016;11(2):e0149065.
Yang S, Shi FT, Leung PC, Huang HF, Fan J. Low Thyroid Hormone in Early Pregnancy Is Associated With an Increased Risk of Gestational Diabetes Mellitus. J Clin Endocrinol Metab. 2016;101(11):4237-4243.
Berta E, Samson L, Lenkey A, Erdei A, Cseke B, Jenei K, Major T, Jakab A, Jenei Z, Paragh G, Nagy EV, Bodor M. Evaluation of the thyroid function of healthy pregnant women by five different hormone assays. Pharmazie.2010;65(6):436-439.
Laurberg P, Andersen SL, Hindersson P, Nohr EA, Olsen J. Dynamics and Predictors of Serum TSH and fT4 Reference Limits in Early Pregnancy: A Study Within the Danish National Birth Cohort. J Clin Endocrinol Metab.2016;101(6):2484-2492.
Price A, Obel O, Cresswell J, Catch I, Rutter S, Barik S, Heller SR, Weetman AP. Comparison of thyroid function in pregnant and non-pregnant Asian and western Caucasian women. Clin Chim Acta. 2001;308(1-2):91-98.
Dhatt GS, Jayasundaram R, Wareth LA, Nagelkerke N, Jayasundaram K, Darwish EA, Lewis A. Thyrotrophin and free thyroxine trimester-specific reference intervals in a mixed ethnic pregnant population in the United Arab Emirates. Clin Chim Acta. 2006;370(1-2):147-151.
La'ulu SL, Roberts WL. Ethnic differences in first-trimester thyroid reference intervals. Clin Chem. 2011;57(6):913-915.
Korevaar TI, Medici M, de Rijke YB, Visser W, de Muinck Keizer-Schrama SM, Jaddoe VW, Hofman A, Ross HA, Visser WE, Hooijkaas H, Steegers EA, Tiemeier H, Bongers-Schokking JJ, Visser TJ, Peeters RP. Ethnic differences in maternal thyroid parameters during pregnancy: the Generation R study. J Clin Endocrinol Metab.2013;98(9):3678-3686.
Medici M, Korevaar TI, Visser WE, Visser TJ, Peeters RP. Thyroid function in pregnancy: what is normal? Clin Chem. 2015;61(5):704-713.
Antonangeli L, Maccherini D, Cavaliere R, Di Giulio C, Reinhardt B, Pinchera A, Aghini-Lombardi F. Comparison of two different doses of iodide in the prevention of gestational goiter in marginal iodine deficiency: a longitudinal study. Eur J Endocrinol. 2002;147(1):29-34.
Moleti M, Di Bella B, Giorgianni G, Mancuso A, De Vivo A, Alibrandi A, Trimarchi F, Vermiglio F. Maternal thyroid function in different conditions of iodine nutrition in pregnant women exposed to mild-moderate iodine deficiency: an observational study. Clin Endocrinol (Oxf). 2011;74(6):762-768.
Shi X, Han C, Li C, Mao J, Wang W, Xie X, Li C, Xu B, Meng T, Du J, Zhang S, Gao Z, Zhang X, Fan C, Shan Z, Teng W. Optimal and safe upper limits of iodine intake for early pregnancy in iodine-sufficient regions: a cross-sectional study of 7190 pregnant women in China. J Clin Endocrinol Metab. 2015;100(4):1630-1638.
Männistö T, Hartikainen AL, Vääräsmäki M, Bloigu A, Surcel HM, Pouta A, Järvelin MR, Ruokonen A, Suvanto E. Smoking and early pregnancy thyroid hormone and anti-thyroid antibody levels in euthyroid mothers of the Northern Finland Birth Cohort 1986. Thyroid. 2012;22(9):944-950.
Lem AJ, de Rijke YB, van Toor H, de Ridder MA, Visser TJ, Hokken-Koelega AC. Serum thyroid hormone levels in healthy children from birth to adulthood and in short children born small for gestational age. J Clin Endocrinol Metab. 2012;97(9):3170-3178.
Chaler EA, Fiorenzano R, Chilelli C, Llinares V, Areny G, Herzovich V, Maceiras M, Lazzati JM, Mendioroz M, Rivarola MA, Belgorosky A. Age-specific thyroid hormone and thyrotropin reference intervals for a pediatric and adolescent population. Clin Chem Lab Med. 2012;50(5):885-890.
La'ulu SL, Rasmussen KJ, Straseski JA. Pediatric Reference Intervals for Free Thyroxine and Free Triiodothyronine by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry. J Clin Res Pediatr Endocrinol. 2016;8(1):26-31.
Soldin SJ, Cheng LL, Lam LY, Werner A, Le AD, Soldin OP. Comparison of FT4 with log TSH on the Abbott Architect ci8200: Pediatric reference intervals for free thyroxine and thyroid-stimulating hormone. Clin Chim Acta.2010;411(3-4):250-252.
Loh TP, Sethi SK, Metz MP. Paediatric reference interval and biological variation trends of thyrotropin (TSH) and free thyroxine (T4) in an Asian population. J Clin Pathol. 2015;68(8):642-647.
Lauffer P, Heinen CA, Goorsenberg AWM, Malekzadeh A, Henneman P, Heijboer AC, Zwaveling-Soonawala N, Boelen A, van Trotsenburg ASP. Analysis of Serum Free Thyroxine Concentrations in Healthy Term Neonates Underlines Need for Local and Laboratory-Specific Reference Interval: A Systematic Review and Meta-Analysis of Individual Participant Data. Thyroid. 2024;34(5):559-565.
Haugen BR. Drugs that suppress TSH or cause central hypothyroidism. Best practice & research Clinical endocrinology & metabolism. 2009;23(6):793-800.
Koulouri O, Moran C, Halsall D, Chatterjee K, Gurnell M. Pitfalls in the measurement and interpretation of thyroid function tests. Best practice & research Clinical endocrinology & metabolism. 2013;27(6):745-762.
Fliers E, Boelen A. An update on non-thyroidal illness syndrome. J Endocrinol Invest. 2021;44(8):1597-1607.
Hamblin PS, SA. D, Mohr VS, Le Grand B, Lim CF, Tuxen DV, Topliss DJ, Stockigt JR. Relationship between thyrotropin and thyroxine changes during recovery from severe hypothyroxinemia of critical illness. J Clin Endocrinol Metab. 1986;62:717-722.
Spencer CA, Eigen A, Shen D, Duda M, Qualls S, Weiss S, Nicoloff JT. Specificity of sensitive assays of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients. Clin Chem. 1987;33:1391-1396.
de Vries EM, Fliers E, Boelen A. The molecular basis of the non-thyroidal illness syndrome. J Endocrinol.2015;225(3):R67-81.
Moura Neto A, Zantut-Wittmann DE. Abnormalities of Thyroid Hormone Metabolism during Systemic Illness: The Low T3 Syndrome in Different Clinical Settings. Int J Endocrinol. 2016;2016:2157583.
LoPresti J, Patil K. Assessing thyroid function in hospitalized patients. New York: Springer.
Rotmensch S, Cole LA. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet. 2000;355(9205):712-715.
Ballieux BE, Weijl NI, Gelderblom H, van Pelt J, Osanto S. False-positive serum human chorionic gonadotropin (HCG) in a male patient with a malignant germ cell tumor of the testis: a case report and review of the literature. Oncologist. 2008;13(11):1149-1154.
Dimeski G. Interference testing. Clin Biochem Rev. 2008;29 Suppl 1(Suppl 1):S43-48.
Henry N, Sebe P, Cussenot O. Inappropriate treatment of prostate cancer caused by heterophilic antibody interference. Nat Clin Pract Urol. 2009;6(3):164-167.
Georges A, Charrié A, Raynaud S, Lombard C, Corcuff JB. Thyroxin overdose due to rheumatoid factor interferences in thyroid-stimulating hormone assays. Clin Chem Lab Med. 2011;49(5):873-875.
Pishdad GR, Pishdad P, Pishdad R. The effect of glucocorticoid therapy on a falsely raised thyrotropin due to heterophilic antibodies. Thyroid. 2013;23(12):1657-1658.
Favresse J, Burlacu MC, Maiter D, Gruson D. Interferences With Thyroid Function Immunoassays: Clinical Implications and Detection Algorithm. Endocr Rev. 2018;39(5):830-850.
Ghazal K, Brabant S, Prie D, Piketty ML. Hormone Immunoassay Interference: A 2021 Update. Ann Lab Med.2022;42(1):3-23.
Campi I, Dell'Acqua M, Stellaria Grassi E, Cristina Vigone M, Persani L. Unusual causes of hyperthyrotropinemia and differential diagnosis of primary hypothyroidism: a revised diagnostic flowchart. Eur Thyroid J. 2023;12(4).
Ismail Y, Ismail AA, Ismail AA. Erroneous laboratory results: what clinicians need to know. Clin Med (Lond).2007;7(4):357-361.
Sturgeon CM, Viljoen A. Analytical error and interference in immunoassay: minimizing risk. Ann Clin Biochem.2011;48(Pt 5):418-432.
Gulbahar O, Konca Degertekin C, Akturk M, Yalcin MM, Kalan I, Atikeler GF, Altinova AE, Yetkin I, Arslan M, Toruner F. A Case With Immunoassay Interferences in the Measurement of Multiple Hormones. J Clin Endocrinol Metab. 2015;100(6):2147-2153.
Massart C, Corcuff JB, Bordenave L. False-positive results corrected by the use of heterophilic antibody-blocking reagent in thyroglobulin immunoassays. Clin Chim Acta. 2008;388(1-2):211-213.
King RI, Florkowski CM. How paraproteins can affect laboratory assays: spurious results and biological effects. Pathology. 2010;42(5):397-401.
LeGatt DF, Higgins TN. Paraprotein interference in immunoassays. Ther Drug Monit. 2015;37(3):417.
Mandal K, Ashorobi D, Lee A, Liao H, Kumar SC, Rosenthal DS. Factitiously Elevated Total Triiodothyronine in a Euthyroid Patient with Multiple Myeloma. Case Rep Endocrinol. 2021;2021:8479193.
Sarkar R. A simple method to overcome paraproteinemic interferences in chemistry and immunoassays. Lab Med.2024.
Sapin R, D'Herbomez M, Schlienger JL. Free thyroxine measured with equilibrium dialysis and nine immunoassays decreases in late pregnancy. Clin Lab. 2004;50(9-10):581-584.
Anckaert E, Poppe K, Van Uytfanghe K, Schiettecatte J, Foulon W, Thienpont LM. FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID-LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations. Clin Chim Acta. 2010;411(17-18):1348-1353.
Guven S, Alver A, Mentese A, Ilhan FC, Calapoglu M, Unsal MA. The novel ischemia marker 'ischemia-modified albumin' is increased in normal pregnancies. Acta Obstet Gynecol Scand. 2009;88(4):479-482.
Cameron SJ, Hagedorn JC, Sokoll LJ, Caturegli P, Ladenson PW. Dysprealbuminemic hyperthyroxinemia in a patient with hyperthyroid graves disease. Clin Chem. 2005;51(6):1065-1069.
Sapin R, Gasser F, Schlienger JL. Familial dysalbuminemic hyperthyroxinemia and thyroid hormone autoantibodies: interference in current free thyroid hormone assays. Horm Res. 1996;45(3-5):139-141.
Kragh-Hansen U, Galliano M, Minchiotti L. Clinical, Genetic, and Protein Structural Aspects of Familial Dysalbuminemic Hyperthyroxinemia and Hypertriiodothyroninemia. Front Endocrinol (Lausanne). 2017;8:297.
Refetoff S, Scherberg NH, Yuan C, Wu W, Wu Z, McPhaul MJ. Free Thyroxine Concentrations in Sera of Individuals with Familial Dysalbuminemic Hyperthyroxinemia: A Comparison of Three Methods of Measurement. Thyroid. 2020;30(1):37-41.
DeCosimo DR, Fang SL, Braverman LE. Prevalence of familial dysalbuminemic hyperthyroxinemia in Hispanics. Ann Intern Med. 1987;107(5):780-781.
Giovanella L, Keller F, Ceriani L, Tozzoli R. Heterophile antibodies may falsely increase or decrease thyroglobulin measurement in patients with differentiated thyroid carcinoma. Clin Chem Lab Med. 2009;47(8):952-954.
Bolstad N, Warren DJ, Nustad K. Heterophilic antibody interference in immunometric assays. Best practice & research Clinical endocrinology & metabolism. 2013;27(5):647-661.
Weber TH, Käpyaho KI, Tanner P. Endogenous interference in immunoassays in clinical chemistry. A review. Scand J Clin Lab Invest Suppl. 1990;201:77-82.
Levinson SS, Miller JJ. Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. Clin Chim Acta. 2002;325(1-2):1-15.
Bjerner J, Olsen KH, Børmer OP, Nustad K. Human heterophilic antibodies display specificity for murine IgG subclasses. Clin Biochem. 2005;38(5):465-472.
Ellis MJ, Livesey JH. Techniques for identifying heterophile antibody interference are assay specific: study of seven analytes on two automated immunoassay analyzers. Clin Chem. 2005;51(3):639-641.
Sun HG, Xu XP, He LQ. Pseudo Elevation of TSH and ACTH Caused by Heterophilic Antibodies: a Case Report and Literature Review. Clin Lab. 2024;70(6).
Astarita G, Gutiérrez S, Kogovsek N, Mormandi E, Otero P, Calabrese C, Alcaraz G, Vázquez A, Abalovich M. False positive in the measurement of thyroglobulin induced by rheumatoid factor. Clin Chim Acta. 2015;447:43-46.
Mongolu S, Armston AE, Mozley E, Nasruddin A. Heterophilic antibody interference affecting multiple hormone assays: Is it due to rheumatoid factor? Scand J Clin Lab Invest. 2016;76(3):240-242.
Preissner CM, O'Kane DJ, Singh RJ, Morris JC, Grebe SKG. Phantoms in the assay tube; Heterophile antibody interferences in serum thyroglobulin assays. J Clin Endocrinol Metab. 2003;88:3069-3074.
Ghosh S, Howlett M, Boag D, Malik I, Collier A. Interference in free thyroxine immunoassay. Eur J Intern Med.2008;19(3):221-222.
Preissner CM, Dodge LA, O'Kane DJ, Singh RJ, Grebe SK. Prevalence of heterophilic antibody interference in eight automated tumor marker immunoassays. Clin Chem. 2005;51(1):208-210.
Marks V. False-positive immunoassay results: a multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries. Clin Chem. 2002;48(11):2008-2016.
Koshida S, Asanuma K, Kuribayashi K, Goto M, Tsuji N, Kobayashi D, Tanaka M, Watanabe N. Prevalence of human anti-mouse antibodies (HAMAs) in routine examinations. Clin Chim Acta. 2010;411(5-6):391-394.
Verburg FA, Wäschle K, Reiners C, Giovanella L, Lentjes EG. Heterophile antibodies rarely influence the measurement of thyroglobulin and thyroglobulin antibodies in differentiated thyroid cancer patients. Horm Metab Res. 2010;42(10):736-739.
Nakano K, Yasuda K, Shibuya H, Moriyama T, Kahata K, Shimizu C. Transient human anti-mouse antibody generated with immune enhancement in a carbohydrate antigen 19-9 immunoassay after surgical resection of recurrent cancer. Ann Clin Biochem. 2016;53(Pt 4):511-515.
Gessl A, Blueml S, Bieglmayer C, Marculescu R. Anti-ruthenium antibodies mimic macro-TSH in electrochemiluminescent immunoassay. Clin Chem Lab Med. 2014;52(11):1589-1594.
Rulander NJ, Cardamone D, Senior M, Snyder PJ, Master SR. Interference from anti-streptavidin antibody. Arch Pathol Lab Med. 2013;137(8):1141-1146.
Dahll LK, Haave EM, Dahl SR, Aas FE, Thorsby PM. Endogenous anti-streptavidin antibodies causing erroneous laboratory results more common than anticipated. Scand J Clin Lab Invest. 2021;81(2):92-103.
Vos MJ, Rondeel JMM, Mijnhout GS, Endert E. Immunoassay interference caused by heterophilic antibodies interacting with biotin. Clin Chem Lab Med. 2017;55(6):e122-e126.
Ylli D, Soldin SJ, Stolze B, Wei B, Nigussie G, Nguyen H, Mendu DR, Mete M, Wu D, Gomes-Lima CJ, Klubo-Gwiezdzinska J, Burman KD, Wartofsky L. Biotin Interference in Assays for Thyroid Hormones, Thyrotropin and Thyroglobulin. Thyroid. 2021;31(8):1160-1170.
McBride M, Dasgupta A. Technical Note: Approach to Identify and Eliminate Biotin Interference in Thyroid Function Tests Using Beckman DXI 800 Analyzer by Taking Advantage of Assay Harmonization with Alinity i Analyzer. Ann Clin Lab Sci. 2023;53(3):482-484.
McBride M, Dasgupta A. Significant Interference of Biotin in Thyroid Function Tests Using Beckman Analyzer: How to Identify such Interferences? Ann Clin Lab Sci. 2023;53(1):130-133.
Countryman B, McBride M, McCracken T, Dasgupta A. Effect of biotin on currently used Beckman thyroglobulin assay and newly reformulated thyroglobulin assay not affected by biotin. Am J Clin Pathol. 2024.
Tang M, Meng X, Ni J, Liu X, Wang X, Li Y, Chai Y, Kou C, Zhang L, Zhang H. The interference of anti-TSH autoantibody on clinical TSH detection. Front Endocrinol (Lausanne). 2024;15:1289923.
Hattori N, Ishihara T, Matsuoka N, Saito T, Shimatsu A. Anti-Thyrotropin Autoantibodies in Patients with Macro-Thyrotropin and Long-Term Changes in Macro-Thyrotropin and Serum Thyrotropin Levels. Thyroid.2017;27(2):138-146.
Chiardi I, Rotondi M, Cantù M, Keller F, Trimboli P. Macro-TSH: An Uncommon Explanation for Persistent TSH Elevation That Thyroidologists Have to Keep in Mind. J Pers Med. 2023;13(10).
Al-Bahadili H, Powers Carson J, Markov A, Jasim S. The Complex Web of Interferences with Thyroid Function Tests. Endocr Pract. 2025;31(1):92-101.
Gangemi S, Saitta S, Lombardo G, Patafi M, Benvenga S. Serum thyroid autoantibodies in patients with idiopathic either acute or chronic urticaria. J Endocrinol Invest. 2009;32(2):107-110.
Colucci R, Lotti F, Dragoni F, Arunachalam M, Lotti T, Benvenga S, Moretti S. High prevalence of circulating autoantibodies against thyroid hormones in vitiligo and correlation with clinical and historical parameters of patients. Br J Dermatol. 2014;171(4):786-798.
Hattori N, Aisaka K, Yamada A, Matsuda T, Shimatsu A. Prevalence and Pathogenesis of Macro-Thyrotropin in Neonates: Analysis of Umbilical Cord Blood from 939 Neonates and Their Mothers. Thyroid. 2023;33(1):45-52.
Newman JD, Bergman PB, Doery JC, Balazs ND. Factitious increase in thyrotropin in a neonate caused by a maternally transmitted interfering substance. Clin Chem. 2006;52(3):541-542.
Benvenga S, Ordookhani A, Pearce EN, Tonacchera M, Azizi F, Braverman LE. Detection of circulating autoantibodies against thyroid hormones in an infant with permanent congenital hypothyroidism and her twin with transient congenital hypothyroidism: possible contribution of thyroid hormone autoantibodies to neonatal and infant hypothyroidism. J Pediatr Endocrinol Metab. 2008;21(10):1011-1020.
Rix M, Laurberg P, Porzig C, Kristensen SR. Elevated thyroid-stimulating hormone level in a euthyroid neonate caused by macro thyrotropin-IgG complex. Acta Paediatr. 2011;100(9):e135-137.
Ni J, Long Y, Zhang L, Yang Q, Kou C, Li S, Li J, Zhang H. High prevalence of thyroid hormone autoantibody and low rate of thyroid hormone detection interference. J Clin Lab Anal. 2022;36(1):e24124.
Giovanella L, Dorizzi RM, Keller F. A hypothyroid patient with increased free thyroid hormones. Clin Chem Lab Med. 2008;46(11):1650-1651.
van der Watt G, Haarburger D, Berman P. Euthyroid patient with elevated serum free thyroxine. Clin Chem.2008;54(7):1239-1241.
Nicoloff J SC. Use and misuse of the sensitive thyrotropin assays. J Clin Endocrinol Metab. 1990;71:553-558.
Zhang X, Higuchi T, Tomonaga H, Lamid-Ochir O, Bhattarai A, Nguyen-Thu H, Taketomi-Takahashi A, Hirasawa H, Tsushima Y. Early detection of progressive disease using thyroglobulin doubling-time in metastatic differentiated thyroid carcinoma treated with radioactive iodine. Nucl Med Commun. 2020;41(4):350-355.
Biondi B. TSH Suppression in Differentiated Thyroid Cancer Patients. Still More Questions than Answers after 30 Years. Thyroid. 2024;34(6):671-673.
Hollowell JG, Staehling NW, Hannon WH, Flanders WD, Gunter EW, Spencer CA, Braverman LE. Serum thyrotropin, thyroxine, and thyroid antibodies in the United States population (1988 to 1994): NHANES III. J Clin Endocrinol Metab. 2002;87:489-499.
De Grande LA, Van Uytfanghe K, Thienpont LM. A Fresh Look at the Relationship between TSH and Free Thyroxine in Cross-Sectional Data. Eur Thyroid J. 2015;4(1):69-70.
Spencer CA, Nicoloff JT. Improved radioimmunoassay for human TSH. Clin Chim Acta. 1980;108:415-424.
Odell WD, Wilber JF, Paul WE. Radioimmunoassay of thyrotropin in human serum. J Clin Endocrinol Metab.1965;25(9):1179-1188.
Hershman J, Pittman J. Utility of the radioimmunoassay of serum thyrotrophin in man Ann Intern Med.1971;74:481-490.
Haigler E, Pittman J, Hershman J, Baugh C. Direct evaluation of pituitary Thyrotropin reserve utilizing synthetic Thyrotropin Releasing Hormone. J Clin Endocrinol Metab. 1971;33:573-581.
Hall R, Ormston B, Besser G, Cryer R. The Thyrotropin-Releasing Hormone test in diseases of the pituitary and hypothalamus. Lancet. 1972;1:7754-7765.
Spencer CA, Schwarzbein D, Guttler RB, LoPresti JS, Nicoloff JT. TRH stimulation test responses employing third and fourth generation TSH assays. J Clin Endocrinol Metab. 1993;76:494-498.
Miles L, CN H. Labeled antibodies and immunological assay systems. Nature. 1968;219:186-189.
Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature.1975;256(5517):495-497.
García de la Rosa I, Feal Carballo S, Almenares Guasch PR, Segura González MT, Pupo Infante M, Frómeta Suárez A, Martínez Beltrán L, Quintana Guerra JM, Lafita Delfino Y, Morejón García G, Pérez Morás PL, Hernández Pérez L, Castells Martínez EM. Generation and characterization of monoclonal antibodies against thyroid-stimulating hormone for newborn screening of congenital hypothyroidism. J Immunoassay Immunochem.2019;40(4):350-366.
Seth J, Kellett H, Caldwell G, Sweeting V, Beckett G, Gow S, Toft A. A sensitive immunoradiometric assay for serum thyroid stimulating hormone: a replacement for the thyrotropin releasing test. Br Med J. 1984;289:1334-1336.
Spencer C, Schwarzbein D, Guttler R, LoPresti J, Nicoloff J. TRH stimulation test responses employing 3rd. and 4th. generation TSH assay technology. J Clin Endocrinol Metab. 1993;76:494-499.
Spencer C, Takeuchi M, Kazarosyan M. Current status and performance goals for serum thyrotropin (TSH) assays. Clinical Chemistry. 1996;42(1):141-145.
Taimela E, R T, P K, Nuutila P FJ, S T, SL K, M V, K I. Ability of two new thyrotropin (TSH) assays to separate hyperthyroid patients from euthyroid patients with low TSH. Clin Chem. 1994;40:101-105.
Moussallieh FM, Ranaivosoa MK, Romain S, Reix N. Analytical validation of two second generation thyroglobulin immunoassays (Roche and Thermo Fisher). Clin Chem Lab Med. 2018;56(12):e302-e305.
Owen WE, Gantzer ML, Lyons JM, Rockwood AL, Roberts WL. Functional sensitivity of seven automated thyroid stimulating hormone immunoassays. Clin Chim Acta. 2011;412(23-24):2336-2339.
Giovanella L, Feldt-Rasmussen U, Verburg FA, Grebe SK, Plebani M, Clark PM. Thyroglobulin measurement by highly sensitive assays: focus on laboratory challenges. Clin Chem Lab Med. 2015;53(9):1301-1314.
Rigo RB, Panyella MG, Bartolomé LR, Ramos PA, Soria PR, Navarro MA. Variations observed for insulin concentrations in an interlaboratory quality control program may be due to interferences between reagents and the matrix of the control materials. Clin Biochem. 2007;40(13-14):1088-1091.
Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29 Suppl 1(Suppl 1):S49-52.
Spencer C, LoPresti J, Fatemi S. How sensitive (second-generation) thyroglobulin measurement is changing paradigms for monitoring patients with differentiated thyroid cancer, in the absence or presence of thyroglobulin autoantibodies. Current opinion in endocrinology, diabetes, and obesity. 2014;21(5):394-404.
Rawlins ML, Roberts WL. Performance characteristics of six third-generation assays for thyroid-stimulating hormone. Clin Chem. 2004;50(12):2338-2344.
Schaaf L, Theodoropoulou M, Gregori A, Leiprecht A, Trojan J, Klostermeier J, Stalla GK. Thyrotropin-releasing hormone time-dependently influences thyrotropin microheterogeneity--an in vivo study in euthyroidism. J Endocrinol. 2000;166(1):137-143.
Donadio S, Morelle W, Pascual A, Romi-Lebrun R, Michalski JC, Ronin C. Both core and terminal glycosylation alter epitope expression in thyrotropin and introduce discordances in hormone measurements. Clin Chem Lab Med. 2005;43(5):519-530.
Estrada JM, Soldin D, Buckey TM, Burman KD, Soldin OP. Thyrotropin isoforms: implications for thyrotropin analysis and clinical practice. Thyroid. 2014;24(3):411-423.
Persani L. Hypothalamic thyrotropin-releasing hormone and thyrotropin biological activity. Thyroid. 1998;8(10):941-946.
Ikegami K, Liao XH, Hoshino Y, Ono H, Ota W, Ito Y, Nishiwaki-Ohkawa T, Sato C, Kitajima K, Iigo M, Shigeyoshi Y, Yamada M, Murata Y, Refetoff S, Yoshimura T. Tissue-specific posttranslational modification allows functional targeting of thyrotropin. Cell Rep. 2014;9(3):801-810.
Persani L, C A, P B-P. Dissociation between immunological and buiological activities of circulating TSH. Exp Clin Endocrinol. 1994;102:38-48.
Oliveira JH, Barbosa ER, Kasamatsu T, Abucham J. Evidence for thyroid hormone as a positive regulator of serum thyrotropin bioactivity. J Clin Endocrinol Metab. 2007;92(8):3108-3113.
Yoshihara A, Noh JY, Watanabe N, Iwaku K, Kunii Y, Ohye H, Suzuki M, Matsumoto M, Suzuki N, Sugino K, Thienpont LM, Hishinuma A, Ito K. Seasonal Changes in Serum Thyrotropin Concentrations Observed from Big Data Obtained During Six Consecutive Years from 2010 to 2015 at a Single Hospital in Japan. Thyroid.2018;28(4):429-436.
Persani L, Asteria C, Tonacchera M, Vitti P, Krishna V, Chatterjee K, Beck-Peccoz P. Evidence for the secretion of thyrotropin with enhanced bioactivity in syndromes of thyroid hormone resistance. J Clin Endocrinol Metab.1994;78:1035-1039.
Persani L, Ferretti E, Borgato S, Faglia G, Beck-Peccoz P. Circulating thyrotropin bioactivity in sporadic central hypothyroidism. J Clin Endocrinol Metab. 2000;85(10):3631-3635.
Pappa T, Johannesen J, Scherberg N, Torrent M, Dumitrescu A, Refetoff S. A TSHβ Variant with Impaired Immunoreactivity but Intact Biological Activity and Its Clinical Implications. Thyroid. 2015;25(8):869-876.
Andersen S, Pedersen KM, Bruun NH, Laurberg P. Narrow individual variations in serum T4 and T3 in normal subjects: a clue to the understanding of subclinical thyroid disease. J Clin Endocrinol Metab. 2002;87:1068-1072.
Boas M, Forman JL, Juul A, Feldt-Rasmussen U, Skakkebaek NE, Hilsted L, Chellakooty M, Larsen T, Larsen JF, Petersen JH, Main KM. Narrow intra-individual variation of maternal thyroid function in pregnancy based on a longitudinal study on 132 women. Eur J Endocrinol. 2009;161(6):903-910.
Meikle AW, Stringham JD, Woodward MG, Nelson JC. Hereditary and environmental influences on the variation of thyroid hormones in normal male twins. J Clin Endocrinol Metab1. 1988;66:588-592.
Hansen PS, Brix TH, Sørensen TI, Kyvik KO, Hegedüs L. Major genetic influence on the regulation of the pituitary-thyroid axis: a study of healthy Danish twins. J Clin Endocrinol Metab. 2004;89(3):1181-1187.
Panicker V, Wilson SG, Spector TD, Brown SJ, Falchi M, Richards JB, Surdulescu GL, Lim EM, Fletcher SJ, Walsh JP. Heritability of serum TSH, free T4 and free T3 concentrations: a study of a large UK twin cohort. Clin Endocrinol (Oxf). 2008;68(4):652-659.
Arnaud-Lopez L, Usala G, Ceresini G, Mitchell BD, Pilia MG, Piras MG, Sestu N, Maschio A, Busonero F, Albai G, Dei M, Lai S, Mulas A, Crisponi L, Tanaka T, Bandinelli S, Guralnik JM, Loi A, Balaci L, Sole G, Prinzis A, Mariotti S, Shuldiner AR, Cao A, Schlessinger D, Uda M, Abecasis GR, Nagaraja R, Sanna S, Naitza S. Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function. American journal of human genetics. 2008;82(6):1270-1280.
Larsen CC, Karaviti LP, Seghers V, Weiss RE, Refetoff S, Dumitrescu AM. A new family with an activating mutation (G431S) in the TSH receptor gene: a phenotype discussion and review of the literature. Int J Pediatr Endocrinol. 2014;2014(1):23.
De Marco G, Agretti P, Camilot M, Teofoli F, Tatò L, Vitti P, Pinchera A, Tonacchera M. Functional studies of new TSH receptor (TSHr) mutations identified in patients affected by hypothyroidism or isolated hyperthyrotrophinaemia. Clin Endocrinol (Oxf). 2009;70(2):335-338.
Alberti L, Proverbio MC, Costagliola S, Romoli R, Boldrighini B, Vigone MC, Weber G, Chiumello G, Beck-Peccoz P, Persani L. Germline mutations of TSH receptor gene as cause of nonautoimmune subclinical hypothyroidism. J Clin Endocrinol Metab. 2002;87(6):2549-2555.
van de Ven AC, Netea-Maier RT, Medici M, Sweep FC, Ross HA, Hofman A, de Graaf J, Kiemeney LA, Hermus AR, Peeters RP, Visser TJ, den Heijer M. Underestimation of effect of thyroid function parameters on morbidity and mortality due to intra-individual variation. J Clin Endocrinol Metab. 2011;96(12):E2014-2017.
Jonklaas J. TSH Reference Intervals: Their Importance and Complexity. Thyroid. 2024;34(8):957-959.
Kuś A, Sterenborg R, Haug EB, Galesloot TE, Visser WE, Smit JWA, Bednarczuk T, Peeters RP, Åsvold BO, Teumer A, Medici M. Towards Personalized TSH Reference Ranges: A Genetic and Population-Based Approach in Three Independent Cohorts. Thyroid. 2024;34(8):969-979.
Biondi B, Cooper DS. The clinical significance of subclinical thyroid dysfunction. Endocr Rev. 2008;29(1):76-131.
Coene KL, Demir AY, Broeren MA, Verschuure P, Lentjes EG, Boer AK. Subclinical hypothyroidism: a 'laboratory-induced' condition? Eur J Endocrinol. 2015;173(4):499-505.
Stöckl D, Van Uytfanghe K, Van Aelst S, Thienpont LM. A statistical basis for harmonization of thyroid stimulating hormone immunoassays using a robust factor analysis model. Clin Chem Lab Med. 2014;52(7):965-972.
Strich D, Karavani G, Levin S, Edri S, Gillis D. Normal limits for serum thyrotropin vary greatly depending on method. Clin Endocrinol (Oxf). 2016;85(1):110-115.
Solberg HE. The IFCC recommendation on estimation of reference intervals. The RefVal program. Clin Chem Lab Med. 2004;42(7):710-714.
Kahapola-Arachchige KM, Hadlow N, Wardrop R, Lim EM, Walsh JP. Age-specific TSH reference ranges have minimal impact on the diagnosis of thyroid dysfunction. Clin Endocrinol (Oxf). 2012;77(5):773-779.
Fan L, Bu Y, Chen S, Wang S, Zhang W, He Y, Sun D. Iodine nutritional status and its associations with thyroid function of pregnant women and neonatal TSH. Front Endocrinol (Lausanne). 2024;15:1394306.
Jensen E, Hyltoft Petersen P, Blaabjerg O, Hansen PS, Brix TH, Kyvik KO, Hegedüs L. Establishment of a serum thyroid stimulating hormone (TSH) reference interval in healthy adults. The importance of environmental factors, including thyroid antibodies. Clin Chem Lab Med. 2004;42(7):824-832.
Spencer CA, Hollowell JG, Kazarosyan M, Braverman LE. National Health and Nutrition Examination Survey III thyroid-stimulating hormone (TSH)-thyroperoxidase antibody relationships demonstrate that TSH upper reference limits may be skewed by occult thyroid dysfunction. J Clin Endocrinol Metab. 2007;92(11):4236-4240.
Surks MI, Hollowell JG. Age-specific distribution of serum thyrotropin and antithyroid antibodies in the US population: implications for the prevalence of subclinical hypothyroidism. J Clin Endocrinol Metab.2007;92(12):4575-4582.
Surks MI, Boucai L. Age- and race-based serum thyrotropin reference limits. J Clin Endocrinol Metab.2010;95(2):496-502.
Sletner L, Jenum AK, Qvigstad E, Hammerstad SS. Thyroid Function During Pregnancy in A Multiethnic Population in Norway. J Endocr Soc. 2021;5(7):bvab078.
Vejbjerg P, Knudsen N, Perrild H, Carlé A, Laurberg P, Pedersen IB, Rasmussen LB, Ovesen L, Jørgensen T. The impact of smoking on thyroid volume and function in relation to a shift towards iodine sufficiency. Eur J Epidemiol.2008;23(6):423-429.
Ittermann T, Khattak RM, Nauck M, Cordova CM, Völzke H. Shift of the TSH reference range with improved iodine supply in Northeast Germany. Eur J Endocrinol. 2015;172(3):261-267.
Nyrnes A, Jorde R, Sundsfjord J. Serum TSH is positively associated with BMI. Int J Obes (Lond). 2006;30(1):100-105.
Duntas LH, Biondi B. The interconnections between obesity, thyroid function, and autoimmunity: the multifold role of leptin. Thyroid. 2013;23(6):646-653.
Soldin OP, Goughenour BE, Gilbert SZ, Landy HJ, Soldin SJ. Thyroid hormone levels associated with active and passive cigarette smoking. Thyroid. 2009;19(8):817-823.
Raverot V, Bonjour M, Abeillon du Payrat J, Perrin P, Roucher-Boulez F, Lasolle H, Subtil F, Borson-Chazot F. Age- and Sex-Specific TSH Upper-Limit Reference Intervals in the General French Population: There Is a Need to Adjust Our Actual Practices. J Clin Med. 2020;9(3).
Meisinger C, Ittermann T, Wallaschofski H, Heier M, Below H, Kramer A, Döring A, Nauck M, Völzke H. Geographic variations in the frequency of thyroid disorders and thyroid peroxidase antibodies in persons without former thyroid disease within Germany. Eur J Endocrinol. 2012;167(3):363-371.
Berghout A, Wiersinga WM, Smits NJ, touber JL. Interrelationships between age, thyroid volume, thyroid nodularity, and thyroid function in patients with sporadic nontoxic goiter. Amer J Med. 1990;89:602-608.
Atzmon G, Barzilai N, Hollowell JG, Surks MI, Gabriely I. Extreme longevity is associated with increased serum thyrotropin. J Clin Endocrinol Metab. 2009;94(4):1251-1254.
Akirov A, Gimbel H, Grossman A, Shochat T, Shimon I. Elevated TSH in adults treated for hypothyroidism is associated with increased mortality. Eur J Endocrinol. 2017;176(1):57-66.
Somwaru LL, Rariy CM, Arnold AM, Cappola AR. The natural history of subclinical hypothyroidism in the elderly: the cardiovascular health study. J Clin Endocrinol Metab. 2012;97(6):1962-1969.
Kapelari K, Kirchlechner C, Högler W, Schweitzer K, Virgolini I, Moncayo R. Pediatric reference intervals for thyroid hormone levels from birth to adulthood: a retrospective study. BMC Endocr Disord. 2008;8:15.
Verburg FA, Kirchgässner C, Hebestreit H, Steigerwald U, Lentjes EG, Ergezinger K, Grelle I, Reiners C, Luster M. Reference ranges for analytes of thyroid function in children. Horm Metab Res. 2011;43(6):422-426.
Goichot B, Sapin R, Schlienger JL. Subclinical hyperthyroidism: considerations in defining the lower limit of the thyrotropin reference interval. Clin Chem. 2009;55(3):420-424.
Biondi B, Cooper DS. Subclinical Hyperthyroidism. N Engl J Med. 2018;379(15):1485-1486.
Kim HJ, McLeod DSA. Subclinical Hyperthyroidism and Cardiovascular Disease. Thyroid. 2024.
Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med. 2000;160:526-534.
Surks MI, Ortiz E, Daniels GH, Sawin CT, Col NF, Cobin RH, Franklyn JA, Hershman JM, Burman KD, Denke MA, Gorman C, Cooper RS, Weissman NJ. Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. Jama. 2004;291(2):228-238.
Efthymiadis A, Henry M, Spinos D, Bourlaki M, Tsikopoulos A, Bourazana A, Bastounis A, Tsikopoulos K. Adequacy of thyroid hormone replacement for people with hypothyroidism in real-world settings: A systematic review and meta-analysis of observational studies. Clin Endocrinol (Oxf). 2024;100(5):488-501.
Yu OHY, Filliter C, Filion KB, Platt RW, Grad R, Renoux C. Levothyroxine Treatment of Subclinical Hypothyroidism and the Risk of Adverse Cardiovascular Events. Thyroid. 2024;34(10):1214-1224.
Blum MR, Bauer DC, Collet TH, Fink HA, Cappola AR, da Costa BR, Wirth CD, Peeters RP, Åsvold BO, den Elzen WP, Luben RN, Imaizumi M, Bremner AP, Gogakos A, Eastell R, Kearney PM, Strotmeyer ES, Wallace ER, Hoff M, Ceresini G, Rivadeneira F, Uitterlinden AG, Stott DJ, Westendorp RG, Khaw KT, Langhammer A, Ferrucci L, Gussekloo J, Williams GR, Walsh JP, Jüni P, Aujesky D, Rodondi N. Subclinical thyroid dysfunction and fracture risk: a meta-analysis. Jama. 2015;313(20):2055-2065.
Collet TH, Gussekloo J, Bauer DC, den Elzen WP, Cappola AR, Balmer P, Iervasi G, Åsvold BO, Sgarbi JA, Völzke H, Gencer B, Maciel RM, Molinaro S, Bremner A, Luben RN, Maisonneuve P, Cornuz J, Newman AB, Khaw KT, Westendorp RG, Franklyn JA, Vittinghoff E, Walsh JP, Rodondi N. Subclinical hyperthyroidism and the risk of coronary heart disease and mortality. Arch Intern Med. 2012;172(10):799-809.
Taylor PN, Razvi S, Pearce SH, Dayan CM. Clinical review: A review of the clinical consequences of variation in thyroid function within the reference range. J Clin Endocrinol Metab. 2013;98(9):3562-3571.
Jay M, Huan P, Cliffe N, Rakoff J, Morris E, Kavsak P, Luthra M, Punthakee Z. Treatment of Subclinical Hyperthyroidism and Incident Atrial Fibrillation. Clin Endocrinol (Oxf). 2024.
Zhang X, Zhang G, Wang S, Jin J, Zhang S, Teng X. The change in thyroid function categories with time in patients with subclinical hypothyroidism: a systematic review and meta-analysis. BMC Endocr Disord.2024;24(1):224.
McQuade C, Skugor M, Brennan DM, Hoar B, Stevenson C, Hoogwerf BJ. Hypothyroidism and moderate subclinical hypothyroidism are associated with increased all-cause mortality independent of coronary heart disease risk factors: a PreCIS database study. Thyroid. 2011;21(8):837-843.
Javed Z, Sathyapalan T. Levothyroxine treatment of mild subclinical hypothyroidism: a review of potential risks and benefits. Ther Adv Endocrinol Metab. 2016;7(1):12-23.
Helfand M. Screening for subclinical thyroid dysfunction in nonpregnant adults: a summary of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med. 2004;140(2):128-141.
Åsvold BO, Vatten LJ, Midthjell K, Bjøro T. Serum TSH within the reference range as a predictor of future hypothyroidism and hyperthyroidism: 11-year follow-up of the HUNT Study in Norway. J Clin Endocrinol Metab.2012;97(1):93-99.
Åsvold BO, Vatten LJ, Bjøro T, Bauer DC, Bremner A, Cappola AR, Ceresini G, den Elzen WP, Ferrucci L, Franco OH, Franklyn JA, Gussekloo J, Iervasi G, Imaizumi M, Kearney PM, Khaw KT, Maciel RM, Newman AB, Peeters RP, Psaty BM, Razvi S, Sgarbi JA, Stott DJ, Trompet S, Vanderpump MP, Völzke H, Walsh JP, Westendorp RG, Rodondi N. Thyroid function within the normal range and risk of coronary heart disease: an individual participant data analysis of 14 cohorts. JAMA Intern Med. 2015;175(6):1037-1047.
Asvold BO, Bjøro T, Vatten LJ. Association of thyroid function with estimated glomerular filtration rate in a population-based study: the HUNT study. Eur J Endocrinol. 2011;164(1):101-105.
Cappola AR, Ladenson PW. Hypothyroidism and atherosclerosis. J Clin Endocrinol Metab. 2003;88(6):2438-2444.
Ochs N, Auer R, Bauer DC, Nanchen D, Gussekloo J, Cornuz J, Rodondi N. Meta-analysis: subclinical thyroid dysfunction and the risk for coronary heart disease and mortality. Ann Intern Med. 2008;148(11):832-845.
Ittermann T, Lorbeer R, Dörr M, Schneider T, Quadrat A, Heßelbarth L, Wenzel M, Lehmphul I, Köhrle J, Mensel B, Völzke H. High levels of thyroid-stimulating hormone are associated with aortic wall thickness in the general population. Eur Radiol. 2016;26(12):4490-4496.
Andersen MN, Olsen AS, Madsen JC, Kristensen SL, Faber J, Torp-Pedersen C, Gislason GH, Selmer C. Long-Term Outcome in Levothyroxine Treated Patients With Subclinical Hypothyroidism and Concomitant Heart Disease. J Clin Endocrinol Metab. 2016;101(11):4170-4177.
Baretella O, Blum MR, Abolhassani N, Alwan H, Wildisen L, Del Giovane C, Tal K, Moutzouri E, Åsvold BO, Cappola AR, Gussekloo J, Iacoviello M, Iervasi G, Imaizumi M, Weiler S, Razvi S, Sgarbi JA, Völzke H, Brown SJ, Walsh JP, Vaes B, Yeap BB, Dullaart RPF, Bakker SJL, Kavousi M, Ceresini G, Ferrucci L, Aujesky D, Peeters RP, Bauer DC, Feller M, Rodondi N. Associations between subclinical thyroid dysfunction and cardiovascular risk factors according to age and sex. J Clin Endocrinol Metab. 2024.
Iqbal A, Jorde R, Figenschau Y. Serum lipid levels in relation to serum thyroid-stimulating hormone and the effect of thyroxine treatment on serum lipid levels in subjects with subclinical hypothyroidism: the Tromsø Study. J Intern Med. 2006;260(1):53-61.
Asvold BO, Bjøro T, Vatten LJ. Associations of TSH levels within the reference range with future blood pressure and lipid concentrations: 11-year follow-up of the HUNT study. Eur J Endocrinol. 2013;169(1):73-82.
Casey BM, Dashe JS, Wells CE, McIntire DD, Leveno KJ, Cunningham FG. Subclinical hyperthyroidism and pregnancy outcomes. Obstet Gynecol. 2006;107(2 Pt 1):337-341.
Ajmani SN, Aggarwal D, Bhatia P, Sharma M, Sarabhai V, Paul M. Prevalence of overt and subclinical thyroid dysfunction among pregnant women and its effect on maternal and fetal outcome. J Obstet Gynaecol India.2014;64(2):105-110.
Debbarma R, Gothwal M, Singh P, Yadav G, Purohit P, Ghuman NK, Gupta N. The Spectrum of Thyroid Dysfunction During Pregnancy and Fetomaternal Outcome, A Study from the Premier Institute of Western India. Indian J Community Med. 2024;49(5):734-738.
Zaccarelli-Marino MA, Dsouki NA, de Carvalho RP, Maciel RMB. Evaluation of Anti-Thyroperoxidase (A-TPO) and Anti-Thyroglobulin (A-Tg) Antibodies in Women with Previous Hashimoto's Thyroiditis during and after Pregnancy. J Clin Med. 2024;13(15).
Tong Z, Xiaowen Z, Baomin C, Aihua L, Yingying Z, Weiping T, Zhongyan S. The Effect of Subclinical Maternal Thyroid Dysfunction and Autoimmunity on Intrauterine Growth Restriction: A Systematic Review and Meta-Analysis. Medicine (Baltimore). 2016;95(19):e3677.
Iijima S. Pitfalls in the assessment of gestational transient thyrotoxicosis. Gynecol Endocrinol. 2020;36(8):662-667.
Brabant G, Peeters RP, Chan SY, Bernal J, Bouchard P, Salvatore D, Boelaert K, Laurberg P. Management of subclinical hypothyroidism in pregnancy: are we too simplistic? Eur J Endocrinol. 2015;173(1):P1-p11.
Negro R, Stagnaro-Green A. Diagnosis and management of subclinical hypothyroidism in pregnancy. Bmj.2014;349:g4929.
Negro R. Thyroid autoimmunity and pre-term delivery: brief review and meta-analysis. J Endocrinol Invest.2011;34(2):155-158.
Negro R, Schwartz A, Stagnaro-Green A. Impact of Levothyroxine in Miscarriage and Preterm Delivery Rates in First Trimester Thyroid Antibody-Positive Women With TSH Less Than 2.5 mIU/L. J Clin Endocrinol Metab.2016;101(10):3685-3690.
Chen X, Jin B, Xia J, Tao X, Huang X, Sun L, Yuan Q. Effects of Thyroid Peroxidase Antibody on Maternal and Neonatal Outcomes in Pregnant Women in an Iodine-Sufficient Area in China. Int J Endocrinol.2016;2016:6461380.
Dhillon-Smith RK, Tobias A, Smith PP, Middleton LJ, Sunner KK, Baker K, Farrell-Carver S, Bender-Atik R, Agrawal R, Bhatia K, Chu JJ, Edi-Osagie E, Ewies A, Ghobara T, Gupta P, Jurkovic D, Khalaf Y, Mulbagal K, Nunes N, Overton C, Quenby S, Rai R, Raine-Fenning N, Robinson L, Ross J, Sizer A, Small R, Underwood M, Kilby MD, Daniels J, Thangaratinam S, Chan S, Boelaert K, Coomarasamy A. The Prevalence of Thyroid Dysfunction and Autoimmunity in Women With History of Miscarriage or Subfertility. J Clin Endocrinol Metab.2020;105(8).
Yu M, Long Y, Wang Y, Zhang R, Tao L. Effect of levothyroxine on the pregnancy outcomes in recurrent pregnancy loss women with subclinical hypothyroidism and thyroperoxidase antibody positivity: a systematic review and meta-analysis. J Matern Fetal Neonatal Med. 2023;36(2):2233039.
Maraka S, Dosiou C. Subclinical Hypothyroidism and Thyroid Autoimmunity in Pregnancy: To Treat or Not to Treat. Endocrinol Metab Clin North Am. 2024;53(3):363-376.
Wilson KL, Casey BM, McIntire DD, Halvorson LM, Cunningham FG. Subclinical thyroid disease and the incidence of hypertension in pregnancy. Obstet Gynecol. 2012;119(2 Pt 1):315-320.
Alavi A, Adabi K, Nekuie S, Jahromi EK, Solati M, Sobhani A, Karmostaji H, Jahanlou AS. Thyroid dysfunction and autoantibodies association with hypertensive disorders during pregnancy. J Pregnancy. 2012;2012:742695.
Nazarpour S, Ramezani Tehrani F, Rahmati M, Azizi F. Prediction of preterm delivery based on thyroid peroxidase antibody levels and other identified risk factors. Eur J Obstet Gynecol Reprod Biol. 2023;284:125-130.
Liu X, Zhang C, Lin Z, Zhu K, He R, Jiang Z, Wu H, Yu J, Luo Q, Sheng J, Fan J, Pan J, Huang H. Association of maternal mild hypothyroidism in the first and third trimesters with obstetric and perinatal outcomes: a prospective cohort study. Am J Obstet Gynecol. 2024.
Haddow JE, Palomaki GE, Allan WC, Williams JR KG, Gagnon J, O'Heir CE, Mitchell ML, Hermos RJ WS, Faix JD, Klein RZ. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. NEJM. 1999;341:549-555.
Li Y, Shan Z, Teng W, Yu X, Li Y, Fan C, Teng X, Guo R, Wang H, Li J, Chen Y, Wang W, Chawinga M, Zhang L, Yang L, Zhao Y, Hua T. Abnormalities of maternal thyroid function during pregnancy affect neuropsychological development of their children at 25-30 months. Clin Endocrinol (Oxf). 2010;72(6):825-829.
Nazarpour S, Ramezani Tehrani F, Simbar M, Tohidi M, Alavi Majd H, Azizi F. Effects of levothyroxine treatment on pregnancy outcomes in pregnant women with autoimmune thyroid disease. Eur J Endocrinol. 2017;176(2):253-265.
Pekonen F, Alfthan H, Stenman UH, Ylikorkala O. Human chorionic gonadotropin (hCG) and thyroid function in early human pregnancy: circadian variation and evidence for intrinsic thyrotropic activity of hCG. J Clin Endocrinol Metab. 1988;66(4):853-856.
Goodwin TM, Montoro M, Mestman JH, Pekary AE, Hershman JM. The role of chorionic gonadotropin in transient hyperthyroidism of hyperemesis gravidarum. J Clin Endocrinol Metab. 1992;75(5):1333-1337.
Korevaar TI, Steegers EA, de Rijke YB, Visser WE, Jaddoe VW, Visser TJ, Medici M, Peeters RP. Placental Angiogenic Factors Are Associated With Maternal Thyroid Function and Modify hCG-Mediated FT4 Stimulation. J Clin Endocrinol Metab. 2015;100(10):E1328-1334.
Grün JP, Meuris S, De Nayer P, Glinoer D. The thyrotrophic role of human chorionic gonadotrophin (hCG) in the early stages of twin (versus single) pregnancies. Clin Endocrinol (Oxf). 1997;46(6):719-725.
Barjaktarovic M, Korevaar TIM, Jaddoe VWV, de Rijke YB, Peeters RP, Steegers EAP. Human chorionic gonadotropin and risk of pre-eclampsia: prospective population-based cohort study. Ultrasound Obstet Gynecol.2019;54(4):477-483.
Männistö T, Surcel HM, Ruokonen A, Vääräsmäki M, Pouta A, Bloigu A, Järvelin MR, Hartikainen AL, Suvanto E. Early pregnancy reference intervals of thyroid hormone concentrations in a thyroid antibody-negative pregnant population. Thyroid. 2011;21(3):291-298.
McNeil AR, Stanford PE. Reporting Thyroid Function Tests in Pregnancy. Clin Biochem Rev. 2015;36(4):109-126.
Tozzoli R, D'Aurizio F, Ferrari A, Castello R, Metus P, Caruso B, Perosa AR, Sirianni F, Stenner E, Steffan A, Villalta D. The upper reference limit for thyroid peroxidase autoantibodies is method-dependent: A collaborative study with biomedical industries. Clin Chim Acta. 2016;452:61-65.
Caruso B, Bovo C, Guidi GC. Causes of Preanalytical Interferences on Laboratory Immunoassays - A Critical Review. Ejifcc. 2020;31(1):70-84.
Roelfsema F, Pijl H, Kok P, Endert E, Fliers E, Biermasz NR, Pereira AM, Veldhuis JD. Thyrotropin secretion in healthy subjects is robust and independent of age and gender, and only weakly dependent on body mass index. J Clin Endocrinol Metab. 2014;99(2):570-578.
Roelfsema F, Boelen A, Kalsbeek A, Fliers E. Regulatory aspects of the human hypothalamus-pituitary-thyroid axis. Best practice & research Clinical endocrinology & metabolism. 2017;31(5):487-503.
van der Spoel E, Roelfsema F, van Heemst D. Within-Person Variation in Serum Thyrotropin Concentrations: Main Sources, Potential Underlying Biological Mechanisms, and Clinical Implications. Front Endocrinol (Lausanne).2021;12:619568.
Beckett G, MacKenzie F. Thyroid guidelines - are thyroid-stimulating hormone assays fit for purpose? Ann Clin Biochem. 2007;44(Pt 3):203-208.
Roelfsema F, Pereira AM, Veldhuis JD, Adriaanse R, Endert E, Fliers E, Romijn JA. Thyrotropin secretion profiles are not different in men and women. J Clin Endocrinol Metab. 2009;94(10):3964-3967.
Walsh JP, Ward LC, Burke V, Bhagat CI, Shiels L, Henley D, Gillett MJ, Gilbert R, Tanner M, Stuckey BG. Small changes in thyroxine dosage do not produce measurable changes in hypothyroid symptoms, well-being, or quality of life: results of a double-blind, randomized clinical trial. J Clin Endocrinol Metab. 2006;91(7):2624-2630.
Jonklaas J, Sarlis NJ, Litofsky D, Ain KB, Bigos ST, Brierley JD, Cooper DS, Haugen BR, Ladenson PW, Magner J, Robbins J, Ross DS, Skarulis M, Maxon HR, Sherman SI. Outcomes of patients with differentiated thyroid carcinoma following initial therapy. Thyroid. 2006;16(12):1229-1242.
Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, Mazzaferri EL, McIver B, Pacini F, Schlumberger M, Sherman SI, Steward DL, Tuttle RM. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009;19(11):1167-1214.
Mebis L, van den Berghe G. The hypothalamus-pituitary-thyroid axis in critical illness. Neth J Med.2009;67(10):332-340.
Van den Berghe G. Non-thyroidal illness in the ICU: a syndrome with different faces. Thyroid. 2014;24(10):1456-1465.
Aidoo ED, Ababio GK, Arko-Boham B, Tagoe EA, Aryee NA. Thyroid dysfunction among patients assessed by thyroid function tests at a tertiary care hospital: a retrospective study. Pan Afr Med J. 2024;49:7.
Stockigt JR. Guidelines for diagnosis and monitoring of thyroid disease: nonthyroidal illness. Clin Chem.1996;42:188-192.
Stathatos N, Levetan C, Burman KD, Wartofsky L. The controversy of the treatment of critically ill patients with thyroid hormone. Best practice & research Clinical endocrinology & metabolism. 2001;15(4):465-478.
Bunevicius R, Steibliene V, Prange AJ, Jr. Thyroid axis function after in-patient treatment of acute psychosis with antipsychotics: a naturalistic study. BMC Psychiatry. 2014;14:279.
Soh SB, Aw TC. Laboratory Testing in Thyroid Conditions - Pitfalls and Clinical Utility. Ann Lab Med. 2019;39(1):3-14.
Uy HL, Reasner CA, Samuels MH. Pattern of recovery of the hypothalamic-pituitary-thyroid axis following radioactive iodine therapy in patients with Graves' disease. Am J Med. 1995;99(2):173-179.
Persani L. Clinical review: Central hypothyroidism: pathogenic, diagnostic, and therapeutic challenges. J Clin Endocrinol Metab. 2012;97:3068-3078.
Lania A, Persani L, Beck-Peccoz P. Central hypothyroidism. Pituitary. 2008;11(2):181-186.
Roelfsema F, Kok S, Kok P, Pereira AM, Biermasz NR, Smit JW, Frolich M, Keenan DM, Veldhuis JD, Romijn JA. Pituitary-hormone secretion by thyrotropinomas. Pituitary. 2009;12(3):200-210.
Beck-Peccoz P, Giavoli C, Lania A. A 2019 update on TSH-secreting pituitary adenomas. J Endocrinol Invest.2019;42(12):1401-1406.
Refetoff S, Weiss RE, Usala SJ. The syndromes of resistance to thyroid hormone. Endocrine Rev. 1993;14:348-399.
Persani L, Rodien P, Moran C, Edward Visser W, Groeneweg S, Peeters R, Refetoff S, Gurnell M, Beck-Peccoz P, Chatterjee K. 2024 European Thyroid Association Guidelines on diagnosis and management of genetic disorders of thyroid hormone transport, metabolism and action. Eur Thyroid J. 2024;13(4).
Dumitrescu AM, Refetoff S. The syndromes of reduced sensitivity to thyroid hormone. Biochim Biophys Acta.2013;1830(7):3987-4003.
Bertalan R, Sallai A, Sólyom J, Lotz G, Szabó I, Kovács B, Szabó E, Patócs A, Rácz K. Hyperthyroidism caused by a germline activating mutation of the thyrotropin receptor gene: difficulties in diagnosis and therapy. Thyroid.2010;20(3):327-332.
Drees JC, Stone JA, Reamer CR, Arboleda VE, Huang K, Hrynkow J, Greene DN, Petrie MS, Hoke C, Lorey TS, Dlott RS. Falsely undetectable TSH in a cohort of South Asian euthyroid patients. J Clin Endocrinol Metab.2014;99(4):1171-1179.
Kalveram L, Kleinau G, Szymańska K, Scheerer P, Rivero-Müller A, Grüters-Kieslich A, Biebermann H. The Pathogenic TSH β-subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR. Int J Mol Sci.2019;20(22).
Emerson JF, Ngo G, Emerson SS. Screening for interference in immunoassays. Clin Chem. 2003;49:1163-1169.
Nayeemuddin SN, Panigrahi A, Bhattacharjee R, Chowdhury S. Heterophilic Interference of Rheumatoid Factor in TSH Immunometric Assay: A Cross-Sectional Observational Study. Indian J Endocrinol Metab. 2024;28(1):29-34.
Czernichow P, Vandalem JL, Hennen G. Transient neonatal hyperthyrotropinemia: a factitious syndrome due to the presence of heterophilic antibodies in the plasma of infants and their mothers. J Clin Endocrinol Metab.1981;53(2):387-393.
Favresse J, Paridaens H, Pirson N, Maiter D, Gruson D. Massive interference in free T4 and free T3 assays misleading clinical judgment. Clin Chem Lab Med. 2017;55(4):e84-e86.
Ohba K, Noh JY, Unno T, Satoh T, Iwahara K, Matsushita A, Sasaki S, Oki Y, Nakamura H. Falsely elevated thyroid hormone levels caused by anti-ruthenium interference in the Elecsys assay resembling the syndrome of inappropriate secretion of thyrotropin. Endocr J. 2012;59(8):663-667.
Takahashi S, Nishikawa M, Nishihara E, Deguchi H, Kohsaka K, Yamaoka H, Hisakado M, Fukata S, Ito M, Miyauchi A, Akamizu T. Interference against a newly labeled substance with ruthenium sulfonate complexes showing discrepant thyroid function test results. Clin Chim Acta. 2024;553:117706.
Elston MS, Sehgal S, Du Toit S, Yarndley T, Conaglen JV. Factitious Graves' Disease Due to Biotin Immunoassay Interference-A Case and Review of the Literature. J Clin Endocrinol Metab. 2016;101(9):3251-3255.
Balzer AHA, Whitehurst CB. An Analysis of the Biotin-(Strept)avidin System in Immunoassays: Interference and Mitigation Strategies. Curr Issues Mol Biol. 2023;45(11):8733-8754.
Lazarus JH, John R, Ginsberg J, Hughes IA, Shewring G, Smith BR, Woodhead JS, Hall R. Transient neonatal hyperthyrotrophinaemia: a serum abnormality due to transplacentally acquired antibody to thyroid stimulating hormone. Br Med J (Clin Res Ed). 1983;286(6365):592-594.
Sazonova DV, Perepelova MA, Shutova AS, Nikankina LV, Kolesnikova GS, Pigarova EA, Dzeranova LK. [Combination of macro-TSH and macroprolactinemia phenomena in a patient with autoimmune thyroiditis and vitiligo]. Probl Endokrinol (Mosk). 2024;70(5):34-39.
Hattori N, Ishihara T, Shimatsu A. Variability in the detection of macro TSH in different immunoassay systems. Eur J Endocrinol. 2016;174(1):9-15.
Verhoye E, Van den Bruel A, Delanghe JR, Debruyne E, Langlois MR. Spuriously high thyrotropin values due to anti-thyrotropin antibodies in adult patients. Clin Chem Lab Med. 2009;47(5):604-606.
Piticchio T, Chiardi I, Tumminia A, Frasca F, Rotondi M, Trimboli P. PEG Precipitation to Detect Macro-TSH in Clinical Practice: A Systematic Review. Clin Endocrinol (Oxf). 2024.
Grünert SC, Schmidts M, Pohlenz J, Kopp MV, Uhl M, Schwab KO. Congenital Central Hypothyroidism due to a Homozygous Mutation in the TSHβ Subunit Gene. Case Rep Pediatr. 2011;2011:369871.
Yeste D, Baz-Redón N, Antolín M, Garcia-Arumí E, Mogas E, Campos-Martorell A, González-Llorens N, Aguilar-Riera C, Soler-Colomer L, Clemente M, Fernández-Cancio M, Camats-Tarruella N. Genetic and Functional Studies of Patients with Thyroid Dyshormonogenesis and Defects in the TSH Receptor (TSHR). Int J Mol Sci. 2024;25(18).
Feldt-Rasmussen U. Analytical and clinical performance goals for thyroid testing: thyroid antibodies. Clin Chem.1996;46:In press.
Saravanan P, Dayan CM. Thyroid autoantibodies. Endocrinol Metab Clin North Am. 2001;30(2):315-337, viii.
McLachlan SM, Rapoport B. Discoveries in Thyroid Autoimmunity in the Past Century. Thyroid. 2023;33(3):278-286.
Massart C, Sapin R, Gibassier J, Agin A, d'Herbomez M. Intermethod variability in TSH-receptor antibody measurement: implication for the diagnosis of Graves disease and for the follow-up of Graves ophthalmopathy. Clin Chem. 2009;55(1):183-186.
D'Aurizio F, Metus P, Ferrari A, Caruso B, Castello R, Villalta D, Steffan A, Gaspardo K, Pesente F, Bizzaro N, Tonutti E, Valverde S, Cosma C, Plebani M, Tozzoli R. Definition of the upper reference limit for thyroglobulin antibodies according to the National Academy of Clinical Biochemistry guidelines: comparison of eleven different automated methods. Auto Immun Highlights. 2017;8(1):8.
Tozzoli R, Bagnasco M, Giavarina D, Bizzaro N. TSH receptor autoantibody immunoassay in patients with Graves' disease: improvement of diagnostic accuracy over different generations of methods. Systematic review and meta-analysis. Autoimmun Rev. 2012;12(2):107-113.
Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM. The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev. 1998;19(6):673-716.
Davies T, Marians R, Latif R. The TSH receptor reveals itself. J Clin Invest. 2002;110(2):161-164.
Barbesino G, Tomer Y. Clinical review: Clinical utility of TSH receptor antibodies. J Clin Endocrinol Metab.2013;98(6):2247-2255.
Rees Smith B, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev.1988;9(1):106-121.
Adams DD. Long-acting thyroid stimulator: how receptor autoimmunity was discovered. Autoimmunity.1988;1(1):3-9.
McKenzie MJ, Zakarija M. Antibodies in autoimmune thyroid disease. 6th. Edition ed. Philadelphia: J B Lippincott.
Ando T, Latif R, Davies TF. Thyrotropin receptor antibodies: new insights into their actions and clinical relevance. Best practice & research Clinical endocrinology & metabolism. 2005;19(1):33-52.
Noh JY, Hamada N, Inoue Y, Abe Y, Ito K, Ito K. Thyroid-stimulating antibody is related to Graves' ophthalmopathy, but thyrotropin-binding inhibitor immunoglobulin is related to hyperthyroidism in patients with Graves' disease. Thyroid. 2000;10(9):809-813.
Mizutori Y, Chen CR, Latrofa F, McLachlan SM, Rapoport B. Evidence that shed thyrotropin receptor A subunits drive affinity maturation of autoantibodies causing Graves' disease. J Clin Endocrinol Metab. 2009;94(3):927-935.
Ko J, Kook KH, Yoon JS, Woo KI, Yang JW. Longitudinal association of thyroid-stimulating immunoglobulin levels with clinical characteristics in thyroid eye disease. BMJ Open. 2022;12(6):e050337.
Abeillon-du Payrat J, Caron P. The key data from the 2022 European Thyroid Association congress: Toward personalized treatment of Graves' orbitopathy. Ann Endocrinol (Paris). 2023;84(6):756-757.
Gupta MK. Thyrotropin-receptor antibodies in thyroid diseases: advances in detection techniques and clinical application. Clin Chem Acta. 2000;293:1-29.
Michalek K, Morshed SA, Latif R, Davies TF. TSH receptor autoantibodies. Autoimmun Rev. 2009;9(2):113-116.
Kahaly GJ. Bioassays for TSH Receptor Antibodies: Quo Vadis? Eur Thyroid J. 2015;4(1):3-5.
Kahaly GJ, Diana T, Glang J, Kanitz M, Pitz S, König J. Thyroid Stimulating Antibodies Are Highly Prevalent in Hashimoto's Thyroiditis and Associated Orbitopathy. J Clin Endocrinol Metab. 2016;101(5):1998-2004.
Morgenthaler NG, Ho SC, Minich WB. Stimulating and blocking thyroid-stimulating hormone (TSH) receptor autoantibodies from patients with Graves' disease and autoimmune hypothyroidism have very similar concentration, TSH receptor affinity, and binding sites. J Clin Endocrinol Metab. 2007;92(3):1058-1065.
Yoshida K, Aizawa Y, Kaise N, Fukazawa H, Kiso Y, Sayama N, Mori K, Hori H, Abe K. Relationship between thyroid-stimulating antibodies and thyrotropin-binding inhibitory immunoglobulins years after administration of radioiodine for Graves' disease: retrospective clinical survey. J Endocrinol Invest. 1996;19(10):682-686.
Quadbeck B, Hoermann R, Hahn S, Roggenbuck U, Mann K, Janssen OE. Binding, stimulating and blocking TSH receptor antibodies to the thyrotropin receptor as predictors of relapse of Graves' disease after withdrawal of antithyroid treatment. Horm Metab Res. 2005;37(12):745-750.
McLachlan SM, Rapoport B. Thyrotropin-blocking autoantibodies and thyroid-stimulating autoantibodies: potential mechanisms involved in the pendulum swinging from hypothyroidism to hyperthyroidism or vice versa. Thyroid.2013;23(1):14-24.
Kamijo K. Shift in Dominance from Blocking to Stimulating Type of Thyrotropin Receptor Antibodies, Resulting in Conversion from Hypothyroidism to Hyperthyroidism during Late Pregnancy. Intern Med. 2024;63(4):521-526.
Seetharamaiah GS, Kurosky A, Desai RK, Dallas JS, Prabhakar BS. A recombinant extracellular domain of the thyrotropin (TSH) receptor binds TSH in the absence of membranes. Endocrinology. 1994;134(2):549-554.
Kung AW, Jones BM. A change from stimulatory to blocking antibody activity in Graves' disease during pregnancy. J Clin Endocrinol Metab. 1998;83(2):514-518.
Davies TF, Ando T, Lin RY, Tomer Y, Latif R. Thyrotropin receptor-associated diseases: from adenomata to Graves disease. J Clin Invest. 2005;115(8):1972-1983.
Morshed SA, Ando T, Latif R, Davies TF. Neutral antibodies to the TSH receptor are present in Graves' disease and regulate selective signaling cascades. Endocrinology. 2010;151(11):5537-5549.
Kamijo K. TSH-receptor antibodies determined by the first, second and third generation assays and thyroid-stimulating antibody in pregnant patients with Graves' disease. Endocr J. 2007;54(4):619-624.
Ajjan RA, Weetman AP. Techniques to quantify TSH receptor antibodies. Nature clinical practice Endocrinology & metabolism. 2008;4(8):461-468.
Diana T, Wüster C, Kanitz M, Kahaly GJ. Highly variable sensitivity of five binding and two bio-assays for TSH-receptor antibodies. J Endocrinol Invest. 2016;39(10):1159-1165.
Zöphel K, Roggenbuck D, Schott M. Clinical review about TRAb assay's history. Autoimmun Rev. 2010;9(10):695-700.
Iida Y, Konishi J, Kasagi K, Kuma K, Torizuka K. Detection of TSH-binding inhibitor immunoglobulins by using the triton-solubilized receptor from human thyroid membranes. Endocrinol Jpn. 1982;29(2):227-231.
Ehlers M, Allelein S, Schott M. TSH-receptor autoantibodies: pathophysiology, assay methods, and clinical applications. Minerva Endocrinol. 2018;43(3):323-332.
Massart C, Gibassier J, d'Herbomez M. Clinical value of M22-based assays for TSH-receptor antibody (TRAb) in the follow-up of antithyroid drug treated Graves' disease: comparison with the second generation human TRAb assay. Clin Chim Acta. 2009;407(1-2):62-66.
Smith BR, Sanders J, Furmaniak J. TSH receptor antibodies. Thyroid. 2007;17(10):923-938.
Feldt-Rasmussen U, Schleusener H, Carayon P. Meta-analysis evaluation of the impact of thyrotropin receptor antibodies on long term remission after medical therapy of Graves' disease. J Clin Endocrinol Metab.1994;78(1):98-102.
Furmaniak J, Sanders J, Sanders P, Miller-Gallacher J, Ryder MM, Rees Smith B. Practical applications of studies on the TSH receptor and TSH receptor autoantibodies. Endocrine. 2020;68(2):261-264.
Evans M, Sanders J, Tagami T, Sanders P, Young S, Roberts E, Wilmot J, Hu X, Kabelis K, Clark J, Holl S, Richards T, Collyer A, Furmaniak J, Smith BR. Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample. Clin Endocrinol (Oxf).2010;73(3):404-412.
Takamura Y, Nakano K, Uruno T, Ito Y, Miya A, Kobayashi K, Yokozawa T, Matsuzuka F, Kuma K, Miyauchi A. Changes in serum TSH receptor antibody (TRAb) values in patients with Graves' disease after total or subtotal thyroidectomy. Endocr J. 2003;50(5):595-601.
Carella C, Mazziotti G, Sorvillo F, Piscopo M, Cioffi M, Pilla P, Nersita R, Iorio S, Amato G, Braverman LE, Roti E. Serum thyrotropin receptor antibodies concentrations in patients with Graves' disease before, at the end of methimazole treatment, and after drug withdrawal: evidence that the activity of thyrotropin receptor antibody and/or thyroid response modify during the observation period. Thyroid. 2006;16(3):295-302.
Giuliani C, Cerrone D, Harii N, Thornton M, Kohn LD, Dagia NM, Bucci I, Carpentieri M, Di Nenno B, Di Blasio A, Vitti P, Monaco F, Napolitano G. A TSHR-LH/CGR chimera that measures functional thyroid-stimulating autoantibodies (TSAb) can predict remission or recurrence in Graves' patients undergoing antithyroid drug (ATD) treatment. J Clin Endocrinol Metab. 2012;97(7):E1080-1087.
McKenzie JM, Zakarija M. Fetal and neonatal hyperthyroidism and hypothyroidism due to maternal TSH receptor antibodies. Thyroid. 1992;2(2):155-159.
Nor Azlin MI, Bakin YD, Mustafa N, Wahab NA, Johari MJ, Kamarudin NA, Jamil MA. Thyroid autoantibodies and associated complications during pregnancy. J Obstet Gynaecol. 2010;30(7):675-678.
Hamada N, Momotani N, Ishikawa N, Yoshimura Noh J, Okamoto Y, Konishi T, Ito K, Ito K. Persistent high TRAb values during pregnancy predict increased risk of neonatal hyperthyroidism following radioiodine therapy for refractory hyperthyroidism. Endocr J. 2011;58(1):55-58.
Heithorn R, Hauffa BP, Reinwein D. Thyroid antibodies in children of mothers with autoimmune thyroid disorders. Eur J Pediatr. 1999;158:24-28.
Abeillon-du Payrat J, Chikh K, Bossard N, Bretones P, Gaucherand P, Claris O, Charrié A, Raverot V, Orgiazzi J, Borson-Chazot F, Bournaud C. Predictive value of maternal second-generation thyroid-binding inhibitory immunoglobulin assay for neonatal autoimmune hyperthyroidism. Eur J Endocrinol. 2014;171(4):451-460.
Tokuda Y, Kasagi K, Iida Y, Hatabu H, Hidaka A, Misaki T, Konishi J. Sensitive, practical bioassay of thyrotropin, with use of FRTL-5 thyroid cells and magnetizable solid-phase-bound antibodies. Clin Chem. 1988;34(11):2360-2364.
Morris JC, 3rd, Hay ID, Nelson RE, Jiang NS. Clinical utility of thyrotropin-receptor antibody assays: comparison of radioreceptor and bioassay methods. Mayo Clin Proc. 1988;63(7):707-717.
Michelangeli VP, Munro DS, Poon CW, Frauman AG, Colman PG. Measurement of thyroid stimulating immunoglobulins in a new cell line transfected with a functional human TSH receptor (JPO9 cells), compared with an assay using FRTL-5 cells. Clin Endocrinol (Oxf). 1994;40(5):645-652.
Michelangeli VP, Poon CW, Arnus EE, Frauman AG, Connelly J, Colman PG. Measurement of TSH receptor blocking immunoglobulins using 3H-adenine incorporation into FRTL-5 and JPO9 cells: use in a child with neonatal hypothyroidism. Clin Endocrinol (Oxf). 1995;42(1):39-44.
Morgenthaler NG, Pampel I, Aust G, Seissler J, Scherbaum WA. Application of a bioassay with CHO cells for the routine detection of stimulating and blocking autoantibodies to the TSH-receptor. Horm Metab Res.1998;30(3):162-168.
McLachlan SM, Rapoport B. Thyroid peroxidase autoantibody epitopes revisited*. Clin Endocrinol (Oxf).2008;69(4):526-527.
Paparodis R, Livadas S, Karvounis E, Bantouna D, Zoupas I, Angelopoulos N, Imam S, Jaume JC. Elevated Preoperative TPO Ab Titers Decrease Risk for DTC in a Linear Fashion: A Retrospective Analysis of 1635 Cases. J Clin Endocrinol Metab. 2023;109(1):e347-e355.
Jaume JC, Burek CL, Hoffman WH, Rose NR, McLachlan SM, Rapoport B. Thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile Hashimoto's thyroiditis: evidence for conservation over time and in families. Clin Exp Immunol. 1996;104(1):115-123.
Ehlers M, Thiel A, Bernecker C, Porwol D, Papewalis C, Willenberg HS, Schinner S, Hautzel H, Scherbaum WA, Schott M. Evidence of a combined cytotoxic thyroglobulin and thyroperoxidase epitope-specific cellular immunity in Hashimoto's thyroiditis. J Clin Endocrinol Metab. 2012;97(4):1347-1354.
Cayzer I, Chalmers SR, Doniach D, Swana G. An evaluation of two new haemagglutination tests for the rapid diagnosis of autoimmune thyroid diseases. J Clin Path. 1978;31:1147-1151.
Mariotti S, Caturegli P, Piccolo P, Barbesino G, Pinchera A. Antithyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab. 1990;71:661-669.
Beever K, Bradbury J, Phillips D, McLachlan SM, Pegg C, Goral A, Overbeck W, Feifel G, Smith BR. Highly sensitive assays of autoantibodies to thyroglobulin and to thyroid peroxidase. Clin Chem. 1989;35:1949-1954.
Laurberg P, Pedersen KM, Vittinghus E, Ekelund S. Sensitive enzyme-linked immunosorbent assay for measurement of autoantibodies to human thyroid peroxidase. Scandinavian Journal Of Clinical And Laboratory Investigation. 1992;52(7):663-669.
La'ulu SL, Slev PR, Roberts WL. Performance characteristics of 5 automated thyroglobulin autoantibody and thyroid peroxidase autoantibody assays. Clin Chim Acta. 2007;376(1-2):88-95.
Kasagi K, Takahashi N, Inoue G, Honda T, Kawachi Y, Izumi Y. Thyroid function in Japanese adults as assessed by a general health checkup system in relation with thyroid-related antibodies and other clinical parameters. Thyroid. 2009;19(9):937-944.
Liu Q, Yang H, Chen Y, He X, Dong L, Zhang X, Yang Y, Tian M, Cheng W, Liu D, Yang G, Li K. Effect of thyroid peroxidase antibody titers trajectories during pregnancy and postpartum on postpartum thyroid dysfunction. Arch Gynecol Obstet. 2024.
Karanikas G, Schuetz M, Wahl K, Paul M, Kontur S, Pietschmann P, Kletter K, Dudczak R, Willheim M. Relation of anti-TPO autoantibody titre and T-lymphocyte cytokine production patterns in Hashimoto's thyroiditis. Clin Endocrinol (Oxf). 2005;63(2):191-196.
Rebuffat SA, Nguyen B, Robert B, Castex F, Peraldi-Roux S. Antithyroperoxidase antibody-dependent cytotoxicity in autoimmune thyroid disease. J Clin Endocrinol Metab. 2008;93(3):929-934.
Carmel R, Spencer CA. Clinical and subclinical thyroid disorders associated with pernicious anemia. Arch Intern Med. 1982;142:1465-1469.
Vanderpump MPJ, Tunbridge WMG, French JM, Appleton D, Bates D, Rodgers H, Evans JG, Clark F, Tunbridge F, Young ET. The incidence of thyroid disorders in the community; a twenty year follow up of the Whickham survey. Clin Endocrinol. 1995;43:55-68.
Hutfless S, Matos P, Talor MV, Caturegli P, Rose NR. Significance of prediagnostic thyroid antibodies in women with autoimmune thyroid disease. J Clin Endocrinol Metab. 2011;96(9):E1466-1471.
Yang S, Huang Z, Zhang Y, Li Y, Zhou Y, Guan H, Fan J. Association of Maternal Thyroglobulin Antibody with Preterm Birth in Euthyroid Women. J Clin Endocrinol Metab. 2025.
Huber G, Staub JJ, Meier C, Mitrache C, Guglielmetti M, Huber P, Braverman LE. Prospective Study of the Spontaneous Course of Subclinical Hypothyroidism: Prognostic Value of Thyrotropin, Thyroid Reserve, and Thyroid Antibodies. J Clin Endocrinol Metab. 2002;87:3221-3226.
Pedersen OM, Aardal NP, Larssen TB, Varhaug JE, Myking O, Vik-Mo H. The value of ultrasonography in predicting autoimmune thyroid disease. Thyroid. 2000;10:251-259.
Mariotti S, Barbesino G, Caturegli P, Atzeni F, Manetti L, Marinò M, Grasso L, Velluzzi F, Loviselli A, Pinchera A, et al. False negative results observed in anti-thyroid peroxidase autoantibody determination by competitive radioimmunoassays using monoclonal antibodies. Eur J Endocrinol. 1994;130(6):552-558.
Schmidt M, Voell M, Rahlff I, Dietlein M, Kobe C, Faust M, Schicha H. Long-term follow-up of antithyroid peroxidase antibodies in patients with chronic autoimmune thyroiditis (Hashimoto's thyroiditis) treated with levothyroxine. Thyroid. 2008;18(7):755-760.
Bell TM, Bansal AS, Shorthouse C, Sandford N, Powell EE. Low titre autoantibodies predict autoimmune disease during interferon alpha treatment of chronic hepatitis C. J Gastroenterol Hepatol. 1999;14:419-422.
Johnston AM, Eagles JM. Lithium-associated clinical hypothyroidism. Prevalence and risk factors. Br J Psychiatry.1999;175:336-339.
Daniels GH. Amiodarone-induced thyrotoxicosis. J Clin Endocrinol Metab. 2001;86(1):3-8.
Bocchetta A, Cocco F, Velluzzi F, Del Zompo M, Mariotti S, Loviselli A. Fifteen-year follow-up of thyroid function in lithium patients. J Endocrinol Invest. 2007;30(5):363-366.
Tsang W, Houlden RL. Amiodarone-induced thyrotoxicosis: a review. Can J Cardiol. 2009;25(7):421-424.
Wang L, Li B, Zhao H, Wu P, Wu Q, Chen K, Mu Y. A systematic review and meta-analysis of endocrine-related adverse events associated with interferon. Front Endocrinol (Lausanne). 2022;13:949003.
Negro R, Žarković M, Attanasio R, Hegedüs L, Nagy EV, Papini E, Akarsu E, Alevizaki M, Ayvaz G, Bednarczuk T, Beleslin BN, Berta E, Bodor M, Borissova AM, Boyanov M, Buffet C, Burlacu MC, Ćirić J, Cohen CA, Díez JJ, Dobnig H, Fadeyev V, Field BCT, Fliers E, Führer D, Galofré JC, Hakala T, Jan J, Kopp P, Krebs M, Kršek M, Kužma M, Leenhardt L, Luchytskiy V, Puga FM, McGowan A, Melo M, Metso S, Moran C, Morgunova T, Niculescu DA, Perić B, Planck T, Poiana C, Robenshtok E, Rosselet PO, Ruchala M, Riis KR, Shepelkevich A, Tronko M, Unuane D, Vardarli I, Visser E, Vryonidou A, Younes YR, Perros P. Use of levothyroxine for euthyroid, thyroid antibody positive women with infertility: Analyses of aggregate data from a survey of European thyroid specialists (Treatment of Hypothyroidism in Europe by Specialists: An International Survey). Clin Endocrinol (Oxf).2024;101(2):180-190.
Mecacci F, Parretti E, Cioni R, Lucchetti R, Magrini A, La Torre P, Mignosa M, Acanfora L, Mello G. Thyroid autoimmunity and its association with non-organ-specific antibodies and subclinical alterations of thyroid function in women with a history of pregnancy loss or preeclampsia. J Reprod Immunol. 2000;46:39-50.
Bussen S, Steck T, Dietl J. Increased prevalence of thyroid antibodies in euthyroid women with a history of recurrent in-vitro fertilization failure. Hum Reprod. 2000;15:545-548.
Poppe K, Glinoer D, tournaye H, Devroey P, Van Steirteghem a, Kaufman L, Velkeniers B. Assisted reproduction and thyroid autoimmunity: an unfortunate combination? J Clin Endocrinol Metab. 2003;88:4149-4152.
Negro R, Formoso G, Coppola L, Presicce G, Mangieri T, Pezzarossa A, Dazzi D. Euthyroid women with autoimmune disease undergoing assisted reproduction technologies: the role of autoimmunity and thyroid function. J Endocrinol Invest. 2007;30(1):3-8.
He X, Wang P, Wang Z, He X, Xu D, Wang B. Thyroid antibodies and risk of preterm delivery: a meta-analysis of prospective cohort studies. Eur J Endocrinol. 2012;167(4):455-464.
Karakosta P, Alegakis D, Georgiou V, Roumeliotaki T, Fthenou E, Vassilaki M, Boumpas D, Castanas E, Kogevinas M, Chatzi L. Thyroid dysfunction and autoantibodies in early pregnancy are associated with increased risk of gestational diabetes and adverse birth outcomes. J Clin Endocrinol Metab. 2012;97(12):4464-4472.
Bucci I, Giuliani C, Di Dalmazi G, Formoso G, Napolitano G. Thyroid Autoimmunity in Female Infertility and Assisted Reproductive Technology Outcome. Front Endocrinol (Lausanne). 2022;13:768363.
McLachlan SM, Rapoport B. Why measure thyroglobulin autoantibodies rather than thyroid peroxidase autoantibodies? Thyroid. 2004;14(7):510-520.
Latrofa F, Ricci D, Montanelli L, Rocchi R, Piaggi P, Sisti E, Grasso L, Basolo F, Ugolini C, Pinchera A, Vitti P. Thyroglobulin autoantibodies in patients with papillary thyroid carcinoma: comparison of different assays and evaluation of causes of discrepancies. J Clin Endocrinol Metab. 2012;97(11):3974-3982.
Pickett AJ, Jones M, Evans C. Causes of discordance between thyroglobulin antibody assays. Ann Clin Biochem.2012;49(Pt 5):463-467.
Spencer CA, Bergoglio LM, Kazarosyan M, Fatemi S, LoPresti JS. Clinical impact of thyroglobulin (Tg) and Tg autoantibody method differences on the management of patients with differentiated thyroid carcinomas. J Clin Endocrinol Metab. 2005;90(10):5566-5575.
Lupoli GA, Okosieme OE, Evans C, Clark PM, Pickett AJ, Premawardhana LD, Lupoli G, Lazarus JH. Prognostic significance of thyroglobulin antibody epitopes in differentiated thyroid cancer. J Clin Endocrinol Metab.2015;100(1):100-108.
Taylor KP, Parkington D, Bradbury S, Simpson HL, Jefferies SJ, Halsall DJ. Concordance between thyroglobulin antibody assays. Ann Clin Biochem. 2011;48(Pt 4):367-369.
Rosario PW, Côrtes MCS, Franco Mourão G. Follow-up of patients with thyroid cancer and antithyroglobulin antibodies: a review for clinicians. Endocr Relat Cancer. 2021;28(4):R111-r119.
van Kinschot CMJ, Peeters RP, van den Berg SAA, Verburg FA, van Noord C, van Ginhoven TM, Visser WE. Thyroglobulin and thyroglobulin antibodies: assay-dependent management consequences in patients with differentiated thyroid carcinoma. Clin Chem Lab Med. 2022;60(5):756-765.
Ruf J, Carayon P, Lissitzky S. Various expressions of a unique anti-human thyroglobulin antibody repertoire in normal state and autoimmune disease. Eur J Immunol. 1985;15(3):268-272.
Aras G, Gültekin SS, Küçük NO. The additive clinical value of combined thyroglobulin and antithyroglobulin antibody measurements to define persistent and recurrent disease in patients with differentiated thyroid cancer. Nucl Med Commun. 2008;29(10):880-884.
Feldt-Rasmussen U, Rasmussen AK. Autoimmunity in differentiated thyroid cancer: significance and related clinical problems. Hormones (Athens). 2010;9(2):109-117.
Zhao Y, Mu Z, Liang D, Zhang T, Zhang X, Sun D, Sun Y, Liang J, Lin Y. Prognostic value of postoperative anti-thyroglobulin antibody in patients with differentiated thyroid cancer. Front Endocrinol (Lausanne).2024;15:1354426.
Spencer CA, Takeuchi M, Kazarosyan M, Wang CC, Guttler RB, Singer PA, Fatemi S, LoPresti JS, Nicoloff JT. Serum thyroglobulin autoantibodies: prevalence, influence on serum thyroglobulin measurement, and prognostic significance in patients with differentiated thyroid carcinoma. J Clin Endocrinol Metab. 1998;83(4):1121-1127.
Görges R, Maniecki M, Jentzen W, Sheu SN, Mann K, Bockisch A, Janssen OE. Development and clinical impact of thyroglobulin antibodies in patients with differentiated thyroid carcinoma during the first 3 years after thyroidectomy. Eur J Endocrinol. 2005;153(1):49-55.
Kim ES, Lim DJ, Baek KH, Lee JM, Kim MK, Kwon HS, Song KH, Kang MI, Cha BY, Lee KW, Son HY. Thyroglobulin antibody is associated with increased cancer risk in thyroid nodules. Thyroid. 2010;20(8):885-891.
Ge H, Chen W, Lin Z, Li Y, Chen S. Analysis of the prognostic value of thyroglobulin antibody change trends during follow-up after (131)I treatment in patients with differentiated thyroid carcinoma. Front Oncol.2025;15:1496594.
Grani G, Calvanese A, Carbotta G, D'Alessandri M, Nesca A, Bianchini M, Del Sordo M, Vitale M, Fumarola A. Thyroid autoimmunity and risk of malignancy in thyroid nodules submitted to fine-needle aspiration cytology. Head Neck. 2015;37(2):260-264.
Karatzas T, Vasileiadis I, Zapanti E, Charitoudis G, Karakostas E, Boutzios G. Thyroglobulin antibodies as a potential predictive marker of papillary thyroid carcinoma in patients with indeterminate cytology. Am J Surg.2016;212(5):946-952.
Pacini F, Schlumberger M, Dralle H, Elisei R, Smit JW, Wiersinga W. European consensus for the management of patients with differentiated thyroid carcinoma of the follicular epithelium. Eur J Endocrinol. 2006;154(6):787-803.
Shin HJ, Lee HS, Kim EK, Moon HJ, Lee JH, Kwak JY. A Study on Serum Antithyroglobulin Antibodies Interference in Thyroglobulin Measurement in Fine-Needle Aspiration for Diagnosing Lymph Node Metastasis in Postoperative Patients. PloS one. 2015;10(6):e0131096.
Gholve C, Damle A, Kulkarni S, Banerjee S, Rajan M. Evaluation of Different Methods for the Detection of Anti- Thyroglobulin Autoantibody: Prevalence of Anti-Thyroglobulin Autoantibody and Anti-Microsomal Autoantibody in Thyroid Cancer Patients. Indian J Clin Biochem. 2022;37(4):473-479.
Chiovato L, Latrofa F, Braverman LE, Pacini F, Capezzone M, Masserini L, Grasso L, Pinchera A. Disappearance of humoral thyroid autoimmunity after complete removal of thyroid antigens. Ann Intern Med. 2003;139(5 Pt 1):346-351.
Kim WG, Yoon JH, Kim WB, Kim TY, Kim EY, Kim JM, Ryu JS, Gong G, Hong SJ, Shong YK. Change of serum antithyroglobulin antibody levels is useful for prediction of clinical recurrence in thyroglobulin-negative patients with differentiated thyroid carcinoma. J Clin Endocrinol Metab. 2008;93(12):4683-4689.
Scappaticcio L, Trimboli P, Verburg FA, Giovanella L. Significance of "de novo" appearance of thyroglobulin antibodies in patients with differentiated thyroid cancer. Int J Biol Markers. 2020;35(3):41-49.
Pacini F, Mariotti S, Formica N, Elisei R. Thyroid autoantibodies in thyroid cancer: Incidence and relationship with tumor outcome. Acta Endocrinol. 1988;119:373-380.
Yamada O, Miyauchi A, Ito Y, Nakayama A, Yabuta T, Masuoka H, Fukushima M, Higashiyama T, Kihara M, Kobayashi K, Miya A. Changes in serum thyroglobulin antibody levels as a dynamic prognostic factor for early-phase recurrence of thyroglobulin antibody-positive papillary thyroid carcinoma after total thyroidectomy. Endocr J.2014;61(10):961-965.
Gianoukakis AG. Thyroglobulin antibody status and differentiated thyroid cancer: what does it mean for prognosis and surveillance? Curr Opin Oncol. 2015;27(1):26-32.
Lee ZJO, Eslick GD, Edirimanne S. Investigating Antithyroglobulin Antibody As a Prognostic Marker for Differentiated Thyroid Cancer: A Meta-Analysis and Systematic Review. Thyroid. 2020;30(11):1601-1612.
Tomer Y, Greenberg D. The thyroglobulin gene as the first thyroid-specific susceptibility gene for autoimmune thyroid disease. Trends Mol Med. 2004;10(7):306-308.
Vejbjerg P, Knudsen N, Perrild H, Laurberg P, Carlé A, Pedersen IB, Rasmussen LB, Ovesen L, Jørgensen T. Thyroglobulin as a marker of iodine nutrition status in the general population. Eur J Endocrinol. 2009;161(3):475-481.
Wang Z, Zhang H, Zhang X, Sun J, Han C, Li C, Li Y, Teng X, Fan C, Liu A, Shan Z, Liu C, Weng J, Teng W. Serum thyroglobulin reference intervals in regions with adequate and more than adequate iodine intake. Medicine (Baltimore). 2016;95(48):e5273.
Bath SC, Pop VJ, Furmidge-Owen VL, Broeren MA, Rayman MP. Thyroglobulin as a Functional Biomarker of Iodine Status in a Cohort Study of Pregnant Women in the United Kingdom. Thyroid. 2017;27(3):426-433.
Vali M, Rose NR, Caturegli P. Thyroglobulin as autoantigen: structure-function relationships. Rev Endocr Metab Disord. 2000;1(1-2):69-77.
Citterio CE, Machiavelli GA, Miras MB, Gruneiro-Papendieck L, Lachlan K, Sobrero G, Chiesa A, Walker J, Munoz L, Testa G, Belforte FS, Gonzalez-Sarmiento R, Rivolta CM, Targovnik HM. New insights into thyroglobulin gene: molecular analysis of seven novel mutations associated with goiter and hypothyroidism. Mol Cell Endocrinol.2013;365(2):277-291.
Persani L, Rurale G, de Filippis T, Galazzi E, Muzza M, Fugazzola L. Genetics and management of congenital hypothyroidism. Best practice & research Clinical endocrinology & metabolism. 2018;32(4):387-396.
Fernández-Cancio M, Antolín M, Clemente M, Campos-Martorell A, Mogas E, Baz-Redón N, Leno-Colorado J, Comas-Armangué G, García-Arumí E, Soler-Colomer L, González-Llorens N, Camats-Tarruella N, Yeste D. Clinical and molecular study of patients with thyroid dyshormogenesis and variants in the thyroglobulin gene. Front Endocrinol (Lausanne). 2024;15:1367808.
Chow E, Siddique F, Gama R. Thyrotoxicosis factitia: role of thyroglobulin. Ann Clin Biochem. 2008;45(Pt 4):447-448; author reply 448.
Jahagirdar VR, Strouhal P, Holder G, Gama R, Singh BM. Thyrotoxicosis factitia masquerading as recurrent Graves' disease: endogenous antibody immunoassay interference, a pitfall for the unwary. Ann Clin Biochem.2008;45(Pt 3):325-327.
Vorasart P, Sriphrapradang C. Factitious thyrotoxicosis: how to find it. Diagnosis (Berl). 2020;7(2):141-145.
Bögershausen LR, Giovanella L, Stief T, Luster M, Verburg FA. Long-term predictive value of highly sensitive thyroglobulin measurement. Clin Endocrinol (Oxf). 2023;98(4):622-628.
Giovanella L, Milan L, Roll W, Weber M, Schenke S, Kreissl M, Vrachimis A, Pabst K, Murat T, Petranović Ovčariček P, Campenni A, Görges R, Ceriani L. Thyroglobulin measurement is the most powerful outcome predictor in differentiated thyroid cancer: a decision tree analysis in a European multicenter series. Clin Chem Lab Med. 2024;62(11):2307-2315.
Giovanella L, D'Aurizio F, Petranović Ovčariček P, Görges R. Diagnostic, Theranostic and Prognostic Value of Thyroglobulin in Thyroid Cancer. J Clin Med. 2024;13(9).
Chindris AM, Diehl NN, Crook JE, Fatourechi V, Smallridge RC. Undetectable sensitive serum thyroglobulin (<0.1 ng/ml) in 163 patients with follicular cell-derived thyroid cancer: results of rhTSH stimulation and neck ultrasonography and long-term biochemical and clinical follow-up. J Clin Endocrinol Metab. 2012;97(8):2714-2723.
Trimboli P, La Torre D, Ceriani L, Condorelli E, Laurenti O, Romanelli F, Ventura C, Signore A, Valabrega S, Giovanella L. High sensitive thyroglobulin assay on thyroxine therapy: can it avoid stimulation test in low and high risk differentiated thyroid carcinoma patients? Horm Metab Res. 2013;45(9):664-668.
Giovanella L, Castellana M, Trimboli P. Unstimulated high-sensitive thyroglobulin is a powerful prognostic predictor in patients with thyroid cancer. Clin Chem Lab Med. 2019;58(1):130-137.
Fernández-Velasco P, Díaz-Soto G, Pérez López P, Torres Torres B, de Luis D. Predictive value and dynamic risk stratification of high sensitive basal or stimulated thyroglobulin assay in a long-term thyroid carcinoma cohort. Endocrine. 2023;81(1):116-122.
Spencer C, Petrovic I, Fatemi S, LoPresti J. Serum thyroglobulin (Tg) monitoring of patients with differentiated thyroid cancer using sensitive (second-generation) immunometric assays can be disrupted by false-negative and false-positive serum thyroglobulin autoantibody misclassifications. J Clin Endocrinol Metab. 2014;99(12):4589-4599.
Latrofa F, Ricci D, Sisti E, Piaggi P, Nencetti C, Marinò M, Vitti P. Significance of Low Levels of Thyroglobulin Autoantibodies Associated with Undetectable Thyroglobulin After Thyroidectomy for Differentiated Thyroid Carcinoma. Thyroid. 2016;26(6):798-806.
Jindal A, Khan U. Is Thyroglobulin Level by Liquid Chromatography Tandem-Mass Spectrometry Always Reliable for Follow-Up of DTC After Thyroidectomy: A Report on Two Patients. Thyroid. 2016;26(9):1334-1335.
Azmat U, Porter K, Senter L, Ringel MD, Nabhan F. Thyroglobulin Liquid Chromatography-Tandem Mass Spectrometry Has a Low Sensitivity for Detecting Structural Disease in Patients with Antithyroglobulin Antibodies. Thyroid. 2017;27(1):74-80.
Spencer CA, Wang CC. Thyroglobulin measurement. Techniques, clinical benefits, and pitfalls. Endocrinol Metab Clin North Am. 1995;24(4):841-863.
Ross HA, Netea-Maier RT, Schakenraad E, Bravenboer B, Hermus AR, Sweep FC. Assay bias may invalidate decision limits and affect comparability of serum thyroglobulin assay methods: an approach to reduce interpretation differences. Clin Chim Acta. 2008;394(1-2):104-109.
Spencer C, Fatemi S, Singer P, Nicoloff J, Lopresti J. Serum Basal thyroglobulin measured by a second-generation assay correlates with the recombinant human thyrotropin-stimulated thyroglobulin response in patients treated for differentiated thyroid cancer. Thyroid. 2010;20(6):587-595.
Giovanella L, Clark PM, Chiovato L, Duntas L, Elisei R, Feldt-Rasmussen U, Leenhardt L, Luster M, Schalin-Jäntti C, Schott M, Seregni E, Rimmele H, Smit J, Verburg FA. Thyroglobulin measurement using highly sensitive assays in patients with differentiated thyroid cancer: a clinical position paper. Eur J Endocrinol. 2014;171(2):R33-46.
Iervasi A, Iervasi G, Ferdeghini M, Solimeo C, Bottoni A, Rossi L, Colato C, Zucchelli GC. Clinical relevance of highly sensitive Tg assay in monitoring patients treated for differentiated thyroid cancer. Clin Endocrinol (Oxf).2007;67(3):434-441.
Bachelot A, Cailleux AF, Klain M, Baudin E, Ricard M, Bellon N, Caillou B, Travagli JP, Schlumberger M. Relationship between tumor burden and serum thyroglobulin level in patients with papillary and follicular thyroid carcinoma. Thyroid. 2002;12(8):707-711.
Giovanella L, Imperiali M, Ferrari A, Palumbo A, Furlani L, Graziani MS, Castello R. Serum thyroglobulin reference values according to NACB criteria in healthy subjects with normal thyroid ultrasound. Clin Chem Lab Med.2012;50(5):891-893.
Sobrero G, Munoz L, Bazzara L, Martin S, Silvano L, Iorkansky S, Bergoglio L, Spencer C, Miras M. Thyroglobulin reference values in a pediatric infant population. Thyroid. 2007;17(11):1049-1054.
Sands NB, Karls S, Rivera J, Tamilia M, Hier MP, Black MJ, Gologan O, Payne RJ. Preoperative serum thyroglobulin as an adjunct to fine-needle aspiration in predicting well-differentiated thyroid cancer. J Otolaryngol Head Neck Surg. 2010;39(6):669-673.
Trimboli P, Treglia G, Giovanella L. Preoperative measurement of serum thyroglobulin to predict malignancy in thyroid nodules: a systematic review. Horm Metab Res. 2015;47(4):247-252.
Jo K, Kim MH, Ha J, Lim Y, Lee S, Bae JS, Jung CK, Kang MI, Cha BY, Lim DJ. Prognostic value of preoperative anti-thyroglobulin antibody in differentiated thyroid cancer. Clin Endocrinol (Oxf). 2017;87(3):292-299.
Jang A, Jin M, Kim CA, Jeon MJ, Lee YM, Sung TY, Kim TY, Kim WB, Shong YK, Kim WG. Serum thyroglobulin testing after thyroid lobectomy in patients with 1-4 cm papillary thyroid carcinoma. Endocrine. 2023;81(2):290-297.
Evans C, Lotz J, Bhandari M, Hellier RT, Wang XY, Lott R, Lackner KJ, Müller R, Kulasingam V. Multi-center evaluation of the highly sensitive Abbott ARCHITECT and Alinity thyroglobulin chemiluminescent microparticle immunoassay. J Clin Lab Anal. 2022;36(9):e24595.
Heilig B, Hufner M, Dorken B, Schmidt-Gayk H. Increased heterogeneity of serum thyroglobulin in thyroid cancer patients as determined by monoclonal antibodies. Klin Wochenschr. 1986;64:776-780.
Schulz R, Bethauser H, Stempka L, Heilig B, Moll A, Hufner M. Evidence for immunological differences between circulating and tissue-derived thyroglobulin in men. Eur J Clin Invest. 1989;19:459-463.
Magro G, Perissinotto D, Schiappacassi M, Goletz S, Otto A, Müller EC, Bisceglia M, Brown G, Ellis T, Grasso S, Colombatti A, Perris R. Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas. Am J Pathol. 2003;163(1):183-196.
Schlumberger M, Hitzel A, Toubert ME, Corone C, Troalen F, Schlageter MH, Claustrat F, Koscielny S, Taieb D, Toubeau M, Bonichon F, Borson-Chazot F, Leenhardt L, Schvartz C, Dejax C, Brenot-Rossi I, Torlontano M, Tenenbaum F, Bardet S, Bussière F, Girard JJ, Morel O, Schneegans O, Schlienger JL, Prost A, So D, Archambeaud F, Ricard M, Benhamou E. Comparison of seven serum thyroglobulin assays in the follow-up of papillary and follicular thyroid cancer patients. J Clin Endocrinol Metab. 2007;92(7):2487-2495.
Shaw JB, Harvey SR, Du C, Xu Z, Edgington RM, Olmedillas E, Saphire EO, Wysocki VH. Protein Complex Heterogeneity and Topology Revealed by Electron Capture Charge Reduction and Surface Induced Dissociation. ACS Cent Sci. 2024;10(8):1537-1547.
Jensen E, Petersen PH, Blaabjerg O, Hegedüs L. Biological variation of thyroid autoantibodies and thyroglobulin. Clin Chem Lab Med. 2007;45(8):1058-1064.
Spencer CA, LoPresti JS. Measuring thyroglobulin and thyroglobulin autoantibody in patients with differentiated thyroid cancer. Nature clinical practice Endocrinology & metabolism. 2008;4(4):223-233.
Grebe S. Soluble thyroid tumor markers – old and new challenges and potential solutions. NZ J Med Lab Science 2013;87:76-87.
Grebe SK. Diagnosis and management of thyroid carcinoma: focus on serum thyroglobulin. Exp Rev Endocrinol Metab. 2009;4:25-43.
Tomoda C, Miyauchi A. Undetectable serum thyroglobulin levels in patients with medullary thyroid carcinoma after total thyroidectomy without radioiodine ablation. Thyroid. 2012;22(7):680-682.
Angell TE, Spencer CA, Rubino BD, Nicoloff JT, LoPresti JS. In search of an unstimulated thyroglobulin baseline value in low-risk papillary thyroid carcinoma patients not receiving radioactive iodine ablation. Thyroid.2014;24(7):1127-1133.
Park S, Jeon MJ, Oh HS, Lee YM, Sung TY, Han M, Han JM, Kim TY, Chung KW, Kim WB, Shong YK, Kim WG. Changes in Serum Thyroglobulin Levels After Lobectomy in Patients with Low-Risk Papillary Thyroid Cancer. Thyroid. 2018;28(8):997-1003.
Feldt-Rasmussen U, Profilis C, Colinet E, Black E, Bornet H, Bourdoux P, Carayon P, Ericsson UB, Koutras DA, Lamas de Leon L, DeNayer P, Pacini F, Palumbo G, Santos A, Schlumberger M, Seidel C, Van Herle AJ, JJM D. Human thyroglobulin reference material (CRM 457) 1st part: Assessment of homogeneity, stability and immunoreactivity. Ann Biol Clin. 1996;54:337-342.
Feldt-Rasmussen U, Profilis C, Colinet E, Black E, Bornet H, Bourdoux P, Carayon P, Ericsson UB, Koutras DA, Lamas de Leon L, DeNayer P, Pacini F, Palumbo G, Santos A, Schlumberger M, Seidel C, Van Herle AJ, JJM D. Human thyroglobulin reference material (CRM 457) 2nd part: Physicochemical characterization and certification. Ann Biol Clin. 1996;54:343-348.
Cubero JM, Rodríguez-Espinosa J, Gelpi C, Estorch M, Corcoy R. Thyroglobulin autoantibody levels below the cut-off for positivity can interfere with thyroglobulin measurement. Thyroid. 2003;13(7):659-661.
Malandrino P, Tumino D, Russo M, Marescalco S, Fulco RA, Frasca F. Surveillance of patients with differentiated thyroid cancer and indeterminate response: a longitudinal study on basal thyroglobulin trend. J Endocrinol Invest.2019;42(10):1223-1230.
Cole TG, Johnson D, Eveland BJ, Nahm MH. Cost-effective method for detection of "hook effect" in tumor marker immunometric assays. Clin Chem. 1993;39:695-696.
Jassam N, Jones CM, Briscoe T, Horner JH. The hook effect: a need for constant vigilance. Ann Clin Biochem.2006;43(Pt 4):314-317.
Leboeuf R, Langlois MF, Martin M, Ahnadi CE, Fink GD. "Hook effect" in calcitonin immunoradiometric assay in patients with metastatic medullary thyroid carcinoma: case report and review of the literature. J Clin Endocrinol Metab. 2006;91(2):361-364.
Hillebrand JJ, Siegelaar SE, Heijboer AC. Falsely decreased thyroglobulin levels in a patient with differentiated thyroid carcinoma. Clin Chim Acta. 2020;509:217-219.
Giovanella L, Ghelfo A. Undetectable serum thyroglobulin due to negative interference of heterophile antibodies in relapsing thyroid carcinoma. Clin Chem. 2007;53(10):1871-1872.
Ross HA, Menheere PP, Thomas CM, Mudde AH, Kouwenberg M, Wolffenbuttel BH. Interference from heterophilic antibodies in seven current TSH assays. Ann Clin Biochem. 2008;45(Pt 6):616.
Ding L, Shankara-Narayana N, Wood C, Ward P, Sidhu S, Clifton-Bligh R. Markedly elevated serum thyroglobulin associated with heterophile antibodies: a cautionary tale. Thyroid. 2013;23(6):771-772.
Netzel BC, Grebe SK, Algeciras-Schimnich A. Usefulness of a thyroglobulin liquid chromatography-tandem mass spectrometry assay for evaluation of suspected heterophile interference. Clin Chem. 2014;60(7):1016-1018.
Guastapaglia L, Chiamolera MI, Viana Lima Junior J, Ferrer CMF, Godoy Viana L, Veiga Chang C, Andrade Siqueira R, Monteiro Barros Maciel R, Henriques Vieira JG, Biscolla RPM. False diagnosis of recurrent thyroid carcinoma: the importance of testing for heterophile antibodies. Archives of endocrinology and metabolism.2024;68:e230115.
Cheng F, Chen J, Wu BL, Yang X, Huang YS. A Rare Case of Falsely Elevated High-Sensitivity Cardiac Troponin T due to Antibody Interference and a Literature Review. Clin Lab. 2023;69(8).
Spencer CA. Recoveries cannot be used to authenticate thyroglobulin (Tg) measurements when sera contain Tg autoantibodies. Clin Chem. 1996;42(5):661-663.
Weigle WO, High GJ. The behaviour of autologous thyroglobulin in the circulation of rabbits immunized with either heterologous or altered homologous thyroglobulin. J Immunol. 1967;98:1105-1114.
Feldt-Rasmussen U. Serum thyroglobulin and thyroglobulin autoantibodies in thyroid diseases. Pathogenic and diagnostic aspects. Allergy. 1983;38(6):369-387.
Sellitti DF, Suzuki K. Intrinsic regulation of thyroid function by thyroglobulin. Thyroid. 2014;24(4):625-638.
Igawa T, Haraya K, Hattori K. Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens from circulation. Immunological reviews. 2016;270(1):132-151.
Latrofa F, Ricci D, Bottai S, Brozzi F, Chiovato L, Piaggi P, Marinò M, Vitti P. Effect of Thyroglobulin Autoantibodies on the Metabolic Clearance of Serum Thyroglobulin. Thyroid. 2018;28(3):288-294.
Tozzoli R, Bizzaro N, Tonutti E, Pradella M, Manoni F, Vilalta D, Bassetti D, Piazza A, Rizzotti P. Immunoassay of anti-thyroid autoantibodies: high analytical variability in second generation methods. Clin Chem Lab Med.2002;40(6):568-573.
Rosário PW, Maia FF, Fagundes TA, Vasconcelos FP, Cardoso LD, Purisch S. Antithyroglobulin antibodies in patients with differentiated thyroid carcinoma: methods of detection, interference with serum thyroglobulin measurement and clinical significance. Arquivos brasileiros de endocrinologia e metabologia. 2004;48(4):487-492.
Feldt-Rasmussen U, Petersen PH, Date J, Madsen CM. Sequential changes in serum thyroglobulin (Tg) and its autoantibodies (TgAb) following subtotal thyroidectomy of patients with preoperatively detectable TgAb. Clin Endocrinol. 1980;12:29-38.
Richards DB, Cookson LM, Berges AC, Barton SV, Lane T, Ritter JM, Fontana M, Moon JC, Pinzani M, Gillmore JD, Hawkins PN, Pepys MB. Therapeutic Clearance of Amyloid by Antibodies to Serum Amyloid P Component. N Engl J Med. 2015;373(12):1106-1114.
Schneider AB, Pervos R. Radioimmunoassay of human thyroglobulin: effect of antithyroglobulin autoantibodies. J Clin Endocrinol Metab. 1978;47:126-137.
Feldt-Rasmussen U, Rasmussen A K. Serum thyroglobulin (Tg)in presence of thyroglobulin autoantibodies (TgAb). Clinical and methodological relevance of the interaction between Tg and TgAb in vivo and in vitro. J Endocrinol Invest. 1985;8:571-576.
Benvenga S, Burek CL, Talor M, Rose NR, Trimarchi F. Heterogeneity of the thyroglobulin epitopes associated with circulating thyroid hormone autoantibodies in hashimoto's thyroiditis and non-autoimmune thyroid diseases. J Endocrinol Invest. 2002;25(11):977-982.
Crane MS, Strachan MW, Toft AD, Beckett GJ. Discordance in thyroglobulin measurements by radioimmunoassay and immunometric assay: a useful means of identifying thyroglobulin assay interference. Ann Clin Biochem.2013;50(Pt 5):421-432.
Spencer C, Fatemi S. Thyroglobulin antibody (TgAb) methods - Strengths, pitfalls and clinical utility for monitoring TgAb-positive patients with differentiated thyroid cancer. Best practice & research Clinical endocrinology & metabolism. 2013;27(5):701-712.
Van Herle AJ, Uller RP. Elevated serum thyroglobulin: a marker of metastases in differentiated thyroid carcinomas. J Clin Invest. 1975;56:272-277.
Spencer CA, Platler BW, Nicoloff JT. The effect of 125-I thyroglobulin tracer heterogeneity on serum Tg RIA measurement. Clin Chim Acta. 1985;153:105-115.
Spencer CA, Platler B, Guttler RB, Nicoloff JT. Heterogeneity of 125-I labelled thyroglobulin preparations. Clin Chim Acta. 1985;151:121-132.
Black EG, Hoffenberg R. Should one measure serum thyroglobulin in the presence of anti-thyroglobulin antibodies? Clin Endocrinol. 1983;19:597-601.
Mariotti S, Barbesino G, Caturegli P, Marino M, Manetti L, Pacini F, Centoni R, Pinchera A. Assay of thyroglobulin in serum with thyroglobulin autoantibodies: an unobtainable goal? J Clin Endocrinol Metab. 1995;80:468-472.
Gholve C, Kumarasamy J, Damle A, Kulkarni S, Venkatesh M, Banerjee S, Rajan MGR. Comparison of Serum Thyroglobulin Levels in Differentiated Thyroid Cancer Patients Using In-House Developed Radioimmunoassay and Immunoradiometric Procedures. Indian J Clin Biochem. 2019;34(4):465-471.
L E M Miles CNH. Labelled antibodies and immunological assay systems. Nature. 1968;219:186-189.
Latrofa F, Ricci D, Grasso L, Vitti P, Masserini L, Basolo F, Ugolini C, Mascia G, Lucacchini A, Pinchera A. Characterization of thyroglobulin epitopes in patients with autoimmune and non-autoimmune thyroid diseases using recombinant human monoclonal thyroglobulin autoantibodies. J Clin Endocrinol Metab. 2008;93(2):591-596.
Shuford CM, Johnson JS, Thompson JW, Holland PL, Hoofnagle AN, Grant RP. More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a µLC-MS/MS system. Clin Mass Spectrom. 2020;15:29-35.
Feldt-Rasmussen U, Verburg FA, Luster M, Cupini C, Chiovato L, Duntas L, Elisei R, Rimmele H, Seregni E, Smit JW, Theimer C, Giovanella L. Thyroglobulin autoantibodies as surrogate biomarkers in the management of patients with differentiated thyroid carcinoma. Curr Med Chem. 2014;21(32):3687-3692.
Hsieh CJ, Wang PW. Sequential changes of serum antithyroglobulin antibody levels are a good predictor of disease activity in thyroglobulin-negative patients with papillary thyroid carcinoma. Thyroid. 2014;24(3):488-493.
Matrone A, Latrofa F, Torregrossa L, Piaggi P, Gambale C, Faranda A, Ricci D, Agate L, Molinaro E, Basolo F, Vitti P, Elisei R. Changing Trend of Thyroglobulin Antibodies in Patients With Differentiated Thyroid Cancer Treated With Total Thyroidectomy Without (131)I Ablation. Thyroid. 2018;28(7):871-879.
Thomas D, Liakos V, Vassiliou E, Hatzimarkou F, Tsatsoulis A, Kaldrimides P. Possible reasons for different pattern disappearance of thyroglobulin and thyroid peroxidase autoantibodies in patients with differentiated thyroid carcinoma following total thyroidectomy and iodine-131 ablation. J Endocrinol Invest. 2007;30(3):173-180.
Hammarlund E, Thomas A, Amanna IJ, Holden LA, Slayden OD, Park B, Gao L, Slifka MK. Plasma cell survival in the absence of B cell memory. Nat Commun. 2017;8(1):1781.
Tsushima Y, Miyauchi A, Ito Y, Kudo T, Masuoka H, Yabuta T, Fukushima M, Kihara M, Higashiyama T, Takamura Y, Kobayashi K, Miya A, Kikumori T, Imai T, Kiuchi T. Prognostic significance of changes in serum thyroglobulin antibody levels of pre- and post-total thyroidectomy in thyroglobulin antibody-positive papillary thyroid carcinoma patients. Endocr J. 2013;60(7):871-876.
Uller RP, Van Herle AJ. Effect of therapy on serum thyroglobulin levels in patients with Graves' disease. J Clin Endocrinol Metab. 1978;46:747-755.
Feldt-Rasmussen U, Blichert-Toft M, Christiansen C, Date J. Serum thyroglobulin and its autoantibody following subtotal thyroid resection of Graves' disease. Eur J Clin Invest. 1982;12(3):203-208.
Benvenga S, Bartolone L, Squadrito S, Trimarchi F. Thyroid hormone autoantibodies elicited by diagnostic fine needle biopsy. J Clin Endocrinol Metab. 1997;82(12):4217-4223.
Polyzos SA, Anastasilakis AD. Alterations in serum thyroid-related constituents after thyroid fine-needle biopsy: a systematic review. Thyroid. 2010;20(3):265-271.
Al-Hilli Z, Strajina V, McKenzie TJ, Thompson GB, Farley DR, Regina Castro M, Algeciras-Schimnich A, Richards ML. Thyroglobulin Measurement in Fine-Needle Aspiration Improves the Diagnosis of Cervical Lymph Node Metastases in Papillary Thyroid Carcinoma. Ann Surg Oncol. 2017;24(3):739-744.
Feldt-Rasmussen U, Bech K, Date J, Hyltoft Pedersen P, Johansen K, Nistrup Madsen S. Thyroid stimulating antibodies, thyroglobulin antibodies and serum proteins during treatment of Graves' disease with radioiodine or propylthiouracil. Allergy. 1982;37(3):161-167.
Feldt-Rasmussen U, Bech K, Date J, Petersen PH, Johansen K. A prospective study of the differential changes in serum thyroglobulin and its autoantibodies during propylthiouracil or radioiodine therapy of patients with Graves' disease. Acta Endocrinol (Copenh). 1982;99(3):379-385.
Yin N, Sherman SI, Pak Y, Litofsky DR, Gianoukakis AG. The De Novo Detection of Anti-Thyroglobulin Antibodies and Differentiated Thyroid Cancer Recurrence. Thyroid. 2020;30(10):1490-1495.
Donegan D, McIver B, Algeciras-Schimnich A. Clinical consequences of a change in anti-thyroglobulin antibody assays during the follow-up of patients with differentiated thyroid cancer. Endocr Pract. 2014;20(10):1032-1036.
Davies L, Welch HG. Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg.2006;135(3):451-457.
Pellegriti G, Frasca F, Regalbuto C, Squatrito S, Vigneri R. Worldwide increasing incidence of thyroid cancer: update on epidemiology and risk factors. J Cancer Epidemiol. 2013;2013:965212.
Lim H, Devesa SS, Sosa JA, Check D, Kitahara CM. Trends in Thyroid Cancer Incidence and Mortality in the United States, 1974-2013. Jama. 2017;317(13):1338-1348.
Bell R, Weinberger DM, Venkatesh M, Fernandes-Taylor S, Francis DO, Davies L. Thyroid Cancer Incidence During 2020 to 2021 COVID-19 Variant Waves. JAMA Otolaryngol Head Neck Surg. 2024;150(11):969-977.
Morosán YJ, Parisi C, Urrutia MA, Rosmarin M, Schnitman M, Serrano L, Luciani W, Faingold C, Pitoia F, Brenta G. Dynamic prediction of the risk of recurrence in patients over 60 years of age with differentiated thyroid carcinoma. Archives of endocrinology and metabolism. 2016;60(4):348-354.
Hay ID, Johnson TR, Kaggal S, Reinalda MS, Iniguez-Ariza NM, Grant CS, Pittock ST, Thompson GB. Papillary Thyroid Carcinoma (PTC) in Children and Adults: Comparison of Initial Presentation and Long-Term Postoperative Outcome in 4432 Patients Consecutively Treated at the Mayo Clinic During Eight Decades (1936-2015). World J Surg. 2018;42(2):329-342.
Sanabria A, Ferraz C, Ku CHC, Padovani R, Palacios K, Paz JL, Roman A, Smulever A, Vaisman F, Pitoia F. Implementing active surveillance for low-risk thyroid carcinoma into clinical practice: collaborative recommendations for Latin America. Archives of endocrinology and metabolism. 2024;68:e230371.
Maniam P, Hey SY, Evans-Harding N, Li L, Conn B, Adamson RM, Hay AJ, Lyall M, Nixon IJ. Practice patterns in management of differentiated thyroid cancer since the 2014 British Thyroid Association (BTA) guidelines. Surgeon.2024;22(1):e54-e60.
Smallridge RC, Diehl N, Bernet V. Practice trends in patients with persistent detectable thyroglobulin and negative diagnostic radioiodine whole body scans: a survey of American Thyroid Association members. Thyroid.2014;24(10):1501-1507.
Rinaldi S, Plummer M, Biessy C, Tsilidis KK, Østergaard JN, Overvad K, Tjønneland A, Halkjaer J, Boutron-Ruault MC, Clavel-Chapelon F, Dossus L, Kaaks R, Lukanova A, Boeing H, Trichopoulou A, Lagiou P, Trichopoulos D, Palli D, Agnoli C, Tumino R, Vineis P, Panico S, Bueno-de-Mesquita HB, Peeters PH, Weiderpass E, Lund E, Quirós JR, Agudo A, Molina E, Larrañaga N, Navarro C, Ardanaz E, Manjer J, Almquist M, Sandström M, Hennings J, Khaw KT, Schmidt J, Travis RC, Byrnes G, Scalbert A, Romieu I, Gunter M, Riboli E, Franceschi S. Thyroid-stimulating hormone, thyroglobulin, and thyroid hormones and risk of differentiated thyroid carcinoma: the EPIC study. J Natl Cancer Inst. 2014;106(6):dju097.
Petric R, Besic H, Besic N. Preoperative serum thyroglobulin concentration as a predictive factor of malignancy in small follicular and Hürthle cell neoplasms of the thyroid gland. World J Surg Oncol. 2014;12:282.
Tian D, Li X, Jia Z. Analysis of Risk Factors and Risk Prediction for Cervical Lymph Node Metastasis in Thyroid Papillary Carcinoma. Cancer Manag Res. 2024;16:1571-1585.
Rosignolo F, Maggisano V, Sponziello M, Celano M, Di Gioia CR, D'Agostino M, Giacomelli L, Verrienti A, Dima M, Pecce V, Durante C. Reduced expression of THRβ in papillary thyroid carcinomas: relationship with BRAF mutation, aggressiveness and miR expression. J Endocrinol Invest. 2015;38(12):1283-1289.
Giovanella L, Ceriani L, Ghelfo A, Maffioli M, Keller F. Preoperative undetectable serum thyroglobulin in differentiated thyroid carcinoma: incidence, causes and management strategy. Clin Endocrinol (Oxf).2007;67(4):547-551.
Durante C, Puxeddu E, Ferretti E, Morisi R, Moretti S, Bruno R, Barbi F, Avenia N, Scipioni A, Verrienti A, Tosi E, Cavaliere A, Gulino A, Filetti S, Russo D. BRAF mutations in papillary thyroid carcinomas inhibit genes involved in iodine metabolism. J Clin Endocrinol Metab. 2007;92(7):2840-2843.
Tallini G, de Biase D, Durante C, Acquaviva G, Bisceglia M, Bruno R, Bacchi Reggiani ML, Casadei GP, Costante G, Cremonini N, Lamartina L, Meringolo D, Nardi F, Pession A, Rhoden KJ, Ronga G, Torlontano M, Verrienti A, Visani M, Filetti S. BRAF V600E and risk stratification of thyroid microcarcinoma: a multicenter pathological and clinical study. Mod Pathol. 2015;28(10):1343-1359.
Haugen BR, Ladenson PW, Cooper DS, Pacini F, Reiners C, Luster M, Schlumberger M, Sherman SI, Samuels M, Graham K, Braverman LE, Skarulis MC, Davies TF, DeGroot L, Mazzaferri EL, Daniels GH, Ross DC, Becker DV, Mazon HR, Cavalieri RR, Spencer CA, McEllin K, Weintraub BD, EC. R. A comparison of Recombinant Human Thyrotropin and Thyroid Hormone Withdrawal for the Detection of Thyroid Remnant or Cancer. J Clin Endocrinol Metab. 1999;84:3877-3885.
Giovanella L, Ceriani L, Suriano S, Ghelfo A, Maffioli M. Thyroglobulin measurement before rhTSH-aided 131I ablation in detecting metastases from differentiated thyroid carcinoma. Clin Endocrinol (Oxf). 2008;69(4):659-663.
Zhu Y, Yang X, Liu Z, Zhang Q, Li Z, Hou X, Zhu H. Predictive value of thyroglobulin after radioiodine therapy for excellent response to treatment in postoperative thyroid cancer. Nucl Med Commun. 2024.
Feldt-Rasmussen U, Petersen PH, Nielsen H, Date J. Thyroglobulin of varying molecular sizes with different disappearence rates in plasma following subtotal thyroidectomy. Clin Endocrinol (Oxf). 1978;9:205-?????
Durante C, Montesano T, Attard M, Torlontano M, Monzani F, Costante G, Meringolo D, Ferdeghini M, Tumino S, Lamartina L, Paciaroni A, Massa M, Giacomelli L, Ronga G, Filetti S. Long-term surveillance of papillary thyroid cancer patients who do not undergo postoperative radioiodine remnant ablation: is there a role for serum thyroglobulin measurement? J Clin Endocrinol Metab. 2012;97(8):2748-2753.
Padovani RP, Robenshtok E, Brokhin M, Tuttle RM. Even without additional therapy, serum thyroglobulin concentrations often decline for years after total thyroidectomy and radioactive remnant ablation in patients with differentiated thyroid cancer. Thyroid. 2012;22(8):778-783.
Tuttle RM, Leboeuf R. Follow up approaches in thyroid cancer: a risk adapted paradigm. Endocrinol Metab Clin North Am. 2008;37(2):419-435, ix-x.
Miyauchi A, Kudo T, Miya A, Kobayashi K, Ito Y, Takamura Y, Higashiyama T, Fukushima M, Kihara M, Inoue H, Tomoda C, Yabuta T, Masuoka H. Prognostic impact of serum thyroglobulin doubling-time under thyrotropin suppression in patients with papillary thyroid carcinoma who underwent total thyroidectomy. Thyroid.2011;21(7):707-716.
Couto JS, Almeida MFO, Trindade VCG, Marone MMS, Scalissi NM, Cury AN, Ferraz C, Padovani RP. A cutoff thyroglobulin value suggestive of distant metastases in differentiated thyroid cancer patients. Braz J Med Biol Res.2020;53(11):e9781.
Pacini F, Sabra MM, Tuttle RM. Clinical relevance of thyroglobulin doubling time in the management of patients with differentiated thyroid cancer. Thyroid. 2011;21(7):691-692.
Miyauchi A, Kudo T, Kihara M, Higashiyama T, Ito Y, Kobayashi K, Miya A. Relationship of biochemically persistent disease and thyroglobulin-doubling time to age at surgery in patients with papillary thyroid carcinoma. Endocr J.2013;60(4):415-421.
Giovanella L, Trimboli P, Verburg FA, Treglia G, Piccardo A, Foppiani L, Ceriani L. Thyroglobulin levels and thyroglobulin doubling time independently predict a positive 18F-FDG PET/CT scan in patients with biochemical recurrence of differentiated thyroid carcinoma. Eur J Nucl Med Mol Imaging. 2013;40(6):874-880.
Giovanella L, Garo ML, Albano D, Görges R, Ceriani L. The role of thyroglobulin doubling time in differentiated thyroid cancer: a meta-analysis. Endocr Connect. 2022;11(4).
Ito Y, Miyauchi A. Prognostic factors of papillary and follicular carcinomas based on pre-, intra-, and post-operative findings. Eur Thyroid J. 2024;13(5).
Schlumberger M CP, Fragu P, Lumbroso J, Parmentier C and Tubiana M,. Circulating thyrotropin and thyroid hormones in patients with metastases of differentiated thyroid carcinoma: relationship to serum thyrotropin levels. J Clin Endocrinol Metab. 1980;51:513-519.
Robbins RJ, Srivastava S, Shaha A, Ghossein R, Larson SM, Fleisher M, Tuttle RM. Factors influencing the basal and recombinant human thyrotropin-stimulated serum thyroglobulin in patients with metastatic thyroid carcinoma. J Clin Endocrinol Metab. 2004;89(12):6010-6016.
Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, Mazzaferri EL, McIver B, Sherman SI, Tuttle RM. Management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid.2006;16(2):109-142.
Pacini F, Castagna MG. Diagnostic and therapeutic use of recombinant human TSH (rhTSH) in differentiated thyroid cancer. Best practice & research Clinical endocrinology & metabolism. 2008;22(6):1009-1021.
Mazzaferri EL, Robbins RJ, Spencer CA, Braverman LE, Pacini F, Wartofsky L, Haugen BR, Sherman SI, Cooper DS, Braunstein GD, Lee S, Davies TF, Arafah BM, Ladenson PW, Pinchera A. A consensus report of the role of serum thyroglobulin as a monitoring method for low-risk patients with papillary thyroid carcinoma. J Clin Endocrinol Metab. 2003;88(4):1433-1441.
Braverman L, Kloos RT, Law B, Jr., Kipnes M, Dionne M, Magner J. Evaluation of various doses of recombinant human thyrotropin in patients with multinodular goiters. Endocr Pract. 2008;14(7):832-839.
Over R, Nsouli-Maktabi H, Burman KD, Jonklaas J. Age modifies the response to recombinant human thyrotropin. Thyroid. 2010;20(12):1377-1384.
Smallridge RC, Meek SE, Morgan MA, Gates GS, Fox TP, Grebe S, Fatourechi V. Monitoring thyroglobulin in a sensitive immunoassay has comparable sensitivity to recombinant human tsh-stimulated thyroglobulin in follow-up of thyroid cancer patients. J Clin Endocrinol Metab. 2007;92(1):82-87.
Mazzaferri EL. Will highly sensitive thyroglobulin assays change the management of thyroid cancer? Clin Endocrinol (Oxf). 2007;67(3):321-323.
Chindris AM, Casler JD, Bernet VJ, Rivera M, Thomas C, Kachergus JM, Necela BM, Hay ID, Westphal SA, Grant CS, Thompson GB, Schlinkert RT, Thompson EA, Smallridge RC. Clinical and molecular features of Hürthle cell carcinoma of the thyroid. J Clin Endocrinol Metab. 2015;100(1):55-62.
Benmoussa JA, Chen K, Najjar S, Applewhite M, Warshaw J. Lateral neck Cystic Mass: The Role of Thyroglobulin Measurement in Fine Needle Aspiration. Endocr Pract. 2018;24(8):767.
Rotman-Pikielny P, Reynolds JC, Barker WC, Yen PM, Skarulis MC, Sarlis NJ. Recombinant human thyrotropin for the diagnosis and treatment of a highly functional metastatic struma ovarii. J Clin Endocrinol Metab.2000;85(1):237-244.
Russo M, Marturano I, Masucci R, Caruso M, Fornito MC, Tumino D, Tavarelli M, Squatrito S, Pellegriti G. Metastatic malignant struma ovarii with coexistence of Hashimoto's thyroiditis. Endocrinol Diabetes Metab Case Rep. 2016;2016:160030.
Trimboli P, Guidobaldi L, Bongiovanni M, Crescenzi A, Alevizaki M, Giovanella L. Use of fine-needle aspirate calcitonin to detect medullary thyroid carcinoma: A systematic review. Diagn Cytopathol. 2016;44(1):45-51.
Randolph GW, Duh QY, Heller KS, LiVolsi VA, Mandel SJ, Steward DL, Tufano RP, Tuttle RM. The prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid. 2012;22(11):1144-1152.
Lin SY, Li MY, Zhou CP, Ao W, Huang WY, Wang SS, Yu JF, Tang ZH, Abdelhamid Ahmed AH, Wang TY, Wang ZH, Hua S, Randolph GW, Zhao WX, Wang B. Accurate preoperative prediction of nodal metastasis in papillary thyroid microcarcinoma: Towards optimal management of patients. Head Neck. 2024;46(5):1009-1019.
Uruno T, Miyauchi A, Shimizu K, Tomoda C, Takamura Y, Ito Y, Miya A, Kobayashi K, Matsuzuka F, Amino N, Kuma K. Usefulness of thyroglobulin measurement in fine-needle aspiration biopsy specimens for diagnosing cervical lymph node metastasis in patients with papillary thyroid cancer. World J Surg. 2005;29(4):483-485.
Boi F, Baghino G, Atzeni F, Lai ML, Faa G, Mariotti S. The diagnostic value for differentiated thyroid carcinoma metastases of thyroglobulin (Tg) measurement in washout fluid from fine-needle aspiration biopsy of neck lymph nodes is maintained in the presence of circulating anti-Tg antibodies. J Clin Endocrinol Metab. 2006;91(4):1364-1369.
Snozek CL, Chambers EP, Reading CC, Sebo TJ, Sistrunk JW, Singh RJ, Grebe SK. Serum thyroglobulin, high-resolution ultrasound, and lymph node thyroglobulin in diagnosis of differentiated thyroid carcinoma nodal metastases. J Clin Endocrinol Metab. 2007;92(11):4278-4281.
Torres MR, Nóbrega Neto SH, Rosas RJ, Martins AL, Ramos AL, da Cruz TR. Thyroglobulin in the washout fluid of lymph-node biopsy: what is its role in the follow-up of differentiated thyroid carcinoma? Thyroid. 2014;24(1):7-18.
Tralongo P, Bruno C, Policardo F, Vegni F, Feraco A, Carlino A, Ferraro G, Milardi D, Navarra E, Pontecorvi A, Lombardi CP, Raffaelli M, Larocca LM, Pantanowitz L, Rossi ED. Diagnostic role of FNA cytology in the evaluation of cervical lymph nodes in thyroid cancers: Combined evaluation of thyroglobulin in eluate from FNA cytology. Cancer Cytopathol. 2023;131(11):693-700.
Grani G, Sponziello M, Filetti S, Durante C. Thyroid nodules: diagnosis and management. Nat Rev Endocrinol.2024;20(12):715-728.
Sakamoto K, Ozawa H, Sato Y, Nakaishi M, Sakanushi A, Matsunobu T, Okubo K, Shinden S. Cutoff value of thyroglobulin in needle aspirates for screening neck masses of thyroid carcinoma. Endocr Relat Cancer.2024;31(12).
García-Molina F, Arense-Gonzalo JJ, Aguera-Sanchez A, Peña-Ros E, Ruiz-Marín M, Matínez-Perez M, Chaves-Benito A, Martínez-Diaz F. [Diagnosis of lymph node metastases from papillary thyroid carcinoma by measuring thyroglobulin in the puncture needle. Calculation of optimal cut-off point in our series]. Rev Esp Patol.2024;57(4):258-264.
Chen J, Lin Z, Xu B, Lu T, Zhang X. The efficacy and assessment value of the level of thyroglobulin wash-out after fine-needle aspiration cytodiagnosis in the evaluation of lymph node metastasis in papillary thyroid carcinoma. World J Surg Oncol. 2024;22(1):149.
Jeon MJ, Kim WG, Jang EK, Choi YM, Lee YM, Sung TY, Yoon JH, Chung KW, Hong SJ, Baek JH, Lee JH, Kim TY, Shong YK, Kim WB. Thyroglobulin level in fine-needle aspirates for preoperative diagnosis of cervical lymph node metastasis in patients with papillary thyroid carcinoma: two different cutoff values according to serum thyroglobulin level. Thyroid. 2015;25(4):410-416.
Boi F, Maurelli I, Pinna G, Atzeni F, Piga M, Lai ML, Mariotti S. Calcitonin measurement in wash-out fluid from fine needle aspiration of neck masses in patients with primary and metastatic medullary thyroid carcinoma. J Clin Endocrinol Metab. 2007;92(6):2115-2118.
Abraham D, Gault PM, Hunt J, Bentz J. Calcitonin estimation in neck lymph node fine-needle aspirate fluid prevents misinterpretation of cytology in patients with metastatic medullary thyroid cancer. Thyroid.2009;19(9):1015-1016.
Ringel DM, Balducci-Silano PL, Anderson JS, Spencer CA, Silverman J, Sparling YH, Francis GL, Burman KD, Wartofsky L, Ladenson PW, Levine MA, Tuttle RM. Quantitative reverse transcriptase polymerase chain reaction of circulating thyroglobulin messenger RNAfor monitoring patients with thyroid carcinoma. J Clin Endocrinol Metab.1999;84:4037-4042.
Barzon L, Boscaro M, Pacenti M, Taccaliti A, Palù G. Evaluation of circulating thyroid-specific transcripts as markers of thyroid cancer relapse. Int J Cancer. 2004;110(6):914-920.
Gupta M, Chia SY. Circulating thyroid cancer markers. Current opinion in endocrinology, diabetes, and obesity.2007;14(5):383-388.
Grammatopoulos D, Elliott Y, Smith SC, Brown I, Grieve RJ, Hillhouse EW, Levine MA, Ringel MD. Measurement of thyroglobulin mRNA in peripheral blood as an adjunctive test for monitoring thyroid cancer. Mol Pathol.2003;56(3):162-166.
Ausavarat S, Sriprapaporn J, Satayaban B, Thongnoppakhun W, Laipiriyakun A, Amornkitticharoen B, Chanachai R, Pattanachak C. Circulating thyrotropin receptor messenger ribonucleic acid is not an effective marker in the follow-up of differentiated thyroid carcinoma. Thyroid Res. 2015;8:11.
Biscolla RP, Cerutti JM, Maciel RM. Detection of recurrent thyroid cancer by sensitive nested reverse transcription-polymerase chain reaction of thyroglobulin and sodium/iodide symporter messenger ribonucleic acid transcripts in peripheral blood. J Clin Endocrinol Metab. 2000;85(10):3623-3627.
Ringel MD. Molecular detection of thyroid cancer: differentiating "signal" and "noise" in clinical assays. J Clin Endocrinol Metab. 2004;89(1):29-32.
Kaufmann S, Schmutzler C, Schomburg L, Körber C, Luster M, Rendl J, Reiners C, Köhrle J. Real time RT-PCR analysis of thyroglobulin mRNA in peripheral blood in patients with congenital athyreosis and with differentiated thyroid carcinoma after stimulation with recombinant human thyrotropin. Endocr Regul. 2004;38(2):41-49.
Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci U S A. 1989;86(8):2617-2621.
Ghossein RA, Bhattacharya S. Molecular detection and characterization of circulating tumor cells and micrometastases in prostatic, urothelial, and renal cell carcinomas. Semin Surg Oncol. 2001;20(4):304-311.
Savagner F, Rodien P, Reynier P, Rohmer V, Bigorgne JC, Malthiery Y. Analysis of Tg transcripts by real-time RT-PCR in the blood of thyroid cancer patients. J Clin Endocrinol Metab. 2002;87(2):635-639.
Elisei R, Vivaldi A, Agate L, Molinaro E, Nencetti C, Grasso L, Pinchera A, Pacini F. Low specificity of blood thyroglobulin messenger ribonucleic acid assay prevents its use in the follow-up of differentiated thyroid cancer patients. J Clin Endocrinol Metab. 2004;89(1):33-39.
Endo T, Kobayashi T. Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis. Am J Physiol Endocrinol Metab. 2008;295(2):E514-518.
Cianfarani F, Baldini E, Cavalli A, Marchioni E, Lembo L, Teson M, Persechino S, Zambruno G, Ulisse S, Odorisio T, D'Armiento M. TSH receptor and thyroid-specific gene expression in human skin. J Invest Dermatol.2010;130(1):93-101.
Campennì A, Aguennouz M, Siracusa M, Alibrandi A, Polito F, Oteri R, Baldari S, Ruggeri RM, Giovanella L. Thyroid Cancer Persistence in Patients with Unreliable Thyroglobulin Measurement: Circulating microRNA as Candidate Alternative Biomarkers. Cancers (Basel). 2022;14(22).
Fagin JA, Nikiforov YE. Progress in Thyroid Cancer Genomics: A 40-Year Journey. Thyroid. 2023;33(11):1271-1286.

Diabetic Retinopathy

ABSTRACT

Diabetic retinopathy is a significant life-altering complication affecting patients with diabetes. Understanding its pathogenesis, prevention, and treatment is critical to delivering effective and comprehensive care for patients with diabetes at all stages. This review discusses the risk factors, epidemiology, pathogenesis, clinical features, and treatment options for diabetic retinopathy, with an emphasis on practical information useful for endocrinologists and other non-ophthalmologists.

INTRODUCTION

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and a leading cause of blindness worldwide and in the US.^1–3 The individual lifetime risk of DR is estimated to be 50–60% in patients with type 2 diabetes and over 90% in patients with type 1 diabetes.⁴ It is one of the most frequent causes of blindness in adults between 20-74 years of age in developed countries.⁵ The same pathologic mechanisms that damage the kidneys and other organs affect the microcirculation of the eye.⁶ With the global epidemic of diabetes, one expects that diabetes will be the leading global cause of vision loss in many countries.^1,2 While DR is specific for diabetes, other eye disorders, such as glaucoma and cataracts, occur earlier and more frequently in people with diabetes.⁵

Often, by the time patients seek ophthalmologic examination and treatment, there are significant alterations of the retinal microvasculature. Therefore, it is important for non-ophthalmologists to recognize the importance of eye disease in patients with diabetes so that appropriate referral to eye-care specialists can be a part of their diabetes management program.

EPIDEMIOLOGY

In the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR), the prevalence of DR in patients with type 1 diabetes was 17% in those with less than 5 years of diabetes vs 98% in those with 15 or more years of diabetes.⁶Proliferative diabetic retinopathy (PDR) was absent in patients with type 1 diabetes of short duration but present in 48% of those with 15 or more years of diabetes. In patients with type 1 diabetes, the 25-year rate of progression of DR was 83%, with progression to PDR occurring in 42% of patients.⁷ Improvement of DR was observed in 18% of patients with type 1 diabetes. In the WESDR, 3.6% of patients with type 1 diabetes were legally blind, and 86% of the blindness was attributable to DR.⁸ The risk of blindness increases with the duration of diabetes.

In the WESDR, patients with type 2 diabetes of less than 5 years had a prevalence of DR of 28%, while in patients with greater than 15 years of diabetes, the prevalence was 78%.⁶ A considerable number of patients with type 2 diabetes (12-19%) have DR at the time of the diagnosis of diabetes.¹ The prevalence of PDR was relatively low in patients with type 2 diabetes (2%) in patients with less than 5 years duration vs 16% in patients with greater than 15 years duration of diabetes.⁶ The prevalence of DR and PDR was greater in the patients with type 2 diabetes using insulin. In the patients with type 2 diabetes, 1.6% were legally blind, and one-third of cases of legal blindness were due to DR.⁸

Of note, the WESDR cohort is 99% white, and data suggest a higher prevalence of DR in Mexican-Americans and African-Americans with type 2 diabetes.^6,9,10 Asians appear to have the same or lower prevalence of DR.^1,10 DR occurs in both males and females with diabetes, but males appear to be at a slightly higher risk.⁹ Diabetic macular edema (DME) occurs more commonly in patients with type 2 diabetes, and with the marked increase in the prevalence of type 2 diabetes, DME is becoming more common.² DME is over two times more prevalent than PDR.⁹

In a pooled analysis of 35 studies between 1980 and 2008, among 22,896 individuals with diabetes, the overall prevalence of DR was 34.6%, PDR 6.96%, DME 6.81%, and vision-threatening DR 10.2%.¹¹ The longer the duration of diabetes, the greater the prevalence of all of these diabetic eye manifestations.¹¹ Moreover, the prevalence of DR, PDR, and DME was greater in patients with type 1 diabetes (77%, 32%, and 14%) compared to patients with type 2 diabetes (32%, 3, and 6%).^1,11

In developed countries the incidence and the risk of progression of DR have greatly declined in patients with type 1 and type 2 diabetes.^1,2,12 The WESDR showed that from 1980 to 2007, the estimated annual incidence of PDR decreased by 77%, and vision impairment decreased by 57% in patients with type 1 diabetes.¹² In an analysis of 28 studies with 27,120 patients, the rates of DR and PDR were lower among participants in 1986-2008 than in 1975-1985.¹³ Thus, patients with recently diagnosed type 1 or type 2 diabetes in developed countries have a much lower risk of PDR, DME, and visual impairment as compared with patients who developed diabetes in the past.^1,12 This marked decrease in the prevalence and incidence of DR and vision impairment is likely due to improved glycemia control, early screening for eye disease, and the more aggressive treatment of blood pressure.¹ However, in countries with limited medical resources, this reduced risk of DR and vision impairment is not occurring.²

In caring for patients with diabetes, health care providers must bear in mind the substantial risks of developing visual loss that these patients face and the treatments that can reduce this risk. For affected patients, diabetes-related visual loss decreases the quality of life and interferes with the performance of daily activities.

RISK FACTORS

Hyperglycemia

The most important treatable risk factor for the development of DR is hyperglycemia. In patients with both type 1 and type 2 diabetes, elevated HbA1c levels are associated with an increased risk and progression of DR.^2,7,14–16 Most importantly, randomized controlled trials comparing intensive glycemic control vs. usual care demonstrated a decrease in DR. A meta-analysis of 6 relatively small randomized trials prior to the publication of the Diabetes Control and Complications Trial (DCCT) reported that after 2 to 5 years of intensive therapy the risk of retinopathy progression was significantly reduced (OR 0.49, P = 0.011).¹⁷ Intensive therapy significantly retarded retinopathy progression to more severe states such as PDR or changes requiring laser treatment (OR 0.44, P = 0.018).¹⁷

The DCCT was a randomized, controlled study of intensive glycemic control (HbA1c approximately 7%) vs. usual care (HbA1c approximately 9%) in 1,441 patients with type 1 diabetes.¹⁸ This study found that intensive glucose control reduced the risk of developing retinopathy by 76% compared to usual care. In patients with pre-existing retinopathy, intensive control slowed progression of the DR by 54%.¹⁸ For every 10% reduction in HbA1c (e.g., 10% to 9% or 9% to 8.1%) the risk of retinopathy progression was reduced on average by 44%.¹⁹ The DCCT participants were followed in an observational Epidemiology of Diabetes Interventions and Complications (EDIC) study. During the EDIC study, the mean HbA1c levels became very similar in the intensive and usual care group, with the HbA1c of the intensive treatment group increasing to approximately 8% and the usual care group HbA1c decreasing to approximately 8%.¹⁹ Despite the similar A1c levels in the 2 groups over 30 years there continued to be an approximately 50% risk reduction of further DR progression and the development of PDR and DME in the original intensive control group, a phenomenon termed metabolic memory.¹⁹ These results indicate the need for early intensive glucose control.

In the Kumamoto study, 110 patients with type 2 diabetes were randomly assigned to a multiple insulin injection treatment group (MIT group) or to a conventional insulin injection treatment group (CIT group) and followed for 6 years.^20,21 HbA1c levels were 7.1% in the MIT group and 9.4% in the CIT group. Moreover, the development of DR after 6 years was 7.7% for the MIT group and 32.0% for the CIT group in the primary-prevention cohort (no microvascular disease at baseline) (P = 0.039), and progression of DR occurred in 19.2% of the MIT group and 44.0% of the CIT group in the secondary-intervention cohort (microvascular disease at baseline) (P = 0.049). This study demonstrated that improved glycemic control reduced DR in patients with type 2 diabetes.

In the UK Prospective Diabetes Study (UKPDS), 3,867 newly diagnosed patients with type 2 diabetes were randomized to diet therapy alone or to sulfonylureas or insulin with the goal of achieving a fasting glucose of 108 mg/dL (6mMol/L) in those treated with sulfonylureas or insulin (intensive group). Over 10 years, HbA1c levels were approximately 7.0% in the patients treated with sulfonylureas/insulin therapy compared with 7.9% in the diet group. This study found a 25% reduction in the risk of microvascular endpoints, including the need for retinal laser treatment, with intensive glucose control.²² A risk reduction of 21% per 1% decrease in HbA1c was observed in this trial. Patients were closely followed after the study ended, and HbA1c levels after one year became similar in the two groups. Similar to the results seen in the DCCT/EDIC study, the benefits on microvascular disease persisted in the intensive control group, confirming the concept of metabolic memory in patients with type 2 diabetes.²³

The ACCORD study was a randomized trial that enrolled 10,251 individuals with type 2 diabetes of a mean duration of 10 years who were at high risk for cardiovascular disease to receive either intensive or standard treatment for glycemia (HbA1c 6.4% vs. 7.5%). A subgroup of 2,856 individuals were evaluated for the effects of intensive vs. standard care at 4 years on the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale. After 4 years, the rates of progression of diabetic retinopathy were 7.3% in the intensive group vs.10.4% in the standard therapy group (odds ratio, 0.67; P=0.003).²⁴ It should be noted that in an analysis of the entire ACCORD study cohort, three-line change in visual acuity was reduced in the intensive control group (HR 0.94, CI 0.89-1.00; p=0.05) but no differences in photocoagulation, vitrectomy, or severe visual loss were observed.²⁵ Four years after the ACCORD trial ended, DR progressed in 5.8% of the intensive treatment group vs.12.7% in the standard treatment group (odds ratio 0.42, P < 0.0001),²⁶ once again confirming the concept of metabolic memory.

It should be noted that two large cardiovascular outcome trials, the ADVANCE trial and the VADT, failed to demonstrate a benefit of intensive glucose control on diabetic retinopathy.^27,28 However, these trials enrolled patients who already had diabetes for several years prior to enrollment, so they likely had not had the opportunity to develop the metabolic memory that could yield better retinopathy outcomes. Additionally, a meta-analysis of the four large cardiovascular outcome studies in patients with type 2 diabetes (UKPDS, ACCORD, ADVANCE, and VADT) found that more intensive glucose control resulted in a decrease in HbA1c of -0.90% and a 13% reduction in the need for retinal photocoagulation therapy or vitrectomy, development of PDR, or progression of DR.²⁹ Another meta-analysis of 7 trials with 10,793 participants reported a 20% decrease in DR with intensive glycemic control (0.80, 0.67 to 0.94; P=0.009).³⁰

Taken together, these results clearly demonstrate that in patients with both type 1 and type 2 diabetes, improvements in glycemic control will reduce the risk of the development and progression of DR.

Rapid Improvement in Glycemic Control

Deterioration of DR, upon initiation of intensive diabetes treatment, was described in the 1980s in patients with type 1 diabetes who were treated intensively with continuous subcutaneous insulin infusions.^31–34 In patients with poor glycemic control and DR, rapidly improving glycemic control can worsen DR and, in some instances, result in PDR or DME. This worsening can occur as soon as 3 months after initiating intensive glycemic control. In the DCCT early worsening was observed at the 6- and/or 12-month visit in 13.1% of patients in the intensive treatment group and in 7.6% of patients assigned to conventional treatment (odds ratio, 2.06; P < .001).³⁵ In the DCCT the most important risk factors for early worsening of DR were a higher HbA1c level and reduction of this level during the first 6 months of treatment.³⁵ It must be recognized that in the DCCT the long-term ophthalmic outcomes in intensively treated patients who had early worsening were similar to or more favorable than outcomes in conventionally treated patients. This early worsening of DR with improved glycemic control has also been described in patients with type 2 diabetes treated with insulin or GLP-1 agonists, following bariatric surgery, in pregnant women with diabetes, and following pancreatic transplants in patients with type 1 diabetes.³⁶ The mechanism(s) leading to early worsening of DR with improvements in glycemic control are unknown (36). The FOCUS study (NCT03811561) is evaluating this further by comparing the rate of 3-step ETDRS severity level progression, and other DR-related outcomes, at 5 years in patients on semaglutide versus placebo.

While this worsening is distressing, it must be recognized that the long-term benefits of improving glycemic control on DR greatly outweigh the risks of early worsening.

Hypertension

In the WESDR, blood pressure (BP) was not related to incidence or progression of retinopathy in the patients with type 2 diabetes using insulin or the type 2 patients not using insulin, but in the patients with type 1 diabetes systolic BP was a significant predictor of the incidence of DR.³⁷ In contrast, in the UKPDS and other studies high BP in patients with type 2 was associated with the development of DR.^2,16,38 In one prospective study the risk of DR increased by 30% for every 10 mm Hg increase in systolic BP at baseline.³⁹

While observational studies can show an association, randomized controlled trials are required to demonstrate causation and the benefits of treatment. A number of studies have examined the effect of lowering BP in patients with hypertension on the development and progression of DR.

STUDIES IN PATIENTS WITH HYPERTENSION

The UKPDS examined the effect of tight vs. less tight BP control in 1,148 hypertensive patients with type 2 diabetes.⁴⁰ In the tight BP control group (captopril and atenolol), BP was significantly reduced compared to the less tight group (144/82 mm Hg vs.154/87 mm Hg; (P<0.0001). After nine years the tight BP control group had a 34% reduction in the deterioration of retinopathy (P=0.0004) and a 47% reduced risk (P=0.004) of deterioration in visual acuity. Additionally, patients in the tight BP group were less likely to undergo photocoagulation (RR, 0.65; P = 0.03), a difference primarily due to a decrease in photocoagulation due to maculopathy (RR, 0.58; P = 0.02).⁴¹ In contrast to glycemic control, the benefits of lowering BP were not sustained when therapy was discontinued and the differences in blood pressure were not maintained, indicating the absence of metabolic memory.⁴²

The HOPE study was a randomized study that compared ramipril vs. placebo in 3,577 participants with diabetes who had a previous cardiovascular event or at least one other cardiovascular risk factor.⁴³ The baseline BP was approximately 142/80 mm Hg, and BP decreased by 1.92/3.3 mm Hg in the ramipril group vs a 0.55 mm Hg increase in systolic BP and 2.30 mm Hg decrease in diastolic BP in the placebo group. This study was not focused on DR but did report that the need for laser was 9.4% in the ramipril group vs. 10.5% in the placebo group (22% decrease; P=0.24). Another ACE inhibitor, lisinopril, was found in a two-year, placebo-controlled trial to be associated with lower rates of retinopathy progression in non-hypertensive patients with type 1 diabetes.⁴⁴

The ADVANCE study examined the effect of BP control on DR in 1,241 patients with type 2 diabetes.⁴⁵ Patients were randomized to BP-lowering agents (perindopril and indapamide) or placebo and followed for approximately 4-5 years. Baseline BP was approximately 143/79 mmHg. In the group randomized to BP medications, a decrease in systolic BP of 6.1 ± 1.2 mmHg and diastolic BP of 2.3 ± 0.6 mmHg was observed (p < 0.001 for both). Fewer patients on BP lowering therapy experienced new or worsening DR compared with those on placebo (OR 0.78; 95% CI 0.57–1.06; p = 0.12), but the difference was not quite statistically significant; as mentioned above, this could be attributable to the fact that patients had diabetes for a long period prior to enrollment. Certain secondary outcomes were significantly reduced (for example DME) in the BP lowering group, but most other eye end points were not significantly decreased compared to the placebo group.

The ACCORD eye study evaluated 2,856 patients with type 2 diabetes for the effect of intensive BP control (BP<120 mm Hg) vs standard BP control (BP<140 mm Hg) on the progression of DR after 4 years of treatment.²⁴ Systolic BP was 117 mm Hg in the intensive-therapy group and 133 mm Hg in the standard-therapy group. The progression of DR was 10.4% with intensive blood-pressure therapy vs. 8.8% with standard therapy (adjusted odds ratio, 1.23; P=0.29).

The Appropriate Blood Pressure Control in Diabetes (ABCD2) Trial was a randomized blinded trial that compared the effects of intensive versus moderate BP control in 470 patients with type 2 diabetes and hypertension.⁴⁶ The intensive group was treated with either nisoldipine or enalapril, while the usual care BP group received placebo. The mean blood pressure achieved was 132/78 mm Hg in the intensive group and 138/86 mm Hg in the moderate group. Over the 5-year follow-up period, there was no difference in the progression of DR between the intensive and moderate groups.

Thus, in patients with hypertension, randomized trials of lowering BP have not consistently shown beneficial effects on DR.

BASIS FOR VARIABILITY

There are numerous possible explanations for the differences in results between these studies. First, the duration of diabetes prior to enrollment in a diabetes-management trial will impact the subsequent outcomes and risk of developing complications. Second, the severity of the hypertension may be important, with greater responses in individuals with higher BP levels. Third, the magnitude of the reduction in BP may be important, with greater benefit with greater decreases in BP. Fourth, the duration of the study may be an important variable, with the longer the study the greater the chances of benefits. Fifth, the presence of DR at baseline and the severity of DR at baseline may influence the response to BP lowering. Sixth, patient variables such as glycemic control, age, diabetes type, duration of diabetes, etc., may influence results. Finally, the drugs used to lower BP may be a key variable as described below.

STUDIES IN PATIENTS WITH NORMAL BP

Because of the potential benefits of angiotensin converting enzyme inhibitors (ACE inhibitors) and angiotensin receptor inhibitors (ARBs) (Renin-Angiotensin System (RAS) inhibitors) on microvascular disease independent of BP effects, a number of studies have explored the effects of these drugs on DR in patients without elevated BP. Below we briefly describe the largest of these studies.

The EUCLID trial was a randomized double-blind placebo-controlled trial in 354 patients with type 1 diabetes who were not hypertensive and were normoalbuminuric (85%) or microalbuminuric.⁴⁴ Study participants were randomized to lisinopril or placebo and followed for 2 years. Systolic BP was 3 mm Hg lower in the lisinopril group than in the placebo group. DR progressed in 23.4% of patients in the placebo group and 13.2% of patients in the lisinopril group (p=0.02). Notably progression to PDR was also reduced in the lisinopril treated group.

The Appropriate Blood Pressure Control in Diabetes (ABCD1) trial was a randomized trial in 480 normotensive type 2 diabetic subjects of more intensive vs. usual BP control.⁴⁷ The intensive group was treated with either nisoldipine or enalapril, while the usual care BP group received placebo. Mean BP in the intensive group was 128/75 mm Hg vs. 137/81 mm Hg in the placebo group (P < 0.0001). After a mean follow-up of 5.4 years, the intensive BP control group demonstrated less progression of diabetic retinopathy (34% vs. 46%, P = 0.019). PDR developed in 0% of patients in the intensive therapy group vs. 3.9% in the placebo group. However, in patients who at baseline did not have DR, the number of patients developing retinopathy was similar in the two groups (39% of patients in the intensive therapy group vs. 42% in the placebo group).

The DIRECT- Prevent 1 trial was a randomized, double-blind, placebo-controlled trial in 1,421 normotensive, normoalbuminuric individuals with type 1 diabetes without retinopathy.⁴⁸ Patients were randomized to candesartan or placebo and followed for 4.7 years. Mean systolic and diastolic BP was reduced by 2.6 mm Hg and 2.7 mm Hg, respectively, in the candesartan group vs. the placebo group. DR developed in 25% of the participants in the candesartan group vs. 31% in the placebo group (18% decrease).

The Direct Protect 1 was a randomized, double-blind, placebo-controlled trial in 1,905 normotensive, normoalbuminuric patients with type 1 diabetes with existing retinopathy.⁴⁸ Patients were randomized to candesartan or placebo and followed for 4.7 years. Mean systolic and diastolic BP was reduced by 3.6 mm Hg and 2.5 mm Hg, respectively, in the candesartan group versus the placebo group. There was an identical 13% progression of DR in the placebo and candesartan groups, and progression to the combined secondary endpoint of PDR or clinically significant DME, or both, did not differ between the two groups.

The DIRECT-Protect 2 trial was a randomized, double-blind, placebo-controlled trial in 1,905 normoalbuminuric, normotensive, or treated hypertensive people with type 2 diabetes with mild to moderately severe retinopathy.⁴⁹ Patients were randomized to candesartan or placebo and followed for 4.7 years. The decrease in systolic/diastolic blood pressure was 4.3/2.5 mm Hg greater in the candesartan group than in the placebo group in individuals who were receiving antihypertensive treatment at baseline (p<0·0001 for both), and for those not on anti-hypertensive therapy at baseline the decrease was 2.9/1.3 mm Hg (p=0.0003/p=0.0045). The risk of progression of retinopathy was non-significantly reduced by 13% in patients on candesartan compared to the placebo group (HR 0.87; p=0.20). However, regression on active treatment was increased by 34% (HR 1.34; p=0.009), and overall change towards less severe retinopathy by the end of the trial was observed in the candesartan group (odds 1.17; p=0.003).

The RASS trial was a controlled trial involving 223 normotensive patients with type 1 diabetes and normoalbuminuria and who were randomly assigned to receive losartan, enalapril, or placebo.⁵⁰ The systolic and diastolic BP during the study were lower in the enalapril group (113/66 mm Hg) and the losartan group (115/66 mm Hg) than in the placebo group (117/68 mm Hg) (P<0.001 for the two systolic and P≤0.02 for the two diastolic comparisons, respectively). After 5 years progression in DR occurred in 38% of patients receiving placebo but only 25% of those receiving enalapril (P=0.02) and 21% of those receiving losartan (P=0.008).

META-ANALYSIS OF ACE INHIBITORS AND ARBS

Many of the studies described above used either an ACE inhibitor or an ARB with variable results on DR. To better understand the effect of RAS inhibitors on DR, a meta-analysis has extensively examined these studies and a number of other trials.⁵¹ In 7 studies with 3,705 participants without DR, RAS inhibitors reduced the development of DR by 27% (p= 0.00006). This decrease in the development of DR was seen in patients with both type 1 and type 2 diabetes and patients who were hypertensive or normotensive. In 16 studies with 9,580 participants with pre-existing DR, RAS inhibitors decreased the progression of DR by 13% (p=0.00006). This decrease in progression of DR was seen in patients with both type 1 and type 2 diabetes and patients who were normotensive. In hypertensive patients there was a trend (7% decrease) that was not statistically significant. It should be noted that in the hypertensive patients RAS inhibitors were compared to other hypertensive drugs, and the number of hypertensive participants was relatively small (n=839). Therefore, the absence of a decrease in progression of DR in hypertensive patients is not definitive. Six studies with 2,624 participants examined the effect of RAS inhibitors on inducing regression of DR. RAS inhibitors increased the regression of DR by 39% (p=0.00002), and this beneficial effect was seen in patients with type 1 and type 2 diabetes. ACE inhibitors were more effective in reducing the development, progression, and regression of DR than ARBs. Thus, with the data available, RAS inhibitors appear to have benefits on DR above and beyond their effects on BP control.

CONCLUSION

Observational studies have shown an association of elevated BP with a higher risk of DR. As should be obvious from the above discussion, the beneficial effects of lowering BP in hypertensive patients on DR have not produced consistent results. Several large carefully carried out studies have failed to demonstrate a beneficial effect of lowering BP on DR (ACCORD, ADVANCE, ABCD2). Potential reasons for this inconsistency were discussed above. It is unlikely that future studies will provide definitive data on this issue, as lowering BP in hypertensive patients with diabetes to prevent cardiovascular disease is essential, and therefore designing clinical trials regarding DR will be very difficult. From the clinician’s viewpoint, treating hypertension in patients with diabetes to prevent cardiovascular disease is standard therapy and may also have beneficial effects DR. Similar to the beneficial effects on renal disease, RAS inhibitors appear to decrease the development and progression of DR, and therefore when treating patients with diabetes who are hypertensive, one should be preferentially consider RAS inhibitors to lower BP in patients with or at high risk of DR. In normotensive patients the available data suggests that RAS inhibition will have beneficial effects on DR, and further studies in this population are possible and would be informative.

Hyperlipidemia

Observational studies of the association of plasma lipids with DR have been inconsistent ⁵² with some studies reporting an increased risk of DR with elevated lipid levels,^53–57 while other studies have not observed a relationship between lipid levels and DR.^{10,38,58–60} Of note a Mendelian randomization study did not demonstrate a causal role of total cholesterol, LDL cholesterol, HDL cholesterol, or triglycerides on DR.⁶¹ From the clinician’s point of view the key question is whether lowering lipid levels will have a beneficial effect on DR.

FIBRATES

Small studies in the 1960’s presented evidence that treatment with clofibrate improved diabetic retinopathy.^62,63 Larger randomized studies have confirmed these observations.

The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study was a randomized trial in patients with Type 2 diabetes. Patients were randomly assigned to receive either fenofibrate 200 mg/day (n=4895) or placebo (n=4900). Laser treatment for retinopathy was significantly lower in the fenofibrate group than in the placebo group (3.4% patients on fenofibrate vs 4.9% on placebo; p=0.0002).⁶⁴ Fenofibrate therapy reduced the need for laser therapy to a similar extent for maculopathy (31% decrease) and for proliferative retinopathy (30% decrease). In the ophthalmology sub-study (n=1012), the primary endpoint of 2-step progression of retinopathy grade did not differ significantly between the fenofibrate and control groups (9.6% patients on fenofibrate vs 12.3% on placebo; p=0.19). In patients without pre-existing retinopathy there was no difference in progression (11.4% vs 11.7%; p=0.87). However, in patients with pre-existing retinopathy, significantly fewer patients on fenofibrate had a 2-step progression than did those on placebo (3.1% patients vs 14.6%; p=0.004). A composite endpoint of 2-step progression of retinopathy grade, macular edema, or laser treatments was significantly reduced in the fenofibrate group (HR 0.66, 95% CI 0.47-0.94; p=0.022).

In the ACCORD Study a subgroup of participants was evaluated for the progression of diabetic retinopathy by 3 or more steps on the Early Treatment Diabetic Retinopathy Study Severity Scale or the development of diabetic retinopathy necessitating laser photocoagulation or vitrectomy over a four-year period.²⁴ At 4 years, the rates of progression of diabetic retinopathy were 6.5% with fenofibrate therapy (n=806) vs. 10.2% with placebo (n=787) (adjusted odds ratio, 0.60; 95% CI, 0.42 to 0.87; P = 0.006). Of note, this reduction in the progression of diabetic retinopathy was of a similar magnitude as intensive glycemic treatment vs. standard therapy.

A double-blind, randomized, placebo-controlled study in 296 patients with type 2 diabetes mellitus and DR evaluated the effect of placebo or etofibrate on DR.⁶⁵ After 12 months an improvement in ocular pathology was more frequent in the etofibrate group vs the placebo group ((46% versus 32%; p< 0.001).

The MacuFen study was a small double-blind, randomized, placebo-controlled study in 110 subjects with DME who did not require immediate photocoagulation or intraocular treatment. Patients were randomized to fenofibrate for placebo for 1 year. Patients treated with fenofibrate acid had a modest improvement in total macular volume that was not statistically significant compared to the placebo group.

Based on these trials a large trial specifically designed to investigate the ocular effects of fenofibrate was carried out (The Lowering Events in Non-proliferative retinopathy in Scotland (LENS) trial).^65a In this trial 1151 patients with diabetic retinopathy or maculopathy were randomized to fenofibrate 145mg (n= 576) or placebo (n= 575) for a median of 4.0 years. Progression of diabetic retinopathy or maculopathy requiring referral or treatment (primary endpoint) occurred in 22.7% of patients in the fenofibrate group and 29.2% of patients in the placebo group (HR 0.73; 95% CI 0.58 to 0.91; P=0.006). Any progression of retinopathy or maculopathy was reduced by 26%, the development of macular edema by 50%, and the need for treatment with intravitreal injection, retinal laser, vitrectomy by 42% in the fenofibrate group.

Taken together these results indicate that fibrates have beneficial effects on the progression of diabetic retinopathy.⁶⁶The mechanisms by which fibrates decrease diabetic retinopathy are unknown, and whether decreases in serum triglyceride levels plays an important role is uncertain. Fibrates activate PPAR alpha, which is expressed in the retina.⁶⁷Diabetic PPARα KO mice developed more severe DR while overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation, suggesting that fibrates could have direct effects on the retina to reduce DR.⁶⁷

STATINS

Several large database studies have suggested that statin use reduces the development of DR.^68–71 Unfortunately, the number of randomized clinical trials testing the hypothesis that statin therapy reduces DR development or progression is very limited.

In a study by Sen and colleagues, 50 patients with diabetes mellitus (Type 1 and 2) with good glycemic control and hypercholesterolemia and having DR were randomized to simvastatin vs. placebo.⁷² Visual acuity improved in four patients using simvastatin and decreased in seven patients in the placebo group and none in the simvastatin group (P = 0.009). Fundus fluorescein angiography and color fundus photography showed improvement in one patient in the simvastatin group, while seven patients showed worsening in the placebo group (P = 0.009).

In a study by Gupta and colleagues, 30 patients with type 2 diabetes with clinically significant macular edema, dyslipidemia, and grade 4 hard exudates were randomized to receive atorvastatin or no lipid lowering drugs.⁷³ All patients received laser therapy. Ten (66.6%) of 15 patients treated with atorvastatin and two (13.3%) of 15 patients in the control group showed a reduction in hard exudates (P =.007). None of the patients treated with atorvastatin and five (33.3%) of 15 in the control group showed subfoveal lipid migration after laser photocoagulation (P =.04). Regression of macular edema was seen in nine eyes in the atorvastatin group and five in the control group (P =.27).

In a study by Narang and colleagues, 30 patients with clinically significant macular edema with a normal lipid profile were randomly treated with atorvastatin or with no lipid lowering drugs. All patients received laser therapy. After a 6-month follow-up visual acuity, macular edema and hard exudates resolution was not significantly different in the two groups.

The data on the benefit of statin therapy on DR are not very strong. Given the current recommendations to prevent cardiovascular disease, most patients with diabetes are treated with statins, and therefore it is unlikely that large, randomized trials of the effect of statin therapy on DR are feasible.

OMEGA-3-FATTY ACIDS

A Study of Cardiovascular Events in Diabetes (ASCEND) was a randomized, placebo controlled, double blind, cardiovascular outcome trial of 1-gram omega-3-fatty acids (400 mg EPA and 300 mg DHA ethyl esters) vs. olive oil placebo in 15,480 patients with diabetes without a history of cardiovascular disease (primary prevention trial).⁷⁴ Total cholesterol, HDL-C, and non-HDL-C levels were not significantly altered by omega-3-fatty acid treatment (changes in TG levels were not reported). After a mean follow-up of 7.4 years the development of retinopathy and the need for laser therapy based on self-report was similar in the omega-3-fatty acid and placebo group. Additionally, there was no difference in patients being referred for retinopathy or maculopathy.^74a Thus, at this time there is no evidence that omega-3-fatty acids influence DR.

NIACIN

It has been estimated that 0.67% of patients treated with niacin develop macular edema.⁷⁵

Pregnancy

Diabetic retinopathy may progress during pregnancy and up to one year postpartum. For additional information on retinopathy during pregnancy see the chapter in Endotext on “Diabetes in Pregnancy.”⁷⁶

Genetics

Some individuals develop DR despite good glycemic control and short duration of disease, while others do not develop DR, even with poor glycemic control and longer duration of diabetes.⁷⁷ Additionally, the strongest environmental factors (duration of diabetes and HbA1c) only explained about 11% of the variation in DR risk in the DCCT trial and 10% in the WESDR study.^12,78 Thus, factors other than glycemic control play an important role. There is a familial relationship in the development of DR, as twin and family studies indicate a genetic basis.^79,80 The differences in the prevalence of DR in different ethnic groups may be related to genetic factors.⁷⁹ Unfortunately, the identification of genetic susceptibility loci for DR through candidate gene approaches, linkage studies, and GWAS has not provided conclusive results.^79–81 From a clinician’s point of view, if there is a family history of DR, one should aggressively control risk factors for DR and ensure close eye follow-up.

SCREENING

The American Academy of Ophthalmology has recommended screening for diabetic retinopathy 5 years after diagnosis in patients with type 1 diabetes, and at the time of diagnosis in patients with type 2 diabetes. Patients without retinopathy should undergo dilated fundus examination annually. If mild non-proliferative diabetic retinopathy (NPDR) is present, exams should be repeated every 9 months. Patients with moderate NPDR should be examined every 6 months. In severe NPDR, exams should be conducted every 3 months. Patients with a new diagnosis of proliferative diabetic retinopathy should be examined every 2 to 3 months, until they are deemed stable, at which point examinations can be performed less frequently. During pregnancy, patients should be examined every 3 months, since retinopathy can progress rapidly in this setting (2019 AAO preferred practice pattern document for monitoring diabetic retinopathy: https://www.aao.org/preferred-practice-pattern/diabetic-retinopathy-ppp).

The American Diabetes Association 2024 guidelines ⁵ recommend the following:

Adults with type 1 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist within 5 years after the onset of diabetes.
Patients with type 2 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist at the time of the diabetes diagnosis.
If there is no evidence of retinopathy for one or more annual eye exams and glycemia is well controlled, then screening every 1–2 years may be considered. If any level of diabetic retinopathy is present, subsequent dilated retinal examinations should be repeated at least annually by an ophthalmologist or optometrist. If retinopathy is progressing or sight-threatening, then examinations will be required more frequently.
Programs that use retinal photography (with remote reading or use of a validated assessment tool) to improve access to diabetic retinopathy screening can be appropriate screening strategies for diabetic retinopathy. Such programs need to provide pathways for timely referral for a comprehensive eye examination when indicated.
Women with preexisting type 1 or type 2 diabetes who are planning pregnancy or who are pregnant should be counseled on the risk of development and/or progression of diabetic retinopathy.
Eye examinations should occur before pregnancy or in the first trimester in patients with preexisting type 1 or type 2 diabetes, and then patients should be monitored every trimester and for 1 year postpartum as indicated by the degree of retinopathy.

PATHOGENESIS

Various mechanisms account for the features of diabetic retinopathy. Histopathologic analysis shows the thickening of capillary basement membranes, microaneurysm formation, loss of pericytes, capillary acellularity, and neovascularization. Microaneurysms, outpouchings of the capillary wall, serve as sites of fluid and lipid leakage, which can lead to the development of diabetic macular edema. Theories on the biochemistry of these end-organ changes include toxic effects from sorbitol accumulation, vascular damage by excessive glycosylation with crosslinking of basement membrane proteins, and activation of protein kinase C-ß2 by vascular endothelial growth factor (VEGF), leading to increased vascular permeability and endothelial cell proliferation. VEGF, produced by the retina in response to hypoxia, is believed to play a central role in the development of neovascularization.^1,82

CLINICAL FEATURES

Non-Proliferative Diabetic Retinopathy (NPDR)

Studies have found that retinopathy in both insulin-dependent and non-insulin-dependent diabetes occurs 3 to 5 years or more after the onset of diabetes. In the WESDR, the prevalence of at least minimal retinopathy was almost 100% after 20 years.⁸³ However, this study was performed prior to the advent of other adjunctive therapies for diabetes that may yield more favorable outcomes over the long term. Another study found that at least 39% of young persons with diabetes developed retinopathy within the first 10 years.⁸⁴ The earliest clinical sign of diabetic retinopathy is the microaneurysm, a red dot seen on ophthalmoscopy that varies from 15 to 60 microns in diameter (Figure 1).

Figure 1. Microaneurysms and intraretinal hemorrhages in nonproliferative retinopathy. (UCSF Department of Ophthalmology)

The lesions can be difficult to distinguish from intraretinal hemorrhages on examination, but with fluorescein angiography microaneurysms can be identified easily as punctate spots of hyperfluorescence (Figure 2, 3). By contrast, hemorrhages block the background fluorescence and therefore appear dark.

Figure 2. Microaneurysms: hyperfluorescent dots in early phase of fluorescein angiogram (arrows). (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology).

Figure 3. Two minutes later, fluorescein leakage from the microaneurysms gives them a hazy appearance. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology)

The severity of NPDR can be graded as mild, moderate, severe, or very severe. In mild disease, microaneurysms are present with hemorrhage or hard exudates (lipid transudates). In moderate NPDR, these findings are associated with cotton-wool spots (focal infarcts of the retinal nerve fiber layer or areas of axoplasmic stasis) or intraretinal microvascular abnormalities (vessels that may be either abnormally dilated and tortuous retinal vessels, or intraretinal neovascularization). The “4-2-1 rule” is used to diagnose severe NPDR: criteria are met if hemorrhages and microaneurysms are present in 4 quadrants, or venous beading (Figure 4) is present in 2 quadrants, or moderate intraretinal microvascular abnormalities are present in 1 quadrant. In very severe NPDR, two of these features are present.

The correct evaluation and staging of NPDR is important as a means of assessing the risk of progression. In the ETDRS, eyes with very severe NPDR had a 60-fold increased risk of developing high-risk proliferative retinopathy after 1 year compared with eyes with mild NPDR.⁸⁵ For eyes with mild or moderate NPDR, early treatment with laser was not warranted, as the benefits in preventing vision loss did not outweigh the side effects (1). By contrast, in very severe NPDR, early laser treatment was often helpful.

Figure 4. Venous beading (arrows) in a case of proliferative diabetic retinopathy. (UCSF Department of Ophthalmology)

Capillary closure can also result in macular ischemia, another cause of vision loss in NPDR. This can be identified clinically as an enlargement of the normal foveal avascular zone on fluorescein angiography (Figure 5).

Figure 5. Capillary dropout around the fovea (white arrow) and in the temporal macula (black arrow). (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology).

Diabetic Macular Edema (DME)

Macular edema may be present at all the stages of diabetic retinopathy and is the most common cause of vision loss in nonproliferative diabetic retinopathy. Because of the increased vascular permeability and breakdown of the blood-retinal barrier, fluid and lipids leak into the retina and cause it to swell. This causes photoreceptor dysfunction, leading to vision loss when the center of the macula, the fovea, is affected. In the ETDRS, diabetic macular edema (DME) was characterized as "clinically significant" if any of the following were noted (Figure 6): retinal thickening within 500 microns of the fovea, hard exudates within 500 microns of the fovea if associated with adjacent retinal thickening, or an area of retinal thickening 1 disc diameter or larger if any part of it is located within 1 disc diameter of the fovea.⁸⁶

Figure 6. Clinically significant macular edema with hard exudates in the fovea. Cotton-wool spots are present near the major vessels. (UCSF Department of Ophthalmology)

Although the cause of the microvascular changes in diabetes is not fully understood, the deficient oxygenation of the retina may induce an overexpression of vascular endothelial growth factor (VEGF), with a consequent increase in vascular leakage and retinal edema.⁸⁷ Besides ischemia, inflammation may also play a role in the development of macular edema in diabetic retinopathy. In fact, elevated levels of extracellular carbonic anhydrase have been discovered in the vitreous of patients with diabetic retinopathy.⁸⁸ Carbonic anhydrase may originate from retinal hemorrhages and erythrocyte lysis and may activate the kallikrein-mediated inflammatory cascade, contributing to the development of DME.

Optical Coherence Tomography (OCT) is a widely used imaging technique that provides high-resolution imaging of the retina (Figure 7).⁸⁹ Working as an “optical ultrasound,” OCT projects a light beam and then acquires the light reflected from the retina to provide a cross-sectional image. Most patients with DME have diffuse retinal thickening or cystoid macular edema (presence of intraretinal cystoid-like spaces). In some patients, DME may be associated with posterior hyaloidal traction, serous retinal detachment or traction retinal detachment.⁹⁰ Cystoid macular edema and posterior hyaloid traction are significantly associated with worse visual acuity.⁹⁰

Figure 7. OCT image showing diabetic macular edema (UCSF Department of Ophthalmology).

Proliferative Diabetic Retinopathy (PDR)

In proliferative diabetic retinopathy, many of the changes seen in NPDR are present in addition to neovascularization that extends along the surface of the retina or into the vitreous cavity (Figure 8). These vessels are in loops that may form a network of radiating spokes or may appear disorganized. In many cases the vessels are first noted on the surface of the optic disc, although they can be easily missed due to their fine caliber. Close inspection often reveals that these new vessels cross over both the normal arteries and the normal veins of the retina, a sign of their unregulated growth.

Figure 8. Active neovascularization in PDR. Fibrovascular proliferation overlies the optic disc (white arrow). Loops of new vessels are especially prominent superior to the disc and extending into the macula, where leakage of fluid has led to deposition of a ring of hard exudate around the neovascular net (black arrow). (UCSF Department of Ophthalmology).

New vessels can also appear on the iris, a condition known as rubeosis iridis (Figure 9). When this occurs, careful inspection of the anterior chamber angle is essential, as growth of neovascularization in this location can obstruct aqueous fluid outflow and cause neovascular glaucoma.

Figure 9. Rubeosis iridis in a case of PDR. Abnormal new vessels are growing along the surface of the iris (arrows). (UCSF Dept. of Ophthalmology).

Neovascularization can remain relatively stable or it can grow rapidly; progression can be noted ophthalmoscopically over a period of weeks. Preretinal new vessels often develop an associated white, fibrous tissue component that can increase in size as the vessels regress. The resulting fibrovascular membrane may then develop new vessels at its edges. This cycle of growth and fibrous transformation of diabetic neovascularization is typical. The proliferation occurs on the anterior surface of the retina, and the vessels extend along the posterior surface of the vitreous body. Fibrous proliferation takes place on the posterior vitreous surface; when the vitreous detaches, the vessels can be pulled forward and the thickened posterior vitreous surface can be seen ophthalmoscopically, highlighted by areas of fibrovascular proliferation.

The severity of PDR can be classified as to the presence or absence of high-risk characteristics. As determined in the Diabetic Retinopathy Study, eyes are classified as high-risk if they have 3 of the following 4 characteristics: the presence of any neovascularization; neovascularization on or within 1-disc diameter of the optic disc; a moderate to severe amount of neovascularization (greater than 1/3 disc area neovascularization of the disc, or greater than 1/2 disc area if elsewhere), or vitreous hemorrhage.

Vision loss in proliferative diabetic retinopathy results from three main causes. First, vitreous hemorrhage occurs because the neovascular tissue is subject to vitreous traction. Coughing or vomiting may also trigger a hemorrhage. Hemorrhage may remain in the preretinal space between the retina and the posterior vitreous surface, in which case it may not cause much vision loss if located away from the macula (Figure 10). In other cases, though, hemorrhage can spread throughout the entire vitreous cavity, causing a diffuse opacification of the visual media with marked vision loss (Figure 11, 12).

Figure 10. Preretinal hemorrhage: blood trapped between the retina and the vitreous in a case of incomplete vitreous detachment. Visual acuity is unaffected. (UCSF Department of Ophthalmology).

Figure 11. Left: moderate vitreous hemorrhage; vision = 20/150. Right: 1 year later after spontaneous clearing of the hemorrhage; vision = 20/30. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology).

Figure 12. Dense vitreous hemorrhage almost completely obscuring the view of the fundus. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology).

Another cause of severe vision loss in PDR is retinal detachment. As the fibrovascular membranes and vitreous contract, their attachments to the retina can cause focal elevations of the retina, resulting in a traction retinal detachment (Figure 13). In other cases the retinal vessels can be avulsed or retinal holes may be created by this traction, leading to a combined traction-rhegmatogenous retinal detachment (Figure 14).

Figure 13. Marked fibrosis with traction exerted on the retina outside the central macula (arrows). The macula does not appear to be elevated centrally. (UCSF Dept. of Ophthalmology).

Figure 14. Traction retinal detachment outside the macula. Note elevation of retinal vessel out of the plane of focus (white arrow). Scatter photocoagulation scars are seen peripherally (black arrow). (UCSF Dept. of Ophthalmology).

Finally, patients with PDR may have macular nonperfusion or coexisting diabetic macular edema that causes vision loss through photoreceptor dysfunction.

TREATMENT

Tight glucose and blood pressure control are critical systemic factors in controlling the progression of diabetic retinopathy. Ocular complications of diabetes are addressed directly through treatment with laser photocoagulation, intravitreal injections, or surgery. Laser treatment has been the primary approach to vision-threatening diabetic retinopathy for decades. Recent randomized clinical trials have demonstrated that intravitreal anti-VEGF agents are more effective than laser under certain conditions.

Laser Photocoagulation for NPDR

Diabetic macular edema is believed to result from fluid and lipid transudation from microaneurysms and telangiectatic capillaries. Focal laser photocoagulation is used to heat and close the microaneurysms, causing them to stop leaking (Figure 15). Macular edema often improves following this form of treatment. Some clinicians apply laser burns in a grid pattern overlying areas of retinal edema without directing treatment to specific microaneurysms; this method can also be effective in reducing retinal thickening. The mechanism by which grid laser treatment achieves these results is not known.

The ETDRS found that the risk of moderate visual loss in eyes with diabetic macular edema was reduced by 50% by photocoagulation.^91,92 At 3 years, 24% of untreated eyes experienced a 3-line decrease in vision compared with 12% of treated eyes. Eyes meeting the criteria for clinically significant macular edema in which the edema was closest to the center were most likely to benefit from treatment. Side effects of laser treatment can include scotomata, noticeable immediately after the procedure, if treatment is performed too close to the fovea. Late enlargement of laser scars can also occur, causing delayed visual loss. Inadvertent photocoagulation of the fovea is a risk of the procedure. Since the amount of energy used is minimal, the treatment is performed under topical anesthesia.

In the ETDRS study, only a very small percentage of eyes improved with focal laser treatment, highlighting the fact that the goal of laser treatment is not to improve vision, but rather to stabilize it and prevent worsening. It is also true that inclusion criteria for that study were based on the presence of “clinically significant” macular edema threatening the macula, even if the visual acuity was not yet reduced. For this reason, it has been argued that the study enrolled patients with excellent visual acuity, making it difficult to demonstrate small improvements in vision after laser treatment.

Due to the recent evidence on the efficacy and safety of anti-VEGF therapy for diabetic macular edema, different modalities of laser therapy have been proposed. Laser may be able to stabilize macular edema and reduce the need for multiple anti-VEGF injections. Modified ETDRS laser techniques include lower intensity laser burns, and they take particular care in maintaining a greater space from the center of the fovea.⁹³ Subthreshold laser therapy and minimalistic fluorescein angiography-guided treatment of microaneurysms may also induce less damage to the macula than the classic ETDRS approach.⁹⁴

Figure 15. Focal laser scars in the macula following treatment for macular edema (arrow). Edema has resolved. (Zuckerberg San Francisco General Hospital, Dept. of Ophthalmology).

Laser Photocoagulation for PDR

Scatter laser photocoagulation, also known as panretinal photocoagulation (PRP), is an important treatment modality for PDR and severe NPDR.⁹² Laser spots are placed from outside the major vascular arcades to the equator of the eye, with burns spaced approximately 1/2 to 1 burn width apart (Figure 16, 17). Although the treatment destroys normal retina, the central vision is unaffected since all spots are placed outside the macula. The theory underlying this treatment is that photocoagulation of the ischemic peripheral retina decreases the elaboration of vasoproliferative factors contributing to PDR. Indeed, VEGF levels in the vitreous are increased in eyes with neovascularization, and they are lower after scatter photocoagulation.⁹⁵ Other factors such as insulin-like growth factor-1 are similarly elevated in the vitreous of eyes with PDR.⁹⁶

Side effects of scatter photocoagulation can include decreased night vision and dark adaptation, and visual field loss. The procedure can be painful, so treatment may be divided into several sessions, and either topical or retrobulbar anesthesia may be used.

Figure 16. Scatter photocoagulation scars in an eye with active PDR. Note that all scatter laser scars are located outside the macula. (UCSF Department of Ophthalmology).

Figure 17. View of laser scars superior to the macula in the same eye. Spots are approximately one-half burn width apart. In the treated area, the retinal vessels are sclerotic (arrows). (UCSF Department of Ophthalmology).

The Diabetic Retinopathy Study evaluated the effects of scatter photocoagulation in over 1700 patients with PDR or severe NPDR. Patients had one eye randomized to treatment and one eye to observation. Treatment was shown to reduce severe visual loss by 50%.⁹⁷ The ETDRS also found a positive risk-benefit ratio for early scatter treatment in patients with severe NPDR or early PDR. Interestingly, a subsequent study demonstrated that scatter laser performed at a single sitting was not worse than treatment divided over four sessions in terms of inducing macular edema or decreasing visual acuity.⁹⁸

Panretinal photocoagulation may induce or aggravate diabetic macular edema, reduce contrast sensitivity and affect the peripheral visual field.⁸⁵ Macular edema can be approached by focal laser or intravitreal injections before or at the time of panretinal photocoagulation. However, it is not recommended to delay panretinal photocoagulation in high-risk PDR.

The Diabetic Retinopathy Clinical Research network (DRCR) study protocol S has shown that intravitreal anti-VEGF agents may be a substitute for panretinal laser treatment.⁹⁹ This multicenter randomized clinical trial compared ranibizumab to PRP in patients with PDR. Mean visual acuity letter improvement at 2 years was +2.8 in the ranibizumab group vs +0.2 in the PRP group (P < 0.001). Mean peripheral visual field sensitivity loss was worse, vitrectomy was more frequent, and DME development was more common in the PRP group. Further studies are needed in order to evaluate the long-term implications of using anti-VEGF agents alone. Ranibizumab may be a reasonable treatment alternative to consider for patients with severe NPDR or non-high-risk PDR who can follow-up regularly.

Corticosteroids for DME

It has been demonstrated that corticosteroids stabilize the blood-retinal barrier, inhibiting leukostasis and modulating the expression of VEGF receptor.¹⁰⁰ On this basis, periocular and intraocular injections and sustained-release steroid implants have been utilized for the treatment of diabetic macular edema. It should be remembered that any of these different methods to deliver corticosteroids to the macula carry a potential risk of increasing the intraocular pressure (glaucoma) and inducing cataract.

The use of intravitreal triamcinolone acetonide has become accepted as a treatment option for diabetic macular edema. Several formulations are available: Kenalog-40, which has a black box warning against intraocular use, and the preservative-free Triesence. Preliminary data from a randomized clinical trial showed that intravitreal corticosteroids induced a noticeable improvement of visual acuity and foveal thickness in patients with severe, refractory DME.¹⁰¹However, intravitreal steroids do not appear to be more efficacious than laser treatment in giving a stable, sustained improvement in vision in the long run, as demonstrated by a recent large study.¹⁰²

A peribulbar corticosteroid injection is of particular interest for eyes with DME that have good visual acuity where the risks of an intravitreal injection may not be justified. Any intravitreal injection through the pars plana, in fact, may directly damage the crystalline lens or cause a severe, sight-threatening infection of the eye (bacterial endophthalmitis). Unfortunately, in 2007 a randomized clinical trial showed that peribulbar triamcinolone, with or without focal photocoagulation, is not effective in cases of mild DME with good visual acuity.¹⁰³

The fact that triamcinolone maintains measurable concentrations in the vitreous cavity for approximately 3 months stimulated further studies on sustained-release or biodegradable intraocular implants that can deliver steroids for a longer period of time.

A fluocinolone acetonide implant (Retisert) was investigated in a multicenter, randomized clinical trial for the treatment of diabetic macular edema. Although the efficacy of this surgically implanted material was demonstrated, it induced cataract in virtually all phakic patients and severe glaucoma needing surgery in 28% of eyes.^104,105

A biodegradable dexamethasone implant (Ozurdex), FDA-approved for the treatment of DME, has demonstrated similar efficacy with more acceptable side effects. At day 90, a visual acuity improvement of 10 letters or more was seen in more eyes in the Ozurdex group (33.3%) than the observation group (12.3%; P = 0.007), but the statistical significance was lost at day 180.¹⁰⁶ The implant was generally well tolerated.

A smaller device releasing fluocinolone acetonide, implantable suturelessly with an office procedure thorough a 25-gauge needle (Iluvien), is also FDA-approved for treatment of DME . This implant has been evaluated in the FAME (Fluocinolone Acetonide in Diabetic Macular Edema) study where 956 patients were randomized worldwide.¹⁰⁷ At month 36, the percentage of patients who gained ≥15 in letter score was 28% compared with 19% (P = 0.018) in the sham group. In patients who reported duration of DME ≥3 years at baseline; the percentage who gained ≥15 in letter score at month 36 was 34.0% compared with 13.4%. Almost all phakic patients in the insert group developed cataract, but their visual benefit after cataract surgery was similar to that in pseudophakic patients. The rate of glaucoma surgery at month 36 was 5%.¹⁰⁸

Anti-VEGF Drugs for DR and DME

Vascular endothelial growth factor (VEGF) is an angiogenic factor that plays a key role in the breakdown of the blood–retina barrier and is significantly elevated in eyes with diabetic macular edema.¹⁰⁹ Antibody fragments that bind VEGF and inhibit angiogenesis were first developed as intraocular injection for the treatment of exudative age-related macular degeneration. These anti-VEGF drugs have been used for the treatment of DR and DME with favorable results.

The first agent that became available was Pegaptanib 0.3 mg (Macugen).¹¹⁰ A randomized trial demonstrated after 2 years of therapy a gain of 6.1 letters in the pegaptanib arm versus 1.3 letters for sham (P<0.01).¹¹¹ Since it is targeted to the isoform VEGF-165 only, it is generally considered very safe but less effective than newer anti-VEGF drugs.

Bevacizumab (Avastin), directed to all the isoforms of VEGF, has been used off-label for the treatment of DME worldwide. The first evidence came from a study on 121 patients with DME followed over 3 months in a phase II randomized clinical trial.¹¹² The BOLT study demonstrated a mean gain of 8.6 letters for bevacizumab versus a mean loss of 0.5 letters when compared to classic macular laser. The patients received a mean of 13 injections over two years, and the treatment was well tolerated with no progression of macular ischemia.¹¹³

Ranibizumab (Lucentis) binds all isoforms of VEGF and is FDA-approved for the treatment of diabetic retinopathy and diabetic macular edema. In the Ranibizumab for Edema of the Macula in Diabetes (READ-2) study, ranibizumab-only was superior to laser and to combined therapy.¹¹⁴ The RESTORE study confirmed that ranibizumab monotherapy and combined with laser was superior to standard laser. At 1 year, no differences were detected between the ranibizumab and ranibizumab plus laser arms.¹¹⁵ A larger DRCR study supported ranibizumab plus prompt or deferred photocoagulation as a mainstay of current therapy for patients with DME.¹¹⁶ In the RESOLVE study, at month 12, mean visual acuity improved from baseline by 10.3±9.1 letters with ranibizumab and declined by 1.4±14.2 letters with sham (P<0.0001).¹¹⁷ The RISE and RIDE studies confirmed the efficacy and the safety of intravitreal monthly injections of ranibizumab with similar results.¹¹⁸ Efficacy against DR progression and improvement of diabetic retinopathy severity was also demonstrated in multiple studies.

Aflibercept 2 mg (Eylea), active against all VEGF-A isoforms, is also FDA-approved for the treatment of DR and DME. In the DA-VINCI study, the different dose regimens of aflibercept demonstrated a mean improvement in visual acuity of 10 to 13 letters versus -1.3 letters for the laser group with a large proportion of eyes (about 40%) gaining 15 or more ETDRS letters at week 52.¹¹⁹ Aflibercept 8 mg (Eylea HD) was also recently approved for DR and DME treatment, based upon the results of the PHOTON trial, offering the possibility of longer duration of action and fewer injections per year, as well as potentially improved efficacy in recalcitrant cases.¹²⁰

The Diabetic Retinopathy Clinical Research Network Protocol T compared bevacizumab, ranibizumab, and aflibercept in the treatment of center-involving DME.¹²¹ When the initial visual-acuity loss was mild, there were no significant differences among study groups. However, at worse levels of initial visual acuity (20/50 or worse), aflibercept was more effective than bevacizumab. The differences between bevacizumab and ranibizumab and between ranibizumab and aflibercept were not statistically significant. Of note, after 6 months of treatment, over 20% of patients in each anti-VEGF treatment group had persistent DME, suggesting that control of other mechanisms beyond VEGF are necessary to achieve optimal outcomes in the treatment of DME.

Bispecific Anti-VEGF and Anti-Angiopoeitin-2 Therapy

Faricimab (Vabysmo) is the first bispecific molecule approved for treatment of retinal disease, offering blockade of both VEGF and Ang2. The Ang1-Tie2 pathway promotes vascular stability; Ang2 interferes with Tie2 signaling, so its blockade is believed to have favorable effects for angiogenic diseases. The YOSEMITE and RHINE studies assessed the effect of faricimab on DME, finding that vision outcomes for faricimab administered on a variable injection schedule known as treat-and-extend were noninferior to aflibercept injected every 8 weeks. Treatment was able to be given every 16 weeks in some patients, offering a possibility of increased convenience and reduced injection-related risks in those patients able to receive fewer injections.¹²²

Currently, on the basis of the above evidence, anti-VEGF or bispecific anti-VEGF and anti-ang2 therapy is first-line therapy for center-involving macular edema, with possible deferred focal laser treatment. It should be mentioned that adverse side effects associated with intravitreal injections are uncommon but severe and include infectious endophthalmitis, cataract formation, retinal detachment, and elevated IOP.

Vitrectomy Surgery for PDR

Surgery may be necessary for eyes in advanced PDR with either vitreous hemorrhage or retinal detachment. In the case of vitreous hemorrhage, many cases will clear spontaneously. For this reason, clinicians often wait 3 to 6 months or more before performing vitrectomy surgery. If surgery is indicated because of persistent non-clearing hemorrhage, retinal detachment involving the macula, or vitreous hemorrhage with neovascularization of the anterior chamber angle (a precursor of neovascular glaucoma), then vitrectomy is performed via a pars plana approach. The vitreous is removed, fibrovascular membranes are dissected away from the retina, retinal detachment is repaired, and scatter laser treatment is applied at the time of surgery via direct intraocular application.

The Diabetic Retinopathy Vitrectomy Study assessed the value of early vitrectomy in patients with severe PDR. The study found that early intervention increased the likelihood of obtaining 20/40 vision or better in eyes with recent severe vitreous hemorrhage or severe PDR. Compared with 15% of control eyes, 25% of treated eyes achieved this level of vision at 2 years.¹⁰⁹ In type 1 diabetes, the benefit of early surgery was even more pronounced, with 36% of treated eyes achieving 20/40 vision compared to 12% of control eyes. The importance of this study, performed between 1976 and 1983 when vitrectomy techniques were much less advanced than they are today, was that it showed conventional “watch and wait” management will not necessarily lead to the best visual outcomes in cases of severe PDR. In practice, clinicians evaluate the risks and benefits of each option before proceeding with scatter photocoagulation, vitrectomy, or observation in such cases.

Recently, the DRCR Protocol D evaluated the effects of pars plana vitrectomy in eyes with moderate vision loss from DME and vitreomacular traction. Although retinal thickness was generally reduced, visual acuity results were less consistent.¹²³ Vitrectomy for refractory, chronic diabetic macular edema in the absence of vitreomacular traction should be reserved to selected cases.

Intravitreal ocriplasmin (Jetrea) is able to induce enzymatic vitreolysis and posterior vitreous detachment and could have a role, eventually associated with vitrectomy, in the treatment of vitreomacular traction and macular edema in diabetic retinopathy.¹²⁴

NOVEL THERAPIES FOR DIABETIC RETINOPATHY

Current therapies are limited in their ability to reverse vision loss in diabetic retinopathy. For example, although focal laser photocoagulation can help stabilize vision by reducing macular edema, it rarely improves vision. Corticosteroids induce cataract progression and intraocular pressure elevation. Anti-VEGF agents do not increase cataract formation rates but they generally need more frequent intravitreal injections, carrying the risk of endophthalmitis; they can temporary increase IOP; they might have systemic adverse effects. For addressing these issues, new sustained-release devices are being designed, and studies are ongoing to test new intravitreal medications.

The development of new treatment modalities is being guided by an understanding of the mechanisms of the disease. From this perspective, researchers are now focusing on the role of inflammation on DME. NSAIDs, anti-TNF agents (Etanercept and Remicade), mecamylamine (an antagonist of nACh receptors), and intravitreal erythropoietin are currently under investigation for the treatment of refractory diabetic macular edema.¹²⁵

In order to create a national taskforce to study and treat diabetic retinopathy, in 2002 the National Eye Institute funded the DRCR, a collaborative network dedicated to design and carry out multicenter clinical trials on diabetic retinopathy and diabetic macular edema. The DRCR network currently includes over 150 participating sites with over 500 physicians throughout the United States.

The DRCR Network has an ongoing project to study genes involved in diabetic retinopathy.

CONCLUSION

Retinopathy remains a challenging complication of diabetes that can adversely affect a patient’s quality of life. Although ophthalmologists can often stabilize the condition or reduce vision loss, prevention and early detection remain the most effective ways to preserve good vision in patients with diabetes. Ensuring tight glucose and blood pressure control and referring patients for ophthalmologic examination are important ways in which internists and other clinicians can help to maximize their patients’ vision and therefore their quality of life. New treatments may offer greater hope for sustained visual improvement in patients with diabetic retinopathy.

REFERENCES

Amoaku WM, Ghanchi F, Bailey C, et al. Diabetic retinopathy and diabetic macular oedema pathways and management: UK Consensus Working Group. Eye Lond Engl. 2020;34:1-51.
Sabanayagam C, Yip W, Ting DS, Tan G, Wong TY. Ten Emerging Trends in the Epidemiology of Diabetic Retinopathy. Ophthalmic Epidemiol. 2016;23:209-222.
Ting DS, Cheung GC, Wong TY. Diabetic retinopathy: global prevalence, major risk factors, screening practices and public health challenges: a review. Clin Experiment Ophthalmol. 2016;44:260-277.
Wong TY, Cheung CM, Larsen M, Sharma S, Simo R. Diabetic retinopathy. Nat Rev Dis Primer. 2016;2:16012.
American Diabetes Association. 11. Microvascular Complications and Foot Care: Standards of Medical Care in Diabetes—2020. Diabetes Care. 2020;43(Suppl 1):S135-S151.
Barrett EJ, Liu Z, Khamaisi M, et al. Diabetic Microvascular Disease: An Endocrine Society Scientific Statement. J Clin Endocrinol Metab. 2017;102:4343-4410.
Klein R, Knudtson MD, Lee KE, Gangnon R, Klein BE. The Wisconsin Epidemiologic Study of Diabetic Retinopathy: XXII the twenty-five-year progression of retinopathy in persons with type 1 diabetes. Ophthalmology. 2008;115:1859-1868.
Fong DS, Aiello L, Gardner TW, et al. Diabetic retinopathy. Diabetes Care. 2003;26:226-229.
Zhang X, Saaddine JB, Chou CF, et al. Prevalence of diabetic retinopathy in the United States, 2005-2008. J Am Med Assoc. 2010;304:649-656.
Wong TY, Klein R, Islam FM, et al. Diabetic retinopathy in a multi-ethnic cohort in the United States. Am J Ophthalmol. 2006;141:446-455.
Yau JW, Rogers SL, Kawasaki R, et al. Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care. 2012;35:556-564.
Antonetti DA, Klein R, Gardner TW. Diabetic retinopathy. N Engl J Med. 2012;366:1227-1239.
Wong TY, Mwamburi M, Klein R, et al. Rates of progression in diabetic retinopathy during different time periods: a systematic review and meta-analysis. Diabetes Care. 2009;32:2307-2313.
Tam VH, Lam EP, Chu BC, Tse KK, Fung LM. Incidence and progression of diabetic retinopathy in Hong Kong Chinese with type 2 diabetes mellitus. J Diabetes Complications. 2009;23:185-193.
Chase HP, Jackson WE, Hoops SL, Cockerham RS, Archer PG, O’Brien D. Glucose control and the renal and retinal complications of insulin-dependent diabetes. J Am Med Assoc. 1989;261:1155-1160.
Stratton IM, Kohner EM, Aldington SJ, et al. UKPDS 50: risk factors for incidence and progression of retinopathy in Type II diabetes over 6 years from diagnosis. Diabetologia. 2001;44:156-163.
Wang PH, Lau J, Chalmers TC. Meta-analysis of effects of intensive blood-glucose control on late complications of type I diabetes. The Lancet. 1993;341:1306-1309.
Diabetes Control Complications Trial Research Group, Nathan DM, Genuth S, et al. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med. 1993;329:977-986.
Nathan DM, Bayless M, Cleary P, et al. Diabetes control and complications trial/epidemiology of diabetes interventions and complications study at 30 years: advances and contributions. Diabetes. 2013;62:3976-3986.
Ohkubo Y, Kishikawa H, Araki E, et al. Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract. 1995;28:103-117.
Shichiri M, Kishikawa H, Ohkubo Y, Wake N. Long-term results of the Kumamoto Study on optimal diabetes control in type 2 diabetic patients. Diabetes Care. 2000;23 Suppl 2:B21-B29.
UK Prospective Diabetes Study Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). The Lancet. 1998;352:837-853.
Holman RR, Paul SK, Bethel MA, Matthews DR, Neil HA. 10-year follow-up of intensive glucose control in type 2 diabetes. N Engl J Med. 2008;359:1577-1589.
ACCORD Study Group, Chew EY, Ambrosius WT, et al. Effects of medical therapies on retinopathy progression in type 2 diabetes. N Engl J Med. 2010;363:233-244.
Ismail-Beigi F, Craven T, Banerji MA, et al. Effect of intensive treatment of hyperglycaemia on microvascular outcomes in type 2 diabetes: an analysis of the ACCORD randomised trial. The Lancet. 2010;376:419-430.
Action to Control Cardiovascular Risk in Diabetes Follow-On Eye Study Group. Persistent Effects of Intensive Glycemic Control on Retinopathy in Type 2 Diabetes in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Follow-On Study. Diabetes Care. 2016;39:1089-1100.
Advance Collaborative Group, Patel A, MacMahon S, et al. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med. 2008;358:2560-2572.
Duckworth W, Abraira C, Moritz T, et al. Glucose control and vascular complications in veterans with type 2 diabetes. N Engl J Med. 2009;360:129-139.
Zoungas S, Arima H, Gerstein HC, et al. Effects of intensive glucose control on microvascular outcomes in patients with type 2 diabetes: a meta-analysis of individual participant data from randomised controlled trials. Lancet Diabetes Endocrinol. 2017;5:431-437.
Hemmingsen B, Lund SS, Gluud C, et al. Intensive glycaemic control for patients with type 2 diabetes: systematic review with meta-analysis and trial sequential analysis of randomised clinical trials. BMJ. 2011;343:d6898.
Hooymans JM, Ballegooie EV, Schweitzer NM, Doorebos H, Reitsma WD, Slutter WJ. Worsening of diabetic retinopathy with strict control of blood sugar. The Lancet. 1982;2:438.
Lauritzen T, Frost-Larsen K, Larsen HW, Deckert T. Two-year experience with continuous subcutaneous insulin infusion in relation to retinopathy and neuropathy. Diabetes. 1985;34 Suppl 3:74-79.
Kroc Collaborative Study Group. Blood glucose control and the evolution of diabetic retinopathy and albuminuria. A preliminary multicenter trial. N Engl J Med. 1984;311:365-372.
Lauritzen T, Frost-Larsen K, Larsen HW, Deckert T. Effect of 1 year of near-normal blood glucose levels on retinopathy in insulin-dependent diabetics. The Lancet. 1983;1:200-204.
Diabetes Control and Complications Trial Research Group. Early worsening of diabetic retinopathy in the Diabetes Control and Complications Trial. Arch Ophthalmol. 1998;116:874-886.
Bain SC, Klufas MA, Ho A, Matthews DR. Worsening of diabetic retinopathy with rapid improvement in systemic glucose control: A review. Diabetes Obes Metab. 2019;21:454-466.
Klein R, Klein BE, Moss SE, Davis MD, DeMets DL. Is blood pressure a predictor of the incidence or progression of diabetic retinopathy? Arch Intern Med. 1989;149:2427-2432.
Tapp RJ, Shaw JE, Harper CA, et al. The prevalence of and factors associated with diabetic retinopathy in the Australian population. Diabetes Care. 2003;26:1731-1737.
Leske MC, Wu SY, Hennis A, et al. Hyperglycemia, blood pressure, and the 9-year incidence of diabetic retinopathy: the Barbados Eye Studies. Ophthalmology. 2005;112:799-805.
UK Prospective Diabetes Study Group. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. BMJ. 1998;317:703-713.
Matthews DR, Stratton IM, Aldington SJ, Holman RR, Kohner EM, Group UPDS. Risks of progression of retinopathy and vision loss related to tight blood pressure control in type 2 diabetes mellitus: UKPDS 69. Arch Ophthalmol. 2004;122:1631-1640.
Holman RR, Paul SK, Bethel MA, Neil HA, Matthews DR. Long-term follow-up after tight control of blood pressure in type 2 diabetes. N Engl J Med. 2008;359:1565-1576.
Heart Outcomes Prevention Evaluation Study Group. Effects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy. The Lancet. 2000;355:253-259.
Chaturvedi N, Sjolie AK, Stephenson JM, et al. Effect of lisinopril on progression of retinopathy in normotensive people with type 1 diabetes. The EUCLID Study Group. EURODIAB Controlled Trial of Lisinopril in Insulin-Dependent Diabetes Mellitus. The Lancet. 1998;351:28-31.
Beulens JW, Patel A, Vingerling JR, et al. Effects of blood pressure lowering and intensive glucose control on the incidence and progression of retinopathy in patients with type 2 diabetes mellitus: a randomised controlled trial. Diabetologia. 2009;52:2027-2036.
Estacio RO, Jeffers BW, Gifford N, Schrier RW. Effect of blood pressure control on diabetic microvascular complications in patients with hypertension and type 2 diabetes. Diabetes Care. 2000;23 Suppl 2:B54-B64.
Schrier RW, Estacio RO, Esler A, Mehler P. Effects of aggressive blood pressure control in normotensive type 2 diabetic patients on albuminuria, retinopathy and strokes. Kidney Int. 2002;61:1086-1097.
Chaturvedi N, Porta M, Klein R, et al. Effect of candesartan on prevention (DIRECT-Prevent 1) and progression (DIRECT-Protect 1) of retinopathy in type 1 diabetes: randomised, placebo-controlled trials. The Lancet. 2008;372:1394-1402.
Sjolie AK, Klein R, Porta M, et al. Effect of candesartan on progression and regression of retinopathy in type 2 diabetes (DIRECT-Protect 2): a randomised placebo-controlled trial. The Lancet. 2008;372:1385-1393.
Mauer M, Zinman B, Gardiner R, et al. Renal and retinal effects of enalapril and losartan in type 1 diabetes. N Engl J Med. 2009;361:40-51.
Wang B, Wang F, Zhang Y, et al. Effects of RAS inhibitors on diabetic retinopathy: a systematic review and meta-analysis. Lancet Diabetes Endocrinol. 2015;3:263-274.
Lim LS, Wong TY. Lipids and diabetic retinopathy. Expert Opin Biol Ther. 2012;12:93-105.
Chew EY, Klein ML, Ferris FL 3rd, et al. Association of elevated serum lipid levels with retinal hard exudate in diabetic retinopathy. Early Treatment Diabetic Retinopathy Study (ETDRS) Report 22. Arch Ophthalmol. 1996;114:1079-1084.
Rema M, Srivastava BK, Anitha B, Deepa R, Mohan V. Association of serum lipids with diabetic retinopathy in urban South Indians--the Chennai Urban Rural Epidemiology Study (CURES) Eye Study--2. Diabet Med. 2006;23:1029-1036.
Salinero-Fort MA, San Andres-Rebollo FJ, de Burgos-Lunar C, Arrieta-Blanco FJ, Gomez-Campelo P, Group M. Four-year incidence of diabetic retinopathy in a Spanish cohort: the MADIABETES study. PLoS ONE. 2013;8:e76417.
Sinav S, Onelge MA, Onelge S, Sinav B. Plasma lipids and lipoproteins in retinopathy of type I (insulin-dependent) diabetic patients. Ann Ophthalmol. 1993;25:64-66.
Lyons TJ, Jenkins AJ, Zheng D, et al. Diabetic retinopathy and serum lipoprotein subclasses in the DCCT/EDIC cohort. Invest Ophthalmol Vis Sci. 2004;45:910-917.
Zhou Y, Wang C, Shi K, Yin X. Relationship between dyslipidemia and diabetic retinopathy: A systematic review and meta-analysis. Medicine (Baltimore). 2018;97:e12283.
Klein BE, Moss SE, Klein R, Surawicz TS. The Wisconsin Epidemiologic Study of Diabetic Retinopathy. XIII. Relationship of serum cholesterol to retinopathy and hard exudate. Ophthalmology. 1991;98:1261-1265.
Klein R, Sharrett AR, Klein BE, et al. The association of atherosclerosis, vascular risk factors, and retinopathy in adults with diabetes: the atherosclerosis risk in communities study. Ophthalmology. 2002;109:1225-1234.
Sobrin L, Chong YH, Fan Q, et al. Genetically Determined Plasma Lipid Levels and Risk of Diabetic Retinopathy: A Mendelian Randomization Study. Diabetes. 2017;66:3130-3141.
Harrold BP, Marmion VJ, Gough KR. A double-blind controlled trial of clofibrate in the treatment of diabetic retinopathy. Diabetes. 1969;18:285-291.
Duncan LJ, Cullen JF, Ireland JT, Nolan J, Clarke BF, Oliver MF. A three-year trial of atromid therapy in exudative diabetic retinopathy. Diabetes. 1968;17:458-467.
Keech AC, Mitchell P, Summanen PA, et al. Effect of fenofibrate on the need for laser treatment for diabetic retinopathy (FIELD study): a randomised controlled trial. The Lancet. 2007;370:1687-1697.
Emmerich KH, Poritis N, Stelmane I, et al. [Efficacy and safety of etofibrate in patients with non-proliferative diabetic retinopathy]. Klin Monatsblätter Für Augenheilkd. 2009;226:561-567.

65a. Preiss D, Logue J, Sammons E, Zayed M, Emberson J, Wade R, Wallendszus K, Stevens W, Cretney R, Harding S, Leese G, Currie G, Armitage J. Effect of Fenofibrate on Progression of Diabetic Retinopathy. NEJM Evid. 2024 Aug;3(8):EVIDoa2400179.

Knickelbein JE, Abbott AB, Chew EY. Fenofibrate and Diabetic Retinopathy. Curr Diab Rep. 2016;16:90.
Hu Y, Chen Y, Ding L, et al. Pathogenic role of diabetes-induced PPAR-alpha down-regulation in microvascular dysfunction. Proc Natl Acad Sci U S A. 2013;110:15401-15406.
Nielsen SF, Nordestgaard BG. Statin use before diabetes diagnosis and risk of microvascular disease: a nationwide nested matched study. Lancet Diabetes Endocrinol. 2014;2:894-900.
Kang EY, Chen TH, Garg SJ, et al. Association of Statin Therapy With Prevention of Vision-Threatening Diabetic Retinopathy. JAMA Ophthalmol. 2019;137:363-371.
Vail D, Callaway NF, Ludwig CA, Saroj N, Moshfeghi DM. Lipid-Lowering Medications Are Associated with Lower Risk of Retinopathy and Ophthalmic Interventions among United States Patients with Diabetes. Am J Ophthalmol. 2019;207:378-384.
Kawasaki R, Kitano S, Sato Y, Yamashita H, Nishimura R, Tajima N. Factors associated with non-proliferative diabetic retinopathy in patients with type 1 and type 2 diabetes: the Japan Diabetes Complication and its Prevention prospective study (JDCP study 4). Diabetol Int. 2019;10:3-11.
Sen K, Misra A, Kumar A, Pandey RM. Simvastatin retards progression of retinopathy in diabetic patients with hypercholesterolemia. Diabetes Res Clin Pract. 2002;56:1-11.
Gupta A, Gupta V, Thapar S, Bhansali A. Lipid-lowering drug atorvastatin as an adjunct in the management of diabetic macular edema. Am J Ophthalmol. 2004;137:675-682.
ASCEND Study Collaborative Group, Bowman L, Mafham M, et al. Effects of n-3 Fatty Acid Supplements in Diabetes Mellitus. N Engl J Med. 2018;379:1540-1550.

74a. Sammons EL, Buck G, Bowman LJ, Stevens WM, Hammami I, Parish S, Armitage J; ASCEND Study Collaborative Group. ASCEND-Eye: Effects of Omega-3 Fatty Acids on Diabetic Retinopathy. Ophthalmology. 2024 May;131(5):526-533.

Domanico D, Verboschi F, Altimari S, Zompatori L, Vingolo EM. Ocular Effects of Niacin: A Review of the Literature. Med Hypothesis Discov Innov Ophthalmol. 2015;4:64-71.
Buschur E, Stetson B, Barbour LA. Diabetes In Pregnancy. Endotext Internet. Published online 2018.
Sun JK, Keenan HA, Cavallerano JD, et al. Protection from retinopathy and other complications in patients with type 1 diabetes of extreme duration: the joslin 50-year medalist study. Diabetes Care. 2011;34:968-974.
Lachin JM, Genuth S, Nathan DM, Zinman B, Rutledge BN, Group DR. Effect of glycemic exposure on the risk of microvascular complications in the diabetes control and complications trial--revisited. Diabetes. 2008;57:995-1001.
Kuo JZ, Wong TY, Rotter JI. Challenges in elucidating the genetics of diabetic retinopathy. JAMA Ophthalmol. 2014;132:96-107.
Cho H, Sobrin L. Genetics of diabetic retinopathy. Curr Diab Rep. 2014;14:515.
Cole JB, Florez JC. Genetics of diabetes mellitus and diabetes complications. Nat Rev Nephrol. 2020;16:377-390.
Behl T, Kotwani A. Exploring the various aspects of the pathological role of vascular endothelial growth factor (VEGF) in diabetic retinopathy. Pharmacol Res. 2015;99:137-148.
Fong DS, Aiello L, Gardner TW, et al. Retinopathy in diabetes. Diabetes Care. 2004;27 Suppl 1:S84-87.
Henricsson M, Nystrom L, Blohme G, et al. The incidence of retinopathy 10 years after diagnosis in young adult people with diabetes: results from the nationwide population-based Diabetes Incidence Study in Sweden (DISS). Diabetes Care. 2003;26:349-354.
Early Treatment Diabetic Retinopathy Study Research Group. Early photocoagulation for diabetic retinopathy. ETDRS report number 9. Ophthalmology. 1991;98:766-785.
Early Treatment Diabetic Retinopathy Study Research Group. Photocoagulation for diabetic macular edema. Early Treatment Diabetic Retinopathy Study report number 1. Arch Ophthalmol. 1985;103:1796-1806.
Caldwell RB, Bartoli M, Behzadian MA, et al. Vascular endothelial growth factor and diabetic retinopathy: role of oxidative stress. Curr Drug Targets. 2005;6:511-524.
Gao BB, Clermont A, Rook S, et al. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med. 2007;13:181-188.
Puliafito CA, Hee MR, Lin CP, et al. Imaging of macular diseases with optical coherence tomography. Ophthalmology. 1995;102:217-229.
Kim BY, Smith SD, Kaiser PK. Optical coherence tomographic patterns of diabetic macular edema. Am J Ophthalmol. 2006;142:405-412.
Relhan N, Flynn Jr H. The Early Treatment Diabetic Retinopathy Study historical review and relevance to today’s management of diabetic macular edema. Curr Opin Ophthalmol. 2017;28:205-212.
Neubauer AS, Ulbig MW. Laser treatment in diabetic retinopathy. Ophthalmologica. 2007;221:95-102.
Writing Committee for the Diabetic Retinopathy Clinical Research Network, Fong DS, Strauber SF, et al. Comparison of the modified Early Treatment Diabetic Retinopathy Study and mild macular grid laser photocoagulation strategies for diabetic macular edema. Arch Ophthalmol. 2007;125:469-480.
Luttrull JK, Dorin G. Subthreshold diode micropulse laser photocoagulation (SDM) as invisible retinal phototherapy for diabetic macular edema: a review. Curr Diabetes Rev. 2012;8:274-284.
Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480-1487.
Simo R, Lecube A, Segura RM, Garcia Arumi J, Hernandez C. Free insulin growth factor-I and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Am J Ophthalmol. 2002;134:376-382.
Diabetic Retinopathy Study Research Group. Preliminary report on effects of photocoagulation therapy. Am J Ophthalmol. 1976;81:383-396.
Diabetic Retinopathy Clinical Research Network, Brucker AJ, Qin H, et al. Observational study of the development of diabetic macular edema following panretinal (scatter) photocoagulation given in 1 or 4 sittings. Arch Ophthalmol. 2009;127:132-140.
Writing Committee for the Diabetic Retinopathy Clinical Research Network, Gross JG, Glassman AR, et al. Panretinal Photocoagulation vs Intravitreous Ranibizumab for Proliferative Diabetic Retinopathy: A Randomized Clinical Trial. J Am Med Assoc. 2015;314:2137-2146.
Cunningham MA, Edelman JL, Kaushal S. Intravitreal steroids for macular edema: the past, the present, and the future. Surv Ophthalmol. 2008;53:139-149.
Gillies MC, Sutter FK, Simpson JM, Larsson J, Ali H, Zhu M. Intravitreal triamcinolone for refractory diabetic macular edema: two-year results of a double-masked, placebo-controlled, randomized clinical trial. Ophthalmology. 2006;113:1533-1538.
Diabetic Retinopathy Clinical Research Network, Beck RW, Edwards AR, et al. Three-year follow-up of a randomized trial comparing focal/grid photocoagulation and intravitreal triamcinolone for diabetic macular edema. Arch Ophthalmol. 2009;127:245-251.
Diabetic Retinopathy Clinical Research Network, Chew E, Strauber S, et al. Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study. Ophthalmology. 2007;114:1190-1196.
Pearson PA, Comstock TL, Ip M, et al. Fluocinolone acetonide intravitreal implant for diabetic macular edema: a 3-year multicenter, randomized, controlled clinical trial. Ophthalmology. 2011;118:1580-1587.
Schwartz SG, Jr FH. Fluocinolone acetonide implantable device for diabetic retinopathy. Curr Pharm Biotechnol. 2011;12:347-351.
Haller JA, Kuppermann BD, Blumenkranz MS, et al. Randomized controlled trial of an intravitreous dexamethasone drug delivery system in patients with diabetic macular edema. Arch Ophthalmol. 2010;128:289-296.
Campochiaro PA, Brown DM, Pearson A, et al. Long-term benefit of sustained-delivery fluocinolone acetonide vitreous inserts for diabetic macular edema. Ophthalmology. 2011;118:626-635.e2.
Campochiaro PA, Brown DM, Pearson A, et al. Sustained delivery fluocinolone acetonide vitreous inserts provide benefit for at least 3 years in patients with diabetic macular edema. Ophthalmology. 2012;119:2125-2132.
Nguyen QD, Tatlipinar S, Shah SM, et al. Vascular endothelial growth factor is a critical stimulus for diabetic macular edema. Am J Ophthalmol. 2006;142:961-969.
Cunningham Jr. E, Adamis AP, Altaweel M, et al. A phase II randomized double-masked trial of pegaptanib, an anti-vascular endothelial growth factor aptamer, for diabetic macular edema. Ophthalmology. 2005;112:1747-1757.
Sultan MB, Zhou D, Loftus J, Dombi T, Ice KS, Group MS. A phase 2/3, multicenter, randomized, double-masked, 2-year trial of pegaptanib sodium for the treatment of diabetic macular edema. Ophthalmology. 2011;118:1107-1118.
Diabetic Retinopathy Clinical Research Network, Scott IU, Edwards AR, et al. A phase II randomized clinical trial of intravitreal bevacizumab for diabetic macular edema. Ophthalmology. 2007;114:1860-1867.
Rajendram R, Fraser-Bell S, Kaines A, et al. A 2-year prospective randomized controlled trial of intravitreal bevacizumab or laser therapy (BOLT) in the management of diabetic macular edema: 24-month data: report 3. Arch Ophthalmol. 2012;130:972-979.
Nguyen QD, Shah SM, Khwaja AA, et al. Two-year outcomes of the ranibizumab for edema of the mAcula in diabetes (READ-2) study. Ophthalmology. 2010;117:2146-2151.
Mitchell P, Bandello F, Schmidt-Erfurth U, et al. The RESTORE study: ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema. Ophthalmology. 2011;118:615-625.
Elman MJ, Bressler NM, Qin H, et al. Expanded 2-year follow-up of ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology. 2011;118:609-614.
Massin P, Bandello F, Garweg JG, et al. Safety and efficacy of ranibizumab in diabetic macular edema (RESOLVE Study): a 12-month, randomized, controlled, double-masked, multicenter phase II study. Diabetes Care. 2010;33:2399-2405.
Nguyen QD, Brown DM, Marcus DM, et al. Ranibizumab for diabetic macular edema: results from 2 phase III randomized trials: RISE and RIDE. Ophthalmology. 2012;119:789-801.
Do DV, Nguyen QD, Boyer D, et al. One-year outcomes of the da Vinci Study of VEGF Trap-Eye in eyes with diabetic macular edema. Ophthalmology. 2012;119:1658-1665.
Brown DM, Boyer DS, Do DV, et al. Intravitreal aflibercept 8 mg in diabetic macular oedema (PHOTON): 48-week results from a randomised, double-masked, non-inferiority, phase 2/3 trial. Lancet Lond Engl. 2024;403(10432):1153-1163. doi:10.1016/S0140-6736(23)02577-1
Wells JA, Glassman AR, Ayala AR, et al. Aflibercept, Bevacizumab, or Ranibizumab for Diabetic Macular Edema: Two-Year Results from a Comparative Effectiveness Randomized Clinical Trial. Ophthalmology. 2016;123:1351-1359.
Wong TY, Haskova Z, Asik K, et al. Faricimab Treat-and-Extend for Diabetic Macular Edema: Two-Year Results from the Randomized Phase 3 YOSEMITE and RHINE Trials. Ophthalmology. 2024;131(6):708-723. doi:10.1016/j.ophtha.2023.12.026
Diabetic Retinopathy Clinical Research Network Writing Committee, Haller JA, Qin H, et al. Vitrectomy outcomes in eyes with diabetic macular edema and vitreomacular traction. Ophthalmology. 2010;117:1087-1093.e3.
Gandorfer A. Enzymatic vitreous disruption. Eye. 2008;22:1273-1277.
Javey G, Schwartz SG, Jr FH. Emerging pharmacotherapies for diabetic macular edema. Exp Diabetes Res. 2012;2012:548732.

Post-Transplant Osteoporosis

ABSTRACT

Organ transplantation has become an established treatment for end-stage diseases, and in recent decades, survival rates have significantly improved. This progress has made diagnosing osteoporosis and other complications essential for prevention, treatment, and enhancing the quality of life for transplant patients. Patients who undergo solid organ transplantation often have risk factors for bone loss and fractures, and these risks can increase after transplantation. Post-transplant fractures have been identified as an independent risk factor for overall mortality in these patients. Preoperative low bone mass increases the likelihood of these complications. Osteoporosis is a significant concern that can develop, worsened by glucocorticoids and immunosuppressive therapy, used after transplantation to prevent organ rejection. A major consequence of this is an elevated risk of fractures in bones with reduced strength and quality, leading to increased morbidity and mortality. Additionally, there are notable differences in bone loss and fracture rates among patients with different types of transplanted organs. Initially, reports indicated that in the first year following transplantation, there was a rapid loss of bone mass and an increased rate of fractures. Unfortunately, bone mass achieved after transplantation remains lower long-term compared to that of healthy individuals. Protocols involving less aggressive use of glucocorticoids and immunosuppressants have been introduced to reduce these complications, along with advancements in infection prevention and treatment, to improve the tolerability of treatments and long-term outcomes. Another strategy has been to optimize bone mass in transplant candidates, administering calcium, vitamin D, and bisphosphonates before surgery. Therefore, the prevention and management of bone loss in both transplant candidates and post-transplant patients should be prioritized to reduce the risk of fractures.

INTRODUCTION

Organ transplantation is a well-accepted procedure for treating end-stage diseases such as kidney disease, chronic liver failure, end-stage pulmonary disease, and heart failure. Over the past decade, advancements in this technique have significantly improved patient survival and quality of life. The number of transplants has steadily increased, rising from 106,879 in 2010 to 157,500 in 2020 (1). However, bone loss is a common complication affecting long-term survival and quality of life during patient follow-up.

After transplantation, rapid and significant bone loss can occur within the first 3-6 months, along with a substantial increase in fracture risk (2,3). The rapid rate of bone loss is likely due to corticosteroids. Greater bone loss has been reported at vertebral and hip sites, along with high rates of fragility fractures. Over half of transplanted patients develop osteoporosis and one-third experience vertebral fractures (4). However, recent studies show a lower rate of bone loss and fractures following transplants, likely due to reduced glucocorticoid doses and modifications in immunosuppression regimens (5,6).

Several risk factors contribute to bone loss in patients including pretransplant disease, aging, hypogonadism, vitamin D deficiency, malabsorption, low body weight, physical inactivity, excessive tobacco or alcohol use, and immunosuppressive therapy (7) (Table 1). Improved management of pretransplant risk factors has led to better bone mineral density (BMD) levels before transplantation.

Table 1. Risk Factors for Bone Disease in Patients with Organ Transplantation
Organ	Potential Risk Factor
Pre-Transplant Factors Affecting All Transplant Patients	-Pre-existing low bone disease -Lower bone mineral density -History of Fractures
Factors Specific to Kidney Transplant Recipients.	-Female gender -Older age -β-microglobulin amyloidosis -Glucocorticoids -Secondary hyperparathyroidism -adynamic bone disease -Chronic metabolic acidosis -Hypogonadism -Vitamin D deficiency -Long-term hemodialysis -Diabetes
Factors Specific to Liver Transplant Recipients.	-Older age -Alcoholism -Hypogonadism -Abnormal vitamin D metabolism -Primary biliary cirrhosis -Cholestasis -Hyperbilirubinemia.
Factors Specific to Heart Transplant Recipients.	-Low levels of vitamin D -Hypogonadism -Long-term heparin -Loops diuretics -Secondary hyperparathyroidism -Physical inactivity, -Therapy with loop diuretics, -Tobacco, alcoholism.
Factors Specific to Lung Transplant Recipients.	-Glucocorticoid therapy -Tobacco -Physical inactivity -Low body weight -Malnutrition -Hypogonadism -Hypercapnia -Hypoxia -Hypogonadism -Cystic Fibrosis
Post-Transplant Factors Affecting All Patients	-Glucocorticoids -Immunosuppressors: cyclosporine, Tacrolimus. -Older age -Kidney dialysis -Diabetes Mellitus -High or low PTH -Cholestatic liver disease, -Primary Biliary Cirrhosis

Table 1. Risk Factors for Bone Disease in Patients with Organ Transplantation

Organ

Potential Risk Factor

Pre-Transplant Factors Affecting All Transplant Patients

-Pre-existing low bone disease

-Lower bone mineral density

-History of Fractures

Factors Specific to Kidney Transplant Recipients.

-Female gender

-Older age

-β-microglobulin amyloidosis

-Glucocorticoids

-Secondary hyperparathyroidism

-adynamic bone disease

-Chronic metabolic acidosis

-Hypogonadism

-Vitamin D deficiency

-Long-term hemodialysis

-Diabetes

Factors Specific to Liver

Transplant Recipients.

-Older age

-Alcoholism

-Hypogonadism

-Abnormal vitamin D metabolism

-Primary biliary cirrhosis

-Cholestasis

-Hyperbilirubinemia.

Factors Specific to Heart

Transplant Recipients.

-Low levels of vitamin D

-Hypogonadism

-Long-term heparin

-Loops diuretics

-Secondary hyperparathyroidism

-Physical inactivity,

-Therapy with loop diuretics,

-Tobacco, alcoholism.

Factors Specific to Lung

Transplant Recipients.

-Glucocorticoid therapy

-Tobacco

-Physical inactivity

-Low body weight

-Malnutrition

-Hypogonadism

-Hypercapnia

-Hypoxia

-Hypogonadism

-Cystic Fibrosis

Post-Transplant Factors Affecting All Patients

-Glucocorticoids

-Immunosuppressors: cyclosporine, Tacrolimus.

-Older age

-Kidney dialysis

-Diabetes Mellitus

-High or low PTH

-Cholestatic liver disease,

-Primary Biliary Cirrhosis

This article will review the causes, prevention, and treatment of post-transplant bone loss and fractures in recipients of major organs involved in transplantation, such as kidney, liver, cardiac, and lung.

BONE AND FRACTURES BEFORE TRANSPLANTATION

Patients referred for solid organ transplantation due to various diseases (kidney, liver, heart, and lung) have a high prevalence of osteoporosis and fractures, with distinct characteristics specific to each transplanted organ (Table 2).

Table 2.- Prevalence of Osteoporosis and Fractures in Patients Pre-Transplantation *
Organ	Osteoporosis		Fracture incidence
Organ	Spine	Hip	Fracture incidence
Renal	22%	20%	24%-38%
Liver	12%-55%	-	22% (5)
Heart	7%-40%	25%	40%
Lung	9%-69%	-	15%-50%

* Using dual X-ray densitometry, Vertebral fracture incidence in the first years post-transplantation. References: 1,2,3,4,5,6,9,120,122,123.

Renal Disease

End-stage renal disease (ESRD) is associated with a form of bone disease known as renal osteodystrophy. This condition develops due to factors such as Vitamin D deficiency, hypercalcemia, hyperphosphatemia, secondary hyperparathyroidism, metabolic acidosis, adynamic bone disease, osteomalacia, and aluminium overload which can lead to low bone BMD. In ESRD, cortical bone is predominantly lost, often resulting in peripheral fractures. The combination of fractures and the classic risk factors of chronic kidney disease significantly increases mortality.

Osteoporosis was found in 27.6% of 221 patients awaiting kidney transplantation and was associated with vascular calcification in 75% and parathyroid hyperplasia in 93.4% of cases. In many of these patients, there is a preference for appendicular fractures, which is different from other solid transplant recipients.

Elevated bone markers of formation (PINP) and resorption markers (B-CTX) were also linked to decreased BMD, confirming a disruption in bone remodelling. This suggests that sustained PTH levels indicate abnormal osteoblast function characterized by high turnover and increased resorption which contributes to a higher fracture risk (11).

Early renal dysfunction is associated with a 38% increase in fracture risk in men over 65 years old. In a cohort of 1477 participants from the Longitudinal Aging Study Amsterdam, followed for six years, patients with chronic kidney disease (CKD) stages 3a and 3b had a 28% and 46% higher fracture risk, respectively, compared to those with stages 1 and 2 (eGFR >60 ml/min/1.73 m²) (12). Early renal dysfunction was linked to lower femoral neck BMD, only in men, likely due to higher PTH levels. Several factors, including hemodialysis and diabetes mellitus further increase fracture risk. Patients with renal insufficiency, low bone turnover, and reduced BMD are at the highest risk for fractures.

Liver Disease

Osteoporosis and osteopenia are frequent complications of chronic liver disease, with a higher prevalence in patients awaiting liver transplantation, particularly in those with cholestatic liver diseases (14,15). Low BMD before transplantation is a major risk factor, influenced by inadequate calcium intake, malabsorption, malnutrition, vitamin D deficiency with secondary hyperparathyroidism, and an abnormal sex hormone ratio. In cirrhotic patients, factors such as hypogonadism, steroid use, and alcoholism can further accelerate bone loss (16). Heavy alcohol consumption affects bone metabolism by modulating Wnt and mTOR signaling [17], leading to decreased bone formation and increased adipogenesis. Additionally unconjugated bilirubin in patients with cholestasis has been shown to exert harmful effects on osteoblasts, reducing their viability. Studies have demonstrated that sera from jaundiced patients can upregulate the RANKL/OPG ratio promoting osteoclastogenesis, while downregulating Runx2, a key transcription factor involved in osteoblast differentiation (18).

Calcium and serum PTH levels are typically normal, but two-thirds of patients may have low levels of 25OHD, due to impaired hepatic hydroxylation of cholecalciferol. Histomorphometric studies have revealed decreased cortical bone volume, low bone formation, poor mineralization, and slightly elevated osteoclastic activity (19)

Cardiac Disease

Bone loss in candidates for cardiac transplantation is associated with the underlying disease and is commonly found in those with congestive heart failure. The prevalence of osteoporosis at the time of cardiac transplantation has been reported to range from 7% to 23% (20). Older studies indicate that 7% of these patients had lumbar osteoporosis and 20% had hip osteoporosis, meaning that fewer than 50% had normal BMD (21). In another study of 51 cardiac transplant candidates, the prevalence of osteoporosis was 27%, with longer waiting times before transplantation identified as a major risk factor for its development. (22).

Interestingly, despite 80% of these patients having vitamin D deficiency, 55% of cardiac transplant candidates, had BMD levels comparable to those of healthy individuals (23). This discrepancy may be explained by seasonal variations and the fact that these patients were ambulatory rather than on the transplant list. It is therefore recommended that patients on the waiting list be evaluated for bone loss prevention. The high prevalence of osteoporosis in this population is linked to chronic illness, poor nutrition, limited mobility, weight loss, gonadal dysfunction, and medications that negatively affect bone health (24). Additionally, patients with congestive heart failure, are often treated with medications such as loop diuretics, which increase urinary calcium losses. Furthermore, azotemia is known to impair vitamin D metabolism leading to elevated PTH levels and further contributing to bone loss.

Lung Disease

Patients who are candidates for lung transplantation are also highly likely to have osteoporosis before surgery. In many cases, chronic exposure to glucocorticoids is the primary risk factor. However, several other risk factors may also contribute to bone loss.

A retrospective study of patients with diffuse parenchymal lung disease referred for lung transplantation found that 30% had lumbar osteoporosis and 49% had femoral osteoporosis (25). Another study reported even higher rates, with 50% of patients having lumbar osteoporosis and 61% having femoral neck osteoporosis (26,27). Additionally, most of these patients had a history of glucocorticoid therapy.

Cystic Fibrosis (CF) in advanced stages, is another lung disease associated with a high prevalence of osteoporosis and fractures in patients awaiting lung transplantation. A meta-analysis found that bone complications are common in CF, with a prevalence of 23.5% for osteoporosis, 14% for vertebral fractures, and 19.7% for non-vertebral fractures (28). In younger patients, potential risks such as calcium and vitamin D malabsorption, malnutrition, delayed puberty, hypogonadism, and glucocorticoid therapy have been identified as contributors to bone loss.

BONE LOSS AND FRACTURES AFTER TRANSPLANTATION

Bone loss and fracture rates are higher in patients who had osteoporosis before transplant (Table 3). In these cohorts, post-transplantation fractures are associated with increased mortality rates (29). The highest fracture risk has been reported in heart and lung recipients, as well as in those with comorbidities such as rheumatoid arthritis, gout, and chronic obstructive pulmonary disease (COPD. Regarding diabetes mellitus, no clear association has been established between this condition and fracture risk in post-transplanted patients.

Table 3.- Prevalence of Osteoporosis and Fractures in Patients Post-Transplantation
Organ	Osteoporosis		Fracture incidence
Organ	Spine	Hip	Fracture incidence
Renal	7%-44%	11%-56%	7-21%)
Liver	11%-52% (6)	-	24-65%
Heart	28%-50%	25%	22%-44%
Lung	31%-84%	-	18-37%

* Using dual X-ray densitometry, Vertebral fracture incidence in the first years post-transplantation. References: 1,2,5,6,7,8,9,10,38,120,121,122,123,124, 125,126.

Bone loss appears to be more significant during the first year after transplantation across all types of organ transplants, primarily affecting trabecular bone, including the vertebrae and peripheral skeleton. When analysing the incidence rate of osteoporosis per 1000 person-years, heart and lung transplants have the highest rates at 6.00, compared to 4.17 and 4.09 for liver and kidney transplants, respectively. A significant increase was also observed in heart and lung transplant recipients, with a rate of 9.36 per 1.000 person-years, compared to 2.44 for liver transplants and 1.98 for kidney transplants. (27). These differences are likely influenced by variations in immunosuppressive regimens and the use of lower steroid doses.

There are inconclusive findings regarding bone mass recovery in transplant recipients beyond the first year, with studies reporting decreases, increases, or stabilization (30,31). After five years of follow-up, hip BMD often remains lower than pre-transplant levels in many patients, similar to trends observed in quality-of-life assessments. (32). There is considerable overlap in BMD values between individuals who develop fractures and those who do not. This suggests factors affecting bone quality -such as geometry, microarchitecture, and intrinsic properties of bone- may play a more significant role in fracture risk than bone quantity alone in transplant patients.

Trabecular Bone Score (TBS) a surrogate of bone quality, has been shown to deteriorate for up to a year after solid organ transplantation, even with therapeutic interventions, such as risedronate, ibandronate, vitamin D, and calcium, and without correlation with BMD. However, after one year, TBS improved and has been identified as a strong and independent predictor of fragility fractures (33,34).

Kidney Disease

As with other organ transplants, BMD loss in the first six months after kidney transplantation primarily affects cortical bones, largely due to persistent hyperparathyroidism and glucocorticoid use. It is estimated that 30% to 50% of patients continued to have hyperparathyroidism post-transplant (35). Specific risk factors for bone loss in kidney transplant recipients include previous chronic kidney disease, duration of dialysis, and hypomagnesemia. Additionally, patients with diabetes mellitus and nephropathy, have an increased risk of bone loss.

Post-transplant treatment factors, such as the use of tacrolimus and steroids, along with older age and elevated body mass index, further contribute to the risk of developing post-transplant diabetes (36). In kidney transplant recipients, those with vitamin D deficiency, have a 2.4 times higher risk of developing post-transplant diabetes compared with those with normal serum 25OHD (>30 ng/ml). Cross-sectional studies report osteoporosis prevalence rates ranging from 17% to 29% at the spine, 11% to 56% at the femoral neck, and 22% to 52% at the radius (11). Although most fractures occur within the first three years post-transplant, the risk continues to increase over time in some patients.

Liver Disease

After liver transplantation, bone density declines rapidly within the first six months, followed by a gradual increase in the subsequent months, with a tendency toward recovery within two years. However, not all patients regain normal BMD levels. Fracture incidence is highest during the first six months post-transplant, particularly in patients with primary biliary cirrhosis. While lumbar BMD tends to improve over time, hip BMD often remains lower for an extended period after transplantation (39).

Previous studies have reported an osteoporosis prevalence of 40.8% in 82 liver transplant recipients with various etiologies who were followed for one year (38). Similarly, a more recent study found a prevalence of 34.5% in 83 patients who were followed for an average of 80 months (39). After the first year, bone loss tends to slow, likely due to reduced use of glucocorticoids and immunosuppressants (40). Additionally, the decrease in bilirubinemia, known to negatively affect osteoblast differentiation and mineralization, may also play a role in this stabilization. However, it is accepted that approximately one-third of liver transplant recipients, still have lumbar spine BMD below the fracture threshold two years post-transplant, despite improvements in survival and quality of life (41). Reported fracture rates after liver transplantation range from 24% to 65%.

Cardiac Disease

Bone loss progresses rapidly after transplantation in these patients, with an estimated decline of 6%-11% at the vertebral site within the first six months, and a similar rate at the hip within the first year (21). Most studies report vertebral fracture incidence ranging from 33 to 36% in the first one to three years post-transplant stabilizing in subsequent years (15). During the initial months after transplantation, bone resorption markers are elevated, while bone formation markers (such as osteocalcin), are reduced. The levels typically return to normal by the end of the first year (38).

Patients with congestive heart failure patients are particularly prone to significant bone loss compared to other cardiac transplant candidates (7). Higher exposure to glucocorticoids, vitamin D deficiency, and testosterone deficiency in men, are linked to reduced bone formation in the first year (Table 3). By the third-year post-transplant, a significant recovery in lumbar BMD is observed. While both men and women experience similar rates of bone loss, women are more prone to fractures due to lower pre-transplantation BMD.

In some patients, bone recovery has not been observed, with a reported fracture prevalence of 40% among 180 individuals who underwent cardiac transplantation over 10 years (44). Furthermore, significant bone loss has been documented, with decreases ranging from 3% to 10% in the lumbar spine, 6% to 11% in the femoral neck, and fracture rates between 12% and 36% within one year. These rates of bone loss are notably higher compared to the 1.41% and 0.35% annual decreases observed at the lumbar spine and femoral neck, respectively, in the healthy population (44,45).

Lung Disease

Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis which can reach 57% to 73% in the first year after lung transplantation (46). Fracture rates continue to rise in some cases, with an average prevalence of 53% by the fifth-year post-transplant (47). Lung transplants have reported the highest fracture rates, likely due to prolonged and intensive immunosuppressive therapy, along with additional life-risk factors. Among lung diseases, patients with COPD are particularly prone to bone loss.

OSTEOPOROSIS AFTER BONE MARROW TRANSPLANTATION

Allogenic or autologous stem cell transplantation is used to treat a variety of hematologic diseases. Advances in histocompatibility testing and improvements in infection control have significantly increased patient survival. Risk factors for osteoporosis include the underlying disease, comorbidities (such as diabetes and obesity), the use of glucocorticoids, and immunosuppressants.

The pathogenesis of bone loss in this context is not well understood. It has been postulated that implanted bone marrow stromal cells may have a reduced capacity to develop into osteogenic lineage. Bone loss is most prominent during the 6 to 12 years following transplantation. Osteoporosis has now been recognized as a common condition in these patients, with a reported prevalence of up to 23% within the first-year post-transplant. Therefore, bone marrow-related osteoporosis following stem cell transplantation appears to be less severe compared to that seen in solid organ transplantation. However, there are still relatively few publications addressing this issue.

PATHOGENESIS OF OSTEOPOROSIS POST-TRANSPLANTATION

Bone loss after solid organ transplantation is caused by numerous factors, including pre-transplant underlying disease, individual risk factors, and the use of glucocorticoids and immunosuppressor drugs. Additional contributors include age, limited mobility, smoking, excessive alcohol consumption, and lifestyle habits.

Glucocorticoids

Glucocorticoids are essential for managing rejection episodes. They are typically administered at high doses initially and then gradually reduced. However, if rejection occurs, the dosage is increased. Recent protocols have aimed to minimize glucocorticoid doses to reduce side effects. High doses of glucocorticoids play a significant role in bone loss. However, it has been shown that even small doses of glucocorticoids are associated with an increased fracture risk (48). The potential impact of glucocorticoid dose on bone loss is supported by the evidence showing no significant bone loss at the lumbar spine and proximal femur in renal transplant patients treated with low doses of steroids and tacrolimus. Additionally, studies have reported that steroid withdrawal in liver transplant patients accelerates lumbar spine bone density recovery without compromising graft tolerance (5,54).

The mechanisms contributing to glucocorticoid-induced bone loss are discussed in the Endotext chapter entitled “An Overview of Glucocorticoid-Induced Osteoporosis” in the Bone Mineral section.

DIRECT EFFECTS OF GLUCOCORTICOIDS ON OSTEOBLASTS AND OSTEOCYTES

Glucocorticoids inhibit bone formation by impairing the proliferation and differentiation of osteoblasts, as well as reducing their lifespan (49,50, 51). This occurs through the inhibition of the canonical Wnt/B catening pathway, and the upregulation of sclerostin and other peptides, which further suppress osteoblast formation.

DIRECT EFFECT OF GLUCOCORTICOIDS ON OSTEOCLASTS

Glucocorticoids increase the production of RANKL (receptor activators of nuclear factor kappa-B ligand) and decrease the production of osteoprotegerin, leading to enhanced bone resorption.

INDIRECT EFFECTS

Similar to other conditions with hypercortisolism, glucocorticoids in post-transplant patients can induce hypogonadism, by directly inhibiting the secretion of estrogens and androgens. They also impair calcium absorption, and negatively affect the synthesis of 25OHD, by inhibiting the 25 hydroxylases.

Calcineurin Inhibitors: Cyclosporine A (CsA) and Tacrolimus

The impact of various immunosuppressor drugs used in post-transplant patients on bone health remains partially unknown and, in some cases, controversial. This uncertainty is likely due to differences in dosage, duration of use, and combination with other medications (Table 4). Among these drugs, two are considered the cornerstone of immunosuppressive therapy for maintaining graft survival. Calcineurin inhibitors work by inhibiting cytokines synthesis, such as interleukin-2, through binding to immunophilin and suppressing the activity of calmodulin-dependent protein phosphatase calcineurin. This suppression reduces by downregulating genes regulatory products, including interleukin 2, interleukin receptors and H-ras and c-myc (55). Studying the effect of these drugs on bone health is challenging due to their frequent coadministration with glucocorticoids. The effects of these drugs are difficult to study, due to their coadministration with glucocorticoids.

Table 4. Effect of Immunosuppressor Drugs on Bone of Post-Transplant Patients
Drug	Effect on Bone
Glucocorticoids	Inhibition of bone formation Stimulation of bone resorption Reduce intestinal calcium absorption Increase urinary calcium excretion Decrease secretion of GH, estrogens and androgens
Calcineurin inhibitors Cyclosporine A & Tacrolimus	Marked stimulation of bone resorption Minor increase in bone formation
Sirolimus (Rapamycin)	No effects on bone volume Inhibits longitudinal growth Decrease bone formation
Everolimus	Decrease bone resorption
Azathioprine	No effect on bone volume
Mycophenolate mofetil	No change in bone volume

Studies in rats have shown that CsA stimulates both osteoblast and osteoclast activity (56). However, administration of CsA in rodents has been associated with severe trabecular bone loss and induced high turnover bone loss, due to increased bone resorption and formation, accompanied by elevated levels of osteocalcin and 1,25(OH)2D3 (57). In a one-month comparative study in rats, both CsA and tacrolimus were found to reduce bone strength. CsA induced high-turnover bone loss by stimulating both bone formation and resorption whereas tacrolimus primarily stimulated bone resorption (58). Additionally, in a small study of renal transplant patients, steroid withdrawal was associated with lower bone loss when CsA was used alone (59). Consequently, the overall effect of CsA on bone density remains unclear.

Tacrolimus has been shown to induce trabecular bone loss without significantly affecting bone formation in the rat (57). Compared to Csa, Tacrolimus-based regimens may allow for a decrease in glucocorticoids use and result in a more modest reduction in BMD. In a study of 350 liver transplant recipients with chronic cholestatic liver disease, patients treated with CsA experienced lower post-transplant bone gain and higher incidence of fractures than those receiving tacrolimus (60). Other studies suggest that tacrolimus induces only a modest reduction in bone mass, while some reports indicate liver transplant recipients treated with tacrolimus had significantly higher femoral neck BMD compared to those receiving CsA (61).

mmTOR Immunosuppressors: Sirolimus and Everolimus

Both drugs, inhibited the activity of the mammalian target of rapamycin (mTOR) a key protein kinase involved in regulating cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis.

Sirolimus, also known as rapamycin, has the advantage of not causing nephrotoxicity. In-vitro, Sirolimus inhibits the proliferation and differentiation of osteoclasts, making it a potential bone-sparing agent (61). Additionally, lower bone resorption markers observed in a study of renal transplant recipients suggest that this drug helps preserve bone mineral density (62). However, potential side effects seen in animal studies, including impaired growth, delayed callus formation, and interference with IGF1- indicating that sirolimus should be used with caution in clinical practice (63).

Everolimus is a derivative of rapamycin, targets the mTOR pathway, and inhibits interleukin-2 (IL”)-induced cell proliferation, thereby suppressing the immune response. Using mouse models everolimus has been shown to act as a potent inhibitor of osteoclast formation and activity (64).

Other Immunosuppressors: Mycophenolate Mofetil (Mn) and Azathioprine

Mn inhibits B and T lymphocyte proliferation while azathioprine, a purine antagonist, reduce lymphocyte count and immunoglobulin synthesis. In animal studies, neither drug has shown an adverse effect on bone mass, and their use may contribute to reduced glucocorticoid co-administration. Currently, many recommended post-transplant regimens consist of a calcineurin inhibitor -such as tacrolimus or cyclosporine A in combination with an antiproliferative agent Mm), with or without low-dose corticosteroids (e.g., prednisolone).

Azathioprine, another purine antagonist, further decreases B and T lymphocytes. The impact of sirolimus, tacrolimus, and Mn on osteoclasts has been studied in cell-cultured systems. The authors detected that the inhibition of osteoclast precursors and proliferation, with Mn and tacrolimus, was lower compared to sirolimus (65). Both drugs are given in protocols combined with other immunosuppressors, which makes it difficult to determine their effects on bone.

POST-TRANSPLANTATION SERUM PTH, VITAMIN D, TESTOSTERONE, AND MAGNESIUM

Changes in serum PTH levels vary following solid organ transplants. No significant alteration in PTH levels has been observed after cardiac transplants, whereas liver transplant recipients often experience a moderate increase. In kidney transplant patients, PTH levels may initially decrease by approximately 50% within the first six months post-transplantation (2). Notably, secondary hyperparathyroidism is observed in some kidney transplant recipients, particularly those with prolonged pre-transplant dialysis duration, reduced glomerular filtration, and low serum 25OHD levels (66). The exact causes of elevated serum PTH levels remain unclear but may be associated with the decline in renal function, which affects approximately 20% of transplant recipients. For a detailed discussion of hyperparathyroidism in patients with renal disease see the Endotext chapter entitled “Hyperparathyroidism in Chronic Kidney Disease” in the Bone and Mineral section.

Serum 25OHD levels are often low before transplantation in all transplant candidates before the procedure and remain low afterward. Following transplantation, 91% of patients experience vitamin D insufficiency, and 55% have a deficiency, with the more severe cases observed in liver transplant recipients (68). However, a tendency toward higher levels is typically seen, likely due to supplementation. This deficiency, along with factors such as immobilization, low sunlight exposure, and inadequate vitamin D intake, contributes to an increased risk of bone loss. Additionally, excess glucocorticoid leads to an increased catabolism of 25OHD.

Furthermore, the frequently observed lower testosterone levels found in transplant patients, which contribute to bone loss, generally recover within one year.

Hypomagnesemia is commonly observed in kidney and cardiac transplant recipients and has been associated with the use of calcineurin inhibitors, particularly tacrolimus. Hypomagnesemia can lead to bone loss by increasing the number of osteoclasts and decreasing osteoblasts, resulting in the deterioration of trabecular bone mass and stiffness, along with elevated PTH levels (127).

POST-TRANSPLANT OSTEOPOROSIS MANAGEMENT

Pre-Transplant Considerations

The evaluation of bone metabolism and fracture risk in candidates for solid organ transplantation should include the following components (Table 5):

-Medical History, Physical Examination, and Assessment of Traditional Osteoporosis Risk Factors: Key elements include age, sex, low body weight, nutritional status, history of fragility fractures, and prior falls. Notably, a history of falls is an important independent risk factor for fractures in the general population (68)

-Evaluation of Potential Secondary Causes of Osteoporosis: These may include endocrinological, nutritional, gastrointestinal, nephrological, rheumatological, hematological, and pharmacological factors (69)

-Bone Turnover Markers (BTMs): Although not diagnostic for osteoporosis, BTMs may serve as surrogate markers for bone remodeling activity and may aid in estimating fracture risk. In a study involving patients with chronic kidney disease (CKD) in the pre-transplant setting, BTMs were inversely associated with BMD, although no significant association with fracture prevalence was observed (70)

-Lumbar Spine Radiography: This modality is useful for detecting vertebral fractures, many of which are asymptomatic (71). Radiographic screening is recommended for all solid organ transplant candidates and is particularly advised in lung transplant recipients, given a reported vertebral fracture prevalence of approximately 25%, often without correlation to BMD measurements (72).

-Dual-energy X-ray absorptiometry (DXA): DXA scanning is recommended for all solid organ transplant candidates. In patients with CKD stages 3–5, DXA has demonstrated predictive value for fracture risk (73). However, its accuracy may be compromised by spinal deformities, degenerative changes, and vascular (e.g., abdominal aorta) or articular calcifications, which can lead to BMD overestimation.

-Fracture Risk Assessment Tool (FRAX): The FRAX algorithm estimates the 10-year probability of hip and other major osteoporotic fractures using clinical risk factors, with or without BMD input (74). Although CKD is not included in the FRAX model, its use is still recommended for renal transplant candidates, as predictive utility has been demonstrated in non-dialysis populations (75).

-Trabecular Bone Score (TBS): Derived from DXA images of the lumbar spine, TBS evaluates bone microarchitecture through texture analysis (76). Its value as an independent predictor of fragility fractures has been confirmed in patients with CKD (77).

-Bone biopsy: In patients with CKD stages 3a–5D, bone biopsy should be considered when there are unexplained fractures, persistent bone pain, hypercalcemia, hypophosphatemia, or suspicion of aluminum toxicity.

Table 5. Clinical Evaluation in Patients with Solid Organ Transplantation
History -Age -History of previous fractures -Gonadal status -Dietary intake calcium/vitamin D -Alcohol abuse -Smoking -Physical Activity: sedentarism, exercise, mobility -Chronic disease: Diabetes, renal osteodystrophy, end-stage pulmonary disease, hepatic diseases. -Medications
Physical examination -Weight -BMI -Presence of imbalance, complications
Laboratory -Serum Ca, P04, Mg -Serum intact PTH -Serum 25OHD -Renal function parameters -Bone mineral density -Trabecular Bone Score -Bone turnover markers (formation/resorption) -Gonadal hormone levels (testosterone in men, estradiol, LH levels in women) -Thyroid function studies -Urinary calcium excretion
Densitometry: DXA Fracture Risk Assessment: FRAX® test

Table 5. Clinical Evaluation in Patients with Solid Organ Transplantation

History

-Age

-History of previous fractures

-Gonadal status

-Dietary intake calcium/vitamin D

-Alcohol abuse

-Smoking

-Physical Activity: sedentarism, exercise, mobility

-Chronic disease: Diabetes, renal osteodystrophy, end-stage pulmonary

disease, hepatic diseases.

-Medications

Physical examination

-Weight

-BMI

-Presence of imbalance, complications

Laboratory

-Serum Ca, P04, Mg

-Serum intact PTH

-Serum 25OHD

-Renal function parameters

-Bone mineral density

-Trabecular Bone Score

-Bone turnover markers (formation/resorption)

-Gonadal hormone levels (testosterone in men, estradiol, LH levels in women)

-Thyroid function studies

-Urinary calcium excretion

Densitometry: DXA

Fracture Risk Assessment: FRAX® test

Preventive Management and Post-Transplant Osteoporosis Treatment

Although numerous studies in solid organ transplant recipients have demonstrated the beneficial effects of antiresorptive agents in preventing bone loss, the majority have been limited by insufficient statistical power to detect significant differences in fracture incidence. Nonetheless, two reports have provided evidence supporting the effectiveness of initiating treatment with bisphosphonates or vitamin D supplementation in reducing the risk of post-transplant fractures (78,79) (Table 6).

Given that patients who have undergone solid organ transplantation are at a higher risk of fractures compared to the general population, particularly within the first year post-transplant, several experts and professional societies recommend preventive treatment during this critical period, especially for heart, lung, and liver transplant recipients. For instance, the International Society for Heart and Lung Transplantation (ISHLT) guidelines recommend preventive therapy for all heart transplant recipients during the first year following transplantation (80). Similarly, the American Association for the Study of Liver Diseases (AASLD) guidelines advocate for the use of bone-protective treatment in all liver transplant recipients (81). In lung transplantation, where the incidence of osteoporosis and osteopenia is significantly higher than in other transplant populations, preventive treatment is also strongly recommended.

In contrast, there is currently no consensus regarding the use of preventive therapy in renal transplantation. Some authors have proposed initiating antiresorptive treatment in patients with osteopenia and a high risk of fracture. They emphasize the importance of a comprehensive fracture risk assessment, which should include factors such as age, sex, history of fragility fractures, bone mineral density (BMD), bone turnover markers, and parathyroid hormone (PTH) levels (82). Preventive treatment is generally recommended for patients with elevated fracture risk and/or evidence of osteopenia.

The management of bone loss in patients undergoing bone marrow transplantation is currently under investigation. A recent meta-analysis suggests that, in patients with a BMD T-score below -1.5, bisphosphonates, particularly zoledronic acid, are effective in preventing bone loss. If renal function is impaired or bisphosphonates are not well tolerated, denosumab is recommended as an alternative. Clinical trials involving teriparatide, abaloparatide, and romosozumab have not yet been published (128).

Table 6. Antiresorptive Therapy with Proven Efficacy in Increasing Bone Mineral Density in Solid Organ Transplant Patients
Medications	Dose and route of administration	Rare possible long-term Adverse Effects
Bisphosphonates -Alendronate -Risedronate -Ibandronate -Zolendronic acid -Pamidronate	70 mg PO/week 5 mg PO/daily 150 mg PO/monthly 3 mg IV/3 months 5 mg IV/year for 5 yrs 30 mg IV/3 months	GI intolerance, hypocalcemia, rare jaw osteonecrosis and atypical femur fracture Same as above Same as above Same as above plus infusion reaction Same as above
Denosumab	60 mg SC/6 months	Liver safe, fractures rebound after cessation
Teriparatide	20 ug/day SC/2 years only	Hypercalcemia

PO = per os; SC = subcutaneously

NON-PHARMACOLOGICAL MEASURES

The general recommendations for all transplant patients are as follows:

Smoking cessation and reduced alcohol consumption (especially in the case of liver transplantation).
Limiting caffeine intake to fewer than 1–2 caffeinated beverages per day.
Nutritional assessment to identify patients at risk of malnutrition or those with established malnutrition, enabling appropriate dietary modifications and initiation of nutritional supplementation if needed. It is also crucial to ensure adequate protein intake, as this helps minimize bone loss, particularly in patients with prior hip fractures.
Avoidance of prolonged immobilization due to the association between sarcopenia, increased risk of falls, and bone fractures. Physical exercise is strongly recommended, including:
- A 30- to 40-minute walk per session (3 to 4 times per week).
- Back and postural exercises for a few minutes per day (3 to 4 times per week).
- Strength training exercises.
Implementation of fall prevention measures, both at home and outdoors.

Finally, to optimize bone metabolism in transplant patients and reduce the risk of developing osteoporosis, it is recommended to appropriately adjust the doses of steroids and immunosuppressants (maintaining these drugs within the therapeutic range and avoiding overdosing) (83).

CALCIUM AND VITAMIN D SUPPLEMENTATION

Although conclusive data in patients with solid organ transplantation is lacking, it is recommended to achieve and maintain normal levels of calcium and vitamin D, with supplementation provided if necessary. Vitamin D deficiency is particularly common in transplant recipients, making supplementation especially important for these patients. The recommended daily intake of calcium ranges from 1000 to 1200 mg, depending on age and sex (84). If dietary calcium intake is insufficient, supplementation should be considered. Vitamin D deficiency leads to inefficient absorption of dietary calcium and phosphorus, as well as secondary hyperparathyroidism, which can impair bone mineralization. An optimal 25(OH)D level of >30 ng/mL is recommended, similar to the target for steroid-induced osteoporosis.

A study involving pre-renal transplant recipients found that cholecalciferol supplementation with vitamin D and calcium, did not lead to a significant improvement in BMD compared to calcium supplementation alone (85). However, a recent study in renal transplant patients showed that taking 4000 IU/day of cholecalciferol for one year reduced lumbar BMD loss compared to placebo (percentage change in BMD: −0.2% with cholecalciferol vs. −1.9% with placebo). The positive effect on BMD was more pronounced in patients who had significant bone mass impairment at the beginning of treatment with more pronounced bone mass impairment at the start of treatment (86)

CALCITRIOL, PARACALCITOL, ALFACALCIDOL

Calcitriol is the active form of vitamin D, while paricalcitol and alfacalcidol are synthetic analogs of vitamin D. It has been demonstrated that these compounds reduce or stabilize parathyroid hormone (PTH) levels and improve bone histology post-transplantation (84,86). Their use has been proposed as a preventive treatment for osteoporosis in transplant patients who may have contraindications to or intolerance of bisphosphonates.

In heart transplantation, a clinical trial comparing alendronate and calcitriol over one year demonstrated that calcitriol significantly reduced bone mass loss in the lumbar spine and femoral neck, with no significant differences between the bisphosphonate and calcitriol groups (87). In renal transplant patients with secondary hyperparathyroidism, supplementation with paricalcitol for six months was associated with reductions in PTH levels and proteinuria, as well as an improvement in bone mass loss (88).

A meta-analysis demonstrated a reduction in vertebral fractures with bisphosphonates or calcitriol during the first year of administration in solid organ transplant recipients (89). It is important to note that this meta-analysis showed considerable heterogeneity across the included studies (e.g., type of transplanted organ, type and dose of bisphosphonate used, and immunosuppressive regimen). Furthermore, only two of the eleven studies included calcitriol as the active comparator. Calcidiol was also found to be an effective therapy in 40 patients following cardiac transplantation. After 18 months of treatment, 12,000 IU weekly of calcidiol led to a 4.9% increase in lumbar BMD, compared to −1.19% and −0.19% with calcitonin and etidronate, respectively. Calcidiol causes less hypercalcemia and hypercalciuria than calcitriol (90).

Despite their potential benefits, calcitriol and synthetic analogues should be used with caution, as their use is associated with an increased risk of hypercalcemia and hypercalciuria. Therefore, periodic monitoring of serum calcium and 24-hour urinary calcium levels is recommended.

BISPHOSPHONATES (BFs)

These drugs have been the most widely used treatment for osteoporosis for over two decades (Table 6). They are analogs of inorganic pyrophosphate that inhibit bone resorption. BFs are generally safe and well-tolerated. They are the initial treatment option for both the prevention and management of post-transplant osteoporosis. Numerous studies have demonstrated improvements in bone density with BFs; however, there is currently no specific recommendation favoring one bisphosphonate over another.

In an early trial, the comparison of salmon calcitonin and sodium etidronate over one year showed that BFs were capable of inducing a greater increase in lumbar BMD (6.4% vs. 8.2%) (91). A more recent retrospective study in renal transplant patients with end-stage renal disease demonstrated that BF treatment for 3.5 years was associated with a significant increase in lumbar spine BMD in recipients 15 years post-transplant (92). Additionally, in renal transplant patients, administration of BFs for 12 months was associated with improvements in lumbar and femoral neck BMD, as well as a reduction in fracture risk (RR 0.62; CI: 0.38–1.01) (86).

Two trials with risedronate have been conducted: one involving 101 patients after kidney transplantation and another with 41 liver transplant patients, both with one year of follow-up. In both trials, there was a significant and early increase in lumbar BMD at 6 months (93,94). In a study of 84 patients with liver or heart transplantation, alendronate and zoledronate administered for one year prevented hip bone loss. However, in heart transplant patients, lumbar BMD remained stable with zoledronate but decreased with alendronate (95).

A more recent meta-analysis reported improvements in BMD and reductions in fracture risk with BFs use in liver transplant patients (96). The results indicate that BFs are associated with superior fracture prevention compared to calcium and vitamin D alone (OR = 0.37; CI: 0.17–0.7). Oral BFs were linked to a lower incidence of vertebral and overall fractures, as well as improvements in lumbar spine and femoral neck BMD, compared to intravenous BFs. The potential superiority of oral BFs may be due to several studies using intravenous zoledronate and ibandronate with doses that did not align with those recommended in clinical guidelines.

The tolerability of BFs is generally acceptable, with the common adverse effect being gastroesophageal reflux (97). Therefore, caution should be exercised in liver transplant patients with pre-existing cirrhosis if esophageal pathology is present, especially in those with pre-transplant esophageal varices. In transplant patients with a history of esophageal injuries or who develop esophagitis with oral bisphosphonate use, zoledronate may be considered, as its parenteral administration is not associated with reflux development. Furthermore, treatment with zoledronate is associated with better patient adherence (98) and may be an option to reduce polypharmacy in transplant patients (95). Another complication associated with BF use is hypercalcemia, typically linked to intravenous zoledronate use and pre-existing vitamin D deficiency (86). Finally, due to its potential to exacerbate adynamic bone disease, BFs are not recommended in patients with glomerular filtration rates below 30 mL/min (96).

DENOSUMAB

Denosumab is a monoclonal antibody against the receptor activator of nuclear factor kappa-B ligand (RANKL), a key factor involved in osteoclast differentiation. By blocking the binding of RANKL to its receptor, RANK, denosumab reduces the formation, function, and survival of osteoclasts. This action decreases bone resorption, increases BMD, and reduces fracture risk in both the short and long term (99).

Therapy with denosumab has demonstrated improvements in BMD in patients undergoing solid organ transplantation, although data are limited. In a study involving 93 renal transplant patients with osteoporosis, after 12 months of denosumab treatment, there was an improvement in bone mineral density of 4.6% (3.3–5.9%) in the lumbar spine and 1.9% (0.1–3.7%) in the total hip (100). Similarly, it was shown that the prevalence of osteoporosis decreased in the lumbar spine from 72% to 50% and in the femoral neck from 78% to 69% in 32 renal transplant recipients (101). High-resolution peripheral quantitative computed tomography has described the beneficial short-term effects of one year of denosumab treatment on bone structure, microarchitecture, and strength in kidney transplant recipients (102).

In a study with various types of transplants (49 renal, 14 liver, and 15 simultaneous kidney-pancreas transplant recipients), denosumab treatment over one year increased lumbar BMD by 11.5 ± 6.2% and femoral neck BMD by 10.4 ± 8.3%, reducing the prevalence of osteoporosis in the spine by 48% and in the proximal femur by 18% (103). In a 4-year trial, denosumab was linked to a significant increase in BMD (9.0 ± 10.7% in the lumbar spine and 3.8 ± 7.9% in the total hip) in renal transplant patients, while the control group showed lower increases in BMD at all sites (104).

Few studies have evaluated whether denosumab improves densitometric outcomes compared to BFs. A clinical trial involving 85 renal transplant patients compared the impact of denosumab versus BFs after more than 3 years and found that denosumab treatment resulted in a significant increase in bone density in both the lumbar spine and femoral neck compared to the BFs group (105). A slight increase in mild urinary tract infections and asymptomatic episodes of hypocalcemia (especially in patients with impaired renal function) has been reported with denosumab. To detect hypocalcemia, it is recommended to measure corrected serum calcium and 25(OH)D levels 2–4 weeks after the denosumab dose (106). No severe complications, such as osteonecrosis of the jaw, have been reported in transplant patients using denosumab. As for vertebral fractures observed in postmenopausal osteoporosis following denosumab discontinuation, this complication has not been evaluated in transplant patients.

In summary, the available studies demonstrate that short- and medium-term use of denosumab is a useful option for treating osteoporosis in patients who have undergone organ transplantation. Its use should be considered, particularly in patients with established chronic kidney disease where bisphosphonates may be contraindicated. Moreover, a sub-analysis of the FREEDOM study showed that the reduction in fracture risk remained similar in patients with chronic kidney disease stages I to IV, suggesting that denosumab could be highly beneficial for this group of patients (107).

TERIPARATIDE

Teriparatide is a fragment of parathyroid hormone comprising amino acids 1–34, retaining the activity of the intact peptide. It is an anabolic agent with proven efficacy in reducing both vertebral and non-vertebral fracture risk in postmenopausal women with osteoporosis (108). However, similar to denosumab, publications evaluating the use of teriparatide in solid organ transplant patients are limited.

In a 6-month clinical trial, 26 renal transplant patients received either teriparatide or a placebo. In the teriparatide-treated group, no improvement in BMD at the femoral neck, lumbar spine, or distal radius was observed; rather, BMD remained stable. In contrast, patients receiving a placebo experienced a decrease in femoral neck bone mass over the 6-month study period (109).

In another study, teriparatide treatment in 18 renal transplant patients was associated with a significant improvement in lumbar spine BMD after 1 year, stability of total hip bone mass, and a significant increase in femoral neck BMD after 2 years of treatment (110). No changes were observed in bone microarchitecture, as assessed by the Trabecular Bone Score.

Teriparatide was generally well tolerated, with isolated episodes of mild and transient hypercalcemia and hypophosphatemia.

In a separate retrospective study of renal transplant patients with osteoporosis, the differences in BMD after 1 year of treatment with alendronate or teriparatide were evaluated. Teriparatide was associated with a significant improvement in BMD at all sites, while bisphosphonate use was linked to a lower rate of complications (111).

SUMMARY OF TREATMENT APPROACHES. WHEN TO START AND STOP ANTIRESORPTIVE THERAPY

The Bone Health and Osteoporosis Foundation has made the following recommendations for initiating pharmacologic therapy:

Patients with lumbar, femoral neck, or total hip BMD T-score ≤-2.5.
Postmenopausal women and men aged ≥50 years with lumbar, femoral neck, or total hip BMD between -1.0 and -2.5 and a 10-year probability of a hip fracture ≥3% or a 10-year probability of a major osteoporosis-related fracture ≥20%.
A hip or vertebral fracture regardless of T-score.
A pelvis, proximal humerus, or distal forearm fracture in a person with low bone mass or osteopenia.

It is recommended that glucocorticoids be administered at the lowest dose possible, and reduced or withdrawn when feasible, in order to minimize early bone loss after transplantation.

Supplementation with calcium and vitamin D is advised. Serum 25OHD levels should be above 50 nmol/L. In kidney transplant recipients; alfacalcidiol or calcitriol can be used due to impaired 1 alpha hydroxylation of this metabolite to reduce secondary hyperparathyroidism.

In the presence of osteoporosis, antiresorptive therapy should be administered. BFs are the most widely used, while denosumab is an alternative, especially in cases of intolerance to bisphosphonates. For patients with suppressed bone turnover markers, BFs should be avoided due to the potential risk of exacerbating low bone turnover or adynamic bone disease. Although denosumab is metabolized hepatically and does not accumulate in renal insufficiency, hypocalcemia and the risk of rebound vertebral fractures upon withdrawal require careful monitoring and consideration of initiating BF therapy. There is limited experience with other drugs, such as romosozumab and abaloparatide, which have demonstrated efficacy in treating adult osteoporosis.

FOLLOW-UP OF POST-TRANSPLANT PATIENTS

After initiating treatment, BMD should be monitored using Dual-energy X-ray absorptiometry (DXA). Although there are no specific recommendations for transplant patients, it is reasonable to repeat DXA after 1 year if denosumab is used, and after 1 or 2 years if bisphosphonates are the treatment (112). If an adequate densitometric response is not observed after the first or second year, considering an alternative treatment would be logical.

The decision to stop treatment should be individualized based on clinical information. After 3 to 5 years of bisphosphonate treatment, patients with a modest fracture risk (T-score >-2.5) may discontinue treatment, while those at high fracture risk (T-score ≤-2.5) should either continue treatment or begin alternative therapy. Research has shown a residual positive skeletal effect even after discontinuing bisphosphonate treatment for several years. Reassessment of fracture risk is recommended after 2–3 years of bisphosphonate therapy. Discontinuation of denosumab treatment is associated with rapid bone loss and multiple vertebral fractures; therefore, bisphosphonates are recommended as an alternative therapy to maintain the gains in bone density (129).

REFERENCES

https://www.statista.com/statistics/398645/global-estimation-of-organ-transplantations/ Access: July 25, 2024.
2. Shane E, Papadopoulos A, Staron RB, Addresso V, Donovan D, McGregor C, Schulman Bone loss and fracture after lung transplantation. Transplantation 1999; 58:220-7
Chen H, Lai YR, Yang Y, Gau SY, Huang CY, Tsai TH, Huang KH, Lee CY. High risk of osteoporosis and fracture following solid organ transplantation: a population-based study. Front Endocrinol 2023; 14:1167574
Maalouf NM, Shane E. Osteoporosis after solid organ transplantation. Journal Clinical Endocrinology and Metabolism, 2005; 90:2456-2459
Martinez GM, Gomez R, Jodar E, Loinaz C, Moreno E, Hawkins F. Long-term follow-up of bone mass after orthotopic liver transplantation: Effect of steroid withdrawal from the immunosuppressive regimen. Osteoporosis Int 2002; 13:147-150
lyer SP, Nikkel LE, Nishiyama KK, Dworakowski E, Cremers S, Zhang C, McMahon D, Boutroy A, Liu XS, Ratner L, Cohen DJ, Guo XE, Shane E, Nickolas T. Kidney transplantation with early corticosteroid withdrawal: paradoxical effects at the central and peripheral skeleton. J Am Soc Nephrol 2014; 25:1331-4.
Kovvuru K, Kanduri SR, Vaitla P, Marathi R, Gosi S, Garcia Anton DF, Vabez Rivera FH. Garla V. Risk factors and management of osteoporosis post-transplant. Medicina 2020; 56(6):302.
Elder G.Current Status of mineral and bone disorders in transplant recipients. Transplantation 2023; 107(10):2107-2119.
Poole KE, Reeve J. Parathyroid hormone -a bone anabolic and catabolic agent. Curr Opin Pharmacol 2005; 5:612-617
Jin Kim K, Ha J, Kim SW, Kim JE, Lee S, Choi HS, Hong N, Kong SH, Ahn SH, Park SY, Back KH on Behalf of Metabolic Bone Disease Study Group of Korean Endocrine Society. Endocrinol Metab 2024; 39:267-282
Lv J, Xie W, Wang S, Zhu Y, Wang Y, Zhang P, Chen J. Associated factor of osteoporosis and vascular calcification in patients awaiting kidney transplantation.International Urology and Nephrology 2023; 55:3217-3224
Chen H, Lips P, Vevloet MG, van Schoor NM, de Jongh T. Association of renal function with bone mineral density and fracture risk in the longitudinal aging study Amsterdam. Osteoporos Int 2018; 29:2129-2138
Hsu S, Bansal N, Denburg M, Ginsberg C, Hoofnagle AN, Isakova T, Ix JH, Robinson-Cohen C, Wolf M, Kestenbaum BR, de Boer IH, Zelnick LR. Risk factors for hip and vertebral fractures in chronic kidney disease: the CRIC Study. J Bone Min Res 2024:39:433-442
Eastell R, Dickson ER, Hodgson SF, Wiesner RH, Porayko MK, Wahner HW, Cedel SL, Riggs BL, Krom RA. Rates of vertebral bone loss before and after liver transplantation in women with primary biliary cirrhosis. Hepatology 1991; 14:296-300
Leidig-Bruckner G, Hosch S, Dodidou P, Rtschel D, Conradt C, Klose C, Otto G, Lange R, Theilmann L, Zimmerman R, Pritsch M, Ziegler R. Frequency and predictors of osteoporotic fractures after cardiac or liver transplantation: a follow-up study. Lancet 2001; 357:342-7
Compston JE. Osteoporosis after liver transplantation. Liver Transpl 2003; 9:321-30.
Luo Z, Liu Y, Liu Y, Chen H, Shi S, Yi Liu Y.Cellular and molecular mechanisms of alcohol-induced osteopenia. Cell Mol Life Sci 2017:74:4443-53
Guañabens N, Parés A. Liver and bone. Arch Biochem Biophys. 2010; 503(1):84-94.
Vedi S, Greer S, Skingle SJ, Garrajam NJ, Ninkovic M, Alexander GA, Compston JE.Mechanism of bone loss after liver transplantation: a histomorphometric analysis. J Bone Miner Res 1999; 13:281-7
Forien M, Coralli R, Verdonk C, Ottaviani S, Ebstein E, Demaria L, Palazzo E, Dorent R, Dieude P. Osteoporosis and risk of fracture in heart transplant patients. Frontiers in Endocrinology 2023; 13:14:1252966
Shane E, Rivas M, McMahon DJ, Staron RB, Silverberg SJ, Seibel MJ, Mancini D, Michler RE, Aaronson K, Addesso V, Lo SH. Bone loss and turnover after cardiac transplantation. J Clin Endocrinol Metab 1997; 821:1497-1506
Garcia Delgado I, Gil-Fraguas L, Robles E, Martinez G, Hawkins F. Clinical factors associated with bone mass loss previous cardiac transplantation. Med Clin (Barc)2000; 114:761-764.
Iqbal N, Ducharme J, Desai S, Chamber S, Terembula K, Chan GW, Shults, Leonard MB, Kumanyka S. Status of bone mineral density in patients selected for cardiac transplantation. Endocrine Practie 2008; 15:704-712
Cohen A, Shane E. Osteoporosis after solid organ and bone marrow transplantation. Osteoporos Int 2003:14:617-30
Shane e, Silverberg SJ, Donovan D, Papadopoulos A, Staron RB, Addesso V, Jorgesen B, McGregor C, Schulman L. Osteoporosis in lung transplantation candidates with end-stage pulmonary disease. Am J Med 1996; 101:262-9
Tschopp O, Boehler A, Speich R, Weder W, Seifert B, Russi EW, Schmid C. Osteoporosis before lung transplantation: association with low body mass index, but not with underlying disease. Am J Transplant 2002; 2:162-72
Kovvuru K, Kanduri SR, Vaitla P, Marathi R, Gosi S, Garcia Anton DF, Cabeza Rivera FH, Garla V. Risk Factors and Management of osteoporosis post-transplant. Medicina 2020; 56(6):302
Paccou J, Zeboulon, Combescure C, Gossee L, Cortet T. The prevalence of osteoporosis, osteopenia and fractures among adults with Cystic Fibrosis: A systematic literature review with meta-analysis. Calcif Tissue 2010; 86:1-7
Chen H, Lai RR, Yang Y, Gau SY, Huan CY, Tsai TH, Huan KH, Lee CY. High risk of osteoporosis following solid organ transplantation: a population-based study. Front Endocrinol 2023: 14: 1167574
Anastasilakis AD, Tsourdi E, Makras P, Polyzos SA, Meter C, McCloskey EV, Pepe J, Zillikens MC. Bone disease following solid organ transplantation: A narrative review and recommendations for management from the European Calcified Society. Bone 2019; 17:401-418
Perez Saez MJ, Herrera D, Prieto Alhambra D, Nogues X, Vera M, Redondo Pachon D, Mir M, Guerri R, Crespo M, Diez Perea A, Pascual J: Bone density, microarchitecture and tissue quality long-term after kidney transplant. Transplantation 2012; 101:1290-4
Monteagudo LJ, Diaz-Guerra GM, Badillo AÁ, Álvarez Martínez CJ, Pablo Gafas A, Gámez García AP, López López E, Arriscado CM, Hawkins Carranza F. Health-Related Quality of Life Long-Term Study in Lung Transplant Patients: A Single-Center Experience. J Surg Res. 2024; 299:313-321.
Librizzi MS, Guadalix S, Martínez-Díaz Guerra G, Allo G, Lora D, Jimenez C, Hawkins F Trabecular bone score in patients with liver transplants after 1 year of risedronate treatment. Transpl Int. 2016 ;29(3):331-7.
Strommen RC, Godang K, Finnes TE, Smerud KT, Reisaeter AV, Hartmann A, Asberg A,Bollerslev J, Pihistrom HK. Trabecular bone score improves early after successful kidney transplantation irrespective of antiresorptive therapy and changes in bone mineral density. Transplantation Direct 2024;10: e1566
Weisinger JR, Carlini RG, Rojas E, Bellorin-Font E. Bone disease after renal transplantation. Clin J Am Soc Nephrol 2006; 1:1300-13
Tej KW, Geailt CM, Lappin DWP. Post-transplant bone disease in kidney transplant recipients: diagnosis and management. Int J Mol Sci 2024; 25(3):1859
Ebeling PR. Primer on Metabolic Bone Diseases and Disorders of Mineral Metabolism. Ninth Edition. Edited byJP Bilezikian,2019, chapter 54 Transplantation Osteoporosis, p- 425-434
Hawkins FG, Leon M, Lopez MB, Valero MA, Larrodera L, Garcia I, Loinaz C, Moreno E. Bone loss and turnover in patients with liver transplantation. Hepato-Gastroenterol 1994; 41:158.161
Li XY, Lew JCH, Kek PC. Bone mineral density following liver transplantation: a 10-year trend analysis. Archives of Osteoporosis 2021; 16:169
Krol CG, Dekkers OM, Kroon HM, Rabelink T, van Hoek B, Hamdy NA. Longitudinal changes in BMD and fracture risk in orthotopic liver transplant recipients not using bone-modifying treatment. J Bone Miner Res 2014; 29(8):1763-9
Hamburg SM, Piers DA, van den Berg AP, Slooff MJH, Haagsma EB. Bone Mineral Density in the long term after liver transplantation. Osteoporos Int 2000; 11:600-606
Rodriguez Aguilar EF, Peez Escobar J, Sanchez Herrera D, Garcia Alanis M, Tapania Y, Anchapaxi I, Gonzalez Flores E, Garcia Juarez I. Bone disease and liver transplatantion a review. Transplant Proc 2021; 53:2346-53
Shane E, Rivas MC, Silverberg SJ, Kim TS, Staron RB, Bilezikian JP. Osteoporosis after cardiac transplantation. Am J Med 1993:94:257-264
Dalle Carbonate L, Zanatta M, Braga V, Sella S, Vilei MT, Feltrin G, Gambino A, Pepe I, Rossini M, Adami S, Giannini S. Densitometric threshold and vertebral fractures in heart transplant patients. Transplantation 2011; 92:106-11
Lofdahl E, Radegran G, Fagher K. Bone health and cardiac transplantation. Best Practice & research clinical rheumatology 2022; 36(3):101770
Yu TM, Li Lin C, Chang SN, Chan Sung F, Huan ST, Kao CH. Osteoporosis and fractures after solid organ transplantation: a nationwide population-based cohort study. Mayo Clin Prac 2014; 89:888-95
Caffarelli C, Tomai Pitinca MD, Alessandri M, Crameli P, Bargagli F, Bennet E, Fossi a, Bernazzali s, Gonnelli S. Timing of osteoporosis vertebral fractures in lung and heart transplantation: a longitudinal study. J Clin Med 2020; 9(9):2941.
Buckley L, Humphrey MB. Glucocorticoid induced osteoporosis. Engl J Med 2018; 379:2547-56
49. Ohnaka K, Tanabe M, Kawate H, Nawata H, Takayanagi R Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem Biophys Res Commun. 2005; 329:177-81.
Hayashi K, Yamaguchi T, Yano S, Kanazawa I, Yamauchi M, Yamamoto M, Sugimoto T. BMP/Wnt antagonists are upregulated by dexamethasone in osteoblasts and reversed by alendronate and PTH: potential therapeutic targets for glucocorticoid-induced osteoporosis. Biochem Biophys Res Commun 2009; 379:261–266.
Mazziotti G, Formenti AM, Adler RA, Bilezikian JP, Grossman A, Sbardella E, Minisola S, Giustina A Glucocorticoid-induced osteoporosis: pathophysiological role of GH/IGF-I and PTH/VITAMIN D axes, treatment options and guidelines. Endocrine 2016; 54:603–611.
Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M, Hakeda Y Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-beta by stimulating the priming of osteoclast progenitors for differentiation into osteoclasts. J Biol Chem 2003; 278:44667–44674.
Meng Chen B, Fua W, Xub H, Chuan-Ju Liua, C. Pathogenic mechanisms of glucocorticoid-induced osteoporosis Cytokine Growth Factor Rev. 2023; 70: 54–66
Epstein S. Postransplantation bone disease: the role of immunosuppressive agents and the skeleton. J Bone Miner Res 1996; 11:1-7
Goffin E, Devogelaer JP, Depresseux G, Squiflflet JP, Pirson Y. Osteoporosis after organ transplantation. Lancet. 2001;357(9268):1623.
SchoslbergM, Movsowitz C, Epstein S, Ismail F, Fallon MD, Thomas S. The effect of cyclosporin A administration and its withdrawal on bone mineral metabolism in the Rat- Endocrinology 1989; 124:2179-2184
Orcel P, Bielakoff J, Modrowski D, Miravet L, de Vernejoul MC. Cyclosporin A induces in vivo inhibition of resorption and stimulation of formation in rt bone. J Bone Miner Res 1989; 4:387-91
Kanda J, Izumo N, Furukawa M, Shimakura T, Yamamoto N, Takahashi E, Asakura T, Wakabayashi H. Effects of calcineurin inhibitors cyclosporine and tacrolimus on bone metabolism in rats. Biomedical Research (Tokyo) 2018; 31:131-139
Movsowitz C, Epstein M, Fallon M, Ismail f, Thomas S. Cyclosporin A in vivo produces severe osteopenia in the rat: effect of dose and duration of administration. Endocrinology 1988; 123:2571-7
Ponticelli V, Aroldi A. Osteoporosis after organ transplantation. The Lancet 2001; 357:1623-24.
Guichelar MM, Schmoll J, Malinchoc M, Hay JE: Fractures and avascular necrosis before and after orthotopic liver transplantation: long-term follow-up and predictive factors. Hepatology 2007; 46:1198-207
Monegal A, Navasa M, Guañabens N, Peris P, Pons F, Martinez de Osaba MJ, Rimola MI, Rodes A, Muñoz Gomez J. Bone mass and mineral metabolism inn liver transplant patients treated with FK506 or cyclosporine A. Calcif Tissue Int 2001; 68:83-86
Campistol JM, Holt DW, Epstein S. Bone metabolism in renal transplant patients treated with cyclosporine or sirolimus. Transplant Int 2005; 18:1028-35
Blaslov K, Katalinic L, Kes P, Spasovki G, Smalcelj R, Basic Jukic N. What is the impact of immunosuppressive treatment on the post-transplant renal osteopathy. Int Urol Nephrol 2014; 46:1019-24
Kelly PA, Gruber SA, Belbod F, Kahan BD. Sirolimus a new potent immunosuppressive agent. Pharmacotherapy 1997; 17:1148-56
Westenfeld T, Schlieper G, Woltje M, Gawilk A, Bandenburg V, Rutkowski P, Floege J, Jahnen-Decjemt W, Ketteler M. Impact of sirolimus, tacrolimus and mycophenolate mofetil on osteoclastogenesis -implications for post-transplantation bone disease. Nephrol Dial Transplant 2011:26:4115-23
Bouquegneau A, Salam S, Delanaye P, Eastell R, Khwaja A. Bone disease after kidney transplantation. Clin J Am Soc Nephrol 2016; 11:1282-96
Stein EM, Shane E. Vitamin D in organ transplantation. Osteoporos Int 2011; 22:2107-2118
Edwards, M.; Jameson, K.; Denison, H.; Harvey, N.; Sayer, A.A.; Dennison, E.; Cooper, C. Clinical risk factors, bone density and fall history in the prediction of incident fracture among men and women. Bone 2013, 52, 541–547.
Arceo-Mendoza RM, Camacho PM. Postmenopausal Osteoporosis: Latest Guidelines. Endocrinol Metab Clin North Am. 2021;50(2):167-178.
Jørgensen HS, Winther S, Bøttcher M, Hauge EM, Rejnmark L, Svensson M, Ivarsen P. Bone turnover markers are associated with bone density, but not with fracture in end stage kidney disease: a cross-sectional study. BMC Nephrol. 2017;6;18(1):284.
Gerdhem P. Osteoporosis and fragility fractures: Vertebral fractures. Best Pract Res Clin Rheumatol. 2013;27(6):743-55.
Lakey WC, Spratt S, Vinson EN, Gesty-Palmer D, Weber T, Palmer S. Osteoporosis in lung transplant candidates compared to matched healthy controls. Clin Transplant. 2011; 25(3):426-35
West, C.E. Lok, L. Langsetmo, A.M. Cheung, E. Szabo, D. Pearce, M. Fusaro, R. Wald, J. Weinstein, S.A. Jamal, Bone mineral density predicts fractures in chronic kidney disease, J. Bone Miner. Res. 30 (5) (2015) 913–919.
Kanis, J.A. Assessment of Osteoporosis at the Primary Health Care Level; World Health Organization Collaborating Centre for Metabolic Bone Diseases, University of Sheffield: Sheffield, UK, 2007; Volume 339, pp. 11224
Whitlock RH, Leslie WD, Shaw J, Rigatto C, Thorlacius L, Komenda P, Collister D, Kanis JA, Tangri N. The Fracture Risk Assessment Tool (FRAX®) predicts fracture risk in patients with chronic kidney disease. Kidney Int. 2019;95(2):447-454.
Pothuaud L, Barthe N, Krieg MA, Mehsen N, Carceller P, Hans D. Evaluation of the potential use of trabecular bone score to complement bone mineral density in the diagnosis of osteoporosis: a preliminary spine BMD–matched, case-control study. J Clin Densitom. 2009;12(2):170–176.
Shevroja E, Lamy O, Hans D. Review on the Utility of Trabecular Bone Score, a Surrogate of Bone Micro-architecture, in the Chronic Kidney Disease Spectrum and in Kidney Transplant Recipients. Front Endocrinol (Lausanne). 2018; 24; 9:561
Stein EM, Ortiz D, Jin Z, McMahon DJ, Shane E. Prevention of fractures after solid organ transplantation: a meta-analysis. J Clin Endocrinol Metab. 2011;96(11):3457-65
Ho OTW, Ng WCA, Ow ZGW, Ho YJ, Lim WH, Yong JN, Wang RS, Wong KL, Ng CH, Muthiah MD, Teo CM. Bisphosphonate therapy after liver transplant improves bone mineral density and reduces fracture rates: an updated systematic review and meta-analysis. Transpl Int. 2021;34(8):1386-1396.
Martin P, DiMartini A, Feng S, Brown R, Fallon M. Evaluation for liver transplantation in adults: 2013 practice guideline by the American Association for the Study of Liver Diseases and the American Society of Transplantation. Hepatology. 2014 ;59(3):1144-65.
Kidney Disease: Improving Global Outcomes (KDIGO) CKD-MBD Update Work Group. KDIGO 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD). Kidney Int Suppl. 2017; 7:1-59.
Mainra R, Elder GJ. Individualized therapy to prevent bone mineral density loss after kidney and kidney-pancreas transplantation. Clin J Am Soc Nephrol. 2010;5(1):117-24.
Anastasilakis AD, Tsourdi E, Makras P, Polyzos SA, Meier C, McCloskey EV, Pepe J, Zillikens MC. Bone disease following solid organ transplantation: A narrative review and recommendations for management from The European Calcified Tissue Society. Bone. 2019; 127:401-418.
Wissing KM, Broeders N, Moreno-Reyes R, Gervy C, Stallenberg B, Abramowicz D. A controlled study of vitamin D3 to prevent bone loss in renal-transplant patients receiving low doses of steroids. Transplantation. 2005;15:79(1):108-15.
Tsujita M, Doi Y, Obi Y, Hamano T, Tomosugi T, Futamura K, Okada M, Hiramitsu T, Goto N, Isaka Y, Takeda A, Narumi S, Watarai Y. Cholecalciferol Supplementation Attenuates Bone Loss in Incident Kidney Transplant Recipients: A Prespecified Secondary Endpoint Analysis of a Randomized Controlled Trial. J Bone Miner Res. 2022; 37(2):303-311.
Palmer SC, Chung EY, McGregor DO, Bachmann F, Strippoli GF. Interventions for preventing bone disease in kidney transplant recipients. Cochrane Database Syst Rev. 2019 Oct 22;10(10): CD005015.
Shane E, Addesso V, Namerow PB, McMahon DJ, Lo SH, Staron RB, Zucker M, Pardi S, Maybaum S, Mancini D. Alendronate versus calcitriol for the prevention of bone loss after cardiac transplantation N Engl J Med 2004; 350:767-776
Trillini M, Cortinovis M, Ruggenenti P, Reyes Loaeza J, Courville K, Ferrer-Siles C, Prandini S, Gaspari F, Cannata A, Villa A, Perna A, Gotti E, Caruso MR, Martinetti D, Remuzzi G, Perico N. Paricalcitol for secondary hyperparathyroidism in renal transplantation. J Am Soc Nephrol. 2015; 26(5):1205-14.
Stein EM, Ortiz D, Jin Z, McMahon DJ, Shane E. Prevention of fractures after solid organ transplantation: a meta-analysis. J Clin Endocrinol Metab. 2011; 96(11):3457-65.
Valero MA; Loinaz C, Larrodera L, Leon M, Moreno E, Hawkins F. Calcitonin and bisphosphonates treatment in bone loss after liver transplantation. Calcif Tissue 1995; 57:15-19
Gilfraguas L, Guadalix s, Martinez g, Jodar E, Vara J, Gomez Sanchez A, Delgado J, De La Cruz J, Lora d, Hawkins F. Bone loss after heart transplant: effect of alendronate, etidronate, calcitonin and calcium plus vitamin D3. Progress in Transplantation 2012; 22; 237-243
Hauck D, Nery L, O'Connell R, Clifton-Bligh R, Mather A, Girgis CM. Bisphosphonates and bone mineral density in patients with end-stage kidney disease and renal transplants: A 15-year single-centre experience. Bone Rep. 2022; 16:101178.
Torregrosa JV, Fuster d, Gentil MA, Marcen R, Guirado L, Zarraga S, Bravo J, Burgos d, Monegal A, Muxi A, Garcia S. Open-label trial: effect of weekly risedronate immediately after transplantation in kidney recipients. Transplantation 2010; 89:1476-81
Guadalix S, Martinez G, Lora D, Vargas C, Gomez M, Cobaleda B, Moreno e, Hawkins F. Effects of early risedronate treatment on bone mineral density and bone turnover markers after liver transplantation: a prospective single-center study. Transplantation International 2011;24: 657-665
Shane E, Cohen A, Stein EM, McMahon DJ, Zhang C, Young P, Pandit K, Staron RB, Verna EC, Brown R, Restaino S, Mancini D. Zoledronic acid versus alendronate for the prevention of bone loss after heart or liver transplantation. J Clin Endocrinol Metab. 2012; 97(12):4481-90.
Ho OTW, Ng WCA, Ow ZGW, Ho YJ, Lim WH, Yong JN, Wang RS, Wong KL, Ng CH, Muthiah MD, Teo CM. Bisphosphonate therapy after liver transplant improves bone mineral density and reduces fracture rates: an updated systematic review and meta-analysis. Transpl Int. 2021; 34(8):1386-1396
Watts N, Freedholm D, Daifotis A. The clinical tolerability profile of alendronate. Int J Clin Pract Suppl. 1999; 101:51-61.
Fobelo Lozano MJ, Sánchez-Fidalgo S. Adherence and preference of intravenous zoledronic acid for osteoporosis versus other bisphosphonates. Eur J Hosp Pharm. 2019;26(1):4-9.
99. Bone HG, Wagman RB, Brandi ML, Brown JP, Chapurlat R, Cummings SR, Czerwiński E, Fahrleitner-Pammer A, Kendler DL, Lippuner K, Reginster JY, Roux C, Malouf J, Bradley MN, Daizadeh NS, Wang A, Dakin P, Pannacciulli N, Dempster DW, Papapoulos S. 10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomized FREEDOM trial and open-label extension. Lancet Diabetes Endocrinol. 2017; 5(7):513-523.
100. Bonani M, Frey D, Brockmann J, Fehr T, Mueller TF, Saleh L, von Eckardstein A, Graf N, Wüthrich RP. Effect of Twice-Yearly Denosumab on Prevention of Bone Mineral Density Loss in De Novo Kidney Transplant Recipients: A Randomized Controlled Trial. Am J Transplant. 2016; 16(6):1882-91
101. Alfieri C, Binda V, Malvica S, Cresseri D, Campise M, Gandolfo MT, Regalia A, Mattinzoli D, Armelloni S, Favi E, Molinari P, Messa P. Bone Effect and Safety of One-Year Denosumab Therapy in a Cohort of Renal Transplanted Patients: An Observational Monocentric Study. J Clin Med. 2021; 10(9):1989.
Bonani M, Meyer U, Frey D, Graf N, Bischoff-Ferrari HA, Wüthrich RP. Effect of Denosumab on Peripheral Compartmental Bone Density, Microarchitecture and Estimated Bone Strength in De Novo Kidney Transplant Recipients. Kidney Blood Press Res. 2016;41(5):614-622.
Brunova J, Kratochvilova S, Stepankova J. Osteoporosis Therapy with Denosumab in Organ Transplant Recipients. Front Endocrinol (Lausanne). 2018; 9:162
Fassio A, Andreola S, Gatti D, Pollastri F, Gatti M, Fabbrini P, Gambaro G, Ferraro PM, Caletti C, Rossini M, Viapiana O, Bixio R, Adami G. Long-Term Bone Mineral Density Changes in Kidney Transplant Recipients Treated with Denosumab: A Retrospective Study with Nonequivalent Control Group. Calcif Tissue Int. 2024 ;115(1):23-30
McKee H, Ioannidis G, Lau A, Treleaven D, Gangji A, Ribic C, Wong-Pack M, Papaioannou A, Adachi JD. Comparison of the clinical effectiveness and safety between the use of denosumab vs bisphosphonates in renal transplant patients. Osteoporos Int. 2020; 31(5):973-980.
Teh JW, Mac Gearailt C, Lappin DWP. Post-Transplant Bone Disease in Kidney Transplant Recipients: Diagnosis and Management. Int J Mol Sci. 2024;25(3):1859.
Jamal SA, Ljunggren O, Stehman-Breen C, Cummings SR, McClung MR, Goemaere S, Ebeling PR, Franek E, Yang YC, Egbuna OI, Boonen S, Miller PD. Effects of denosumab on fracture and bone mineral density by level of kidney function. J Bone Miner Res. 2011;26(8):1829-35
Ebina K, Etani Y, Noguchi T, Nakata K, Okada S. Clinical effects of teriparatide, abaloparatide, and romosozumab in postmenopausal osteoporosis. J Bone Miner Metab. 2024. doi: 10.1007/s00774-024-01536-0. Epub ahead of print.
Cejka D, Benesch T, Krestan C, Roschger P, Klaushofer K, Pietschmann P, Haas M. Effect of teriparatide on early bone loss after kidney transplantation. Am J Transplant. 2008;8(9):1864-70.
Vetrano D, Aguanno F, Passaseo A, Barbuto S, Tondolo F, Catalano V, Zavatta G, Pagotto U, La Manna G, Cianciolo G. Efficacy and safety of teriparatide in kidney transplant recipients with osteoporosis and low bone turnover: a real-world experience. Int Urol Nephrol. 2025. doi: 10.1007/s11255-025-04383-8 Epub ahead of print.
Qiu Z, Lin C, Zhang Y, Lin C, Deng J, Wu M. Analysis of the Efficacy and Safety of Teriparatide and Alendronate in the Treatment of Osteoporosis After Renal Transplantation. Iran J Kidney Dis. 2021;15(6):451-456.
Bruyere O, Reginster JY. Monitoring osteoporosis therapy. Best Pract Res Clin Endocrinol Metabol. 2014; 28:835–841
Ninkovic M, Skingle S, Beacroft PWP, Bishop N, Alexander GJM, Compston JE. Incidence of vertebral fracture in the first three months after orthotopic liver transplantation. Eur J Gastroenterol Hepato 2000; 12:931-4
Atsumi K, Kushida K, Yamazaki K, Shimisu S, Ohmura A, Inoue T. Risk factors for vertebral fractures in renal osteodystrophy. Am J Kidney Dis 1999; 33:287-93
Alem AM, Sherrard DJ, Gillen DL, Weiss NS, Beresford SA, Heckbert SR, Wong C, Stehman-Breen C. Increase risk of fractures among patients with end stage renal disease. Kidney In t2000; 58(1):396-9
Majumdar SR, Ezekowitz JA, Lix LM, Leslie WD. Heart failure is a clinical and densitometrically independent and novel risk factor for major osteoporotic fractures: Population-based cohort study of 45.000 subjects. J Clin Endocrinol Metab2012; 87:1179-86
Monegal A, M. Navasa M, Guanabens N, Peris P, Pons F, Martinez de Osaba MJ, Ordi J, Rimola A, Rodes J and Munoz-Gomez J. Bone Disease After Liver Transplantation: A Long-Term Prospective Study of Bone Mass Changes, Hormonal Status and Histomorphometric Characteristics.Osteoporos Int (2001) 12:484–
Cohen A, Sambrook P, Shane E. Management of bone loss after organ transplantation 2004; J Bone miner Rese19:1919-32
Wang TK, O'Sullivan S, Gamble GD, Ruygrok, PN. Bone density in heart or lung transplant recipients’ longitudinal study. Transplant Proc 2013; 45(6):2357-65.
Spira A, Gutiérrez C, Chaparro C, Hutcheon MA, Chan CK. Osteoporosis and lung transplantation: a prospective study. Chest 2000; 117:476-81
Gong-bin Lan, Xu-biao Xie, Long-kai Peng, Lei Liu, Lei Song, He-long Dai. Current status of research on osteoporosis after solid organ transplantation: Pathogenesis and Management. Biomed Res Int 2015:413169.
Muchmore JS, Cooper DK, Ye Y, Schlegel VT, Zuhdi N. Loss of vertebral bone density in heart transplant patients. Transplantation Proc. 1991 ;23(1 Pt 2):1184-5.
Carbonare LD, Zanatta M, Braga V, Sella S, Vilei MT, Giuseppe, Gambino FA, Pepe I, Rossini M, Adami S, Giannini S. Densitometric threshold and vertebral fractures in heart transplant patients. Transplantation 2011; 92:106-111
Graat-Verboom L, Wouters EFM, Smeenk FWJM, van den Borne BEE, Lunde R, Spruit MA. Current Status of research on osteoporosis in COPD: a systematic review. Eur Respir J 2009 Jul;34(1):209-18
Ferrari SL, Rizzoli SR, Nicod L. Osteoporosis in patients undergoing lung transplantation. Eur Respir J 1996; 9:2378-82
Harriman A, Alex C, Heroux A, Camacho P. Incidence of fractures after cardiac and lung transplantation: a single center experience. Journal of Osteoporosis 2014; 2014:2014:573041
Van Laecke S, Van Biesen W. Hipomagnesaemia in kidney transplantation. Transplantation Reviews 2015; 29:154-150
Kendler DL, Body JJ, Brandi ML, Broady r, Cannat-Andia J, Maghraoui EI, Guglielmi G, Hadji P, Pierroz DD, Viller TJ, Ebeling PR; Rizzoli R, for the International Osteoporosis Foundation Committee of Scientific Advisors Working Group on Cancer and Bone Disease. Osteoporosis management in hematologic stem cell transplant recipients: Executive summary.Journal of Bone Oncology 2021; 28:100361
LeBoff MS,Greenspan, SL, Insogna KL, Lewiecki EM, Saag KG, Singer AJ, Siris ES . Consensus Statement. The clinician’s guide to prevention and treatment of osteoporosis. Osteoporosis International 2022: 33:2049–2102

Endocrine Disruptor Chemicals

Appendix updated May 2, 2025

ABSTRACT

Endocrine Disrupting Chemicals (EDCs) impact health and disease. Scientific research conducted over the last few decades has solidified our knowledge of the health impacts of these chemicals. Intrauterine exposure of EDCs can have transgenerational effects, thus laying the foundation for disease in later life, when exposure may not be documentable. The meticulously orchestrated endocrine system is often a target for these chemicals. As the endocrine system is central to the body’s physiological and biological functions, EDCs can lead to perturbations in the functioning of an individual. Exposure to EDCs can occur right from children’s products to personal care products, food containers to pesticides and herbicides. Moreover, there are many unsuspected chemicals which may be contributing to the disease burden in the society, which have never been studied. The dose response relationship may not always be predictable for the different EDCs as even low-level exposures that may occur in everyday life can have significant effects in a susceptible individual. Although individual compounds have been studied in detail, the effects of a combination of these chemicals are yet to be studied in order to understand the real-life situation, where human beings are exposed to a cocktail of these EDCs. This chapter aims to summarize the available literature regarding these EDCs and their effects on endocrine physiology.

INTRODUCTION

Endocrine Disrupting Chemicals (EDCs) are a ubiquitous problem. This is a global issue and health hazard not well addressed due to lack of evidence and testing. Only a few EDCs are known and the others are suspected or yet to be explored (1). EDCs represent a broad class of natural or synthetic chemicals which are widely dispersed in the environment. This can be ingested or consumed or inhaled and may be found in larger quantities or trace amounts in serum, placenta, fat, umbilical cord blood etc. Exposure to EDCs can occur as early as in gestational period or childhood and can impact later stages of life. EDCs can alter normal physiological mechanisms in our body leading to a myriad of endocrinological problems both in children and adults.

The Endocrine Society defined EDC as “an exogenous chemical, or mixture of chemicals, that interfere with any aspect of hormone action.” In other words, the chemical substances that can affect the endocrine system resulting in adverse effects are called Endocrine Disruptor Chemicals (EDCs) (2). These chemicals often bind to the endogenous receptors (e.g.: estrogen receptor, steroid receptor) and interfere with the normal function of brain, reproductive organs, development, immune system, and other organs (3).

The common EDCs are bisphenol A (BPA), perchlorate, dioxins, phthalates, phytoestrogens, polychlorinated biphenyls (PCB), polybrominated diphenyl ethers (PBDE), triclosan, perfluoroalkyl and polyfluoroalkyl substances (PFAS), pesticides like dichlorodiphenyldichloroethylene (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE), organophosphorus compounds, alkylphenols(surfactants), parabans, methoxychlor, diethylstilbestrol (DES), fungicide vinclozolin, and natural hormones (2) (4) (5). Among these, BPA is the most commonly encountered EDC, which has both estrogenic and antiandrogenic properties. EDCs are mostly lipophilic in nature and resistant to metabolism (6). EDCs are usually present in food, beverages, pesticides, or air. People who get exposed to any of these EDCs may have hormonal imbalance. Even a small amount of EDC consumed can result in hormonal imbalance especially in children (2). Sometimes they are stored in body fats, and transferred to the developing fetus via the placenta (6).

Studies on animal models and humans reveal that the mechanisms through which the EDCs act involve divergent pathways. The EDC`s can act like endogenous hormones and thereby increase or decrease the cellular response. Also, they can block the effects of hormones and stimulate or inhibit the production of hormones. They can thus interfere with synthesis, transport, action, and degradation of hormones (7). EDCs can act via nuclear receptors, nonsteroidal receptors, transcription coactivators, and certain enzymatic pathways (5).

HISTORY OF EDCs

The effect of EDCs was first noticed by pig farmers in USA. Farmers observed pigs fed on moldy grain did not reproduce. Later it was found that moldy grain contained mycoestrogens. Several other incidents with such EDCs were noticed by farmers in other parts of the world. In 1940, diethylstilbestestrol (DES), a synthetic estrogen, was prescribed to women in their first trimester of pregnancy to prevent threatened miscarriage. Later in 1971, a rare vaginal cancer in daughters born to mothers who had taken DES was noted. All these events inspired Rachel Carlson to write a book named ‘Silent Spring’. In this book the author warned about long- term consequences of the use of pesticides and herbicides. In another book ‘Our Stolen Future ‘by Theo Colbron, Dianne Dumankosi, and John Peterson Meyers additional evidence on EDC was described. The hypothesis and evidence generated by this book was used for future research on EDC. This booked paved the path for the US regulators to create the United States Environment Protect Agency.

ARE HORMONES AND EDCs THE SAME?

EDCs are not the same as hormones but they can mimic hormones, and produce ill effects in the body.

Table 1. The Difference Between Hormone and Endocrine Disruptor Chemicals. (4)(8)
Hormones	EDCs
(1) These are chemical substances produced by the body and transported via bloodstream to the cells and organs which carry receptors for the hormone and on which it has a specific regulatory effect.	(1) Exogenous substance that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or populations.
(2) They act via specific receptors and produces class effects	(2) They act via hormone and other receptors and produces abnormal functions and interactions.
(3) No bio accumulation	(3) Results in bioaccumulation
(4) Non-linear dose response with saturable kinetics	(4) Non-linear dose response with saturable kinetics
E.g.; steroid hormones, thyroid hormones	E.g.; Perchlorate, Dioxins, Phthalates

EDCs AND HUMAN HEALTH

EDCs can affect several systems in our body resulting in many ill health effects. There is evidence showing various diseases are linked to EDCs as shown in Table 2.

Table 2. Examples of EDCs and Their Possible Mechanisms Resulting in Clinical Conditions. (4)(9)(10)
EDCs	Main Sources	Possible Mechanism	Clinical condition
Alkylphenols	Detergents Shampoos Pesticide	Mimics estrogen	Breast cancer
Phthalates	Plastic products Personal care products (perfume, moisturizer)	Not yet known	Testicular and ovarian toxicants
Polychlorinated biphenyls (chlorinated/ halogenated/ TBBPA)	Paints Plastics Lubricants Electrical applications	Estrogenic and anti-androgenic activity Indirectly regulate circulating gonadal hormones. Inducers of CYP1A and CYPIIB Decreased NMDA receptor binding in striatum, frontal cortex and hippocampus, cerebellum Reduced glutamate and dopamine Acts at AhR signaling pathways resulting in cytotoxic effects	Neurobehavioral defects like cognitive deficits in children Neurotoxicity Thyroid toxicity Susceptibility to infections Cancers (especially Breast Cancer) Infertility

TBBPA- Tetrabromobisphenol A, CYP - cytochrome P450 enzymes, NMDA- N-methyl-D-aspartate, AhR - aryl hydrocarbon receptor

EFFECT OF EDCs ON ENDOCRINE SYSTEM

Neuro- Hypothalamic Effects

According to recent studies one in eight children 2-9 years of age suffer from neurodevelopmental disorders (NDDs) in India. NDDs include speech and language disorders, autism, cerebral palsy, epilepsy, vision impairment, ADHD, learning disorders, etc. EDCs are one among other risk factors associated with development of NDDs in children. NDD burden can be lessened by eliminating the causative factors or by preventing exposure to them. The major EDCs associated with NDDs are PCB and polybrominated diphenyl ethers (PBDEs). Other EDCs that are linked to NDDs but lack firm evidence are brominated flame retardants, perfluorinated compounds, and pesticides. Animal studies reveal that EDCs can alter or affect neuronal development, synaptic organization, neurotransmitter synthesis and release, and structural development of the brain (11). Studies of pregnant women who lived near Lake Michigan, with high levels of exposure to PCBs, revealed that children of mothers with the highest exposure levels were much more likely to have lower average IQ levels and poorer performance on reading comprehension (12). BPA and phthalates have also been shown to be associated with behavioral problems in children, including anxiety and depression (13,14). Prenatal pesticide exposure has been linked to increased likelihood of children having autism spectrum disorder or developmental delay (15).

EDCs can cause perturbations of the neuroendocrine processes originating in the hypothalamus, and can also act on the steroid hormone receptors and other signaling pathways that occur widely throughout the brain. The critical period of exposure is important because even minor alterations in hormones can alter the neurobiological outcome during development. Our knowledge in this area is predominantly derived from animal studies as human studies (postmortem studies, accurate measurement of hypothalamic releasing hormones) are not feasible. Animal studies have shown the variable effects of BPA exposure on ER α and β protein and mRNA expression in different areas of the brain (16,17,18,19). Treatment of adult male and female rats for 4 days with low-dose BPA had significant effects on mRNAs for aromatase (increased in both sexes) and 5α-reductase 1 (decreased in females) in the prefrontal cortex (20). Although we know that developmental EDC exposure can alter the expression of genes and proteins for steroid hormone receptors, we cannot draw generalized conclusions from these animal models and future research should target especially this area of early EDC exposure.

EDC exposure can also have neuroendocrine effects. Animal studies have reported on the stimulatory as well as inhibitory actions of BPA on GnRH and kisspeptin systems (21,22). Studies on PCBs and phthalates have shown mixed results. Animal studies have shed some light on the effect of EDCs on the developing hypothalamic pituitary adrenal (HPA) axis. BPA exposure has been found to be associated with an increase in adrenal weight and an attenuated stress response (23). Basal corticosterone, as well as CRH- or ACTH-induced corticosterone release, has been found to be significantly suppressed in PCB exposed rats (24). These effects of EDCs on the HPA axis leading to aberrant stress response needs to be evaluated further in humans. Animal studies have opened up some new and interesting possibilities of EDC exposure with changes in AVP and oxytocin levels and social behavior (25,26).

Thyroid Function

EDCs can interfere with thyroid hormone synthesis, release, transport, metabolism and clearance.

Table 3. EDCs Effect on Thyroid Function (27,28)
EDC	Source	Possible Outcome
Perchlorate	Oxidant in solid rocket propellants, fireworks, airbag deployment systems, etc.	Interferes with the uptake of iodide into the thyrocyte by sodium/iodide symporter (NIS)
Thiocyanates	Cigarettes	Interferes with the uptake of iodide
Isoflavones (Phytoestrogens)	Soy protein	TPO inhibitors resulting in goiter in children
PCB	Paints Plastics	They can act as TR agonist or antagonist, or reduce circulating levels of T4 resulting in relative hypothyroidism, increase in expression of glial fibrillar acidic protein leading to neurotoxicity in children.
BPA	Plastics Food cans Dental sealants	Binds to TRb and antagonizesT3 activation. It can block T3-induced oligodendrocyte development from precursor cells, resulting in ADHD. Halogenated BPA can act as TR agonists, TBBPA bind to TR and induces GH3 cell proliferation and GH production.

PCB - Polychlorinated biphenyls, BPA - Bisphenol A, ADHD- Attention Deficit Hyperactivity Disorder, TBBPA - Tetrabromobisphenol A, T4 - tetraiodothyronine (thyroxine), T3 - triiodothyronine, TPO – Thyroperoxidase, TR – Thyroid Receptor, TH - Thyroid hormone, GH- Growth Hormone

Adipose Tissue and Metabolic Disorders

OBESITY

Obesity can result in the metabolic syndrome, reproductive problems, and cardiovascular risk factors. “Obesogens” are defined as “xenobiotic chemicals that can disrupt the normal developmental and homeostatic controls over adipogenesis and/or energy balance” (29). The “obesogen hypothesis” suggests that prenatal or early-life exposure to certain EDCs compounded by sedentary lifestyle and improper nutrition predisposes certain individuals to become obese later in life (30).

In DES exposed mice an increase in body fat, leptin, adiponectin, interleukin (IL) - 6, triglyceride (TG) was observed. EDCs cause upregulation of gene expression involved in adipocyte differentiation and lipid metabolism resulting in fat accumulation (31). PPARg (peroxisome proliferator-activated receptors), a major regulator of adipogenesis, are expressed in adipocytes. It promotes adipocyte differentiation and the induction of lipogenic enzymes. During activation, PPARg along with retinoid X receptor (RXR), forms a heterodimer complex which then binds to PPAR response elements for regulation of fatty acids and repression of lipolysis. EDCs like tributyltin (TBT) and triphenyltin acts as PPARg and RXR agonists and increases adipose tissue mass.

Phytoestrogens mimic endogenous estrogens and exert various biological actions. They can bind to estrogen receptor (ER)a and estrogen receptor (ER)b and influence lipogenesis. One of the major sources of phytoestrogens is soy protein which contains genistein, a phytoestrogen. At low doses genistein inhibits lipogenesis whereas at high doses it can promote lipogenesis (27). EDCs like BPA, phthalates, dioxins perfluorinated compounds, and some pesticides are emerging as potential obesogens warranting further research.

Table 4. Potential Obesogenic Actions of EDCs

· Agonist at PPARᵧ and RXRα (32)

· Promotion of adipogenesis through ERs (33)

· Increase in enzymatic activity of 11-β hydroxysteroid dehydrogenase type 1 (11-β HSD type 1) (34)

· Increase in insulin stimulated lipogenesis (35)

· Alterations in blood levels of insulin, leptin, and adiponectin (36)

· Alteration of central energy regulatory pathways (37)

· Decreased TRH expression and type 4 melanocortin receptors in the paraventricular nucleus of the hypothalamus and stimulation of orexigenic pathways (38)

· Epigenetic transgenerational inheritance of adult-onset obesity (39)

DIABETES AND GLUCOSE HOMEOSTASIS

EDCs can disrupt glucose homeostasis in our body by affecting both insulin- and glucagon-secretory cells. Any toxic chemical that kills β cells or disrupts their function has been termed a “diabetogen”. The “diabetogen hypothesis” suggests that “every EDC circulating in plasma able to produce insulin resistance, independently of its obesogenic potential and its accumulation in adipocytes, may be considered a risk factor for metabolic syndrome and type 2 diabetes” (40). The obesogenic EDCs are risk factors for type 2 diabetes as well and lead to the dangerous combination of obesity and diabetes or “Diabesity”. However, certain EDCs may directly cause insulin resistance and defects in insulin production and secretion, without significantly affecting the weight of the individual. Studies have shown that acute treatment with BPA causes a temporary hyperinsulinemia, whereas longer-term exposure suppresses adiponectin release, and aggravates insulin resistance, obesity related syndromes, and development of diabetes mellitus. The hyperinsulinemia is attributed to the very rapid closure of ATP-sensitive K+ channels, potentiation of glucose-stimulated Ca2+ signals, and release of insulin via binding at extranuclear ER (41). Low doses of DES have been shown to impair the molecular signaling that regulates glucagon production through non genomic mechanism (27). POPs have been demonstrated to have direct effects on insulin signaling (42). They can lead to insulin resistance by causing adipose tissue inflammation. Heavy metals such as arsenic and mercury have also been considered as potential diabetogens. Intake of a high fat diet along with exposure to a cocktail of these EDCs (DEHP, BPA, PCB153, and TCDD) has been found to have sex specific alterations in the metabolic milieu in offspring. In males, there was alteration in the cholesterol metabolism whereas in females, there was pronounced effect on the glucose metabolism through a decrease in ER α expression and estrogen target genes (43).

The causal relationship between EDCs and type 1 diabetes is an area warranting research as animal studies have shown exposure to EDCs associated with insulitis (44).

Reproductive System

Over the past few decades there has been a surge in the incidence of reproductive system related disorders among both the males and females. EDCs can be attributed to this surge. Exposure to EDCs especially phytoestrogens have resulted in early menarche and polycystic ovarian diseases (PCODs) in adolescent girls. Infertility affects up to 15% of couples in the reproductive age group worldwide. The EDCs and their effect on reproductive system is summarized in Table 5.

Table 5. Effects of EDCs on the Reproductive System (27)(9)

EDC

Possible Mechanism

Possible Clinical Condition

Males

Females

Vinclozolin

Epigenetic (altered DNA methylation in germ cell lines)

AR antagonism

Hypospadias

Undescended testes

Delayed puberty

Prostate disease/cancer

Dysregulates the gland development Formation of

mammary tumor

DES

Increased ER expression in

Epididymis

Epigenetic silencing of

mRNA

Hypospadias Cryptorchidism Micropenis

Epididymal cysts

Vaginal adenocarcinoma

Ectopic pregnancy

Infertility

DDT/DDE

Antiandrogen

Antiprogestin

Induction of aromatase

Reduced insulin-like factor

Cryptorchidism

Infertility

Risk of breast cancer in females

Precocious and early puberty

Infertility

PCB

Estrogen agonist / Estrogen antagonist / antiandrogenic activity

Prostate cancer

Early onset of menarche

Delayed pubertal

development

Accumulates in breast adipose tissue

Phthalates

ER agonist/antagonist

Antiandrogen and decreases testosterone synthesis

Reduced anogenital distance and

Leydig cell function

HypospadiasCryptorchidism

Increased cell proliferation in

the breast

BPA

ER agonist

Antiandrogen

Inhibition of apoptotic activity in breast

Increased number of progesterone receptor positive

epithelial cells

Nongenomic activation of ERK1/2

Reduced sulfotransferase inactivation of estradiol

Prostate cancer

Testicular cancer in fetus

Altered breast development

Early puberty

Dioxins

ER agonist

Antiandrogen

Interfere with sex-steroid

synthesis

Inhibition of cyclooxygenase2 via AhR

Cryptorchidism

Premature thelarche

Endometriosis

Breast cancer

DES – Diethylstilbestrol, DDT – dichlorodiphenyldichloroethylene, DDE - dichlorodiphenyldichloroethylen, DNA – Deoxyribonucleicacid, AR – Androgen Receptor, ER – Estrogen Receptor, AhR - aryl hydrocarbon receptor, ERK1/2 - extracellular signal-regulated kinase 2, mRNA – messenger RNA

EFFECTS ON THE FEMALE REPRODUCTIVE SYSTEM

In vivo animal studies and thereafter in vitro studies indicate that exposure to BPA (1–30M) impairs meiotic progression in human fetal oocytes, increased levels of recombination, and induces epigenetic changes that may contribute to chromosome congression failure (45,46). Studies in rats have shown that neonatal BPA exposure decreased the numbers of all follicle types and increased atretic follicles during adulthood (47). In vitro animal studies have demonstrated the toxic effect of phthalates on the follicle growth and inhibition of estradiol production (48). Similar toxic effects of pesticides and environmental pollutants on gene expression, follicle growth and oocyte quality have been confirmed in animal studies. BPA and phthalates have also been implicated in altered steroidogenesis in the gonads (49). Findings of alteration in uterine structure and function after exposure to EDCs is more concerning as it may lead to abnormalities in implantation and recurrent abortions (50). BPA exposure has been associated with increased risk of implantation failure and miscarriages (51). Animal studies have also pointed towards the transgenerational effect of prenatal BPA exposure on female fertility (52). Experimental studies have shown an association between phthalate exposure and reduced fertility (53). The findings of these studies need to be confirmed in the human ovary to fully understand the impact of these EDCs on fertility and reproductive health as well as the transgenerational impact. EDCs have also been found to have adverse effects on menstrual cyclicity in women. Fungicide exposure has been associated with a significant decrease in bleeding (54). BPA and pesticides may accelerate ovarian failure and may lead to premature menopause in women (55). In utero exposure to DES increases the lifetime risk of premature menopause (56). Propyl paraben, a preservative in personal care product, was associated with lower antral follicle counts as well as higher day-3 FSH levels indicating accelerated ovarian aging (57). It may not be exposure to just a single EDC and more often than not it may be a cocktail of these that could lead to early reproductive senescence. In animal studies, late gestational exposure to DES causes ovarian hyperandrogenism and menstrual abnormalities similar to those in women with PCOS (58). A few epidemiological studies have pointed towards an association between phthalate exposure and risk of endometriosis, possibly due to increased viability of cells (59). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure may disrupt cannabinoid signaling in the human endometrium and lead to increased inflammation in the endometrium (60). TCCD exposure can cause a progesterone-resistant phenotype that may persist over multiple generations, suggesting that TCDD exposure has transgenerational effects on endometriosis (61). TCDD increases the expression of thymus-expressed chemokine and promotes the invasiveness of endometrial stromal cells by increasing the expression of matrix metalloproteinase-2 and -9(62). TCDD also reduces the expression of CD82 (a wide-spectrum tumor metastasis suppressor that inhibits the mobility and invasiveness of cells), and increases the expression of CCL2-CCR2, which recruits macrophages and further down-regulates CD82 (63). Pesticides like fenvalerate stimulate the growth of uterine fibroid cells by enhancing cell cycle progression and inhibiting apoptosis through an ER-independent pathway (64). DES exposure has been shown to increase the occurrence of early onset fibroids in the Sister Study and Nurses’ Health Study II (65,66). Given the multiplicity of effects of EDCs on the female reproductive system, there remains an urgent need for future studies to confirm the findings of experimental and animal studies and understand the underlying mechanisms.

EFFECTS ON THE MALE REPRODUCTIVE SYSTEM

EDCs, by virtue of their antiandrogenic and estrogenic effects can have a profound influence on the male reproductive physiology. Studies on the causative effect of EDCs on hypospadias have not given consistent results due to the small number of subjects studied. Levels of chlorinated pesticides have been found to be higher in breast milk of mothers with cryptorchid boys (77). Studies on the incidence of cryptorchidism with xenoestrogen exposure showed detectable levels of lindane and mirex in placenta with higher cryptorchidism risk (78). Higher dioxin levels in breast milk and dibutyltin concentrations in placenta were associated with cryptorchidism in Danish boys (79). Dioxins may have estrogenic effects through interaction of the dioxin-AhR nuclear translocator complex with estrogen receptor. High exposure to DDE and PCBs also has a higher risk of cryptorchidism (80). Environmental factors play an important role in the development of testicular cancers. Cryptorchidism and hypospadias are well-characterized risk factors of testicular germ cell cancers (TGCC). Although TGCC is probably a condition with fetal origins, it has been practically difficult to prove the association between pre and postnatal exposure of EDCs and TGCC, given the long lag time between exposure and effect. A positive association of TGCC with DDE (81) and chlordane exposure (cis-nonachlor and trans-nonachlor) has been found (82). Intra uterine exposure to EDCs that affect the spermatogonial stem cells or Sertoli cells, can cause irreversible changes that result in permanently low adult sperm number. PCB exposure may affect sperm DNA integrity and motility (83). DDE exposure has been inversely associated with sperm motility and total sperm count (84), and positively correlated with defects in sperm chromatin condensation and morphology (85). Fetal and perinatal exposure via breast-feeding to dioxin in the Seveso accident was associated with reductions in sperm concentration, number of motile sperm, and total sperm number (86). PBDEs used as flame retardants have been found to negatively affect sperm concentration, testicular size and sperm motility (87). The major impediment to establishing a causal role of these effects of EDCs is the long lag time between the critical exposure and the manifestation of the adverse outcomes.

EFFECTS ON PUBERTY

There has been a decrease in the age of breast development but the age of menarche has not changed significantly. This finding alerted researchers about the possible interfering role of EDCs in pubertal mechanisms. Results of epidemiological studies have been equivocal on the effects of BPA and phthalates on pubertal onset (67,68). Studies have pointed towards higher kisspeptin levels in girls exposed to phthalates, which may promote precocious puberty (69). There have been inconsistencies between animal and human studies and hence, inconclusive data on the effects of other EDCs like pesticides and environmental contaminants on puberty. Apparently innocuous substances like lavender oil and tea tree oil present in lotions and creams can lead to prepubertal gynecomastia by their estrogenic effects (70). A very interesting hypothesis has been put forward to explain the role of EDCs in precocious puberty seen in immigrant girls from developing countries. Early and temporary exposure to weakly estrogenic dichlorodiphenyltrichloroethane (DDT) in developing countries, where the exposure is still high, could stimulate hypothalamic maturation while the pituitary gonadotrophins are inhibited via a negative feedback that prevents manifestation of central maturation. This negative feedback disappears after withdrawal from the exposure, as happens when the child migrates to a different environment. This could precipitate precocious puberty in these migrant children (71). High exposure to endosulfan has been shown to be associated with pubertal delay, due to its antisteroidogenic properties (72). Dioxins act through aryl hydrocarbon receptors and thereby interact with other nuclear receptors. Exposure in boys has been associated with delayed puberty and in girls with delayed thelarche due to its antiestrogenic effects (73,74). Lead exposure has been implicated in delayed puberty in both boys and girls (75,76). Endocrine disrupters may alter the levels of endogenous hormones and their ratios by influencing their production, secretion, binding to carriers, metabolism and excretion. When studying these compounds, one needs to keep in mind about their active metabolites and the multiplicity of effects on the complex endocrine milieu.

Hormone Responsive Cancer

Most cancer occur due to genetic predisposition or exposure to environmental or occupational hazards. EDCs can alter the genes and result in uncontrolled proliferation of cells. Almost all the EDCs identified are known to cause cancer. People working in certain industries like coal, steel, rubber, textile, paper manufacturing, paint are at higher risk of developing cancers due to increased exposure to these EDCs. Studies have shown that early exposure to these EDCs BPA, PCBs, perflourinated compounds, phthalates, and some pesticides can increase cancer risk(1).Several EDCs that mimic endogenous estrogens are potential carcinogens. The estrogen-responsive cancers including breast, endometrial, ovarian, and prostate cancers are caused due to several chemical xenoestrogens and phytoestrogens (88). EDC exposure during the critical periods of mammary gland development like gestation, puberty, and pregnancy may predispose to carcinogenesis. Dioxin exposure, especially TCDD has been found to increase the incidence of breast cancer (89). Inconsistent results have been obtained with regards to pesticide exposure and breast cancer risk, possibly due to individual chemicals studied whereas in real life, humans are often exposed to a mixture of them. Breast cancer patients present more frequently with a combination of aldrin, DDE, and DDD, and this mixture has not been found in healthy women (90). Exposure to diethyl phthalate, the parent may be associated with a 2-fold increase in breast cancer risk (91). EDCs may influence other estrogen dependent cancers as well. In women previously exposed to chlorotriazine herbicides, there was a significant 2.7-fold increased risk for ovarian neoplasms (92). Higher PFOA levels are associated with ovarian cancer (93). In males, those EDCs that can interfere with androgen and estrogen signaling pathways can increase the risk of prostate cancer. A classic example of developmental exposure and onset of latent disease is the progeny of mothers exposed to DES during pregnancy. Although prostatic structural abnormalities have been documented in this cohort (94), the exact effect on prostate cancer is yet to be ascertained as the cohort is still being followed up. Pesticide exposure and carcinogenesis has garnered much interest after the Agricultural Health Study (AHS) in the United States. Specific organophosphate insecticides like fonofos, malathion, terbufos, and aldrin) have been associated with increased risk of aggressive prostate cancer (95). Certain organophosphates like coumaphos and organochlorine (aldrin) pesticides increase prostate cancer risk in men with a family history of the disease (96). Compounds like chlorpyrifos, coumaphos, fonofos, and phorate strongly inhibit the hepatic CYP1A2 and CYP3A4 enzymes that metabolize testosterone, estradiol, and estrone (97) and thereby act as EDCs apart from causing DNA damage by oxidative stress. TCDD, the most toxin dioxin in the Agent Orange herbicide spray has been found to have a strong positive association in the incidence and aggressiveness of prostate cancer in the Vietnam veterans (98). Trace elements like arsenic and cadmium, have been classified as EDCs due to their ability to act as a ligand and/or interact with members of the steroid receptor superfamily and have been implicated in prostate cancer although more conclusive studies are needed.

Effect on The Adrenals

The adrenal gland is probably one of the most ignored glands in toxicology, despite it being very sensitive to toxins. By virtue of its intense vascularity, its capacity for uptake and storage of lipophilic agents and high local concentrations of enzymes of CYP family with potential for bioactivation of toxins, the adrenals are very susceptible to the toxic effects of EDCs. The results of toxicological research on adrenals may not always be straightforward because of the dynamic nature of the HPA axis. Thus, even in the face of compromised adrenal steroidogenesis, it is not surprising to find relatively normal levels of circulating cortisol, albeit with an increased ACTH drive. Hence, scientists studying the toxic effect of EDCs on the adrenals, need to take into account the ACTH and cortisol levels as well as the adrenal weight. One of the earliest evidences for an adrenal disruptor was the use of the anesthetic, etomidate, which inhibits CYP11B1, leading to adrenal insufficiency. Another direct inhibitor of adrenal steroidogenic enzymes is a derivative of the pesticide DDD, mitotane (o,p’-DDD), which is used to treat Cushing’s syndrome. Polychlorinated biphenyl 126 (PCB126) causes an increase in aldosterone biosynthesis by increasing expression of CYP11B2, the enzyme which catalyzes the final step of aldosterone biosynthesis. High concentrations PCB126 has been shown to increase expression of the Angiotensin 1 (AT1) receptor, enhancing angiotensin II responsiveness of adrenal cells. Lead has also been reported to increase aldosterone synthesis by a mechanism consistent with upregulation of CYP11B2. It has also been reported that a class of herbicides (2-chloro-s-triazine herbicides) increase the expression of CYP19, which encodes aromatase, raising the possibility of increased adrenal estrogen secretion (99). The lack of a clear understanding of the adrenal toxicology can be overcome by the use of sophisticated endocrine studies, which take into account the dynamicity of the HPA axis.

EFFECT OF EDCs DURING PREGNANCY

Studies on animals have shown that EDCs can affect germ cell lines. In a cohort study of 47,540 women with history of exposure to diethylstilbestrol (DES) during pregnancy and ADHD diagnosis were followed up to three generations (F0, F1, F2) to know consequences of exposure to DES. This study revealed that the progeny of mothers who used DES in the 1^st trimester of pregnancy had higher risk of developing ADHD. BPA is another EDC which can lead to neuroendocrinal problems (100). This highlights the ill effects of EDCs in vertical transmission. EDCs like perfluorooctanoic acid have been implicated in pregnancy induced hypertension. There have been some pointers towards an association between BPA and preterm birth but it has not been conclusively proven in experimental animal studies (101).

Phthalate exposure during pregnancy may be associated with increased odds of prematurity (102). The possible mechanisms are interference with the placental function via effects on trophoblast differentiation and placental steroidogenesis which could increase the risk of preterm birth. Similar genetic effects of pesticides have also been shown to result in increased prematurity and preterm birth. This risk has been shown to be magnified in those with certain genetic mutations, highlighting the gene- environment interaction (103). Environmental contaminants like TCCD exert pro-inflammatory effects on the placenta, leading to infection-mediated preterm birth (104). EDCs have also been implicated in adverse birth outcomes. In the Generation R study in The Netherlands, prenatal BPA exposure was associated with reduced fetal weight and head circumference (105). The same study also showed that maternal phthalate exposure was associated with an increased time-to-pregnancy (106) and impaired fetal growth during pregnancy and decreased placental weight (107). In a similar Japanese study, maternal urinary MEHP levels were negatively associated with anogenital distance (AGD) in male offspring (108). Pesticide exposure during the second trimester of pregnancy have been negatively associated with birth weight, birth length, and head circumference as shown in the data from Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) (109). Increased incidence of infants being born as small for gestational age has also been reported in mothers who were exposed to pesticides (110). A sex dependent nature of these adverse birth outcomes has been demonstrated in a Chinese study with a decrease in gestational duration in girls but not boys (111). Similarly, in the Hokkaido Study on Environment and Children’s Health, an ongoing cohort study in Japan, PCDF and PCDD exposures were negatively associated with birth weight and infant development, with males being more susceptible than females (112). However, not all studies are shown these consistent adverse effects of EDCs. Hence future studies should confirm these preliminary findings and also study certain EDCs which have never been studied so far in experimental and epidemiological studies.

DETECTION OF EDCs

EDCs may be in complex forms or in trace amounts in biological fluids or environment which makes it difficult to identify or detect them. The methods used for the detection of these compounds should be highly sensitive and specific. These include liquid chromatography, gas chromatography and capillary electrophoresis. The bioassay techniques (Receptor binding assay, Receptor gene assay, DNA binding assay) are either qualitative or quantitative and can be helpful to know the biological effects of the complex samples. Due to the complexities and trace amount of the EDC, preconcentration is required (5). However, the limitations in sensitivity, reproductivity, difficulty in separation, and affordability still remain.

FUTURE TRENDS

Newer methods are being explored to predict the effect of chemical disruptors using artificial intelligence (AI). Combining artificial neural network (ANN) and chemical similarity approaches, a significant role of AI in chemical endocrine receptor disruption prediction has been demonstrated. For example, isoflavone genistein, a phytoestrogenfrom soy was found to be active or disruptive whereas isoflavone daidzein from the soy was predicted to be inactive or non-disruptive (113). ANN can be used to predict chemical activity against estrogen and androgen receptors. Machine learning and ANN can more accurately and precisely predict EDCs in future.

Biosensors are newer devices which can detect chemicals up to femtomolar limit of detection. Aptasensors, Nanotubes, Molecularly imprinted polymer (MIP)-based sensors are the emerging EDC detectors (114). Recently a device called ‘Tethys’ has been invented to detect presence of lead in water. Lead is known to affect the hormone signaling and central nervous system. This device works on the basis of nanocarbon tubes and could send water quality information via Bluetooth (115).

Among several computer aided approaches, invitro and in silico predictions are now used to predict large number of chemical disruptors in the environment (116). Also the ligand-based models, like QSAR models which can predict biological activities of EDC and structure-based models can be combined with Artificial Intelligence technology for more accurate EDC predictions (117).

EDCs IN THE TROPICS

Pesticide use has increased over the years due to intensification of agricultural practices in the tropical countries. While the developed countries do have a well-established legal framework for pesticide environmental risk assessments, such requirements are either not available or inadequately implemented in tropical countries. Added to these woes are the fact that cheap compounds that are environmentally persistent and highly toxic, banned from agriculture use in developed countries, still remain popular in developing countries (118). These may lead to soil and water contamination with pesticide residues. The effect of these compounds on the applicators as well as the consumers are manyfold. In a multi-centric study to assess the pesticide residues in selected food commodities (Surveillance of Food Contaminants in India, 1993), DDT residues were found in about 82% of the 2205 samples of bovine milk. Data on 186 samples of 20 commercial brands of infants’ formulae showed the presence of residues of DDT and Hexachlorocyclohexane (HCH) isomers in about 70 and 94% of the samples with their maximum level of 4.3 and 5.7 mg/kg respectively (119). The average daily intake of HCH and DDT by Indians was reported to be 115 and 48 mg per person respectively, which were higher than those observed in most of the developed countries (120). Over these continuous levels of exposure through food, water and soil are the occasional spillovers and accidents that lead to greater exposure. Although these exposures have been documented well in literature, there are sparse studies from the tropical areas on the long-term effects, especially in relation to the endocrine system. Although there are compelling social and economic benefits for the rampant use of EDCs, the policymakers need to be made aware of the long term and sometimes transgenerational effects of these molecules.

CONCLUSION

EDCs are an emerging global health problem that requires urgent attention and action. The most common EDCs that we encounter in our day-to-day life are BPA, PCBs, paraben etc. This results in endocrinological problems in all the age groups. There is an urgent need of novel biomarkers, detectors or assays using novel technologies for the early detection of EDCs. The novel technologies like Artificial Intelligence, OMICS (Genomics, Epigenomics, Mitochondriomics) and Nano technology are the new-way forward in this regard. Food and Health authorities play a vital role in curbing this problem. Food and safety laws should be more stringent and higher throughput screening for EDCs should be done prior to approval of any products. BPA free, paraben free products should be encouraged. Industrialists and others manufacturers must make sure not to pollute the water with the industrial wastes. All these measures will help in eliminating EDCs related health problems.

REFERENCES

Gore AC, Crews D, Doan LL, Merrill ML, Patisaul H, Zota A. INTRODUCTION TO ENDOCRINE DISRUPTING CHEMICALS (EDCs). :76.
Endocrine Disruptors [Internet]. National Institute of Environmental Health Sciences. [cited 2021 Feb 7]. Available from: https://www.niehs.nih.gov/health/topics/agents/endocrine/index.cfm
Research NC for T. Endocrine Disruptor Knowledge Base [Internet]. FDA. FDA; 2019 [cited 2021 Feb 7]. Available from: https://www.fda.gov/science-research/bioinformatics-tools/endocrine-disruptor-knowledge-base
WHO | Global assessment of the state-of-the-science of endocrine disruptors [Internet]. WHO. World Health Organization; [cited 2021 Feb 8]. Available from: https://www.who.int/ipcs/publications/new_issues/endocrine_disruptors/en/
Jones L, Regan F. Endocrine Disrupting Chemicals. In: Reference Module in Chemistry, Molecular Sciences and Chemical Engineering [Internet]. Elsevier; 2018 [cited 2021 Feb 7]. p. B9780124095472144000. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780124095472145123
Elobeid MA, Allison DB. Putative environmental-endocrine disruptors and obesity: a review. Curr Opin Endocrinol Diabetes Obes. 2008 Oct;15(5):403–8.
Schneider M, Pons J-L, Labesse G, Bourguet W. In Silico Predictions of Endocrine Disruptors Properties. Endocrinology. 2019 Nov 1;160(11):2709–16.
Bergman Å, United Nations Environment Programme, World Health Organization. State of the science of endocrine disrupting chemicals - 2012 an assessment of the state of the science of endocrine disruptors [Internet]. Geneva: WHO : UNEP; 2013 [cited 2021 Feb 7]. Available from: http://www.who.int/ceh/publications/endocrine/en/index.html
Lucaccioni L, Trevisani V, Marrozzini L, Bertoncelli N, Predieri B, Lugli L, et al. Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence. Int J Mol Sci. 2020 Mar 18;21(6):2078.
Bell MR. Endocrine-disrupting actions of PCBs on brain development and social and reproductive behaviors. Curr Opin Pharmacol. 2014 Dec;19:134–44.
Arora NK, Nair MKC, Gulati S, Deshmukh V, Mohapatra A, Mishra D, et al. Neurodevelopmental disorders in children aged 2–9 years: Population-based burden estimates across five regions in India. PLOS Med. 2018 Jul 24;15(7):e1002615.
Jacobson JL, Jacobson SW. Intellectual impairment in children exposed to polychlorinated biphenyls in utero. N Engl J Med. 1996;335:783–789.
Harley KG, Gunier RB, Kogut K, et al. Prenatal and early childhood bisphenol A concentrations and behavior in school-aged children. Environ Res. 2013;126:43–50.
Braun JM, Kalkbrenner AE, Calafat AM, et al. Impact of early-life bisphenol A exposure on behavior and executive function in children. Pediatrics. 2011;128:873–882.
Shelton JF, Geraghty EM, Tancredi DJ, et al. Neurodevelopmental disorders and prenatal residential proximity to agricultural pesticides: the CHARGE Study. Environ Health Perspect. 2014;122:1103–1109.
Chen F, Zhou L, Bai Y, Zhou R, Chen L. Sex differences in the adult HPA axis and affective behaviors are altered by perinatal exposure to a low dose of bisphenol A. Brain Res. 2014;1571:12–24.
Cao J, Rebuli ME, Rogers J, et al. Prenatal bisphenol A exposure alters sex-specific estrogen receptor expression in the neonatal rat hypothalamus and amygdala. Toxicol Sci. 2013;133:157–173.
Monje L, Varayoud J, Muñoz-de-Toro M, Luque EH, Ramos JG. Exposure of neonatal female rats to bisphenol A disrupts hypothalamic LHRH pre-mRNA processing and estrogen receptor expression in nuclei controlling estrous cyclicity. Reprod Toxicol. 2010;30:625–634.
Yu B, Chen QF, Liu ZP, et al. Estrogen receptor and expressions in hypothalamus-pituitary-ovary axis in rats exposed lactationally to soy isoflavones and bisphenol A. Biomed Environ Sci. 2010;23:357–362.
Castro B, Sánchez P, Torres JM, Preda O, del Moral RG, Ortega E. Bisphenol A exposure during adulthood alters expression of aromatase and 5 alpha-reductase isozymes in rat prostate. PLoS One. 2013;8:e55905.
Bai Y, Chang F, Zhou R, et al. Increase of anteroventral periventricular kisspeptin neurons and generation of E2- induced LH-surge system in male rats exposed perinatally to environmental dose of bisphenol-A. Endocrinology.2011;152:1562–1571.
Patisaul HB, Todd KL, Mickens JA, Adewale HB. Impact of neonatal exposure to the ER agonist CD-1, bisphenol- A or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats. Neurotoxicology.2009;30:350–357.
Panagiotidou E, Zerva S, Mitsiou DJ, AlexisMN,Kitraki E. Perinatal exposure to low-dose bisphenol A affects the neuroendocrine stress response in rats. J Endocrinol.2014;220:207–218.
Meserve LA, Murray BA, Landis JA. Influence of maternal ingestion of Aroclor 1254 (PCB) or FireMaster BP-6 (PBB) on unstimulated and stimulated corticosterone levels in young rats. Bull Environ Contam Toxicol. 1992;48:715–720.
Wolstenholme JT, Taylor JA, Shetty SR, Edwards M, Connelly JJ, Rissman EF. Gestational exposure to low dose bisphenol A alters social behavior in juvenile mice. PLoS One. 2011;6:e25448.
Sullivan AW, Beach EC, Stetzik LA, et al. A novel model for neuroendocrine toxicology: neurobehavioral effects of BPA exposure in a prosocial species, the prairie vole (Microtus ochrogaster). Endocrinology.2014;155:3867–3881.
Diamanti-Kandarakis E, Bourguignon J-P, Giudice LC, Hauser R, Prins GS, Soto AM, et al. Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement. Endocr Rev. 2009 Jun 1;30(4):293–342.
Lauretta R, Sansone A, Sansone M, Romanelli F, Appetecchia M. Endocrine Disrupting Chemicals: Effects on Endocrine Glands. Front Endocrinol. 2019 Mar 21;10:178.
Grün F, Blumberg B. Environmental obesogens: organotins and endocrine disruption via nuclear receptor signaling. Endocrinology. 2006;147:S50–S55.
Grün F, Watanabe H, Zamanian Z, et al. Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates. Mol Endocrinol. 2006;20:2141–2155.
Newbold RR, Padilla-Banks E, Snyder RJ, Phillips TM, Jefferson WN. Developmental exposure to endocrine disruptors and the obesity epidemic. Reprod Toxicol Elmsford N. 2007 May;23(3):290–6.
Kanayama T, Kobayashi N, Mamiya S, Nakanishi T, Nishikaw J. Organotin compounds promote adipocyte differentiation as agonists of the peroxisome proliferator activated receptor /retinoid X receptor pathway. Mol Pharmacol. 2005;67:766–774.
Masuno H, Iwanami J, Kidani T, Sakayama K, Honda K. Bisphenol A accelerates terminal differentiation of 3T3–L1 cells into adipocytes through the phosphatidylinositol 3-kinase pathway. Toxicol Sci. 2005;84:319–327.
Wang J, Sun B, Hou M, Pan X, Li X. The environmental obesogen bisphenol A promotes adipogenesis by increasing the amount of 11ẞ-hydroxysteroid dehydrogenase type 1 in the adipose tissue of children. Int J Obes (Lond).2013;37:999–1005.
Neel BA, Brady MJ, SargisRM.The endocrine disrupting chemical tolylfluanid alters adipocyte metabolism via glucocorticoid receptor activation. Mol Endocrinol. 2013;27:394–406.
Angle BM, Do RP, Ponzi D, et al. Metabolic disruption in male mice due to fetal exposure to low but not high doses of bisphenolA(BPA): evidence for effects on body weight,food intake, adipocytes, leptin, adiponectin, insulin and glucose regulation. Reprod Toxicol. 2013;42:256–268.
Batista TM, Alonso-Magdalena P, Vieira E, et al. Shortterm treatment with bisphenol-A leads to metabolic abnormalities in adult male mice. PLoS One. 2012;7:e33814.
Decherf S, Demeneix BA. The obesogen hypothesis: a shift of focus from the periphery to the hypothalamus. J Toxicol Environ Health B Crit Rev. 2011;14:423–448.
Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK. Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations. PLoS One. 2013;8:e55387.
Alonso-Magdalena P, Quesada I, Nadal A. Endocrine disruptors in the etiology of type 2 diabetes mellitus. Nat Rev Endocrinol. 2011;7:346–353.
Soriano S, Alonso-Magdalena P, García-Arévalo M, et al.Rapid insulinotropic action of low doses of bisphenol-A on mouse and human islets of Langerhans: role of estrogen receptor . PLoS One. 2012;7:e31109.
Ruzzin J, Petersen R, Meugnier E, et al. Persistent organic pollutant exposure leads to insulin resistance syndrome. Environ Health Perspect. 2010;118:465–471.
Naville D, Pinteur C, Vega N, et al. Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice. FASEB J. 2013;27:3860–3870.
Bodin J, Bølling AK, Becher R, Kuper F, Løvik M, Nygaard UC. Transmaternal bisphenol A exposure accelerates diabetes type 1 development in NOD mice. Toxicol Sci. 2014;137:311–323.
Brieño-Enríquez MA, Reig-Viader R, Cabero L, et al. Gene expression is altered after bisphenol A exposure in human fetal oocytes in vitro. Mol Hum Reprod. 2012;18:171–183.
Trapphoff T, Heiligentag M, El Hajj N, Haaf T, Eichenlaub-Ritter U. Chronic exposure to a low concentration of bisphenolA during follicle culture affects the epigenetic status of germinal vesicles and metaphase II oocytes. Fertil Steril. 2013;100:1758–1767.
Li Y, Zhang W, Liu J, et al. Prepubertal bisphenol A exposure interferes with ovarian follicle development and its relevant gene expression. Reprod Toxicol. 2014;44:33– 40.
Craig ZR, Hannon PR, Wang W, Ziv-Gal A, Flaws JA. Di-n-butyl phthalate disrupts the expression of genes involved in cell cycle and apoptotic pathways in mouse ovarian antral follicles. Biol Reprod. 2013;88:23.
Peretz J, Gupta RK, Singh J, Hernández-Ochoa I, Flaws JA. Bisphenol A impairs follicle growth, inhibits steroidogenesis, and downregulates rate-limiting enzymes in the estradiol biosynthesis pathway. Toxicol Sci. 2011;119:209–217.
Chang CC, Hsieh YY, Hsu KH, Tsai HD, Lin WH, Lin CS. Deleterious effects of arsenic, benomyl and carbendazim on human endometrial cell proliferation in vitro. Taiwan J Obstet Gynecol. 2010;49:449–454.
Ehrlich S, Williams PL, Missmer SA, et al. Urinary bisphenol A concentrations and implantation failure among women undergoing in vitro fertilization. Environ Health Perspect. 2012;120:978–983.
Ziv-Gal A, Wang W, Zhou C, Flaws JA. The effects of in utero bisphenol A exposure on reproductive capacity in several generations of mice. Toxicol Appl Pharmacol.2015;284:354–362.
Schmidt JS, Schaedlich K, Fiandanese N, Pocar P, Fischer B. Effects of di(2-ethylhexyl) phthalate (DEHP) on female fertility and adipogenesis inC3H/N mice. Environ Health Perspect. 2012;120:1123–1129.
Buck Louis GM, Rios LI, McLain A, Cooney MA, Kostyniak PJ, Sundaram R. Persistent organochlorine pollutants and menstrual cycle characteristics. Chemosphere. 2011;85:1742–1748.
Grindler NM, Allsworth JE, Macones GA, Kannan K, Roehl KA, Cooper AR. Persistent organic pollutants and early menopause in U.S. women. PLoS One. 2015;10:e0116057.
Hoover RN, Hyer M, Pfeiffer RM, et al. Adverse health outcomes in women exposed in utero to diethylstilbestrol. N Engl J Med. 2011;365:1304–1314.
Smith KW, Souter I, Dimitriadis I, et al. Urinary paraben concentrations and ovarian aging among women from a fertility center. Environ Health Perspect. 2013;121:1299–1305.
Abbott DH, Nicol LE, Levine JE, Xu N, Goodarzi MO, Dumesic DA. Nonhuman primate models of polycystic ovary syndrome. Mol Cell Endocrinol. 2013;373:21–28.
Kim SH, Chun S, Jang JY, Chae HD, Kim CH, Kang BM. Increased plasma levels of phthalate esters in women with advanced-stage endometriosis: a prospective case-control study. Fertil Steril. 2011;95:357–359.
Resuehr D, Glore DR, Taylor HS, Bruner-Tran KL, Osteen KG. Progesterone-dependent regulation of endometrial cannabinoid receptor type 1 (CB1-R) expression is disrupted in women with endometriosis and in isolated stromal cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Fertil Steril. 2012;98:948–956.e1.
Bruner-Tran KL, Ding T, Osteen KG. Dioxin and endometrial progesterone resistance. Semin Reprod Med. 2010;28:59–68.
Wang Y, Yu J, Luo X, et al. Abnormal regulation of chemokine TECK and its receptor CCR9 in the endometriotic milieu is involved in pathogenesis of endometriosis by way of enhancing invasiveness of endometrial stromal cells. Cell Mol Immunol. 2010;7:51–60.
Li MQ, Hou XF, Lv SJ, et al. CD82 gene suppression in endometrial stromal cells leads to increase of the cell invasiveness in the endometriotic milieu. J Mol Endocrinol. 2011;47:195–208.
Gao X, Yu L, Castro L, et al. An endocrine-disrupting chemical, fenvalerate, induces cell cycle progression and collagen type I expression in human uterine leiomyoma and myometrial cells. Toxicol Lett. 2010;196:133–141.
D’Aloisio AA, Baird DD, DeRoo LA, Sandler DP. Association of intrauterine and early-life exposures with diagnosis of uterine leiomyomata by 35 years of age in the Sister Study. Environ Health Perspect. 2010;118:375–381.
Mahalingaiah S, Hart JE, Wise LA, Terry KL, Boynton-Jarrett R, Missmer SA. Prenatal diethylstilbestrol exposure and risk of uterine leiomyomata in the Nurses’ Health Study II. Am J Epidemiol. 2014;179:186–191.
Peretz J, Vrooman L, Ricke WA, et al. Bisphenol A and reproductive health: update of experimental and human evidence, 2007–2013. Environ Health Perspect. 2014;122:775–786.
Chakraborty TR, Alicea E, Chakraborty S. Relationships between urinary biomarkers of phytoestrogens, phthalates, phenols, and pubertal stages in girls. Adolesc Health Med Ther. 2012;3:17–26.
Chen CY, Chou YY, Wu YM, Lin CC, Lin SJ, Lee CC. Phthalates may promote female puberty by increasing kisspeptin activity. Hum Reprod. 2013;28:2765–2773.
Henley, D.V., Lipson, N., Korach, K.S., Bloch, C.A., 2007. Prepubertal gynecomastia linked to lavender and tea tree oils. N. Engl. J. Med. 356, 479–485.
Rasier, G., Toppari, J., Parent, A.-S., Bourguignon, J.-P., 2006. Female sexual maturation and reproduction after prepubertal exposure to estrogens and endocrine disrupting chemicals: a review of rodent and human data. Mol. Cell. Endo.254–255, 187–201.
Saiyed, H., Dewan, A., Bhatnagar, V., Shenoy, U., Shenoy, R., Rajmohan, H., Patel, K., Kashyap, R., Kulkarni, P., Rajan, B., Lakkad, B., 2003. Effect of endosulfan on male reproductive development. Environ. Health Perspect. 111, 1958–1962.
. Guo, Y.L., Lambert, G.H., Hsu, C.C., Hsu, M.M., 2004. Yucheng: health effects of prenatal exposure to polychlorinated biphenyls and dibenzofurans. Int. Arch. Occup. Environ. Health 77, 153–158.
Leijs,M.M., Koppe, J.G., Olie, K., van Aalderen,W.M.C., de Voogt, P., Vulsma, T.,Westra, M., ten Tusscher, G.W., 2008. Delayed initiation of breast development in girls with higher prenatal dioxin exposure; a longitudinal cohort study. Chemosphere 73, 999–1004.
Hauser, R., Sergeyev, O., Korrick, S., Lee,M.M., Revich, B., Gitin, E., Burns, J.S.,Williams, P.L., 2008. Association of blood lead levels with onset of puberty in Russian boys. Environ. Health Perspect. 116, 976–980.
Selevan, S.G., Rice, D.C., Hogan, K.A., Euling, S.Y., Pfahles-Hutchens, A., Bethel, J., 2003. Blood lead concentration and delayed puberty in girls. N. Engl. J. Med.348, 1527–1536.
Damgaard IN, Skakkebaek NE, Toppari J, et al. Persistent pesticides in human breast milk and cryptorchidism. Environ Health Perspect. 2006;114:1133–1138.
Fernandez MF, Olmos B, Granada A, et al. Human exposure to endocrine-disrupting chemicals and prenatal risk factors for cryptorchidism and hypospadias: a nested case-control study. Environ Health Perspect. 2007;115(suppl 1):8 –14.
Virtanen HE, Koskenniemi JJ, Sundqvist E, et al. Associations between congenital cryptorchidism in newborn boys and levels of dioxins and PCBs in placenta. Int J Androl. 2012;35:283–293.
Brucker-Davis F, Wagner-Mahler K, Delattre I, et al. Cryptorchidism at birth in Nice area (France) is associated with higher prenatal exposure to PCBs and DDE, as assessed by colostrum concentrations. Hum Reprod.2008;23:1708–1718.
Cook MB, Trabert B, McGlynn KA. Organochlorine compounds and testicular dysgenesis syndrome: human data. Int J Androl. 2011;34:e68–e84; discussion e84–e65.
Trabert B, Longnecker MP, Brock JW, Klebanoff MA, McGlynn KA. Maternal pregnancy levels of trans-nonachlor and oxychlordane and prevalence of cryptorchidism and hypospadias in boys. Environ Health Perspect.2012;120:478–482.
Vested A, Giwercman A, Bonde JP, Toft G. Persistent organic pollutants and male reproductive health. Asian J Androl. 2014;16:71–80.
Aneck-Hahn NH, Schulenburg GW, Bornman MS, Farias P, de Jager C. Impaired semen quality associated with environmental DDT exposure in young men living in a malaria area in the Limpopo Province, South Africa. J Androl. 2007;28:423–434.
De Jager C, Farias P, Barraza-Villarreal A, et al. Reduced seminal parameters associated with environmental DDT exposure and p,p’-DDE concentrations in men in Chiapas, Mexico: a cross-sectional study. J Androl. 2006;27:16–27.
Mocarelli P, Gerthoux PM, Needham LL, et al. Perinatal exposure to low doses of dioxin can permanently impair human semen quality. Environ Health Perspect. 2011;119:713–718.
Abdelouahab N, Ainmelk Y, Takser L. Polybrominated diphenyl ethers and sperm quality. Reprod Toxicol.2011;31:546–550.
Hwang K-A, Choi K-C. Endocrine-Disrupting Chemicals with Estrogenicity Posing the Risk of Cancer Progression in Estrogen-Responsive Organs. In: Advances in Molecular Toxicology [Internet]. Elsevier; 2015 [cited 2021 Feb 7]. p. 1–33. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780128022290000013.
Warner M, Eskenazi B, Mocarelli P, et al. Serum dioxin concentrations and breast cancer risk in the Seveso Women’s Health Study. Environ Health Perspect. 2002;110:625–628.
Boada LD,ZumbadoM,Henríquez-Hernández LA, et al. Complex organochlorine pesticide mixtures as determinant factor for breast cancer risk: a population-based case-control study in the Canary Islands (Spain). Environ Health. 2012;11:28.
López-Carrillo L, Hernández-Ramírez RU, Calafat AM, et al. Exposure to phthalates and breast cancer risk in northern Mexico. Environ Health Perspect. 2010;118:539–544.
Donna A, Crosignani P, Robutti F, et al. Triazine herbicides and ovarian epithelial neoplasms. Scand J Work Environ Health. 1989;15:47–53.
VieiraVM,HoffmanK, ShinHM,Weinberg JM, Webster TF, Fletcher T. Perfluorooctanoic acid exposure and cancer cancer outcomes in a contaminated community: a geographic analysis. Environ Health Perspect. 2013;121:318–323.
Driscoll SG, Taylor SH. Effects of prenatal maternal estrogen on the male urogenital system. Obstet Gynecol.1980;56:537–542.
Koutros S, Beane Freeman LE, Lubin JH, et al. Risk of total and aggressive prostate cancer and pesticide use in the Agricultural Health Study. Am J Epidemiol. 2013;177:59–74.
ChristensenCH,Platz EA, Andreotti G, et al. Coumaphos exposure and incident cancer among male participants in the Agricultural Health Study (AHS). Environ Health Perspect. 2010;118:92–96.
Usmani KA, ChoTM,Rose RL, Hodgson E. Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals. Drug Metab Dispos.2006;34:1606–1614.
Institute of Medicine. Veterans and Agent Orange: Update 2012. Washington DC: The National Academy of Sciences; 2014.
Hinson, J. P., & Raven, P. W. (2006). Effects of endocrine-disrupting chemicals on adrenal function. Best Practice & Research Clinical Endocrinology & Metabolism, 20(1), 111–120.
Kioumourtzoglou M-A, Coull BA, O’Reilly ÉJ, Ascherio A, Weisskopf MG. Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits. JAMA Pediatr. 2018 Jul 1;172(7):670–7.
Cantonwine D, Meeker JD, Hu H, et al. Bisphenol A exposure in Mexico City and risk of prematurity: a pilot nested case control study. Environ Health. 2010;9:62.
Ferguson KK, McElrath TF, Meeker JD. Environmental phthalate exposure and preterm birth. JAMA Pediatr.2014;168:61–67.
Mustafa MD, Banerjee BD, Ahmed RS, Tripathi AK, Guleria K. Gene-environment interaction in preterm delivery with special reference to organochlorine pesticides. Mol Hum Reprod. 2013;19:35–42.
Peltier MR, Arita Y, Klimova NG, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances placental inflammation. J Reprod Immunol. 2013;98:10–20.
Snijder CA, Heederik D, Pierik FH, et al. Fetal growth and prenatal exposure to bisphenol A: the generationRstudy. Environ Health Perspect. 2013;121:393–398.
Burdorf A, Brand T, Jaddoe VW, Hofman A, Mackenbach JP, Steegers EA. The effects of work-related maternal risk factors on time to pregnancy, preterm birth and birth weight: the Generation R Study. Occup Environ Med. 2011;68:197–204.
Snijder CA, te Velde E, Roeleveld N, Burdorf A. Occupational exposure to chemical substances and time to pregnancy: a systematic review. Hum Reprod Update. 2012;18:284–300.
Suzuki Y, Yoshinaga J, Mizumoto Y, Serizawa S, Shiraishi H. Foetal exposure to phthalate esters and anogenital distance in male newborns. Int J Androl. 2012;35:236–244.
Gemmill A, Gunier RB, Bradman A, Eskenazi B, Harley KG. Residential proximity to methyl bromide use and birth outcomes in an agricultural population in California. Environ Health Perspect. 2013;121:737–743.
Migeot V, Albouy-Llaty M, Carles C, et al. Drinking water exposure to a mixture of nitrate and low-dose atrazine metabolites and small-for-gestational age (SGA) babies: a historic cohort study. Environ Res. 2013;122:58–64.
Wang P, Tian Y, Wang XJ, et al. Organophosphate pesticide exposure and perinatal outcomes in Shanghai, China. Environ Int. 2012;42:100–104.
Kishi R, Sasaki S, Yoshioka E, Yuasa M, Sata F, Saijo Y, Kurahashi N, Tamaki J, Endo T, Sengoku K, Nonomura K, Minakami H; Hokkaido Study on Environment and Children's Health. Cohort profile: the Hokkaido study on environment and children's health in Japan. Int J Epidemiol. 2011 Jun;40(3):611-8.
Paulose R, Jegatheesan K, Balakrishnan GS. A big data approach with artificial neural network and molecular similarity for chemical data mining and endocrine disruption prediction. Indian J Pharmacol. 2018 Jul 1;50(4):169.
Jaffrezic-Renault N, Kou J, Tan D, Guo Z. New trends in the electrochemical detection of endocrine disruptors in complex media. Anal Bioanal Chem. 2020 Sep;412(24):5913–23.
Street ME, Audouze K, Legler J, Sone H, Palanza P. Endocrine Disrupting Chemicals: Current Understanding, New Testing Strategies and Future Research Needs. Int J Mol Sci. 2021 Jan 19;22(2):933.
Artificial Intelligence Facilitates Chemical Toxicity Evaluation: the Case of Bisphenol S [Internet]. Newsroom | Inserm. 2019 [cited 2021 Feb 8]. Available from: https://presse.inserm.fr/en/artificial-intelligence-facilitates-chemical-toxicity-evaluation-the-case-of-bisphenol-s/34525/
Porta N, ra Roncaglioni A, Marzo M, Benfenati E. QSAR Methods to Screen Endocrine Disruptors. Nucl Recept Res [Internet]. 2016 [cited 2021 Feb 17];3. Available from: http://www.kenzpub.com/journals/nurr/2016/101203/
Carvalho, F.P., 2006. Agriculture, pesticides, food security and food safety. Environ. Sci.Policy 9, 685–692.
Toteja G.S, Dasgupta J, Saxena B.N, Kalra R.L, editors. Report of an ICMR Task Force Study (Part 1). New Delhi: Indian Council of Medical Research; 1993. Surveillance of Food Contaminants in India.
Kannan K, Tanabe S, Ramesh A, Subramanian A, Tatsukawa R. Persistent organochlorine residues in food stuffs from India and their implications on human dietary exposure. J Agric Food Chem. 1992;40:518.

APPENDIX

RECENT UPDATES ON ENDOCRINE DISRUPTING CHEMICALS (EDCs)

Updated May 2, 2025

PRENATAL EXPOSURE AND FETAL DEVELOPMENT

Recent studies have shown that prenatal exposure to endocrine disrupting chemicals (EDCs) can significantly impact fetal development. The HPP-3D (Human Placental Plasticity–3D) study found a negative association between maternal phthalate levels and fetal liver volume, with changes comparable to a 5 kg/m² difference in parental Body Mass Index suggesting early structural alterations with potential lifelong metabolic consequences (1). In the Ko-CHENS (Korean Children’s Environmental Health Study ) study, personal care product use was linked to higher levels of monoethyl phthalate (MEP), and cooking with plastic was associated with increased mono-n-butyl phthalate (MNBP) levels (2). The Let's R.O.A.R (Let’s Reclaim Our Ancestral Roots) pilot intervention demonstrated that culturally tailored strategies could reduce low-molecular weight phthalate metabolites by over 5% in Black women, particularly dibutyl phthalate (3).

Neurodevelopment and Behavioral Outcomes

EDCs have also been implicated in neurodevelopmental and behavioral disorders. The PELAGIE (Perturbateurs Endocriniens: Étude Longitudinale sur les Anomalies de Grossesse, l’Infertilité et l’Enfance) cohort from France found that exposure to perfluorinated compounds such as perfluorooctanoic acid (PFOA), (perfluorononanoic acid) PFNA, and perfluorodecanoic acid (PFDA) was associated with externalizing and internalizing behaviors in children (4). Bisphenol A (BPA) was found to be associated with aromatase gene methylation and autism spectrum disorder (ASD) traits in boys, with reversibility shown in mouse models using !0-hydroxy-2-decenoic acid(10HDA), suggesting a potential therapeutic pathway (5). Another study identified BPA interaction with 35 of 77 ASD-related genes in transcriptomic analysis (6). A systematic review concluded that EDC exposure, especially to metals, phthalates, and PFAS is linked to poorer cognitive, language, and motor development in children, with girls being more susceptible (7).

FEMALE REPRODUCTIVE HEALTH AND OVARIAN FUNCTION

The Study of Women’s Health Across the Nation (SWAN) found that exposure to heavy metals like arsenic, cadmium, and mercury was associated with lower anti-Müllerian hormone (AMH) levels and a faster premenopausal decline (8). A human ovarian model exposed to diethylstilbestrol (DES) and ketoconazole (KTZ) showed altered follicle survival and steroidogenesis, with upregulation of stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase, indicating potential biomarkers for ovarian toxicity (9). Microplastics (MPs) have been detected for the first time in human ovarian follicular fluid, in 14 out of 18 women undergoing IVF, with an average of 2,191 particles/mL. A significant correlation has been observed between MP levels and FSH, suggesting potential effects on ovarian function. While no link has been found with fertilization or pregnancy outcomes, the findings highlight a concerning new avenue for understanding the reproductive impact of microplastic exposure (10). In a rat model, di(2-ethylhexyl) phthalate (DEHP) exposure induced polycystic ovary syndrome (PCOS)-like changes, insulin resistance, and oxidative stress via the PPARγ pathway (11).

COGNITIVE AGING

EDC exposure may also affect cognitive aging. Analysis of NHANES data (2011–2014) linked exposure to 47 EDCs, including PFNA, PCB-199, and PCB-206, with worse verbal fluency and global cognition, though delayed recall effects were mixed (12).

PUBERTY AND HORMONAL PATHWAYS

In vitro analysis using the Tox21 10K library identified musk ambrette and methacholine analogs as agonists of KISS1R and GnRHR, suggesting that they may promote early puberty through hormonal activation (13).

TOXICOLOGY AND RISK ASSESSMENT

Regulatory and technological advancements in EDC detection and safety evaluation are ongoing. The European Food Safety Authority (EFSA) revised the tolerable daily intake for BPA from 4 µg/kg/day to 0.2 ng/kg/day, indicating increased concern over low-dose effects (14). New Approach Methods (NAMs), including in vitro and in silico tools, were used to prioritize over 200 low-data chemicals for further study (15). An electrochemical sensor using a 2D-Al quasicrystal structure detected PFOA with high sensitivity (16). Concerns have been raised regarding the toxicity and potential endocrine disrupting effects of UV filters used in sunscreens. Six commonly used organic UV filters were assessed using the ToxCast/Tox21 database and found that they exhibited low biological activity, with most effects occurring at concentrations above cytotoxic levels. Except for oxybenzone, human plasma levels were significantly lower than those causing activity in assays. Overall, these UV filters showed weak or negligible endocrine-disrupting potential, supporting their low risk to human health(17).

CONCLUSION

The growing body of evidence highlights the pervasive impact of EDCs on female reproductive and metabolic health across the life course. From fetal development to menopause, EDCs such as phthalates, bisphenol A, perfluoroalkyl substances, and heavy metals disrupt hormonal pathways, with long-term health implications. Advances in biomonitoring, mechanistic studies, and NAMs are enhancing our understanding and risk assessment of these exposures. Continued interdisciplinary research and policy actions are critical to mitigate risks and safeguard public health.

REFERENCES

Stevens DR, Sinkovskaya E, Przybylska A, Nehme L, Diab Y, Onishi K, Saade G, Abuhamad A, Ferguson KK. Gestational exposure to endocrine disrupting chemicals and fetal liver development: Findings from the HPP-3D study. ISEE Conference Abstracts. 2024;2024(1)]. doi: 10.1289/isee.2024.0691.
Surabhi S, Bang Y, Oh J, Shin J, Park E, Ha E. Pregnant women lifestyle and exposure to endocrine disrupting chemicals. *ISEE Conference Abstracts*. 2023 Sep 17;2023(1). doi:10.1289/isee.2023.SA-087. Available from: https://ehp.niehs.nih.gov/doi/full/10.1289/isee.2023.SA-087.
McDonald JA, Vilfranc CL, Wang X, Tsui F, Franklin J, Walker DAH, Martinez M, Shepard P, Terry MB, Llanos AAM, Barrett ES, Houghton LC, Pennell K. Hair care product exposure among pregnant women of color in New York City: Feasibility of a mixed-methods educational intervention study. ISEE Conference Abstracts. 2024;2024(1).
Hélène Tillaut, Christine Monfort, Florence Rouget, Fabienne Pelé, Fabrice Lainé, Eric Gaudreau et al.Prenatal Exposure to Perfluoroalkyl Substances and Child Behavior at Age 12: A PELAGIE Mother–Child Cohort Study.Environmental Health Perspectives 131:11.CID: 117009. https://doi.org/10.1289/EHP12540.3. Let’s R.O.A.R. Pilot – Reduction in phthalate metabolites through culturally tailored interventions.
Symeonides, C., Vacy, K., Thomson, S. et al. Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype. Nat Commun 15, 6367 (2024). https://doi.org/10.1038/s41467-024-48897-8.
Santos JX, Rasga C, Marques AR, et al. A role for gene-environment interactions in autism spectrum disorder is supported by variants in genes regulating the effects of exposure to xenobiotics. Front Neurosci. 2022;16:862315. doi: 10.3389/fnins.2022.862315.
Yang Z, Zhang J, Wang M, Wang X, Liu H, Zhang F, Fan H. Prenatal endocrine-disrupting chemicals exposure and impact on offspring neurodevelopment: A systematic review and meta-analysis. Neurotoxicology. 2024 Jul;103:335-357. doi: 10.1016/j.neuro.2024.07.006. Epub 2024 Jul 14. PMID: 39013523.
Ding N, Wang X, Harlow SD, Randolph JF Jr, Gold EB, Park SK. Heavy Metals and Trajectories of Anti-Müllerian Hormone During the Menopausal Transition. J Clin Endocrinol Metab. 2024 Oct 15;109(11):e2057-e2064. doi: 10.1210/clinem/dgad756. PMID: 38271266.
Li T, Vazakidou P, Leonards PEG, Damdimopoulos A, Panagiotou EM, Arnelo C, Jansson K, Pettersson K, Papaikonomou K, van Duursen M, Damdimopoulou P. Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using in vitro models. *Toxicol. 2023*;485:153425.
Montano L, Raimondo S, Piscopo M, Ricciardi M, Guglielmino A, Chamayou S, et al. First evidence of microplastics in human ovarian follicular fluid: An emerging threat to female fertility. Ecotoxicol Environ Saf. 2025;291:117868. doi:10.1016/j.ecoenv.2025.117868.
Wang S, Xu K, Du W, Gao X, Ma P, Yang X, Chen M. Exposure to environmental doses of DEHP causes phenotypes of polycystic ovary syndrome. *Toxicol.* 2024 Dec;509:153952. doi: 10.1016/j.tox.2024.153952.
Zuo Q, Gao X, Fu X, Song L, Cen M, Qin S, Wu J. Association between mixed exposure to endocrine-disrupting chemicals and cognitive function in elderly Americans. Public Health. 2024 Mar;228:36-42. doi: 10.1016/j.puhe.2023.12.021. Epub 2024 Jan 22. PMID: 38262207.
Shu Yang, Li Zhang, Kamal Khan, Jameson Travers, Ruili Huang, Vukasin M Jovanovic, Rithvik Veeramachaneni, Srilatha Sakamuru, Carlos A Tristan, Erica E Davis, Carleen Klumpp-Thomas, Kristine L Witt, Anton Simeonov, Natalie D Shaw, Menghang Xia, Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R, Endocrinology, Volume 165, Issue 10, October 2024, bqae103, https://doi.org/10.1210/endocr/bqae103.
EFSA CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids), Lambré C, Barat Baviera JM, Bolognesi C, Chesson A, Cocconcelli PS, Crebelli R, et al. Scientific Opinion on the re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs. EFSA J. 2023;21(4):6857. doi: 10.2903/j.efsa.2023.6857
Katie Paul Friedman, Russell S Thomas, John F Wambaugh, Joshua A Harrill, Richard S Judson, Timothy J Shafer, Antony J Williams, Jia-Ying Joey Lee, Lit-Hsin Loo, Matthew Gagné, Alexandra S Long, Tara S Barton-Maclaren, Maurice Whelan, Mounir Bouhifd, Mike Rasenberg, Ulla Simanainen, Tomasz Sobanski, Integration of new approach methods for the assessment of data-poor chemicals, Toxicological Sciences, 2025;, kfaf019, https://doi.org/10.1093/toxsci/kfaf019.
Chakraborty A, Tromer R, Yadav TP, Mukhopadhyay NK, Lahiri B, Rao R, Roy A, Aich N, Woellner CF, Galvao DS, Tiwary CS. Ultrasensitive Electrochemical Sensor for Perfluorooctanoic Acid Detection Using Two-dimensional Aluminium Quasicrystal. arXiv preprint arXiv:2501.07587. 2025 Jan 6.
David O Onyango, Bastian G Selman, Jane L Rose, Corie A Ellison, J F Nash, Comparison between endocrine activity assessed using ToxCast/Tox21 database and human plasma concentration of sunscreen active ingredients/UV filters, Toxicological Sciences, Volume 196, Issue 1, November 2023, Pages 25–37, https://doi.org/10.1093/toxsci/kfad082.

Dyslipidemia in Chronic Kidney Disease

ABSTRACT

Chronic kidney disease (CKD) is associated with a dyslipidemia comprising high triglycerides, low HDL-C, and altered lipoprotein composition. Cardiovascular diseases are the leading cause of mortality in CKD, especially in end stage renal disease patients. Thus, therapies to reduce cardiovascular risk are urgently needed in CKD. Robust clinical trial evidence has found that the use of statins in pre-end stage CKD patients, as well as in renal transplant recipients, can decrease cardiovascular events; however, providers need to be aware of dose restrictions for statin therapy in CKD subjects. Furthermore, statin therapy does not reduce cardiovascular events in dialysis patients, nor does statin therapy confer any protection against the progression of renal disease. Niacin and fibrates are effective in lipid lowering in CKD and appear to have some cardiovascular benefit, but further study is needed to clearly define their role. Novel therapies with PCSK 9 inhibitors, bempedoic acid, and inclisiran have all been shown to improve LDL-C levels but there is currently limited data for reduction of cardiovascular events or mortality in patients with CKD/ESRD. This article reviews the epidemiology of CKD, association of CKD with cardiovascular events, and the effects of CKD on lipid levels and metabolism. The chapter discusses clinical trial evidence for and against statin and non-statin lipid lowering therapy in CKD patients.

CKD EPIDEMIOLOGY

Chronic kidney disease (CKD) is defined as renal impairment for greater than 3 months duration that results in an estimated glomerular filtration rate (eGFR) < 60ml/min/1.73m². CKD is classified into 5 stages based on the eGFR (Table 1) and albuminuria category (Table 2). CKD is a worldwide health problem with a rising incidence and prevalence. CKD, especially in the early stages, is often asymptomatic; thus, the actual prevalence may be even higher than estimated. End stage renal disease (ESRD) is defined as needing dialysis or transplant, and the prevalence and incidence of ESRD have doubled over the past 10 years (1). The annual mortality rate of dialysis patients is greater than 20%. The burden of co-morbidities and the cost of caring for CKD patients is high, and thus a major focus is increased screening and early detection of CKD when interventions to delay or prevent progression to ESRD may be effective. There are multiple causes of CKD with the most common causes in Westernized nations being hypertension and diabetes; however, a wide range of etiologies including infectious, auto-immune, genetic, obstructive, and ischemic injury are all prevalent.

Table 1. Stages of CKD Based on eGFR
GFR category	GFR (ml/min/1.73 m²)	Terms
G1	≥ 90	Normal or High
G2	60-89	Mildly decreased
G3a	45-59	Mildly to moderately decreased
G3b	30-44	Moderately to severely decreased
G4	15-29	Severely decreased
G5	<15	Kidney failure

In the absence of evidence of kidney damage, neither G1 nor G2 fulfills the criteria for CKD.

Table 2. Stages of CKD based on Albuminuria
CKD stage	AER (mg/24h)	ACR (mg/mmol)	ACR (mg/g)
A1	<30	<3	<30
A2	30-300	3-30	30-300
A3	>300	>30	>300

AER: albumin excretion rate; ACR: albumin to creatinine ratio.

While the burden of CKD itself is significant, the leading causes of morbidity and mortality in CKD are cardiovascular diseases (CVD), primarily atherosclerotic coronary artery disease. Risk factors for CVD in CKD include the traditional risk factors – dyslipidemia, hypertension, sex, age, smoking, and family history and CKD patients appear to benefit similar to non-CKD patients from therapies targeting these risk factors. Regardless of the cause of CKD, patients with CKD are at increased risk for CVD, which has led to the National Kidney Foundation classifying all patients with CKD as “highest risk” for CVD regardless of their levels of traditional CVD risk factors. Per the 2022 ACC consensus for non-statin therapies, CKD is considered an ASCVD risk enhancer (2). The focus of this chapter is on the dyslipidemia of CKD and the risk of CVD in CKD.

Nephrotic Syndrome

Nephrotic syndrome differs from other types of CKD in its presentation and risks. Nephrotic syndrome is comprised of significant proteinuria (typically > 3g/24h), hypoalbuminemia, peripheral (+/- central) edema, and significant hyperlipidemia and lipiduria may also be seen. It is frequently seen in children, and the etiology includes minimal change disease (up to 85%), focal segmental glomerulosclerosis (up to 15%) and secondary causes (rare) including systemic lupus erythrematosis, Henoch Schonlein Purpura, or membrano-proliferative glomerulopathy. In adults, the etiology is more likely to involve a systemic disease such as diabetes, amyloidosis, or lupus. Nephrotic syndrome may be transient or persistent. Most (approximately 80% of children) cases of nephrotic syndrome are successfully treated with glucocorticoids with resolution of all features including hyperlipidemia; however, steroid-resistant nephrotic syndrome patients often have persistent dyslipidemia, which may place them at increased risk for CVD. For example, a small study found increased CVD markers including pulse wave velocity, carotid artery intima-media thickness, and left ventricular mass in patients with steroid-resistant nephrotic syndrome compared to controls (3), implying increased risk for CVD events. Treatment of nephrotic syndrome dyslipidemia includes therapies specifically targeting the renal disease (primarily glucocorticoids, but also renin-angiotensin system antagonists which can help decrease proteinuria) and lipid lowering agents.

CVD IN CKD

CVD accounts for 40-50% of all deaths in ESRD patients, with CVD mortality rates approximately 15 times that seen in the general population (4). However, CVD is highly prevalent in patients who progress to ESRD implying that earlier stages of CKD increase the development of CVD. A number of factors have been proposed as risk factors for CVD in CKD including proteinuria, inflammation, anemia, malnutrition, oxidative stress, and uremic toxins (5). Ongoing research is investigating whether these (and other) markers may be therapeutic targets. Interestingly, proteinuria correlates with blood pressure, total cholesterol, TGs, and inversely correlates with HDL-C (6). Thus, it remains unclear if proteinuria itself is a risk factor (e.g. a cause of CVD) or a biomarker. Meta-analyses of the general population and high risk population cohorts found that both lower eGFR (<60 ml/min/1.73 m²) and higher albuminuria (>10 mg/g creatinine) are predictors of total mortality and CVD mortality; furthermore, eGFR and albuminuria are independent of each other and of traditional CVD risk factors (7, 8). A meta-analysis that assessed individual participant data of over 22 million individuals from 64 global cohorts estimated the risk of myocardial infarction up to 6-fold higher for those with urine albumin/creatinine ratio over 300 mg/g and eGFR < 15 mL/min/1.73m²; similar estimates were conducted for other CVD outcomes such as stroke, CVD mortality, heart failure and others (9). Estimated GFR > 60 ml/min/1.73 m² alone is not a risk factor for CVD or total mortality.

Dyslipidemia in CKD

EFFECT OF CKD ON LIPID LEVELS

CKD is associated with a dyslipidemia comprised of elevated TGs and low HDL-C. Levels of LDL-C (and thus, total cholesterol) are generally not elevated; however, proteinuria correlates with cholesterol and TGs. CKD leads to a down regulation of lipoprotein lipase and the LDL receptor, and increased TGs in CKD are due to delayed catabolism of TG rich lipoproteins, with no differences in production rate (10). CKD is associated with lower levels of apoA-I (due to decreased hepatic expression (11)) and higher apoB/apoA-I ratio. Decreased lecithin-cholesterol acyltransferase (LCAT) activity and increased cholesteryl ester transfer protein (CETP) activity contribute to decreased HDL-C levels. Beyond decreased HDL-C levels, the HDL in CKD is less effective in its anti-oxidative and anti-inflammatory functions [for review see (12)].

As CKD progresses the dyslipidemia often worsens. In an evaluation of 2001-2010 National Health and Nutrition Examination Survey (NHANES), the prevalence of dyslipidemia increased from 45.5% in CKD stage 1 (albuminuria with an eGFR ≥ 90 mL/min/1.73 m²) to 67.8% in CKD stage 4 (eGFR 15-29 mL/min/1.73 m²); similarly, the use of lipid lowering agents increased from 18.1% in CKD stage 1 to 44.7% in CKD stage 4 (13). Of more than 1000 hemodialysis patients studied only 20% had “normal” lipid levels (defined as LDL-C <130 mg/dl, HDL-C > 40 and TGs < 150); of 317 peritoneal dialysis patients only 15% had “normal” lipid levels (14). A larger study evaluating dyslipidemia in > 21,000 incident dialysis patients found 82% prevalence of dyslipidemia and suggested a threshold of non-HDL-C > 100 mg/dl (2.6mmol/L) to identify dyslipidemia in CKD stage 5 subjects (15). Peritoneal dialysis is associated with higher cholesterol levels than hemodialysis, although the reasons aren’t fully understood. In subjects who switched from peritoneal dialysis to hemodialysis there was a decrease in cholesterol levels of almost 20% following transition (16). The National Kidney Foundation recommends routine screening of all adults and adolescents with CKD using a standard fasting lipid profile (total cholesterol, LDL-C, HDL-C and TGs), and follows the classification of the National Cholesterol Education Panel for levels (desirable, borderline or high). Although some studies have found associations between Lp(a) and dialysis patients, this is not well defined and there is no current indication for routine screening of Lp(a).

EFFECT OF CKD ON LIPOPROTEIN COMPOSITION

Beyond simply measuring lipid levels, emerging evidence implies that lipoprotein particle size and composition is altered in CKD, with increased small dense LDL and decreased larger LDL particles in CKD subjects compared to controls (17). Small dense LDL is thought to be more atherogenic than larger LDL particles. An emerging theory is that beyond lipid levels or lipoprotein size, lipoprotein particle “cargo” can affect atherosclerosis development and progression. Lipoprotein particles transport numerous bioactive lipids, microRNAs, other small RNAs, proteins, hormones, etc. For example, a recent study compared LDL particle composition between subjects with stage 4/5 CKD and non-CKD controls, and found similar total lipid and cholesterol content, but altered content of various lipid subclasses, for example decreased phosphatidylcholines, sulfatides, and ceramides and increased N-acyltaurines (18). Many of these lipid species are known to have either pro- or anti-atherogenic properties and thus could directly affect atherogenesis.

EFFECT OF RENAL TRANSPLANTATION ON LIPID LEVELS

Dyslipidemia is frequently seen in renal transplant recipients, including increased total cholesterol, LDL-C, and TGs, and decreased HDL-C. The dyslipidemia may have existed pre-transplant or be related to transplantation associated factors. Cyclosporine increases LDL-C via both increased production and decreased clearance. Corticosteroids increase both cholesterol and TG levels in a dose-dependent manner. The adverse effects of cyclosporine and corticosteroids on lipid levels appear to be additive (19). Tacrolimus and azathioprine appear to have less induction of dyslipidemia than cyclosporine (20). Sirolimus increases both cholesterol and TGs, in part due to decreased LDL clearance (21).

EFFECT OF NEPHROTIC SYNDROME ON LIPID LEVELS

The dyslipidemia in nephrotic syndrome can be striking with significant elevations of cholesterol, LDL-C, TGs and lipoprotein(a); HDL-C is often low, especially HDL2. The cause of elevated lipid levels is multi-factorial, including reduction in oncotic pressure which stimulates hepatic apoB synthesis (although the exact mechanism by which this occurs is not known), decreased metabolism of lipoproteins, and decreased clearance. Patients with nephrotic syndrome have decreased LDL receptor activity and increased acyl-CoA cholesterol acytransferase (ACAT) and HMG-CoA reductase activity leading to increased LDL-C levels (22, 23). Low HDL-C is thought to be due at least in part to LCAT deficiency secondary to accelerated renal loss of LCAT (24). TGs are elevated due to impaired clearance of chylomicrons and TG-rich lipoproteins, as well as increased TG production (25).

EVIDENCE FOR/AGAINST LIPID LOWERING THERAPY IN CKD FOR CVD OUTCOMES

Given the high prevalence of CVD in CKD, and the robust clinical evidence in non-CKD subjects that lipid lowering reduces CVD outcomes, there is great interest in using lipid lowering therapy in CKD subjects. Statins are the most commonly used lipid-lowering medications and thus far have been shown to reduce CVD events and/or mortality in virtually every population studied. However, CKD patients seem to be a unique population in that at present there is no evidence of benefit for CVD outcomes in dialysis patients with statin therapy. The Canadian Journal of Cardiology lists CKD as a statin indicated condition in its newest guidelines published in 2021(26) while AHA/ACC lists CKD as a risk enhancer but not a high-risk condition based on 2018 guidelines (27). Despite growing evidence to support CKD as a CVD risk equivalent, the use of statin therapy in CKD does not appear to be rising more than in the non-CKD population based on data from Mefford et al looking at trends in statin use amongst US adults with CKD from 1999-2014 (28). As discussed below it appears that statins can reduce CVD events in pre-end stage CKD subjects, and in post-renal transplant subjects, but not in dialysis patients (Table 3). The Kidney Disease: Improving Global Outcomes (KDIGO) 2024 clinical practice guideline recommends using statin or statin/ezetimibe combination therapy for adults ≥ 50 years old with eGFR < 60 ml/min/1.73 m² (29). Additionally, they recommend that in adults aged18–49 years with CKD but not treated with chronic dialysis or kidney transplantation, that statin treatment be used if the following risk factors are present; known coronary disease, diabetes, prior ischemic stroke, or estimated 10-year incidence of coronary death or nonfatal myocardial infarction >10%.

Use of Statins in Pre-ESRD CKD Patients

Although many of the initial statin CVD studies did not include many CKD patients, evidence from sub-group analyses of large statin studies suggested that CKD subjects had similar benefits to non-CKD individuals. For example, the Heart Protection Study (HPS) which assessed >20,000 subjects at high risk of CVD included a subgroup of 1,329 subjects with impaired kidney function. In this subgroup those that received simvastatin had a 28% proportional risk reduction and an 11% absolute risk reduction of a major cardiovascular event compared to those randomized to placebo, which was similar to the effect on the overall cohort (30). Further, in the Pravastatin Pooling Project, 4,991 subjects with CKD3 were examined and a 23% reduction in cardiovascular events was seen in the pravastatin group (31). In a retrospective study with 47,200 subjects followed through the Department of Veterans Affairs, starting statin therapy 12 months prior to transitioning to ESRD conferred a reduction in 12 month all-cause mortality (HR 0.79), cardiovascular events (HR 0.83) and hospitalization rate (HR 0.89) (32). Several other studies or meta-analyses similarly predicted that CKD subjects would have reduction in CVD with statin therapy. For example, a meta-analysis of 38 studies with >37,000 participants with CKD but not yet on dialysis found a consistent reduction in major cardiovascular events, all-cause mortality, cardiovascular death and myocardial infarction in statin users compared to placebo groups. There was no clear effect of statin on stroke, nor was there any effect of statin use on progression of the renal disease (33). Another meta-analysis similarly reported efficacy of statin therapy, but that the relative reductions in CVD evens with statin therapy declined with lower eGFR, to the point of no benefit in dialysis patients (34). Thus, CKD patients with pre-end stage renal disease statins effectively lower total cholesterol and LDL-C levels and decrease CVD risk. The different statins have different degrees of renal involvement in their metabolism, and providers should be aware of dose restrictions in CKD (Table 4).

Unclear Whether to Use Statins in Subjects with Nephrotic Syndrome

Several small clinical studies have investigated the use of lipid lowering therapies in nephrotic syndrome, but data is only available for statins and fibrates, and no CVD outcome data is available. Several small studies using statins have found efficacy in lowering LDL-C and that statins were safe and well tolerated (35, 36). Two recent small studies suggest that statin therapy in nephrotic syndrome may reduce CVD risk (37, 38). Thus, the use of statins in nephrotic syndrome appears to be safe and efficacious in terms of lipid lowering; however, it remains unclear if statins should be recommended for benefit on either CVD or renal outcomes.

Use of Statins in Subjects with only Microalbuminuria

The Prevention of Renal and Vascular Endstage Disease Intervention Trial (PREVEND IT) randomized 864 subjects with persistent microalbuminuria (urinary albumin of 15-300mg/24h x 2 samples) to fosinopril (an angiotensin converting enzyme inhibitor) or placebo and to pravastatin 20 mg or placebo. Inclusion criteria for the study included blood pressure <160/100 mm Hg and no use of antihypertensive medications and total cholesterol < 300 mg/dl (8 mmol/L) or < 192 mg/dl (5 mmol/L) if patient had known CVD and no use of lipid lowering medications. Although diabetes was not an exclusion criteria, <3% of the subjects had diabetes (39). The use of statin did not affect either urinary albumin excretion or cardiovascular events; however, the use of fosinopril significantly decreased albuminuria and had a trend to reduce cardiovascular events. Thus, in the absence of other indications for statin therapy, this study suggests no benefit in subjects that solely have microalbuminuria; however the study was limited by small size and few CVD events. A subsequent analysis found that the subjects with isolated microalbuminuria had an increased risk for CVD events and mortality compared to those without risk factors (40); thus isolated microalbuminuria appears to indicate high risk and further study is needed to determine effective therapies to reduce risk.

No Benefit of Statins in Dialysis Patients

Studies specifically examining the role of statins in ESRD subjects have not found a benefit. The Deutsche Diabetes Dialyse Studie (4D) randomized 1255 type 2 diabetic subjects on maintenance hemodialysis to either 20 mg atorvastatin or placebo daily. The cholesterol and LDL-C reduction was similar to that seen in non-dialysis patients; however, unlike non-CKD subjects there was no significant reduction in cardiovascular death, nonfatal myocardial infarction, or stroke with atorvastatin compared to placebo (41). A long-term follow-up of the 4D study population found similar effects after 11.5 years as were found at the end of the original study: no CVD benefit, but also no evidence of harm (42). Similarly, A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis (AURORA) randomized 2776 subjects on maintenance hemodialysis to rosuvastatin 10 mg or placebo. Again, the LDL-C lowering in dialysis patients was similar to that seen in other studies in non-dialysis patients, but there was no significant effect on the primary endpoint of cardiovascular death, nonfatal myocardial infarction, or stroke (43). The Study of Heart and Renal Protection (SHARP) randomized 9270 CKD patients (3023 on dialysis) to simvastatin plus ezetimibe versus placebo. The SHARP study did report a significant reduction in major atherosclerotic events in the simvastatin plus ezetimibe group but was not powered to compare non-dialysis and dialysis patients (44). However, a meta-analysis of 25 studies involving 8289 dialysis patients found no benefit of statin therapy on major cardiovascular events, cardiovascular mortality, all-cause mortality, or myocardial infarction, despite efficacious lipid lowering (45). Nevertheless, a post-hoc analysis of the 4D study did demonstrate a benefit of statin therapy in the subgroup that had LDL-C > 145 mg/dl (3.76mmol/l) (46). Although the use of statins in dialysis patients does not clearly cause harm, at present there is no indication for use in dialysis patients, with the exception of a possible benefit in those with a significant elevation in LDL-C.

WHY IS STATIN THERAPY INEFFECTIVE IN DIALYSIS SUBJECTS?

Given the robust data demonstrating statin efficacy in CVD risk reduction in virtually all other populations studied, the lack of efficacy in ESRD subjects is perplexing. However, it may be due to different mechanisms of disease progression in ESRD populations compared to other populations. In ESRD subjects there is increased inflammation and oxidative stress as well as increased non-lipid-associated pro-atherogenic factors, which may be the major cause of atherosclerosis development or progression in CKD subjects [for review see (47)]. Therefore, the relative impact of dyslipidemia on CVD development and progression in ESRD subjects may be less than in other CKD and non-CKD subjects, and thus the potential benefit of lipid lowering therapy is reduced. In ESRD subjects with significant hyperlipidemia (such as genetic hyperlipidemias) there may still be a role for statins or other lipid lowering therapies. Furthermore, while no benefit has been found for statins in dialysis subjects, there is no evidence of increased harm, and thus consideration of lipid lowering medications in particular individuals with ESRD is warranted.

Use of Statins in Renal Transplant Recipients

The Assessment of Lescol in Renal Transplant (ALERT) study randomized 2102 renal transplant recipients to fluvastatin or placebo. There was a non-significant 17% reduction in the combined primary endpoint (cardiac mortality, nonfatal myocardial infarction, or coronary intervention procedures) but a significant reduction in cardiac death or myocardial infarction (48, 49). Furthermore, a post hoc analysis suggested that earlier initiation of statins post-transplant was associated with greater benefit (50). A small study found no benefit of statin therapy on coronary calcification in renal transplant patients (51) albeit coronary calcium scores are not a good index of the benefits of statins (52). Furthermore, as with pre-end stage CKD patients there did not appear to be any benefit from statin therapy on progression of renal disease or graft loss in statin treated transplant recipients (53). Thus, renal transplant patients should be considered for statin therapy for CVD risk reduction, but not for graft preservation. Several of the statins have drug interactions, particularly with cyclosporine, thus providers must be aware of dose and drug restrictions (Table 4).

Table 3. Use of Statins in Various CKD Subgroups
Patient population	Statin indicated? Yes/no
Microalbuminuria*	Unclear
CKD 1-4	Yes
Nephrotic syndrome	Unclear
Dialysis patients	No
Renal transplant recipients	Yes

* in the absence of any other indication

EVIDENCE FOR/AGAINST LIPID LOWERING THERAPY IN CKD FOR RENAL OUTCOMES

Given the evidence that renal lipid deposition is associated with progression of renal disease itself, there has been an ongoing interest in whether targeting dyslipidemia in CKD can help delay the progression of the renal disease. The dyslipidemia in CKD is associated not only with increased CVD but also with adverse renal prognosis (54, 55). Biopsy studies have found that the amount of renal apoB/apoE is correlated with increased progression of the renal disease itself (56). Animal studies have supported this concept. A meta-analysis of several small, older studies suggested that the rate of decline in GFR was decreased in subjects receiving a lipid-lowering agent (the included studies mainly used statins but the meta-analysis also included a study using gemfibrozil and another using probucol) (57). However, the relationship between lipid levels and renal disease is unclear, as prospective cohort studies have not found any relationship of lipid levels to progression of kidney disease (58). Furthermore, the SHARP study, which included subjects with earlier stages of CKD (stages 3-5 were included) found no benefit of lipid lowering therapy on the progression of renal disease. A meta-analysis of statins in pre-end stage CKD patients found no overall effect of statins on renal disease progression (33) and the ALERT study found no benefit of statin use on renal graft or renal disease parameters (53). Thus, there does not appear to be any use for statins to improve renal function or CKD itself.

SAFETY OF STATINS IN CKD

Statin Safety– Renal Outcomes

An observational study using administrative databases containing information on > 2 million patients suggested that the use of high potency statins was associated with acute kidney injury, especially within the first 120 days of statin use (59). However, a subsequent analysis of 24 placebo-controlled statin studies and 2 high versus low-dose statin studies found no evidence of renal injury from statin use (60). These discrepant results can be explained by the quality of the data: in randomized controlled trials, albeit not designed or powered to look at renal injury, data quality tends to be higher than that in administrative data sets, which often contain bias for selection, ascertainment, and classification. Furthermore, statins appear to have a nephron-protective role in the prevention of contrast induced acute kidney injury. A meta-analysis of 15 trials examining the effect of statin pre-treatment before coronary angiography found a significant reduction in acute kidney injury in those treated with high dose statin compared to controls treated with either placebo or low dose statin (61). One study specifically examined the use of statins in subjects with diabetes and existing CKD undergoing angiography and found a benefit to statin pre-treatment in reducing the risk of contrast induced acute renal injury (62). As discussed above, the use of statins in pre-end stage CKD or post-renal transplant patients demonstrates neither benefit nor harm on renal outcomes. Thus, based on available evidence there does not seem to be any renal harm from statin use, and the presence of CKD should not be a contra-indication to statin use, although some statins require dose restrictions in CKD (Table 4).

Statin Safety – Diabetes Outcomes

As a class, emerging evidence demonstrates that statins increase new diagnoses of diabetes (63). As diabetes can lead to or exacerbate renal injury, this is another potential harm of statins. However, there is no evidence that statin therapy acutely raises normal fasting glucose into the diabetic range and rather the evidence from clinical trials suggests that statin therapy instead leads individuals at high risk of diabetes to progress to diabetes diagnosis sooner than may have happened without statin therapy. A subsequent meta-analysis of 5 statin trials with >32,000 patients without diabetes at baseline found that high dose statin was associated with increased risk for new diabetes diagnosis compared to low or moderate dose statin therapy (64). However, the number needed to harm (induce diabetes) is 498 whereas the number needed to treat (prevent cardiovascular events) is 155 for intensive statin therapy; implying that despite the increased risk of new onset diabetes, statin therapy’s benefits outweigh the risks.

Which Statins to use in CKD?

The various statins have different degrees of renal clearance; thus, with CKD patients it is important to be aware of the metabolism of the agent of interest and understand if/when dose adjustments are needed. Most statins are primarily metabolized through hepatic pathways, and dose adjustment in early CKD is typically not needed (eGFR> 30 ml/min). However, with more advanced CKD, eGFR< 30 ml/min (or ESRD, although statins are not indicated in this population) most agents have maximum dose restrictions (Table 4).

Table 4. Statin Dosing in CKD
Statin	Usual dose range (mg/d)	Clearance route	Dose range for CKD stages1-3	Dose range for CKD stages4-5	Use with cyclosporine
Atorvastatin	10-80	Liver	10-80	10-80	Avoid use with cyclosporine
Fluvastatin	20-80	Liver	20-80	20-40	Max dose 20 mg/d with cyclosporine
Lovastatin	10-80	Liver	10-80	10-20	Avoid use with cyclosporine
Pitavastatin	1-4	Liver/Kidney	1-2	1-2	Avoid use with cyclosporine
Pravastatin	10-80	Liver/Kidney	10-80	10-20	Max dose 20 mg/d when used with cyclosporine
Rosuvastatin	10-40	Liver/Kidney	5-40	5-10	Max dose 5 mg/d with cyclosporine
Simvastatin	5-40	Liver	5-40	5-40	Avoid use with cyclosporine

BEYOND STATINS

There has been relatively little research into the use of non-statin lipid lowering agents in CKD. There is an emerging interest in niacin in CKD patients for its phosphorus-lowering effects, and niacin has similar lipid-altering efficacy in CKD as compared to non-CKD subjects. Fibrates are metabolized via the kidney and thus are generally contraindicated in CKD. Ezetimibe has been shown to be safe and effective in reducing LDL-C in patients with CKD; however, studies have typically compared treatment with ezetimibe added to statin therapy vs. control and few studies compare ezetimibe monotherapy vs. control. PCSK9-inhibitors have been shown to be safe in CKD and efficacious in lowering LDL-C but there remains limited data regarding morbidity and mortality outcomes with this therapy. Newer therapies include bempedoic acid and inclisiran both remain relatively unstudied in CKD/ESRD. The following sections summarize the available data on the use of other lipid lowering agents in CKD (Table 5).

Niacin

As niacin is not cleared via the kidney it is theoretically safe in CKD; however, its use is limited due to side effects (predominantly flushing) and a lack of data. Several short-term studies have evaluated niacin in CKD patients, and it is efficacious in lipid lowering. There is an emerging interest in the use of niacin or its analog niacinamide in CKD and ESRD patients for their effects to decrease phosphate levels. A meta-analysis of randomized controlled trials of niacin and niacinamide in dialysis patients found that niacin reduced serum phosphorus but did not change serum calcium levels; furthermore niacin increased HDL-C levels but had no significant effect on LDL-C, TGs, or total cholesterol levels; no CVD outcomes data were provided (65). Animal studies have suggested a beneficial effect of niacin on renal outcomes (66), and clinical literature is suggestive that this may occur in humans (67). Kang et al treated patients with CKD stages 2-4 with niacin 500mg/d x 6 months; niacin led to increased HDL-C and decreased TG levels, and improved GFR compared to baseline levels (68). Laropiprant has been developed as an inhibitor of prostaglandin-medicated niacin-induced flushing. In a sub-study examining the use of niacin with laropiprant in dyslipidemic subjects with impaired renal function, the use of niacin resulted in a mean decrease in serum phosphorus of 11% with similar effects between those with eGFR above or below 60 ml/min/1.73 m²(69); the parent study reported a significant reduction in lipid parameters including a decrease in LDL-C of 18%, decrease in TGs of 25%, and an increase in HDL-C of 20% (70). Thus, there may be an indication for the use of niacin in CKD subjects beyond lipid lowering considerations. However, cardiovascular outcome studies evaluating the combination of statin plus niacin in patient without kidney disease have not found any additional benefit compared to statin alone (71, 72); thus, at this time further research is needed in CKD subjects to determine if niacin may be more beneficial than statins, or if the addition of niacin to statin may confer non-CVD benefit, for example, phosphorus lowering.

Fibrates

Fibric acid derivatives are used primarily to raise HDL-C and lower TGs; thus, they target two major components of CKD associated dyslipidemia. However, fibrates are known to decrease renal blood flow and glomerular filtration and they are cleared renally (73); therefore, there is significant concern regarding their use in CKD. Furthermore, fibric acid derivatives raise serum creatinine levels and thus trigger medical investigations into renal disease progression. Thus, there is concern regarding their use in CKD. However, there is a potential for fibric acid derivatives to improve both CVD and CKD outcomes. The acute changes in serum creatinine do not necessarily indicate adverse renal effects. A meta-analysis (74) examined the use of fibrates in CKD subjects and reported beneficial effects to reduce total cholesterol and TG levels and raise HDL-C levels with no effect on LDL-C levels. In addition, 3 trials reporting on > 14,000 patients reported that fibrates reduced risk of albuminuria progression in diabetic subjects, with 2 trials (>2,000 patients) reporting albuminuria regression (75-77). This was associated with a reduction in major cardiovascular events, CVD death, stroke and all-cause mortality in subjects with moderate renal dysfunction, but not in those with eGFR > 60 ml/min/1.73m². Thus, despite the elevations in serum creatinine seen with fibrates, there is the potential for both cardiac and renal benefit, and further studies specifically designed to evaluate these outcomes in CKD subjects are needed. At this point, providers are encouraged to consider fibrate therapy for appropriate subjects, especially if statins are not tolerated or are contra-indicated.

Ezetimibe

Ezetimibe is presently the only member of the class of cholesterol absorption inhibitors. As monotherapy it can lower LDL-C approximately 15-20%; however, the majority of research has examined ezetimibe in combination with a statin (primarily simvastatin) where the addition of ezetimibe can induce a further 20-25% lowering of LDL-C. Ezetimibe is metabolized through intestinal and hepatic metabolism and does not require any dose adjustment in CKD or ESRD, making it a potentially attractive therapy in CKD. The Improved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE IT) study demonstrated that the combination of a statin + ezetimibe led to further LDL-C lowering and improved CVD outcomes compared to statin alone in high-risk patients (78). A secondary analysis of this study evaluating outcomes based on eGFR showed that compared to statin alone, the combination of statin + ezetimibe was more effective in reducing risk of CVD outcomes in those with eGFR < 60/ml/min/1.73m² (79). The Study of Heart and Renal Protection (SHARP) compared CVD and renal effects in CKD patients treated with statin + ezetimibe versus placebo. There was a reduction in CVD events (44); however, there was no effect on renal disease progression (80). Note, neither of these studies included an ezetimibe only arm; thus, the effects of ezetimibe monotherapy on outcomes are unknown, although it can be expected to reduce CVD events in proportion to its degree of LDL-C lowering. A small study evaluating ezetimibe monotherapy in CKD patients found it safe and effective (81). Thus, the use of ezetimibe with or without statin is likely to benefit pre-end stage CKD patients in terms of CVD outcomes. Given that the impact of ezetimibe is on lowering LDL-C we can anticipate lack of CVD benefit in ESRD subjects based on the statin studies and SHARP.

Fish Oil

Omega-3 polyunsaturated fatty acids can lower TG levels, making them a potential therapy in CKD. The role of fish oil/ omega-3 supplements in the general population for prevention of CVD events remains unclear, with some studies suggesting benefit but others finding no CVD protection. A recent meta-analysis found no evidence for CVD protection (82) while a meta-analysis of thirteen randomized control trials involving 127,477 patients demonstrated marine omega-3 supplementation was associated with small but significantly lower risk of MI, CHD death, total CHD, CVD death, and total CVD with linear relationship to dose (83). In CKD patients there is little data to support the use of fish oil and much of the data is conflicting. A small, randomized study evaluated omega-3 fish oil supplements, coenzyme Q10, or both in subjects with CKD stage 3 for 8 weeks. The group that received the omega-3 supplements had decreased heart rate and blood pressure and TGs, but there was no effect on renal function (eGFR, or albuminuria) (84). Conversely, a study evaluating dietary omega-3 intake found that higher consumption was associated with reduced likelihood of CKD (85). A randomized controlled trial in patients with CKD and microalbuminuria showed that omega-3 fatty acid supplementation had no effect on urine albumin excretion; however, there was a beneficial effect on serum TG levels and pulse wave velocity (86). Fish oil supplementation has not been found to have any clear benefit on hemodialysis arteriovenous graft function (87, 88) or on cardiovascular events or mortality in hemodialysis patients (89). Thus, there is no clear benefit for the use of fish oil supplements in CKD, but further research is needed.

Bile Acid Resins

The bile acid resins tend to be used less commonly than other classes of lipid lowering agents overall, and their use in CKD is limited by a lack of data. Bile acid resins as a class can lower LDL-C by 10-20% so they are less effective than statins; furthermore, they can raise TG levels and their use is contraindicated with elevated TG levels, for example > 400-500 mg/dl (>4.5 – 5.6 mmol/L). Thus, overall bile acid resins are rarely used in CKD patients. However, their metabolism is intestinal and thus there are no required modifications for their use in mild-moderate CKD. Although there are no theoretical concerns regarding their use in ESRD there is no data to address safety or efficacy.

PCSK9 Inhibitors

Monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9) have been developed and approved for patients with clinical atherosclerotic CVD not meeting lipid goals despite maximally tolerated statin therapy. Statins can cause higher PCSK9 levels through activation of sterol regulatory element-binding protein-2 with co-expression of LDL receptors & PCSK9 (90). PCSK9 inhibitors lower LDL-C in addition to statin-mediated lowering and have been shown to decrease CVD events in outcome studies in secondary prevention populations (91). Two PCSK9 monoclonal antibodies are presently available in the US – evolucumab and alirocumab. PCSK9 plasma levels are not influenced by eGFR in CKD patients (92) but are increased in nephrotic syndrome (93). The German Chronic Kidney Disease study (GCKD) investigated the association between PCSK9 and cardiovascular disease in patients with moderate CKD (eGFR >30 ml/min/1.73 m² or eGFR >60 ml/min/1.73 m² with UACR >300 mg/g). GCKD showed no association of PCSK9 concentrations with eGFR or UACR (except for those with nephrotic-range albuminuria) but did show that higher baseline PCSK9 concentrations increased the odds of baseline cardiovascular disease (90). As monoclonal antibodies are not cleared by the kidney and thus are approved for use in CKD and ESRD with no dose adjustment. The ODYSSEY OUTCOME trial randomized post-acute coronary syndrome patients with LDL-C > 70mg/dL on maximally tolerated statin to placebo vs alirocumab; the intervention arm with alirocumab had nearly twice the absolute reduction in cardiovascular events (94). Of note patients with eGFR < 30 ml/min/m² were excluded from the ODYSSEY OUTCOME trial. However, a later sub analysis looked at the effect of alirocumab on major adverse cardiovascular events based on renal function. The sub analysis showed that irrespective of eGFR alirocumab was efficacious in reducing LDL-C. Further, annualized incidence rates of major adverse cardiovascular events and death increased with decreasing eGFR but rates were lower in the alirocumab group compared to placebo and there were no significant difference in incidence of major adverse events between treatment groups with eGFR < 60 ml/min/m²(95). Further, data from a pooled analysis of nine trials comparing alirocumab vs control showed that among patients with ASCVD and LDL-C > 100 mg/dL those with additional risk factors including CKD had the greatest absolute cardiovascular benefit from alirocumab therapy in addition to maximally tolerated statin compared to placebo (96). A recent systematic review of 7 studies including 5 RCTs and 2 review studies showed safety of PCSK-9 inhibitors in mild-moderate CKD. However, this conclusion is somewhat limited as patients with an eGFR <20 ml/min/m² were not included in the trials (97). Furthermore, the relationship between PCSK-9 inhibitors’ lipid lowering and lower cardiovascular risks resulting in improved morbidity and mortality is altered with severe CKD due to non-thrombotic causes of morbidity and mortality. Studies remain ongoing to further look at mortality and morbidity outcome in PCSK-9 inhibitors specifically in patients with CKD 3 or higher. The ALIDIAL study examined the safety and efficacy of alirocumab in patients receiving dialysis and the dialysis patients had a similar response at the same alirocumab dose with reduced cholesterol levels and no unexpected adverse events when compared to the patients not receiving dialysis (98). There remains very limited data in patients with ESRD and PCSK-9 inhibitor use as monotherapy for dyslipidemia.

Bempedoic Acid

Currently approved for use in combination with maximally tolerated statin, bempedoic acid facilitates further LDL-C reduction by inhibiting cholesterol synthesis in the liver through blocking adenosine triphosphate-citrate lyase (ACL). Currently, use in CKD is approved without dosage adjustment for eGFR > 30ml/minute/1.73m^2; however, below this eGFR threshold there is insufficient data to guide its use. As bempedoic acid has hepatic metabolism it is presumably safe in CKD. Bempedoic acid increases serum creatinine and uric acid levels through interference with tubular secretion (99). A 52-week study in very high-risk CVD patients demonstrated that bempedoic acid added to maximally tolerated statin therapy was safe and led to a significant reduction in LDL-C levels (100). Further, combination with ezetimibe is safe and can increase the cholesterol-lowering effect more than either agent alone when added to standard therapy (101). The Cholesterol Lowering via bempedoic acid, an ACL-Inhibiting Regimen (CLEAR) Outcomes trial, a cardiovascular outcome study that was published March 2023, demonstrated a decrease in cardiovascular events but excluded patients with eGFR <30 ml/minute/1.73 m2 as well as nephritic or nephrotic syndrome (102). At this time, the data remains limited regarding the benefit and use of bempedoic acid in ESRD.

Inclisiran

Newest to the market, inclisiran is a small interfering RNA (siRNA) that acts in hepatocytes to break down mRNA for PCSK-9 which increases LDL receptor recycling thus increasing LDL cholesterol uptake. It is FDA approved for use in heterozygous familial hypercholesterolemia and in secondary prevention of cardiovascular events as an adjunct to lifestyle and maximally tolerated statin. It is administered by subcutaneous injections at 3 and then 6-month intervals. There are no cardiovascular outcomes studies yet available. There is no recommended dosage adjustment in CKD, but there have been no studies done in patients with ESRD. An analysis of the ORION-1 and ORION-7 studies compared inclisiran in patients with renal impairment and those with normal renal function found similar safety and efficacy, suggesting no dose adjustment is needed in CKD (103). However, no patients on dialysis were studied in these trials. ORION-8 is a 3-year extension of the preceding ORION-3, ORION-9, ORION-10 and ORION-11 studies that examined long-term efficacy and safety in regard to treatment-emergent adverse events (TEAEs) and treatment-emergent severe adverse events (TESAEs). More than 70% of patients at each visit in ORION-8 achieved the preset LDL-C goals. Almost 78% of patients had TEAEs and 30% had TESAEs. It is unclear if patients with CKD were included as there is no clear renal exclusion criteria (many subjective criteria) or stratification of patients by renal function (104). A post-hoc analysis of 7 clinical trials of inclisiran from 2023 showed no detection of safety signals related to kidney TEAEs and found inclisiran to be well-tolerated for up to 6 years (105). The ORION-4 trial is investigating the impact of inclisiran on MACE but results will not be available until 2026. Further studies will be required to assess the safety of inclisiran use in CKD and ESRD.

Table 5. Non-Statin Treatments
Agent	Usual dose range (mg/d)	Clearance route	Dose range for CKD stages1-3	Dose range for CKD stages 4-5	Use with cyclosporine
Niaspan	500-2000	Hepatic/renal	No data	No data	No data
Gemfibrozil	1200	Renal	Avoid if creatinine > 2.0 mg/dl	Avoid if creatinine > 2.0 mg/dl	Cautious use
Fenofibrate	40-200	renal	40-60	avoid	Cautious use
Ezetimibe	10	Intestinal/hepatic	10	10	Cautious use
Colsevelam	3750 (6 x 625 mg tablets daily)	Intestinal	No change	unknown	May reduce levels of cyclosporine
Fish oil	4000		No change	Caution	No data
PCSK9 inhibitors	Alirocumab 75-150mg SC q 2 weeks Evolocumab 140mg weekly SC - 420mg monthly SC	Unknown	No change	Potentially safe and effective in dialysis	No data
Bempedoic acid	180 mg daily	Hepatic	No change	Not defined	No data
Inclisiran	284 mg SC at 0 and 3 months then every 6 months	Nucleases	No change	Not defined	No data

SUMMARY

CVD is the leading cause of mortality in CKD, and as with the non-CKD population dyslipidemia is a significant contributor. The dyslipidemia of CKD comprises primarily high TG levels and low HDL-C levels; however, emerging data suggests that the composition of the lipoprotein particles is altered by CKD, and that altered composition and/or lipoprotein cargo may be a cause of the increased CVD in CKD. The use of statins has been shown to be safe and efficacious in lipid lowering in CKD, and of benefit in reducing CVD events in individuals with pre-end stage CKD, or post renal transplant, but not in dialysis patients. The various available agents have different clearance routes, and some statins need dose adjustment in CKD. In patients that cannot tolerate or who have contra-indications to statin therapy, there may be some benefit from use of PCSK9 inhibitors, ezetimibe, fibrates, niacin, or newer therapies such as bempedoic acid and inclisiran, but further studies are needed to better investigate their use.

REFERENCES

National Kidney, F. 2012. KDOQI Clinical Practice Guideline for Diabetes and CKD: 2012 Update. Am J Kidney Dis 60: 850-886.
Writing, C., D. M. Lloyd-Jones, P. B. Morris, C. M. Ballantyne, K. K. Birtcher, A. M. Covington, S. M. DePalma, M. B. Minissian, C. E. Orringer, S. C. Smith, Jr., A. A. Waring, and J. T. Wilkins. 2022. 2022 ACC Expert Consensus Decision Pathway on the Role of Nonstatin Therapies for LDL-Cholesterol Lowering in the Management of Atherosclerotic Cardiovascular Disease Risk: A Report of the American College of Cardiology Solution Set Oversight Committee. J Am Coll Cardiol 80: 1366-1418.
Candan, C., N. Canpolat, S. Gokalp, N. Yildiz, P. Turhan, M. Tasdemir, L. Sever, and S. Caliskan. 2014. Subclinical cardiovascular disease and its association with risk factors in children with steroid-resistant nephrotic syndrome. Pediatric nephrology 29: 95-102.
Foley, R. N., P. S. Parfrey, and M. J. Sarnak. 1998. Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 32: S112-119.
Parfrey, P. S., and R. N. Foley. 1999. The clinical epidemiology of cardiac disease in chronic renal failure. J Am Soc Nephrol 10: 1606-1615.
Sarnak, M. J., B. E. Coronado, T. Greene, S. R. Wang, J. W. Kusek, G. J. Beck, and A. S. Levey. 2002. Cardiovascular disease risk factors in chronic renal insufficiency. Clin Nephrol 57: 327-335.
Chronic Kidney Disease Prognosis, C., K. Matsushita, M. van der Velde, B. C. Astor, M. Woodward, A. S. Levey, P. E. de Jong, J. Coresh, and R. T. Gansevoort. 2010. Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative meta-analysis. Lancet 375: 2073-2081.
van der Velde, M., K. Matsushita, J. Coresh, B. C. Astor, M. Woodward, A. Levey, P. de Jong, R. T. Gansevoort, C. Chronic Kidney Disease Prognosis, M. van der Velde, K. Matsushita, J. Coresh, B. C. Astor, M. Woodward, A. S. Levey, P. E. de Jong, R. T. Gansevoort, A. Levey, M. El-Nahas, K. U. Eckardt, B. L. Kasiske, T. Ninomiya, J. Chalmers, S. Macmahon, M. Tonelli, B. Hemmelgarn, F. Sacks, G. Curhan, A. J. Collins, S. Li, S. C. Chen, K. P. Hawaii Cohort, B. J. Lee, A. Ishani, J. Neaton, K. Svendsen, J. F. Mann, S. Yusuf, K. K. Teo, P. Gao, R. G. Nelson, W. C. Knowler, H. J. Bilo, H. Joosten, N. Kleefstra, K. H. Groenier, P. Auguste, K. Veldhuis, Y. Wang, L. Camarata, B. Thomas, and T. Manley. 2011. Lower estimated glomerular filtration rate and higher albuminuria are associated with all-cause and cardiovascular mortality. A collaborative meta-analysis of high-risk population cohorts. Kidney Int 79: 1341-1352.
Writing Group for the, C. K. D. P. C., M. E. Grams, J. Coresh, K. Matsushita, S. H. Ballew, Y. Sang, A. Surapaneni, N. Alencar de Pinho, A. Anderson, L. J. Appel, J. Arnlov, F. Azizi, N. Bansal, S. Bell, H. J. G. Bilo, N. J. Brunskill, J. J. Carrero, S. Chadban, J. Chalmers, J. Chen, E. Ciemins, M. Cirillo, N. Ebert, M. Evans, A. Ferreiro, E. L. Fu, M. Fukagawa, J. A. Green, O. M. Gutierrez, W. G. Herrington, S. J. Hwang, L. A. Inker, K. Iseki, T. Jafar, S. K. Jassal, V. Jha, A. Kadota, R. Katz, A. Kottgen, T. Konta, F. Kronenberg, B. J. Lee, J. Lees, A. Levin, H. C. Looker, R. Major, C. Melzer Cohen, M. Mieno, M. Miyazaki, O. Moranne, I. Muraki, D. Naimark, D. Nitsch, W. Oh, M. Pena, T. S. Purnell, C. Sabanayagam, M. Satoh, S. Sawhney, E. Schaeffner, B. Schottker, J. I. Shen, M. G. Shlipak, S. Sinha, B. Stengel, K. Sumida, M. Tonelli, J. M. Valdivielso, A. D. van Zuilen, F. L. J. Visseren, A. Y. Wang, C. P. Wen, D. C. Wheeler, H. Yatsuya, K. Yamagata, J. W. Yang, A. Young, H. Zhang, L. Zhang, A. S. Levey, and R. T. Gansevoort. 2023. Estimated Glomerular Filtration Rate, Albuminuria, and Adverse Outcomes: An Individual-Participant Data Meta-Analysis. JAMA 330: 1266-1277.
Chan, D. T., G. K. Dogra, A. B. Irish, E. M. Ooi, P. H. Barrett, D. C. Chan, and G. F. Watts. 2009. Chronic kidney disease delays VLDL-apoB-100 particle catabolism: potential role of apolipoprotein C-III. J Lipid Res 50: 2524-2531.
Vaziri, N. D., G. Deng, and K. Liang. 1999. Hepatic HDL receptor, SR-B1 and Apo A-I expression in chronic renal failure. Nephrol Dial Transplant 14: 1462-1466.
Schuchardt, M., M. Tolle, and M. van der Giet. 2015. High-density lipoprotein: structural and functional changes under uremic conditions and the therapeutic consequences. Handbook of experimental pharmacology 224: 423-453.
Kuznik, A., J. Mardekian, and L. Tarasenko. 2013. Evaluation of cardiovascular disease burden and therapeutic goal attainment in US adults with chronic kidney disease: an analysis of national health and nutritional examination survey data, 2001-2010. BMC nephrology 14: 132.
Longenecker, J. C., J. Coresh, N. R. Powe, A. S. Levey, N. E. Fink, A. Martin, and M. J. Klag. 2002. Traditional cardiovascular disease risk factors in dialysis patients compared with the general population: the CHOICE Study. J Am Soc Nephrol 13: 1918-1927.
Pennell, P., B. Leclercq, M. I. Delahunty, and B. A. Walters. 2006. The utility of non-HDL in managing dyslipidemia of stage 5 chronic kidney disease. Clin Nephrol 66: 336-347.
Rao, R., D. Ansell, J. A. Gilg, S. J. Davies, E. J. Lamb, and C. R. Tomson. 2009. Effect of change in renal replacement therapy modality on laboratory variables: a cohort study from the UK Renal Registry. Nephrol Dial Transplant 24: 2877-2882.
Chu, M., A. Y. Wang, I. H. Chan, S. H. Chui, and C. W. Lam. 2012. Serum small-dense LDL abnormalities in chronic renal disease patients. British journal of biomedical science 69: 99-102.
Reis, A., A. Rudnitskaya, P. Chariyavilaskul, N. Dhaun, V. Melville, J. Goddard, D. J. Webb, A. R. Pitt, and C. M. Spickett. 2014. Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease. J Lipid Res.
Hricik, D. E., J. T. Mayes, and J. A. Schulak. 1991. Independent effects of cyclosporine and prednisone on posttransplant hypercholesterolemia. Am J Kidney Dis 18: 353-358.
Marcen, R., J. Chahin, A. Alarcon, and J. Bravo. 2006. Conversion from cyclosporine microemulsion to tacrolimus in stable kidney transplant patients with hypercholesterolemia is related to an improvement in cardiovascular risk profile: a prospective study. Transplantation proceedings 38: 2427-2430.
Ma, K. L., X. Z. Ruan, S. H. Powis, Y. Chen, J. F. Moorhead, and Z. Varghese. 2007. Sirolimus modifies cholesterol homeostasis in hepatic cells: a potential molecular mechanism for sirolimus-associated dyslipidemia. Transplantation 84: 1029-1036.
Vaziri, N. D. 2003. Molecular mechanisms of lipid disorders in nephrotic syndrome. Kidney Int 63: 1964-1976.
Vaziri, N. D., T. Sato, and K. Liang. 2003. Molecular mechanisms of altered cholesterol metabolism in rats with spontaneous focal glomerulosclerosis. Kidney Int 63: 1756-1763.
Vaziri, N. D., K. Liang, and J. S. Parks. 2001. Acquired lecithin-cholesterol acyltransferase deficiency in nephrotic syndrome. Am J Physiol Renal Physiol 280: F823-828.
Vaziri, N. D., C. H. Kim, D. Phan, S. Kim, and K. Liang. 2004. Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome. Kidney Int 66: 262-267.
Pearson, G. J., G. Thanassoulis, T. J. Anderson, A. R. Barry, P. Couture, N. Dayan, G. A. Francis, J. Genest, J. Gregoire, S. A. Grover, M. Gupta, R. A. Hegele, D. Lau, L. A. Leiter, A. A. Leung, E. Lonn, G. B. J. Mancini, P. Manjoo, R. McPherson, D. Ngui, M. E. Piche, P. Poirier, J. Sievenpiper, J. Stone, R. Ward, and W. Wray. 2021. 2021 Canadian Cardiovascular Society Guidelines for the Management of Dyslipidemia for the Prevention of Cardiovascular Disease in Adults. The Canadian journal of cardiology 37: 1129-1150.
Grundy, S. M., N. J. Stone, A. L. Bailey, C. Beam, K. K. Birtcher, R. S. Blumenthal, L. T. Braun, S. de Ferranti, J. Faiella-Tommasino, D. E. Forman, R. Goldberg, P. A. Heidenreich, M. A. Hlatky, D. W. Jones, D. Lloyd-Jones, N. Lopez-Pajares, C. E. Ndumele, C. E. Orringer, C. A. Peralta, J. J. Saseen, S. C. Smith, Jr., L. Sperling, S. S. Virani, and J. Yeboah. 2019. 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol 73: e285-e350.
Mefford, M. T., R. S. Rosenson, L. Deng, R. M. Tanner, V. Bittner, M. M. Safford, B. Coll, K. E. Mues, K. L. Monda, and P. Muntner. 2019. Trends in Statin Use Among US Adults With Chronic Kidney Disease, 1999-2014. Journal of the American Heart Association 8: e010640.
Kidney Disease: Improving Global Outcomes, C. K. D. W. G. 2024. KDIGO 2024 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney Int 105: S117-S314.
. 2002. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 360: 7-22.
Tonelli, M., C. Isles, G. C. Curhan, A. Tonkin, M. A. Pfeffer, J. Shepherd, F. M. Sacks, C. Furberg, S. M. Cobbe, J. Simes, T. Craven, and M. West. 2004. Effect of pravastatin on cardiovascular events in people with chronic kidney disease. Circulation 110: 1557-1563.
Soohoo, M., H. Moradi, Y. Obi, C. M. Rhee, E. O. Gosmanova, M. Z. Molnar, M. L. Kashyap, D. L. Gillen, C. P. Kovesdy, K. Kalantar-Zadeh, and E. Streja. 2019. Statin Therapy Before Transition to End-Stage Renal Disease With Posttransition Outcomes. Journal of the American Heart Association 8: e011869.
Palmer, S. C., S. D. Navaneethan, J. C. Craig, D. W. Johnson, V. Perkovic, J. Hegbrant, and G. F. Strippoli. 2014. HMG CoA reductase inhibitors (statins) for people with chronic kidney disease not requiring dialysis. Cochrane Database Syst Rev 5: CD007784.
Cholesterol Treatment Trialists, C., W. G. Herrington, J. Emberson, B. Mihaylova, L. Blackwell, C. Reith, M. D. Solbu, P. B. Mark, B. Fellstrom, A. G. Jardine, C. Wanner, H. Holdaas, J. Fulcher, R. Haynes, M. J. Landray, A. Keech, J. Simes, R. Collins, and C. Baigent. 2016. Impact of renal function on the effects of LDL cholesterol lowering with statin-based regimens: a meta-analysis of individual participant data from 28 randomised trials. Lancet Diabetes Endocrinol 4: 829-839.
Toto, R. D., S. M. Grundy, and G. L. Vega. 2000. Pravastatin treatment of very low density, intermediate density and low density lipoproteins in hypercholesterolemia and combined hyperlipidemia secondary to the nephrotic syndrome. Am J Nephrol 20: 12-17.
Matzkies, F. K., U. Bahner, M. Teschner, H. Hohage, A. Heidland, and R. M. Schaefer. 1999. Efficiency of 1-year treatment with fluvastatin in hyperlipidemic patients with nephrotic syndrome. Am J Nephrol 19: 492-494.
Busuioc, R., G. Stefan, S. Stancu, A. Zugravu, and G. Mircescu. 2023. Nephrotic Syndrome and Statin Therapy: An Outcome Analysis. Medicina (Kaunas) 59.
Zou, X., L. Nie, Y. Liao, Z. Liu, W. Zheng, X. Qu, X. Xu, H. Qin, H. Wang, J. Liu, G. He, and T. Jing. 2022. Effects of statin therapy and treatment duration on cardiovascular disease risk in patients with nephrotic syndrome: A nested case-control study. Pharmacotherapy 42: 311-319.
Asselbergs, F. W., G. F. Diercks, H. L. Hillege, A. J. van Boven, W. M. Janssen, A. A. Voors, D. de Zeeuw, P. E. de Jong, D. J. van Veldhuisen, W. H. van Gilst, R. Prevention of, and I. Vascular Endstage Disease Intervention Trial. 2004. Effects of fosinopril and pravastatin on cardiovascular events in subjects with microalbuminuria. Circulation 110: 2809-2816.
Scheven, L., M. Van der Velde, H. J. Lambers Heerspink, P. E. De Jong, and R. T. Gansevoort. 2013. Isolated microalbuminuria indicates a poor medical prognosis. Nephrol Dial Transplant 28: 1794-1801.
Wanner, C., V. Krane, W. Marz, M. Olschewski, J. F. Mann, G. Ruf, and E. Ritz. 2005. Atorvastatin in patients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med 353: 238-248.
Krane, V., K. R. Schmidt, L. J. Gutjahr-Lengsfeld, J. F. Mann, W. Marz, F. Swoboda, C. Wanner, and D. S. Investigators. 2016. Long-term effects following 4 years of randomized treatment with atorvastatin in patients with type 2 diabetes mellitus on hemodialysis. Kidney Int 89: 1380-1387.
Fellstrom, B. C., A. G. Jardine, R. E. Schmieder, H. Holdaas, K. Bannister, J. Beutler, D. W. Chae, A. Chevaile, S. M. Cobbe, C. Gronhagen-Riska, J. J. De Lima, R. Lins, G. Mayer, A. W. McMahon, H. H. Parving, G. Remuzzi, O. Samuelsson, S. Sonkodi, D. Sci, G. Suleymanlar, D. Tsakiris, V. Tesar, V. Todorov, A. Wiecek, R. P. Wuthrich, M. Gottlow, E. Johnsson, and F. Zannad. 2009. Rosuvastatin and cardiovascular events in patients undergoing hemodialysis. N Engl J Med 360: 1395-1407.
Baigent, C., M. J. Landray, C. Reith, J. Emberson, D. C. Wheeler, C. Tomson, C. Wanner, V. Krane, A. Cass, J. Craig, B. Neal, L. Jiang, L. S. Hooi, A. Levin, L. Agodoa, M. Gaziano, B. Kasiske, R. Walker, Z. A. Massy, B. Feldt-Rasmussen, U. Krairittichai, V. Ophascharoensuk, B. Fellstrom, H. Holdaas, V. Tesar, A. Wiecek, D. Grobbee, D. de Zeeuw, C. Gronhagen-Riska, T. Dasgupta, D. Lewis, W. Herrington, M. Mafham, W. Majoni, K. Wallendszus, R. Grimm, T. Pedersen, J. Tobert, J. Armitage, A. Baxter, C. Bray, Y. Chen, Z. Chen, M. Hill, C. Knott, S. Parish, D. Simpson, P. Sleight, A. Young, and R. Collins. 2011. The effects of lowering LDL cholesterol with simvastatin plus ezetimibe in patients with chronic kidney disease (Study of Heart and Renal Protection): a randomised placebo-controlled trial. Lancet 377: 2181-2192.
Palmer, S. C., S. D. Navaneethan, J. C. Craig, D. W. Johnson, V. Perkovic, S. U. Nigwekar, J. Hegbrant, and G. F. Strippoli. 2013. HMG CoA reductase inhibitors (statins) for dialysis patients. Cochrane Database Syst Rev 9: CD004289.
Marz, W., B. Genser, C. Drechsler, V. Krane, T. B. Grammer, E. Ritz, T. Stojakovic, H. Scharnagl, K. Winkler, I. Holme, H. Holdaas, C. Wanner, D. German, and I. Dialysis Study. 2011. Atorvastatin and low-density lipoprotein cholesterol in type 2 diabetes mellitus patients on hemodialysis. Clin J Am Soc Nephrol 6: 1316-1325.
Vaziri, N. D., and K. C. Norris. 2013. Reasons for the lack of salutary effects of cholesterol-lowering interventions in end-stage renal disease populations. Blood purification 35: 31-36.
Holdaas, H., B. Fellstrom, A. G. Jardine, I. Holme, G. Nyberg, P. Fauchald, C. Gronhagen-Riska, S. Madsen, H. H. Neumayer, E. Cole, B. Maes, P. Ambuhl, A. G. Olsson, A. Hartmann, D. O. Solbu, and T. R. Pedersen. 2003. Effect of fluvastatin on cardiac outcomes in renal transplant recipients: a multicentre, randomised, placebo-controlled trial. Lancet 361: 2024-2031.
Jardine, A. G., H. Holdaas, B. Fellstrom, E. Cole, G. Nyberg, C. Gronhagen-Riska, S. Madsen, H. H. Neumayer, B. Maes, P. Ambuhl, A. G. Olsson, I. Holme, P. Fauchald, C. Gimpelwicz, T. R. Pedersen, and A. S. Investigators. 2004. fluvastatin prevents cardiac death and myocardial infarction in renal transplant recipients: post-hoc subgroup analyses of the ALERT Study. Am J Transplant 4: 988-995.
Holdaas, H., B. Fellstrom, A. G. Jardine, G. Nyberg, C. Gronhagen-Riska, S. Madsen, H. H. Neumayer, E. Cole, B. Maes, P. Ambuhl, J. O. Logan, B. Staffler, C. Gimpelewicz, and A. S. Group. 2005. Beneficial effect of early initiation of lipid-lowering therapy following renal transplantation. Nephrol Dial Transplant 20: 974-980.
Yazbek, D. C., A. B. de Carvalho, C. S. Barros, J. O. Medina Pestana, and M. E. Canziani. 2016. Effect of Statins on the Progression of Coronary Calcification in Kidney Transplant Recipients. PLoS One 11: e0151797.
Kambalapalli, S., M. Bhandari, N. Punnanithinont, B. Iskander, M. A. Khan, and M. Budoff. 2025. Bridging Prevention and Imaging: The Influence of Statins on CAC and CCTA Findings. Curr Atheroscler Rep 27: 50.
Fellstrom, B., H. Holdaas, A. G. Jardine, I. Holme, G. Nyberg, P. Fauchald, C. Gronhagen-Riska, S. Madsen, H. H. Neumayer, E. Cole, B. Maes, P. Ambuhl, A. G. Olsson, A. Hartmann, J. O. Logan, T. R. Pedersen, and I. Assessment of Lescol in Renal Transplantation Study. 2004. Effect of fluvastatin on renal end points in the Assessment of Lescol in Renal Transplant (ALERT) trial. Kidney Int 66: 1549-1555.
Samuelsson, O., H. Mulec, C. Knight-Gibson, P. O. Attman, B. Kron, R. Larsson, L. Weiss, H. Wedel, and P. Alaupovic. 1997. Lipoprotein abnormalities are associated with increased rate of progression of human chronic renal insufficiency. Nephrol Dial Transplant 12: 1908-1915.
Samuelsson, O., P. O. Attman, C. Knight-Gibson, R. Larsson, H. Mulec, L. Weiss, and P. Alaupovic. 1998. Complex apolipoprotein B-containing lipoprotein particles are associated with a higher rate of progression of human chronic renal insufficiency. J Am Soc Nephrol 9: 1482-1488.
Sato, H., S. Suzuki, H. Kobayashi, S. Ogino, A. Inomata, and M. Arakawa. 1991. Immunohistological localization of apolipoproteins in the glomeruli in renal disease: specifically apoB and apoE. Clin Nephrol 36: 127-133.
Fried, L. F., T. J. Orchard, and B. L. Kasiske. 2001. Effect of lipid reduction on the progression of renal disease: a meta-analysis. Kidney Int 59: 260-269.
Rahman, M., W. Yang, S. Akkina, A. Alper, A. H. Anderson, L. J. Appel, J. He, D. S. Raj, J. Schelling, L. Strauss, V. Teal, D. J. Rader, and C. S. Investigators. 2014. Relation of serum lipids and lipoproteins with progression of CKD: The CRIC study. Clin J Am Soc Nephrol 9: 1190-1198.
Dormuth, C. R., B. R. Hemmelgarn, J. M. Paterson, M. T. James, G. F. Teare, C. B. Raymond, J. P. Lafrance, A. Levy, A. X. Garg, P. Ernst, and S. Canadian Network for Observational Drug Effect. 2013. Use of high potency statins and rates of admission for acute kidney injury: multicenter, retrospective observational analysis of administrative databases. BMJ 346: f880.
Bangalore, S., R. Fayyad, G. K. Hovingh, R. Laskey, L. Vogt, D. A. DeMicco, D. D. Waters, C. Treating to New Targets Steering, and Investigators. 2014. Statin and the risk of renal-related serious adverse events: Analysis from the IDEAL, TNT, CARDS, ASPEN, SPARCL, and other placebo-controlled trials. Am J Cardiol 113: 2018-2020.
Lee, J. M., J. Park, K. H. Jeon, J. H. Jung, S. E. Lee, J. K. Han, H. L. Kim, H. M. Yang, K. W. Park, H. J. Kang, B. K. Koo, S. H. Jo, and H. S. Kim. 2014. Efficacy of short-term high-dose statin pretreatment in prevention of contrast-induced acute kidney injury: updated study-level meta-analysis of 13 randomized controlled trials. PLoS One 9: e111397.
Han, Y., G. Zhu, L. Han, F. Hou, W. Huang, H. Liu, J. Gan, T. Jiang, X. Li, W. Wang, S. Ding, S. Jia, W. Shen, D. Wang, L. Sun, J. Qiu, X. Wang, Y. Li, J. Deng, J. Li, K. Xu, B. Xu, R. Mehran, and Y. Huo. 2014. Short-term rosuvastatin therapy for prevention of contrast-induced acute kidney injury in patients with diabetes and chronic kidney disease. J Am Coll Cardiol 63: 62-70.
Sattar, N., D. Preiss, H. M. Murray, P. Welsh, B. M. Buckley, A. J. de Craen, S. R. Seshasai, J. J. McMurray, D. J. Freeman, J. W. Jukema, P. W. Macfarlane, C. J. Packard, D. J. Stott, R. G. Westendorp, J. Shepherd, B. R. Davis, S. L. Pressel, R. Marchioli, R. M. Marfisi, A. P. Maggioni, L. Tavazzi, G. Tognoni, J. Kjekshus, T. R. Pedersen, T. J. Cook, A. M. Gotto, M. B. Clearfield, J. R. Downs, H. Nakamura, Y. Ohashi, K. Mizuno, K. K. Ray, and I. Ford. 2010. Statins and risk of incident diabetes: a collaborative meta-analysis of randomised statin trials. Lancet 375: 735-742.
Preiss, D., S. R. Seshasai, P. Welsh, S. A. Murphy, J. E. Ho, D. D. Waters, D. A. DeMicco, P. Barter, C. P. Cannon, M. S. Sabatine, E. Braunwald, J. J. Kastelein, J. A. de Lemos, M. A. Blazing, T. R. Pedersen, M. J. Tikkanen, N. Sattar, and K. K. Ray. 2011. Risk of incident diabetes with intensive-dose compared with moderate-dose statin therapy: a meta-analysis. JAMA 305: 2556-2564.
He, Y. M., L. Feng, D. M. Huo, Z. H. Yang, and Y. H. Liao. 2014. Benefits and harm of niacin and its analog for renal dialysis patients: a systematic review and meta-analysis. Int Urol Nephrol 46: 433-442.
Cho, K. H., H. J. Kim, V. S. Kamanna, and N. D. Vaziri. 2010. Niacin improves renal lipid metabolism and slows progression in chronic kidney disease. Biochim Biophys Acta 1800: 6-15.
Streja, E., C. P. Kovesdy, D. A. Streja, H. Moradi, K. Kalantar-Zadeh, and M. L. Kashyap. 2015. Niacin and progression of CKD. Am J Kidney Dis 65: 785-798.
Kang, H. L., D. Y. Kim, S. M. Lee, K. H. Kim, S. H. Han, H. K. Nam, K. H. Kim, S. E. Kim, Y. K. Son, and W. S. An. 2013. Effect of low-dose niacin on dyslipidemia, serum phophorus levels and adverse effects in patients with chornic kidney disease. Kidney Res Clin Pract 32: 21-26.
Maccubbin, D., D. Tipping, O. Kuznetsova, W. A. Hanlon, and A. G. Bostom. 2010. Hypophosphatemic effect of niacin in patients without renal failure: a randomized trial. Clin J Am Soc Nephrol 5: 582-589.
Maccubbin, D., H. E. Bays, A. G. Olsson, V. Elinoff, A. Elis, Y. Mitchel, W. Sirah, A. Betteridge, R. Reyes, Q. Yu, O. Kuznetsova, C. M. Sisk, R. C. Pasternak, and J. F. Paolini. 2008. Lipid-modifying efficacy and tolerability of extended-release niacin/laropiprant in patients with primary hypercholesterolaemia or mixed dyslipidaemia. International journal of clinical practice 62: 1959-1970.
Boden, W. E., J. L. Probstfield, T. Anderson, B. R. Chaitman, P. Desvignes-Nickens, K. Koprowicz, R. McBride, K. Teo, and W. Weintraub. 2011. Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. N Engl J Med 365: 2255-2267.
Group, H. T. C., M. J. Landray, R. Haynes, J. C. Hopewell, S. Parish, T. Aung, J. Tomson, K. Wallendszus, M. Craig, L. Jiang, R. Collins, and J. Armitage. 2014. Effects of extended-release niacin with laropiprant in high-risk patients. N Engl J Med 371: 203-212.
Sica, D. A. 2009. Fibrate therapy and renal function. Curr Atheroscler Rep 11: 338-342.
Jun, M., B. Zhu, M. Tonelli, M. J. Jardine, A. Patel, B. Neal, T. Liyanage, A. Keech, A. Cass, and V. Perkovic. 2012. Effects of fibrates in kidney disease: a systematic review and meta-analysis. J Am Coll Cardiol 60: 2061-2071.
Davis, T. M., R. Ting, J. D. Best, M. W. Donoghoe, P. L. Drury, D. R. Sullivan, A. J. Jenkins, R. L. O'Connell, M. J. Whiting, P. P. Glasziou, R. J. Simes, Y. A. Kesaniemi, V. J. Gebski, R. S. Scott, A. C. Keech, I. Fenofibrate, and i. Event Lowering in Diabetes Study. 2011. Effects of fenofibrate on renal function in patients with type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study. Diabetologia 54: 280-290.
Tonelli, M., D. Collins, S. Robins, H. Bloomfield, and G. C. Curhan. 2004. Gemfibrozil for secondary prevention of cardiovascular events in mild to moderate chronic renal insufficiency. Kidney Int 66: 1123-1130.
Ting, R. D., A. C. Keech, P. L. Drury, M. W. Donoghoe, J. Hedley, A. J. Jenkins, T. M. Davis, S. Lehto, D. Celermajer, R. J. Simes, K. Rajamani, and K. Stanton. 2012. Benefits and safety of long-term fenofibrate therapy in people with type 2 diabetes and renal impairment: the FIELD Study. Diabetes Care 35: 218-225.
Cannon, C. P., M. A. Blazing, R. P. Giugliano, A. McCagg, J. A. White, P. Theroux, H. Darius, B. S. Lewis, T. O. Ophuis, J. W. Jukema, G. M. De Ferrari, W. Ruzyllo, P. De Lucca, K. Im, E. A. Bohula, C. Reist, S. D. Wiviott, A. M. Tershakovec, T. A. Musliner, E. Braunwald, R. M. Califf, and I.-I. Investigators. 2015. Ezetimibe Added to Statin Therapy after Acute Coronary Syndromes. N Engl J Med 372: 2387-2397.
Stanifer, J. W., D. M. Charytan, J. White, Y. Lokhnygina, C. P. Cannon, M. T. Roe, and M. A. Blazing. 2017. Benefit of Ezetimibe Added to Simvastatin in Reduced Kidney Function. J Am Soc Nephrol 28: 3034-3043.
Haynes, R., D. Lewis, J. Emberson, C. Reith, L. Agodoa, A. Cass, J. C. Craig, D. de Zeeuw, B. Feldt-Rasmussen, B. Fellstrom, A. Levin, D. C. Wheeler, R. Walker, W. G. Herrington, C. Baigent, M. J. Landray, S. C. Group, and S. C. Group. 2014. Effects of lowering LDL cholesterol on progression of kidney disease. J Am Soc Nephrol 25: 1825-1833.
Morita, T., S. Morimoto, C. Nakano, R. Kubo, Y. Okuno, M. Seo, K. Someya, M. Nakahigashi, H. Ueda, N. Toyoda, M. Kusabe, F. Jo, N. Takahashi, T. Iwasaka, and I. Shiojima. 2014. Renal and vascular protective effects of ezetimibe in chronic kidney disease. Internal medicine 53: 307-314.
Rizos, E. C., E. E. Ntzani, E. Bika, M. S. Kostapanos, and M. S. Elisaf. 2012. Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic review and meta-analysis. JAMA 308: 1024-1033.
Hu, Y., F. B. Hu, and J. E. Manson. 2019. Marine Omega-3 Supplementation and Cardiovascular Disease: An Updated Meta-Analysis of 13 Randomized Controlled Trials Involving 127 477 Participants. Journal of the American Heart Association 8: e013543.
Mori, T. A., V. Burke, I. Puddey, A. Irish, C. A. Cowpland, L. Beilin, G. Dogra, and G. F. Watts. 2009. The effects of [omega]3 fatty acids and coenzyme Q10 on blood pressure and heart rate in chronic kidney disease: a randomized controlled trial. J Hypertens 27: 1863-1872.
Gopinath, B., D. C. Harris, V. M. Flood, G. Burlutsky, and P. Mitchell. 2011. Consumption of long-chain n-3 PUFA, alpha-linolenic acid and fish is associated with the prevalence of chronic kidney disease. The British journal of nutrition 105: 1361-1368.
Bunout, D., G. Barrera, S. Hirsch, and E. Lorca. 2021. A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of an Omega-3 Fatty Acid Supplement in Patients With Predialysis Chronic Kidney Disease. J Ren Nutr 31: 64-72.
Lok, C. E., L. Moist, B. R. Hemmelgarn, M. Tonelli, M. A. Vazquez, M. Dorval, M. Oliver, S. Donnelly, M. Allon, and K. Stanley. 2012. Effect of fish oil supplementation on graft patency and cardiovascular events among patients with new synthetic arteriovenous hemodialysis grafts: a randomized controlled trial. Jama 307: 1809-1816.
Irish, A. B., A. K. Viecelli, C. M. Hawley, L. S. Hooi, E. M. Pascoe, P. A. Paul-Brent, S. V. Badve, T. A. Mori, A. Cass, P. G. Kerr, D. Voss, L. M. Ong, K. R. Polkinghorne, A. Omega-3 Fatty, and G. Aspirin in Vascular Access Outcomes in Renal Disease Study Collaborative. 2017. Effect of Fish Oil Supplementation and Aspirin Use on Arteriovenous Fistula Failure in Patients Requiring Hemodialysis: A Randomized Clinical Trial. JAMA Intern Med 177: 184-193.
Svensson, M., E. B. Schmidt, K. A. Jorgensen, and J. H. Christensen. 2006. N-3 fatty acids as secondary prevention against cardiovascular events in patients who undergo chronic hemodialysis: a randomized, placebo-controlled intervention trial. Clin J Am Soc Nephrol 1: 780-786.
Kheirkhah, A., C. Lamina, B. Kollerits, J. F. Schachtl-Riess, U. T. Schultheiss, L. Forer, P. Sekula, F. Kotsis, K. U. Eckardt, F. Kronenberg, and G. Investigators. 2022. PCSK9 and Cardiovascular Disease in Individuals with Moderately Decreased Kidney Function. Clin J Am Soc Nephrol 17: 809-818.
Sabatine, M. S., R. P. Giugliano, A. C. Keech, N. Honarpour, S. D. Wiviott, S. A. Murphy, J. F. Kuder, H. Wang, T. Liu, S. M. Wasserman, P. S. Sever, T. R. Pedersen, F. S. Committee, and Investigators. 2017. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. N Engl J Med 376: 1713-1722.
Morena, M., C. Le May, L. Chenine, L. Arnaud, A. M. Dupuy, M. Pichelin, H. Leray-Moragues, L. Chalabi, B. Canaud, J. P. Cristol, and B. Cariou. 2017. Plasma PCSK9 concentrations during the course of nondiabetic chronic kidney disease: Relationship with glomerular filtration rate and lipid metabolism. J Clin Lipidol 11: 87-93.
Pavlakou, P., E. Liberopoulos, E. Dounousi, and M. Elisaf. 2017. PCSK9 in chronic kidney disease. Int Urol Nephrol 49: 1015-1024.
Schwartz, G. G., P. G. Steg, M. Szarek, D. L. Bhatt, V. A. Bittner, R. Diaz, J. M. Edelberg, S. G. Goodman, C. Hanotin, R. A. Harrington, J. W. Jukema, G. Lecorps, K. W. Mahaffey, A. Moryusef, R. Pordy, K. Quintero, M. T. Roe, W. J. Sasiela, J. F. Tamby, P. Tricoci, H. D. White, A. M. Zeiher, O. O. Committees, and Investigators. 2018. Alirocumab and Cardiovascular Outcomes after Acute Coronary Syndrome. N Engl J Med 379: 2097-2107.
Tunon, J., P. G. Steg, D. L. Bhatt, V. A. Bittner, R. Diaz, S. G. Goodman, J. W. Jukema, Y. U. Kim, Q. H. Li, C. Mueller, A. Parkhomenko, R. Pordy, P. Sritara, M. Szarek, H. D. White, A. M. Zeiher, G. G. Schwartz, and O. O. Investigators. 2020. Effect of alirocumab on major adverse cardiovascular events according to renal function in patients with a recent acute coronary syndrome: prespecified analysis from the ODYSSEY OUTCOMES randomized clinical trial. Eur Heart J 41: 4114-4123.
Vallejo-Vaz, A. J., K. K. Ray, H. N. Ginsberg, M. H. Davidson, R. H. Eckel, L. V. Lee, L. Bessac, R. Pordy, A. Letierce, and C. P. Cannon. 2019. Associations between lower levels of low-density lipoprotein cholesterol and cardiovascular events in very high-risk patients: Pooled analysis of nine ODYSSEY trials of alirocumab versus control. Atherosclerosis 288: 85-93.
Igweonu-Nwakile, E. O., S. Ali, S. Paul, S. Yakkali, S. Teresa Selvin, S. Thomas, V. Bikeyeva, A. Abdullah, A. Radivojevic, A. A. Abu Jad, A. Ravanavena, C. Ravindra, and P. Balani. 2022. A Systematic Review on the Safety and Efficacy of PCSK9 Inhibitors in Lowering Cardiovascular Risks in Patients With Chronic Kidney Disease. Cureus 14: e29140.
East, C., K. Bass, A. Mehta, G. Rahimighazikalayed, S. Zurawski, and T. Bottiglieri. 2022. Alirocumab and Lipid Levels, Inflammatory Biomarkers, Metabolomics, and Safety in Patients Receiving Maintenance Dialysis: The ALIrocumab in DIALysis Study (A Phase 3 Trial to Evaluate the Efficacy and Safety of Biweekly Alirocumab in Patients on a Stable Dialysis Regimen). Kidney Med 4: 100483.
Mayer, G., D. Dobrev, J. C. Kaski, A. G. Semb, K. Huber, A. Zirlik, S. Agewall, and H. Drexel. 2024. Management of dyslipidaemia in patients with comorbidities: facing the challenge. Eur Heart J Cardiovasc Pharmacother 10: 608-613.
Ray, K. K., H. E. Bays, A. L. Catapano, N. D. Lalwani, L. T. Bloedon, L. R. Sterling, P. L. Robinson, C. M. Ballantyne, and C. H. Trial. 2019. Safety and Efficacy of Bempedoic Acid to Reduce LDL Cholesterol. N Engl J Med 380: 1022-1032.
Powell, J., and C. Piszczatoski. 2021. Bempedoic Acid: A New Tool in the Battle Against Hyperlipidemia. Clin Ther 43: 410-420.
Nissen, S. E., A. M. Lincoff, D. Brennan, K. K. Ray, D. Mason, J. J. P. Kastelein, P. D. Thompson, P. Libby, L. Cho, J. Plutzky, H. E. Bays, P. M. Moriarty, V. Menon, D. E. Grobbee, M. J. Louie, C. F. Chen, N. Li, L. Bloedon, P. Robinson, M. Horner, W. J. Sasiela, J. McCluskey, D. Davey, P. Fajardo-Campos, P. Petrovic, J. Fedacko, W. Zmuda, Y. Lukyanov, S. J. Nicholls, and C. O. Investigators. 2023. Bempedoic Acid and Cardiovascular Outcomes in Statin-Intolerant Patients. N Engl J Med 388: 1353-1364.
Wright, R. S., M. G. Collins, R. M. Stoekenbroek, R. Robson, P. L. J. Wijngaard, U. Landmesser, L. A. Leiter, J. J. P. Kastelein, K. K. Ray, and D. Kallend. 2020. Effects of Renal Impairment on the Pharmacokinetics, Efficacy, and Safety of Inclisiran: An Analysis of the ORION-7 and ORION-1 Studies. Mayo Clin Proc 95: 77-89.
Wright, R. S., F. J. Raal, W. Koenig, U. Landmesser, L. A. Leiter, S. Vikarunnessa, A. Lesogor, P. Maheux, Z. Talloczy, X. Zang, G. G. Schwartz, and K. K. Ray. 2024. Inclisiran administration potently and durably lowers LDL-C over an extended-term follow-up: the ORION-8 trial. Cardiovasc Res 120: 1400-1410.
Wright, R. S., W. Koenig, U. Landmesser, L. A. Leiter, F. J. Raal, G. G. Schwartz, A. Lesogor, P. Maheux, C. Stratz, X. Zang, and K. K. Ray. 2023. Safety and Tolerability of Inclisiran for Treatment of Hypercholesterolemia in 7 Clinical Trials. J Am Coll Cardiol 82: 2251-2261.

Mineralocorticoid Defects in Children

ABSTRACT

Isolated aldosterone deficiency in children related either to impaired secretion by the adrenal gland or to aldosterone resistance in target tissues is rare. The incidence is estimated to be <1:1,000,000 for congenital isolated primary hypoaldosteronism and 1:66,000 to 1:166,000 for congenital aldosterone resistance (1). Children may present with salt wasting, hyponatremia, hypotension, hyperkalemia, metabolic acidosis, and failure to thrive. There is a wide phenotypic spectrum based on the severity and etiology of aldosterone deficiency or action. In this chapter, we briefly discuss the physiology of mineralocorticoids in newborns, categorize the causes of isolated hypoaldosteronism, and review the etiologies to guide clinical and laboratory evaluation and treatment.

INTRODUCTION

Mineralocorticoids are a class of steroids produced in the zona glomerulosa in the adrenal cortex that regulate sodium, potassium and water balance; aldosterone is the primary mineralocorticoid. Its synthesis involves several enzymes within the adrenal, the final step regulated by aldosterone synthase (CYP11B2) (Figure 1). Aldosterone secretion involves an intricate feedback loop involving multiple organs including the adrenal glands, kidneys, liver, lungs, and blood vessels. The major regulators of aldosterone synthesis and secretion are the renin-angiotensin-aldosterone (RAA) axis and potassium (Figure 2). Aldosterone binds to the mineralocorticoid receptor at the kidney to activate specific amiloride-sensitive sodium (ENaC) channels and a Na-K- ATPase pump. Through these actions, aldosterone promotes sodium reabsorption and urinary potassium excretion (Figure 2).

Mineralocorticoid deficiency (also referred to as hypoaldosteronism) refers to compromised aldosterone secretion from the adrenal glands or its cellular action. Hypoaldosteronism is observed as part of global adrenal cortex dysfunction in both congenital and acquired disorders, such as primary adrenal insufficiency (PAI), adrenal hypoplasia congenita (AHC), and congenital adrenal hyperplasia (CAH). In these disorders, hypoaldosteronism occurs together with glucocorticoid deficiency (i.e., adrenal insufficiency) and/or other deficient or dysregulated adrenal steroid secretion. While rare in children, hypoaldosteronism may occur as an isolated condition, either congenital or acquired, and can be classified into 1) defective aldosterone biosynthesis 2) disturbances in stimulation of aldosterone secretion, and 3) impaired aldosterone action at the target tissue, mainly the kidneys (resistance) (2). The latter is also referred to as “pseudohypoaldosteronism” since circulating aldosterone levels are elevated despite clinical symptoms and signs of mineralocorticoid deficiency due to dysfunctional mineralocorticoid receptor or its downstream effects (2). In this chapter, we discuss isolated aldosterone-deficient conditions other than PAI, AHC, and CAH. In depth coverage of adrenal insufficiency can be found in Endotext.org chapter: Adrenal Insufficiency in Children (3).

Normal aldosterone production, regulation, and action are essential in neonates, infants, and children for salt balance and overall growth. If untreated, defects in aldosterone secretion or action in children may lead to salt wasting, hypotension, hyperkalemia, metabolic acidosis, and failure to thrive. Severe hyponatremia (salt wasting) and metabolic acidosis can be life-threatening in newborns and infants. In depth coverage of mineralocorticoid deficiency and resistance can be found in Endotext.org chapter: Aldosterone Deficiency and Resistance (4). Our chapter focuses on isolated aldosterone defects in the pediatric population.

Figure 1. Enzyme defects related to aldosterone synthesis. Schematic of adrenal steroidogenesis demonstrating the various enzymes involved in aldosterone synthesis (large black box). The red lines indicate the specific enzymatic defects that result in defects in aldosterone synthesis. Cortisol circulates in the bloodstream at higher concentrations than aldosterone and it also interacts with MR. However, within the kidney and target tissues, there is selectivity of MR by aldosterone due to the enzyme 11βHSD2 that converts active cortisol to inactive cortisone (small black box). SCC: side-chain cleavage. HSD: hydroxysteroid dehydrogenase. MR: mineralocorticoid receptor. DHEA: dehydroepiandrosterone. Aldo: aldosterone.

PATHOPHYSIOLOGY

Aldosterone Synthesis

Aldosterone biosynthesis occurs at the zona glomerulosa, the outermost layer of the adrenal cortex, via the action of several enzymes: cholesterol desmolase [also known as cholesterol side-chain cleavage enzyme] (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3B2), 21-hydroxylase (CYP21A2), 11-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) (Figure1). The first four enzymes are also expressed in the zona fasciculata and are involved in cortisol biosynthesis. Defects in any of these enzymes may lead to combined aldosterone/cortisol deficiencies as part of the syndromes seen in Congenital Adrenal Hyperplasia. Aldosterone synthase encoded by CYP11B2, the last enzymatic step in aldosterone biosynthesis, is expressed only at the zona glomerulosa and genetic defects in this gene result in isolated aldosterone deficiency (Figure 1).

Aldosterone synthesis involves two steps. The first includes the 18-hydroxylation of corticosterone to form 18-hydroxycorticosterone (18OH corticosterone) and the second is the 18-oxidation of 18OH corticosterone to form aldosterone. Although it was previously considered that these two steps are catalyzed by two different enzymes, it is now known to involve the same enzyme, aldosterone synthase (5). Based on the two final steps in aldosterone synthesis, two subtypes of aldosterone synthase deficiency (ASD) have been described; however, with further clarification of the enzymatic process this is now thought to be an overlapping clinical spectrum, depending on the degree of enzyme deficiency (5).

Aldosterone Regulation and Action

Serum potassium concentrations and the Renin, Angiotensin, Aldosterone (RAA) axis are the main regulators of aldosterone synthesis. Hyperkalemia has a direct stimulating effect independent of RAA axis (2). The RAA axis is a feedback loop that regulates sodium, potassium, water, fluid volume, and blood pressure (2). The cells in the macula densa of the juxtaglomerular apparatus are triggered to release renin in response to a drop in perfusion. Angiotensinogen is a protein produced from the liver that is cleaved to angiotensin I (Ang I) by renin (2). Angiotensin-converting enzyme (ACE) in vascular endothelium rapidly converts Ang I to Angiotensin II (Ang II). Ang II is the most potent stimulus for aldosterone production and release (2). Of note, tissue and plasma peptidase inactivate angiotensin within minutes and circulating renin levels are the rate-limiting factor of this process (1).

Aldosterone mediates its effects by binding to the mineralocorticoid receptor (MR; aka NR3C2) at the distal convoluted tubules and collecting duct epithelial cells of the kidneys (Figure 2). The MR is a member of the nuclear receptor family, and along with the glucocorticoid and androgen receptors, forms the steroid receptor subfamily. In its unliganded state, the MR is located in the cytoplasm. Upon binding with its ligand, MR is translocated into the nucleus, where it modulates the transcription of several genes, such as those that encode the ENaC subunits (1). Mutations that inactivate the MR result in aldosterone resistance or pseudo-hypoaldosteronism type 1 (PHA1).

Aldosterone, 11-deoxycorticosterone (DOC), and cortisol are all endogenous agonists of the MR. Specifically, cortisol and aldosterone have an equal affinity for the mineralocorticoid receptor (2); however, selectivity of MR receptor for aldosterone is ensured in epithelial target tissues by 11βHSD2 enzyme that converts active cortisol to inactive cortisone (1) (Figure 1). This is of particular importance as cortisol circulates at concentrations 100 to 1,000-fold higher than aldosterone. Loss-of-function mutations of the kidney 11βHSD2 result in excessive cortisol-dependent MR activation and cause an autosomal recessive form of familial hypertension called apparent mineralocorticoid excess (6).

After binding to MR, aldosterone activates ENaC gene transcription, decreases ENaC degradation, and activates Na-K ATPase pump. ENaC, located at the apical membrane of epithelial cells, plays a crucial role in sodium reabsorption, potassium secretion, and subsequent volume expansion. ENaC consists of 3 subunits (a, b, and g) that are encoded by unique genes (SCNN1A, SCNN1B, SCNN1G, respectively) (1). Defects in these genes can impair ENaC function and lead also to aldosterone resistance or pseudo hypoaldosteronism type Ib. In addition to the epithelial cells of the distal convoluted tubule, ENaC is expressed at the epithelial cells of other tissues that are involved in salt conservation, such as colon, sweat glands, and lungs. Dysfunction of ENaC, therefore, has systemic manifestations from muti-organ water and salt loss.

Figure 2. Physiology of aldosterone secretion and action. The figure demonstrates the renin-angiotensin-aldosterone (RAA) system and its effects on sodium and potassium homeostasis, and blood pressure. Aldosterone secretion is regulated by decreased blood volume and hyponatremia via activation of the RAA axis, and indirectly, by hyperkalemia. Aldosterone then binds to the mineralocorticoid receptor (MR; aka NR3C2) at the distal convoluted tubules and collecting duct of the kidneys. Upon binding with aldosterone, MR translocate into the nucleus, where it modulates the transcription of the genes that encode the epithelial sodium channel (ENaC). ENaC is a sodium-selective ion channel that plays a crucial role in sodium reabsorption. Aldosterone action results in urinary potassium excretion and sodium reabsorption, and thus, increased blood volume.

Aldosterone Secretion in the Newborn

There are limited studies in infants investigating the interaction between water, sodium, and the renin-angiotensin-aldosterone system. Various changes related to water turnover, sodium metabolism, and kidney adaptation to extrauterine life occur in the neonatal period (1). The immediate postnatal phase in the first week of life is characterized by oliguria followed by a diuretic phase with extracellular contraction and net loss of sodium and water (1). Maximum weight loss occurs during this period (up to 10% of birth weight is considered normal). Kidneys in the neonate exhibit tubular immaturity resulting in sodium wasting and impaired ability to reabsorb water (1). Additionally, aldosterone and renin concentrations are higher in the newborn period, whereas expression of renal MR is reduced, leading to transient renal resistance to aldosterone (7). In very preterm infants, there is decreased activity of 11β-hydroxylase (CYP11B1) and low aldosterone synthase (CYP11B2) activity, possibly due to immaturity of these enzymes in the fetal adrenal cortex, leading to deficient aldosterone secretion (8, 9). After the first week of life, water losses decrease, and positive sodium balance is important for growth (1). It is essential to acknowledge these physiologic changes when evaluating mineralocorticoid function in the neonatal period.

CLINICAL PRESENTATION

The clinical presentation of aldosterone deficiency is variable depending on the etiology. Broadly, the signs of hypoaldosteronism include hypotension, hyponatremia (salt wasting), hyperkalemia, and metabolic acidosis. The symptoms that can be seen in infants and children related to these electrolyte derangements are dehydration, vomiting, irritability, weakness, seizures, and failure to thrive.

ETIOLOGY OF ISOLATED HYPOALDOSTERONISM

Isolated aldosterone disorders can be classified into disorders of defective synthesis, aldosterone resistance and diminished stimulation (Table 1).

Defective Aldosterone Synthesis

This refers to hyperreninemic hypoaldosteronism in which the renin production is intact, and the defect is at the level of the adrenal gland. The etiology of defective aldosterone synthesis can be separated into congenital and acquired causes. It is important to note that aldosterone deficiency due to defect in synthesis can be the first presenting sign of adrenal cortex failure and later progress to involve insufficient cortisol production. For descriptions of disorders involving adrenal cortical failure such as congenital adrenal hyperplasia and Addison’s disease, see Endotext.org chapter: Adrenal Insufficiency in Children (3).

CONGENITAL CAUSES

Prematurity

Very preterm infants (<33 weeks’ gestation) have deficient aldosterone concentrations, thought to be related to both the general immaturity of the fetal adrenal cortex and specifically a defect in aldosterone production, perhaps due to low aldosterone synthase activity (10). This also aligns with other defects in adrenal steroidogenesis seen in preterm infants (e.g. low 11β-hydroxylation leading to high 17OHP and false positive on the newborn screening) (11).

Aldosterone Synthase Deficiency

Variants in the CYP11B2 gene result in variable loss of enzyme activity and aldosterone deficiency. As seen in Figure 1, aldosterone synthase is responsible for the hydroxylation of corticosterone to 18-hydroxycorticosterone followed by oxidation from 18-hydroxycorticosterone to aldosterone. Previously, these steps were thought to be controlled by 2 different enzymes and this disorder was called corticosterone methyl-oxidase (CMO) deficiency with 2 subtypes described (CMOI and CMOII) based on the aldosterone and precursor relative concentrations. These subtypes are now thought to be a spectrum of severity (12). Due to continued production of DOC and corticosterone, there is some mineralocorticoid activity. However, this may be insufficient in the setting of aldosterone resistance of the neonate and salt loss may occur in infancy. Children are more affected than adults who may even have normal renin levels as the mineralocorticoid sensitivity improves and exogenous salt from table food intake increases.

ACQUIRED CAUSES

Critical Illness

Despite intact ACTH and renin secretion as well as angiotensin II production, a portion of critically ill patients may have low aldosterone levels (13, 14). This is considered to represent a shift in the adrenal cortex prioritizing cortisol production to aid in recovery.

Adrenalectomy

Typically, unilateral adrenalectomy would not be expected to lead to a glucocorticoid or mineralocorticoid defect; however, this may occur in the setting of a hyperfunctioning defect in one adrenal with contralateral atrophy. In the case of mineralocorticoid function, a patient with Conn’s syndrome (also known as primary hyperaldosteronism) who undergoes unilateral adrenalectomy can experience signs and symptoms of hypoaldosteronism including hyperkalemia, with reports indicating this occurrence in 6-62% of patients (15-17). Post surgical monitoring is recommended, although few patients require medical treatment.

Medication Induced

While other medications may lead to diminished aldosterone stimulation or resistance, heparin is known to reduce aldosterone synthesis leading to natriuresis and hyperkalemia without an impact on corticosteroid production (18).

Aldosterone Resistance

This refers to impaired action of aldosterone at the level of the target tissue and can further be categorized into congenital and acquired causes.

CONGENITAL CAUSES

Pseudo-Hypoaldosteronism (PHA) Type 1

The genetic form of aldosterone resistance occurs due to a mutation impacting the mineralocorticoid receptor (19). Despite the prevalent consideration of PHA1 as a genetic form of type IV renal tubular acidosis (RTA), the biochemical profile can differ. Hyperkalemia, hyponatremia, and acidosis are universal; however, while RTA type IV involves a hyperchloremic non-anion gap acidosis, there are descriptions of both hyper and hypochloremia as well as an anion gap acidosis in PHA1 (20). PHA can be either autosomal dominant or recessive. The autosomal recessive disease (PHA1b) occurs due to a mutation in the genes encoding one of the 3 ENaC subunits (SCNN1A, SCNN1B, SCNN1G) (21). The presentation of PHA1b is often severe given the systemic nature of ENaC outside of the kidney and in the epithelial cells of other tissues including colon, sweat glands, and lungs. The autosomal dominant disease (PHA1a) occurs due to a mutation in the gene encoding the mineralocorticoid receptor (NR3C2) and is restricted to the kidney (21, 22). This form is milder and tends to improve during childhood. However, despite its isolation to the kidney, the hyperkalemia that results can be devastating if not identified and treated early; cases are described involving cardiac arrest and hypoxic ischemic encephalopathy as an outcome (23).

Despite the classification of PHA1 based on mutation (PHA1a vs. PHA1b), some individuals with features of PHA1 do not have identifiable molecular defects.

ACQUIRED CAUSES

Secondary Pseudo-Hypoaldosteronism (PHA Type 3)

PHA Type 3 is often associated with urinary tract infections (UTI) and/or related to underlying urinary anomalies, primarily urinary tract obstruction, resulting in decreased aldosterone responsiveness (24). PHA Type 3 occurs frequently in male infants; a recent systematic review identified 80% of cases in male babies under 4 months of age (25). Presentation can include failure to thrive and vomiting and laboratory evaluation reveals hyperreninemic, hyperaldosteronism with impaired responsiveness, and hyponatremic, hyperkalemic metabolic acidosis. Early identification allows for prevention of electrolyte related morbidity and expedited resolution of urinary obstruction through surgical management in over 40% of cases (24, 25).

Medications that block the ENaC channel (amiloride) or MR (spironolactone) will cause aldosterone resistance. These medications are used therapeutically in resistant hypertension and to prevent hypokalemia seen with other diuretics. Spironolactone is also used for its anti-androgenic properties and the potential for dehydration and hyperkalemia should be considered and monitored. Other ENaC blockers include triamterene, trimethoprim, and pentamidine, while other aldosterone antagonists include synthetic progestins and calcineurin inhibitors.

Diminished Stimulation

Decreased renin or angiotensin II results in decreased aldosterone production due to diminished adrenal stimulation. When this hyporeninemic hypoaldosteronism occurs with hyperchloremia and non-anion gap metabolic acidosis, it is called Type 4 RTA. In adults, this is most often associated with nephropathy (diabetes, autonomic neuropathy, sickle cell disease, HIV, SLE) and medications (beta blockers, ACE inhibitors) (26). In children, Gordon Syndrome (Familial Hyperkalemic hypertension or pseudo-hypoaldosteronism type II [PHA2]) is rare and associated with low renin/low aldosterone (or inappropriately normal for degree of hyperkalemia) state with normal glomerular filtration. This is thought to be due to abnormal thiazide-sensitive sodium-chloride co-transporter in the distal nephron (mutations in WNK1, WNK4, CUL3, or KLHL3 genes) (27). The increased sodium and chloride reabsorption leads to hypertension, volume expansion, and decreased potassium and hydrogen excretion resulting in hyperkalemia and metabolic acidosis. In contrast to PHA1, PHA2 does have a biochemical profile that aligns with Type IV RTA including hyponatremic, hyperkalemic, and hyperchloremic non-anion gap metabolic acidosis (28). These causes of defective aldosterone stimulation are rare in the pediatric population, so when identified, affected children should be referred to the appropriate subspecialities such as nephrology for evaluation and treatment. Given the rare nature and lack of a primary endocrine etiology, these causes are not reviewed in the table below.

Table 1. CAUSES OF ISOLATED ALDOSTERONE DEFECTS IN CHILDREN
	Condition	Cause	Presentation
Congenital – Aldosterone Synthesis	Prematurity (transient)	Immaturity of aldosterone synthase in very premature infants	HYPERreninemic,HYPOaldosteronism Hyponatremia and hyperkalemia Increased corticosterone
Congenital – Aldosterone Synthesis	Aldosterone synthase deficiency Formerly divided into: CMO I deficiency (low 18-OH corticosterone) -CMO II deficiency (high 18-OH corticosterone)	Autosomal recessive or autosomal dominant (mixed penetrance) variant in CYP11B2
Acquired – Aldosterone Synthesis	Critical illness	Thought to represent a shift in the adrenal cortex prioritizing cortisol production to aid in recovery
	Adrenalectomy	Can occur in the setting of a hyperfunctioning lesion with contralateral atrophy
	Medication induced	Heparin
Congenital – Aldosterone Resistance	Systemic Pseudo-hypoaldosteronism (PHA1b)	Autosomal recessive variant in ENaC gene (SCNN1A, SCNN1B, SCNN1G)	HYPERreninemicHYPERaldosteronism (pseudo-hypoaldosteronism) Hyponatremia and hyperkalemia (electrolytes may be normal in mild cases)
Congenital – Aldosterone Resistance	Renal Pseudo-hypoaldosteronism (PHA1a)	Autosomal dominant variant in MR receptor gene (NR3C2)
Acquired – Aldosterone Resistance	Secondary Pseudo-hypoaldosteronism (type 3 PHA)	Associated with urinary tract infections
Acquired – Aldosterone Resistance	Secondary (medication induced) pseudo-hypoaldosteronism	Meds that block ENaC (amiloride, triamterene, trimethoprim, pentamidine), meds that block MR receptor (spironolactone, synthetic progestins, calcineurin inhibitors)

DIAGNOSTIC APPROACH

Defects of aldosterone synthesis or action in children should be suspected in the setting of dehydration, hyponatremia (salt wasting), and hyperkalemia. The clinical phenotype varies depending on the etiology and some infants or children may present only with mild electrolyte abnormalities and failure to thrive. Additionally, the causes of hyponatremia in children are broad, and may include iatrogenic causes due to hypotonic fluid, central nervous system or lung disease causing syndrome of inappropriate antidiuretic hormone (SIADH), excess ingestion of free water, and high salt losses due to diarrhea. Determining volume status and urinary sodium content are starting points for refining the etiology of hyponatremia. Mineralocorticoid deficiency is characterized by hypovolemic hyponatremia with high urine sodium. The other causes of hyponatremia will not be discussed in this chapter.

Differential Diagnosis and Laboratory Evaluation

The first step in the evaluation of a child with suspected mineralocorticoid deficiency is to determine whether there is associated adrenal insufficiency (figure 3). The evaluation for adrenal insufficiency includes measurement of serum cortisol (ideally morning level depending on the clinical scenario and age of patient), ACTH, 17-hydroxyprogesterone (17OHP), and possible provocative testing (ACTH stimulation test). Plasma renin activity and serum aldosterone should be measured to evaluate for mineralocorticoid deficiency. If there is global adrenal dysfunction resulting in both cortisol and aldosterone deficiency, the differential diagnosis can be narrowed to causes of primary adrenal insufficiency (see Endotext: Adrenal Insufficiency in Children) (3). It is critical to identify primary adrenal insufficiency, especially in infants, and promptly treat with hydrocortisone to avoid adrenal crisis.

If an isolated aldosterone defect is considered, the second step is to evaluate whether the defect is at the level of the adrenal glands or kidneys. High renin and low aldosterone points to a defect at the level of the adrenal glands (defective aldosterone synthesis). High renin and high aldosterone points to a defect at the level of the kidneys causing resistance to aldosterone. The various causes of aldosterone resistance are detailed above in the section “Etiology”. Briefly, these include congenital (mutations in MR or ENaC channel) and acquired causes (medications, transient resistance in the setting of UTI, or renal tubular dysfunction). Low renin and low aldosterone states do not commonly occur in children, as they are often the consequence of chronic illness causing type IV RTA (i.e. in adults with diabetic nephropathy); however, there is also a genetic form, Gordon Syndrome or pseudo-hypoaldosteronism type 2 which is characterized by hypertension, hyperkalemia, and metabolic acidosis.

As stated above, measurement of renin and aldosterone at the time of hyponatremia and hyperkalemia are important biochemical markers to differentiate the etiology of hypoaldosteronism. Furthermore, if there is suspicion for aldosterone synthase deficiency, corticosterone and 18-hydroxycorticosterone measurements can be useful (see figure 1). Values need to be interpreted according to age of the patient. Hemolyzed lblood may result in a falsely elevated potassium level and must be repeated to ensure accuracy of test values.

Figure 3. A proposed approach in the differential and diagnostic evaluation of children with suspected aldosterone deficiency.

Genetic Testing

In addition to biochemical evaluation, genetic testing is an invaluable tool to help guide treatment and prognosis, especially in infanta and children where the clinical manifestations of aldosterone defects vary widely (29). Genetic testing including whole exome sequencing or gene panels (for pseudo-hypoaldosteronism) may clarify the diagnosis, treatment, and prognosis. Genes associated with hypoaldosteronism and pseudo-hypoaldosteronism include CYP11B2, NR3C2, SCNN1A, SCNN1B, SCNN1G, WNK1, WNK4, CUL3, KLHL3 (2).

TREATMENT

The initial management depends upon severity of presentation and etiology of the mineralocorticoid defect. Infants or children who are acutely ill with salt-wasting crisis must undergo fluid resuscitation to correct salt and water losses. It is essential to give stress dose corticosteroids (intramuscular or intravenous hydrocortisone 100mg/m2) if co-existing glucocorticoid deficiency exists. Hydrocortisone at high doses has mineralocorticoid effect, and fludrocortisone tablets may be added once hydrocortisone is weaned to be below about 50-60 mg/m2/day (3).

Oral treatment options for children with aldosterone defects include mineralocorticoid replacement (fludrocortisone), sodium chloride tablets, and sodium bicarbonate. The management plan depends on the underlying mineralocorticoid defect and is separated according to those children who are not able to produce aldosterone, and those who have resistance to its action.

Primary Hypoaldosteronism

Children with primary hypoaldosteronism (including those with adrenal insufficiency such as Addison’s Disease or CAH) should start mineralocorticoid replacement (fludrocortisone 0.05-0.2 mg/day). Infants and young children usually need higher doses of fludrocortisone in addition to sodium chloride supplementation due to renal resistance and general diet that is lower in salt. Sodium chloride is weaned over time as renin activity normalizes, and salt is incorporated into the diet. Fludrocortisone is continued for mineralocorticoid replacement and titrated based on normalization of blood pressure, electrolytes, and renin levels.

Aldosterone Resistance

Children with autosomal recessive (PHA1b) and autosomal dominant (PHA1a) pseudo-hypoaldosteronism are usually treated with high dose sodium chloride supplementation. Those who have PHA1a (mild form only affecting the kidneys) usually need lower doses of salt supplementation with gradual clinical improvement (typically no need for salt supplementation by 1-3 years of age) (30). Infants and children with the severe/systemic form (PHA1b) are more difficult to manage given the need for higher doses of salt supplementation, potassium lowering agents, and potential for recurrent pulmonary infections (31). Some of these children might need gastrostomy tubes to allow for consistent high dose salt supplementation which is not always tolerated by mouth. Sodium bicarbonate is another medication used to improve metabolic acidosis which can impact growth and development if acidosis persists. Given the rarity of pseudo-hypoaldosteronism, the doses of sodium chloride and sodium bicarbonate are not well established and must be titrated based on serum sodium and bicarbonate concentrations.

Table 2. SUMMARY OF TREATMENT OPTIONS FOR CHILDREN WITH ALDOSTERONE DEFECTS:
Treatment	Dose	Considerations
Fludrocortisone	0.05-0.2 mg/day	Once or twice daily Doses titrated based on blood pressure, electrolytes, and renin levels.
Sodium chloride (salt tablets)	2 g/day or 2-5 mEq/kg daily	1-gram NaCl tablets = 17mEq Higher/more frequent doses in babies and weaned down as they get older. Doses titrated based on sodium levels.
Sodium bicarbonate or sodium citrate/citric acid	2-3 mEq/kg daily	Titrate based on bicarbonate levels

CONCLUSION

Isolated defects in aldosterone synthesis or action are rare in children; however, it is important to identify these disorders to prevent life-threatening complications. Infants may present with salt wasting crisis while older children may present with failure to thrive, mild hyponatremia, and metabolic acidosis. The two major categories of isolated hypoaldosteronism include aldosterone synthesis defects and aldosterone resistance. There are several genes associated with isolated hypoaldosteronism, and genetic testing is an important diagnostic tool. Treatment and prognosis depend on the underlying etiology.

REFERENCES

Bizzarri C, Pedicelli S, Cappa M, Cianfarani S. Water Balance and ‘Salt Wasting' in the First Year of Life: The Role of Aldosterone-Signaling Defects. Hormone Research in Paediatrics. 2016;86(3):143-53.
Rajkumar V WM. Hypoaldosteronism. Treasure Island (FL): StatPearls Publishing; 2023.
Kilberg M, Vogiatzi M. Adrenal Insufficiency in Children. EndoText. 2024.
Arai K, Papadopoulou-Marketou N, Chrousos G. Aldosterone Deficiency and Resistance. EndoText. 2020.
White PC. Aldosterone synthase deficiency and related disorders. Mol Cell Endocrinol. 2004;217(1-2):81-7.
Carvajal CA, Tapia-Castillo A, Vecchiola A, Baudrand R, Fardella CE. Classic and Nonclassic Apparent Mineralocorticoid Excess Syndrome. J Clin Endocrinol Metab. 2020;105(4).
Martinerie L, Viengchareun S, Delezoide AL, Jaubert F, Sinico M, Prevot S, et al. Low renal mineralocorticoid receptor expression at birth contributes to partial aldosterone resistance in neonates. Endocrinology. 2009;150(9):4414-24.
Martinerie L, Pussard E, Yousef N, Cosson C, Lema I, Husseini K, et al. Aldosterone-Signaling Defect Exacerbates Sodium Wasting in Very Preterm Neonates: The Premaldo Study. The Journal of Clinical Endocrinology & Metabolism. 2015;100(11):4074-81.
Travers S, Martinerie L, Boileau P, Lombes M, Pussard E. Alterations of adrenal steroidomic profiles in preterm infants at birth. Arch Dis Child Fetal Neonatal Ed. 2018;103(2):F143-F51.
Martinerie L, Pussard E, Yousef N, Cosson C, Lema I, Husseini K, et al. Aldosterone-Signaling Defect Exacerbates Sodium Wasting in Very Preterm Neonates: The Premaldo Study. J Clin Endocrinol Metab. 2015;100(11):4074-81.
Kamrath C, Hartmann MF, Boettcher C, Wudy SA. Reduced activity of 11beta-hydroxylase accounts for elevated 17alpha-hydroxyprogesterone in preterms. J Pediatr. 2014;165(2):280-4.
Miller WL. MECHANISMS IN ENDOCRINOLOGY: Rare defects in adrenal steroidogenesis. Eur J Endocrinol. 2018;179(3):R125-R41.
Findling JW, Waters VO, Raff H. The dissociation of renin and aldosterone during critical illness. J Clin Endocrinol Metab. 1987;64(3):592-5.
Trimarchi T. Endocrine problems in critically ill children: an overview. AACN Clin Issues. 2006;17(1):66-78.
Starker LF, Christakis I, Julien JS, Schwarz K, Graham P, Grubbs EG, et al. Considering Postoperative Functional Hypoaldosteronism after Unilateral Adrenalectomy. Am Surg. 2017;83(6):598-604.
Queiroz NL, Stumpf MAM, Souza VCM, Maciel AAW, Fagundes GFC, Okubo J, et al. Renal Function Evolution and Hypoaldosteronism Risk After Unilateral Adrenalectomy for Primary Aldosteronism. Horm Metab Res. 2024;56(5):350-7.
Shariq OA, Bancos I, Cronin PA, Farley DR, Richards ML, Thompson GB, et al. Contralateral suppression of aldosterone at adrenal venous sampling predicts hyperkalemia following adrenalectomy for primary aldosteronism. Surgery. 2018;163(1):183-90.
Oster JR, Singer I, Fishman LM. Heparin-induced aldosterone suppression and hyperkalemia. Am J Med. 1995;98(6):575-86.
Tajima T, Morikawa S, Nakamura A. Clinical features and molecular basis of pseudohypoaldosteronism type 1. Clin Pediatr Endocrinol. 2017;26(3):109-17.
Adachi M, Nagahara K, Ochi A, Toyoda J, Muroya K, Mizuno K. Acid-Base Imbalance in Pseudohypoaldosteronism Type 1 in Comparison With Type IV Renal Tubular Acidosis. J Endocr Soc. 2022;6(12):bvac147.
Furgeson SB, Linas S. Mechanisms of type I and type II pseudohypoaldosteronism. J Am Soc Nephrol. 2010;21(11):1842-5.
Geller DS, Rodriguez-Soriano J, Vallo Boado A, Schifter S, Bayer M, Chang SS, et al. Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat Genet. 1998;19(3):279-81.
Tauber KA, Ermacor K, Listman J. Cardiac arrest in a newborn: A case of pseudohypoaldosteronism. Clin Case Rep. 2024;12(2):e8265.
Moreno Sanchez A, Garcia Atares A, Molina Herranz D, Antonanzas Torres I, Romero Salas Y, Ruiz Del Olmo Izuzquiza JI. Secondary pseudohypoaldosteronism: a 15-year experience and a literature review. Pediatr Nephrol. 2024;39(11):3233-9.
Betti C, Lavagno C, Bianchetti MG, Kottanattu L, Lava SAG, Schera F, et al. Transient secondary pseudo-hypoaldosteronism in infants with urinary tract infections: systematic literature review. Eur J Pediatr. 2024;183(10):4205-14.
Mustaqeem R AA. Renal Tubular Acidosis. Treasure Island (FL): StatPearls Publishing. Available from: https://www.ncbi.nlm.nih.gov/books/NBK519044/.
Manas F, Singh S. Pseudohypoaldosteronism Type II or Gordon Syndrome: A Rare Syndrome of Hyperkalemia and Hypertension With Normal Renal Function. Cureus. 2024 Jan 19;16(1).
Adachi M, Motegi S, Nagahara K, Ochi A, Toyoda J, Mizuno K. Classification of pseudohypoaldosteronism type II as type IV renal tubular acidosis: results of a literature review. Endocr J. 2023;70(7):723-9.
Turan I, Kotan LD, Tastan M, Gurbuz F, Topaloglu AK, Yuksel B. Molecular genetic studies in a case series of isolated hypoaldosteronism due to biosynthesis defects or aldosterone resistance. Clin Endocrinol (Oxf). 2018;88(6):799-805.
Krishna S, Augustian M. Autosomal Dominant Pseudohypoaldosteronism Type 1 in a Newborn With Failure to Thrive. Cureus. 2024;16(4):e59356.
Karacan Küçükali G ÇS, Tunç G, Oğuz MM, Çelik N, Akkaş KY, Şenel S, Güleray Lafcı N, Savaş Erdeve Ş. Clinical Management in Systemic Type Pseudohypoaldosteronism Due to SCNN1B Variant and Literature Review. J Clin Res Pediatr Endocrinol. 2021;13(4):446-51.

Normal Physiology of Growth Hormone in Adults

ABSTRACT

Growth hormone (GH) is an ancestral hormone, secreted episodically from somatotroph cells in the anterior pituitary. Since the recognition of its multiple and complex effects in the early 1960s, the physiology and regulation of GH has become a major area of research interest in the field of endocrinology. In adulthood its main role is to regulate metabolism. Pituitary synthesis and secretion of GH is stimulated by episodic hypothalamic secretion of GH releasing factor and inhibited by somatostatin. Insulin-like Growth Factor I (IGF-I) inhibits GH secretion by a negative loop at both hypothalamic and pituitary levels. In addition, age, gender, pubertal status, food, exercise, fasting, sleep, and body composition play important regulatory roles. GH acts both directly through its own receptors and indirectly through the induced production of IGF-I. Their effects may be synergic (stimulate growth) or antagonistic as for the effect on glucose metabolism: GH stimulates lipolysis and promotes insulin resistance, whereas IGF-I acts as an insulin agonist. The bioactivity of IGF-I is tightly controlled by a multitude of IGF-I binding globulins. The mechanisms to explain the insulin antagonist effect of GH in humans are causally linked to lipolysis and ensuing elevated levels of circulating free fatty acids (FFA). The nitrogen retaining properties of GH predominantly involve stimulation of protein synthesis, which could be either direct or mediated through IGF-I, insulin, or lipid intermediates. In this chapter the normal physiology of GH secretion and the effects of GH on intermediary metabolism throughout adulthood, focusing on human studies, are presented.

INTRODUCTION

Harvey Cushing proposed in 1912 in his monograph "The Pituitary Gland" the existence of a "hormone of growth” and was thereby among the first to indicate that the primary action of growth hormone (GH) was to control and promote skeletal growth. In clinical medicine GH (also called somatotrophin) was previously known for its role on promoting growth of hypopituitary children, and for its adverse effects in connection with hypersecretion as observed in acromegaly. The multiple and complex actions of human GH were, however, acknowledged shortly after the advent of pituitary derived preparation of the hormone in the late fifties, as reviewed by Raben in 1962 (1).

In the present chapter we will briefly review the normal physiology of GH secretion and the effects of GH on intermediary metabolism throughout adulthood. Other important physiological effects of GH are presented in the review on GH replacement in adults.

GROWTH HORMONE

GH is a single chain protein with 191 amino-acids and two disulfide bonds. The human GH gene is located on chromosome 17q22 as part of a locus that comprises five genes. In addition to two GH related genes (GH1 that codes for the main adult growth hormone, produced in the somatotrophic cells found in the anterior pituitary gland and, to a minor extent, in lymphocytes, and GH2 that codes for the placental GH), there are three genes coding for chorionic somatomammotropin (CSH1, CSH2 and CSHL) (also known as placental lactogen) genes (2,3). The GH1 gene encodes two distinct GH isoforms (22 kDa and 20 kDa). The principal and most abundant GH form in pituitary and blood is monomeric 22K-GH isoform, representing also the recombinant GH available for therapeutic use (and subsequently for doping purposes) (3). Administration of recombinant 22K-GH exogenously leads to a decrease in the 20K-GH isoform and testing both isoforms is used to detect GH doping in sports (4).

As already mentioned, GH is secreted by the somatotroph cells located primarily in the lateral wings of the anterior pituitary. A recent single cell RNA sequencing study performed in mice showed that GH-expressing cells, representing the somatotrophs, are the most abundant cell population in adult pituitary gland (5). The differentiation of somatotroph cell is governed by the pituitary transcription factor 1 (Pit-1). Data in mice suggests that the pituitary holds regenerative competence, the GH-producing cells being regenerated from the pituitary’s stem cells in young animals after a period of 5 months (6).

Physiological Regulation of GH Secretion

The morphological characteristics and number of somatotrophs are remarkably constant throughout life, while the secretion pattern changes. GH secretion occurs in a pulsatile fashion, and in a circadian rhythm with a maximal release in the second half of the night. So, sleep is an important physiological factor that increases GH release. Interestingly, the maximum GH levels occur within minutes of the onset of slow wave sleep and there is marked sexual dimorphism of the nocturnal GH increase in humans, constituting only a fraction of the total daily GH release in women, but the bulk of GH output in men (7).

GH secretion is also gender, pubertal status, and age dependent (Figure 1) (8). Integrated 24 h GH concentration is significantly greater in women than in men and greater in the young than in the old adults. The serum concentration of free estradiol, but not free testosterone, correlates with GH and when correcting for the effects of estradiol, neither gender nor age influence GH concentration. This suggests that estrogens play a crucial role in modulating GH secretion (8). During puberty, a 3-fold increase in pulsatile GH secretion occurs that peaks around the age of 15 years (yr) in girls and 1 yr later in boys (9).

Figure 1. The secretory pattern of GH in young and old females and males. In young individuals the GH pulses are larger and more frequent and females secrete more GH than men (modified from (8)).

Pituitary synthesis and secretion of GH is stimulated by episodic hypothalamic hormones. Growth hormone releasing hormone (GHRH) stimulates while somatostatin (SST) inhibits GH production and release. GH stimulates IGF-I production which in turn inhibits GH secretion at both hypothalamic and pituitary levels. The gastric peptide ghrelin is also a potent GH secretagogue, which acts to boost hypothalamic GHRH secretion and synergize with its pituitary GH-stimulating effects (Figure 2) (10). Interestingly, recently germline or somatic duplication of GPR101 constitutively activates the cAMP pathway in the absence of a ligand, leading to GH release. Although GPR101 physiology is unclear it is worth mentioning it since it clearly has an effect on GH physiology (11).

In addition, a multitude of other factors may impact the GH axis most probably due to interaction with GHRH, somatostatin, and ghrelin. Estrogens stimulate the secretion of GH but inhibit the action of GH on the liver by suppressing GH receptor (GHR) signaling. In contrast, androgens enhance the peripheral actions of GH (12). Exogenous estrogens potentiate pituitary GH responses to submaximal effective pulses of exogenous GHRH (13) and mutes inhibition by exogenous SST (14). Also, exogenous estrogen potentiates ghrelin action (15).

GH release correlates inversely with intraabdominal visceral adiposity via mechanisms that may depend on increased FFA flux, elevated insulin, or free IGF-I.

Figure 2. Factors that stimulate and suppress GH secretion under physiological conditions.

GROWTH HORMONE RELEASING HORMONE (GHRH)

GHRH is a 44 amino-acid polypeptide produced in the arcuate nucleus of the hypothalamus. These neuronal terminals secrete GHRH to reach the anterior pituitary somatotrophs via the portal venous system, which leads to GH transcription and secretion. Moreover, animal studies have demonstrated that GHRH plays a vital role in the proliferation of somatotrophs in the anterior pituitary, whereas the absence of GHRH leads to anterior pituitary hypoplasia (16). In addition, GHRH upregulates GH gene expression and stimulates GH release (17). The secretion of GHRH is stimulated by several factors including depolarization, α2-adrenergic stimulation, hypophysectomy, thyroidectomy, and hypoglycemia and it is inhibited by somatostatin, IGF-I, and activation of GABAergic neurons.

GHRH acts on the somatotrophs via a seven trans-membrane G protein-coupled stimulatory cell-surface receptor. This receptor has been extensively studied over the last decade leading to the identification of several important mutations. Point mutations in the GHRH receptors, as illustrated by studies done on the lit/lit dwarf mice, showed a profound impact on subsequent somatotroph proliferation leading to anterior pituitary hypoplasia (18). Unlike the mutations in the Pit-1 and PROP-1 genes, which lead to multiple pituitary hormone deficiencies and anterior pituitary hypoplasia, mutations in the GHRH receptor leads to profound GH deficiency with anterior pituitary hypoplasia. Subsequently to the first GHRH receptor mutation described in 1996 (19) an array of familial GHRH receptor mutations have been recognized over the last decade. These mutations account for almost 10% of the familial isolated GH deficiencies. An affected individual will present with short stature and a hypoplastic anterior pituitary. However, they lack certain typical features of GH deficiency such as midfacial hypoplasia, microphallus, and neonatal hypoglycemia (20).

SOMATOSTATIN (SST)

SST is a cyclic peptide, encoded by a single gene in humans, which mostly exerts inhibitory effects on endocrine and exocrine secretions. Many cells in the body, including specialized cells in the anterior periventricular nucleus and arcuate nucleus, produce SST. These neurons secrete SST into the adenohypophyseal portal venous system, via the median eminence, to exert effect on the anterior pituitary. SST has a short half-life of approximately 2 minutes as it is rapidly inactivated by tissue peptidase in humans. The secretion of SST by the hypothalamic neurons is inhibited by high blood glucose and is stimulated by serum GH/IGF-I level, exercise, and immobilization (21).

SST acts via a seven trans-membrane, G protein coupled receptor and, thus far, five subtypes of the receptor have been identified in humans (SSTR1-5). Although all five receptor subtypes are expressed in the human fetal pituitary, adult pituitary only express 4 subtypes (SSTR1, SSTR2, SSTR3, SSTR5). Out of these four subtypes, somatotrophs exhibit more sensitivity to SSTR2 and SSTR5 ligands in inhibiting the secretion of GH in a synergistic manner (22).

GHRELIN

Ghrelin is a 28 amino-acid peptide that is the natural ligand for the GH secretagogue receptor. In fact, ghrelin and GHRH have a synergistic effect in increasing circulating GH levels (7). Ghrelin is primarily secreted by stomach and may be involved in the GH response to fasting and food intake.

Clinical Implications

GH LEVELS- INFLUENCE ON BODY COMPOSISTION, PHYSICAL FITNESS, AND AGE

With the introduction of dependable radioimmunological assays, it was recognized that circulating GH is blunted in obese subjects, and that normal aging is accompanied by a gradual decline in GH levels (23,24). It has been hypothesized that many of the senescent changes in body composition and organ function are related to or caused by decreased GH (25), also known as "the somatopause".

Studies done in the late 90s have uniformly documented that adults with severe GH deficiency are characterized by increased fat mass and reduced lean body mass (LBM) (26). It is also known that normal GH levels can be restored in obese subjects following massive weight loss (27), and that GH substitution in GH-deficient adults normalizes body composition. What remains unknown is the cause-effect relationship between decreased GH levels and senescent changes in body composition. Is the propensity for gaining fat and losing lean body mass initiated or preceded by a primary age-dependent decline in GH secretion and action? Alternatively, accumulation of fat mass secondary to non-GH dependent factors (e.g., lifestyle, dietary habits) results in a feedback inhibition of GH secretion. Moreover, little is known about possible age-associated changes in GH pharmacokinetics and bioactivity.

Cross sectional studies done to assess the association between body composition and stimulated GH release in healthy subjects show that, adult people (mean age 50 yr) have a lower peak GH response to secretagogues (clonidine and arginine), and females had a higher response to arginine when compared to males. Multiple regression analysis, however, reveal that intra-abdominal fat mass is the most important and negative predictor of peak GH levels as previously mentioned (Figure 3) (28). In the same population, 24-h spontaneous GH levels also predominantly correlated inversely with intra-abdominal fat mass (29).

Figure 3. Correlation between intra-abdominal fat mass and 24-hour GH secretion.

A detailed analysis of GH secretion in relation to body composition in elderly subjects has, to our knowledge, not been performed. Instead, serum IGF-I has been used as a surrogate or proxy for GH status in several studies of elderly men (30-32). These studies comprise large populations of ambulatory, community-dwelling males aged between 50-90 yr. As expected, the serum IGF-I declined with age (Figure 4), but IGF-I failed to show any significant association with body composition or physical performance.

Figure 4. Changes in serum IGF-I with age; modified from (33).

GH ACTION - INFLUENCE OF AGE, SEX, AND BODY COMPOSITION

Considering the great interest in the actions of GH in adults, surprisingly few studies have addressed possible age associated differences in the responsiveness or sensitivity to GH. In normal adults the senescent decline in GH levels is paralleled by a decline in serum IGF-I, suggesting a down-regulation of the GH-IGF-I axis. Administration of GH to elderly healthy adults has generally been associated with predictable, albeit modest, effects on body composition and side effects in terms of fluid retention and modest insulin resistance (34). Whether this reflects an unfavorable balance between effects and side effects in older people or employment of excessive doses of GH is uncertain, but it is evident that older subjects are not resistant to GH. Short-term dose response studies clearly demonstrate that older patients require a lower GH dose to maintain a given serum IGF-I level (35,36), and it has been observed that serum IGF-I increases in individual patients on long-term therapy if the GH dosage remains constant. Moreover, patients with GH deficiency older than 60 yr are highly responsive to even a small dose of GH (37). Interestingly, there is a gender difference in response to GH treatment with men being more responsive in terms of IGF-I generation and fat loss during therapy, most probably due to men having lower estrogen levels that negatively impact the effect of GH on IGF-I generation in the liver (38).

The pharmacokinetics and short-term metabolic effects of a near physiological intravenous GH bolus (200 micrograms) were compared in a group of young (30 yr) and older (50 yr) healthy adults (39). The area under the GH curve was significantly lower in older subjects, whereas the elimination half-life was similar in the two groups, suggesting both an increased metabolic clearance rate and apparent distribution volume of GH in older subjects. Both parameters showed a strong positive correlation with fat mass, although multiple regression analysis revealed the age to be an independent positive predictor. The short-term lipolytic response to the GH bolus was higher in young as compared to older subjects. Interestingly, the same study showed that the GH binding protein (GHBP) correlated strongly and positively with abdominal fat mass (40).

A prospective long-term study of normal adults with serial concomitant estimations of GH status and adiposity would provide useful information about the cause-effect relationship between GH status and body composition as a function of age. In the meantime, the following hypothesis is proposed (Figure 5): 1.Changes in life-style and genetic predispositions promote accumulation of body fat with aging; 2. The increased fat mass increases FFA availability, inducing insulin resistance and hyperinsulinemia; 3. High insulin levels suppress IGF binding protein (BP)-1 resulting in a relative increase in free IGF-I levels; 4. Systemic elevations of FFA, insulin, and free IGF-I suppresses pituitary GH release, which further increases fat mass; 5. Endogenous GH is cleared more rapidly in subjects with high amount of fat tissue.

At present it is not justified to treat the age-associated deterioration in body composition and physical performance with GH also due to concern that the ensuing elevation of IGF-I levels may increase the risk for the development of neoplastic disease.

Figure 5. Hypothetical model for the association between low GH levels and increased visceral fat adults.

LIFE- LONG GH DEFICIENCY

A real-life model for the GH effects in human physiology is provided by the subjects with life-long severe reduction in GH signaling due to GHRH or GHRH receptor mutations, combined deficiency of GH, prolactin, and TSH, or global deletion of GHR. They show short stature, doll facies, high-pitched voices, central obesity, and are fertile (41). Despite central obesity and increased liver fat, they are insulin sensitive, partially protected from cancer, and present a major reduction in pro-aging signaling and perhaps increased longevity (42). The decrease in cancer risk in life-long GH deficiency together with reports on the GH permissive role for neoplastic colon growth (43), preneoplastic mammary lesions (44), and progression of prostate cancer (45) demands, at least, a careful tailoring of GH substitution dosage in the GH deficient patients. However, recent evidence suggests that the GH produced locally by the colon tumor cells, and not pituitary GH, acts in an autocrine and paracrine manner to suppress the tumor suppressor proteins and to increase nuclear β-catenin accumulation and epithelial–mesenchymal transition potentially participating in tumor progression (46,47).

GH AND IMMUNE SYSTEM

Although the majority of data on the relation between GH and the immune system are from animal studies, it seems that GH may pose immunomodulatory actions. Immune cells express receptors for growth hormone, and respond to GH stimulation (48). The GHR is expressed by several lymphocyte subpopulations. GH stimulates in vitro T and B-cell proliferation and immunoglobulin synthesis, enhances human myeloid progenitor cell maturation, and modulates in vivoTh1/Th2 (8) and humoral immune responses (49). It has been shown that GH can induce de novo T cell production and enhance CD4 recovery in HIV+ patients. Another study with possible clinical relevance showed that sustained GH expression reduced prodromal disease symptoms and eliminated progression to overt diabetes in mouse model of type 1 diabetes, a T-cell–mediated autoimmune disease. GH altered the cytokine environment, triggered anti-inflammatory macrophage (M2) polarization, maintained activity of the suppressor T-cell population, and limited Th17 cell plasticity (49). JAK/STAT signaling, the principal mediator of GHR activation, is well-known to be involved in the modulation of the immune system, so it is tempting to assume that GH may have a role too, but clear data in humans are needed.

Growth Hormone Signaling in Humans

GROWTH RECEPTOR ACTIVATION

GH receptor signaling is a separate and prolific research field by itself (50), so this section will focus on recent data obtained in human models.

GHR have been identified in many tissues including fat, lymphocytes, liver, muscle, heart, kidney, brain, and pancreas (51,52). Activation of receptor-associated Janus kinase (JAK) 2 is the critical step in initiating GH signaling. One GH molecule binds to two GHR molecules that exist as preformed homodimers. Following GH binding, the intracellular domains of the GHR dimer undergo rotation, which brings together the two intracellular domains, each of which bind one JAK2 molecule. This in turn induces cross-phosphorylation of tyrosine residues in the kinase domain of each JAK2 molecule followed by tyrosine phosphorylation of the GHR (51,53). Phosphorylated residues on GHR and JAK2 form docking sites for different signaling molecules including signal transducers and activators of transcription (STAT) 1, 3, 5a and 5b. STATs bound to the activated GHR-JAK2 complex are subsequently phosphorylated on a single tyrosine by JAK2 after which they dimerize and translocate to the nucleus, where they bind to DNA and act as gene transcription factors. A STAT5b binding site has been characterized in the IGF-I gene promoter region, which mediates GH-stimulated IGF-I gene activation (54). Attenuation of JAK2-associated GH signaling is mediated by a family of cytokine-inducible suppressors of cytokine signaling (SOCS) (55). SOCS proteins bind to phosphotyrosine residues on the GHR or JAK2 and suppress GH signaling by inhibiting JAK2 activity and competing with STATs for binding on the GHR. As an example, it has been reported that the inhibitory effect of estrogen on hepatic IGF-I production seems to be mediated via up regulation of SOCS-2 (56).

GH SIGNALING

Data on GHR signaling derive mainly from rodent models and experimental cell lines, although GH-induced activation of the JAK2/STAT5b and the MAPK pathways have been recorded in cultured human fibroblasts from healthy human subjects (57). STAT5b in human subjects is critical for GH-induced IGF-I expression and growth promotion as demonstrated by the identification of mutations in the STAT5b gene of patients presenting with severe GH insensitivity in the presence of normal GHR (58). GHR signaling in human models in vivo has been reported in a study in healthy young male subjects exposed to an intravenous GH bolus vs. saline (59). In muscle and fat biopsies significant tyrosine phosphorylation of STAT5b was recorded after GH exposure at 30-60 minutes. There was no evidence of GH-induced activation of PI 3-kinase, Akt/PKB, or MAPK in either tissue (59).

GH AND INSULIN SIGNALING

There is animal and in vitro evidence to suggest that insulin and GH share post-receptor signaling pathways (60). Convergence has been reported at the levels of STAT5 and SOCS3 (61) as well as on the major insulin signaling pathway: insulin receptor substrates (IRS) 1 and 2, PI 3-kinase, Akt, and extracellular regulated kinases (ERK) 1 and 2 (62,63). Studies in rodent models suggest that the insulin-antagonistic effects of GH in adipose and skeletal muscle involve suppression of insulin-stimulated PI3-kinase activity (60,64). One study assessed the impact of a GH infusion on insulin sensitivity and the activity of PI3-kinase as well as PKB/AKt in skeletal muscle in a controlled design involving healthy young subjects (65). The infusion of GH induced a sustained increase in FFA levels and subsequently insulin resistance as assessed by the euglycemic clamp technique, as expected, but was not associated with any changes in the insulin-stimulated increase in either IRS-1 associated PI3-kinase or PKB/Akt activity. It was subsequently assessed that insulin had no impact on GH-induced STAT5b activation or SOCS3 mRNA expression (66).

INSULIN-LIKE GROWTH FACTOR-I

Physiology of IGF-I

GH acts both directly through its own receptor and indirectly through the induced production of IGF-I. GH stimulates synthesis of IGF-I in the liver and many other GH target tissues (Figure 6); about 75% of circulating IGF-I is liver-derived.IGF-I is a 70 amino-acid peptide, found in the circulation, 99% bound to transport proteins.

Following the initial discovery of IGF-I, it was thought, that GH governs somatic growth only by IGF-I produced by the liver (67). However, in the 1980s this hypothesis was changed by the identification of IGF-I production in numerous tissues (Figure 6). IGF-I is known as a global and tissue-specific growth factor as well as an endocrine factor. In some tissues IGF-I acts as a potent inhibitor of cellular apoptosis.

Figure 6. GH is produced in the pituitary gland. In the periphery, GH acts directly and indirectly through stimulation of IGF-I production. In the circulation, the liver is the most important source of IGF-I (75%) but other tissues (e.g. brain, adipose tissue, kidney, bone, and muscles) may contribute. Under GH stimulation the muscle, adipose tissue, and bone have been shown to secrete IGF-I that has a paracrine/autocrine effect.

Interestingly, insulin and IGF-I share many structural and functional similarities implying that they have originated from the same ancestral molecule. Both molecules could have been part of the cycle of food intake and consequent tissue growth. The IGF-I gene is a member of the insulin gene family and the IGF-I receptor is structurally similar to the insulin receptor in its tetrametric structure, with 2 alpha and 2 beta subunits (68). The alpha subunit binds IGF-I, IGF-II, and insulin; however, the subunit has a higher affinity towards IGF-I compared to IGF-II and insulin. Although insulin and IGF-I share many similarities, during evolution, the functionality of the two molecules has become more divergent, where insulin plays a more metabolic role and IGF-I plays a role in cell growth.

The IGF-I receptor is expressed in many tissues in the body. However, the receptor number on each cell is strictly regulated by several systemic and tissue factors including circulating GH, iodothyronines, platelet-derived growth factor, and fibroblast growth factor. Following the binding of the IGF-I molecule, the receptor undergoes a conformational change, which activates tyrosine kinase, leading to auto-phosphorylation of tyrosine. The activated receptor phosphorylates “insulin receptor substrate-2” (IRS-2), which in-turn activates the RAS activating protein SOS. This complex activates the mitogen activated protein kinase (MAP kinase) pathway. Thus activation of the MAP kinase pathway becomes vital in the stimulation of cell growth by IGF-I (69,70).

IGF-I is bound almost 100% to a family of binding proteins (IGFBP) in the circulation. The IGFBP family comprises six binding proteins (IGFBP 1-6) with a high affinity towards IGF-I and II. Apart from regulating the free plasma IGF fraction, IGFBPs also play an important role in the transport of IGF into different tissues and extravascular space. IGFBP-3 and IGFBP-2 are the most abundant forms seen in plasma and are saturated with IGF-I due to their high affinity. 75% of IGF-I is bound to IGFBP-3. Interestingly, similar to IGF-I, IGFBP-3 production is also regulated by GH. In the plasma, IGFBP-3 is bound to a protein called acid labile subunit (ALS), which stabilizes the “IGFBP3-IGF-I” complex, prolonging its half-life to approximately 16 hours (71). IGFBP-1, on the other hand, is present in lower concentration in plasma than IGFBP-2 and 3. However, due to lower affinity for IGF-I, IGFBP-1 is usually in an unsaturated state and changing plasma concentrations of IGFBP-1 becomes important in determining the unbound fraction of IGF-I. A recently new discovered player in the regulation of IGF-I bioavailability is the pregnancy-associated plasma protein-A2 (PAPP-A2) that cleaves IGFBP3 and 5 and releases IGF-I. Homozygous mutations in PAPP-A2 result in growth failure with elevated total but low free IGF-I (72). Low IGF-I bioavailability impairs growth and glucose metabolism in a mouse model of human PAPP-A2 deficiency and treatment with recombinant human IGF-I in PAPP-A2 deficient patients improves growth and bone mass and ameliorates glucose metabolism (72,73).

Effects of IGF-I

Studies on hypophysectomized animals overexpressing IGF-I demonstrate the independent anabolic effects of IGF-I (74). IGF-I plays a key role in growth, where it acts not only as a determinant of postnatal growth, but also as an intra-uterine growth promoter. Total inactivation of the IGF-I gene in mice produce a perinatal mortality of 80% with the surviving animal showing significant growth retardation compared to controls (75). Human IGF-I deficiency can be either due to GH deficiency, GHR inactivation, or IGF-I gene mutation. Interestingly, infants with congenital GH deficiency and GHR mutations present with only minor growth retardation, whereas the rare patient with IGF-I deficiency, secondary to a homozygous partial deletion of the IGF-I gene, presents with severe pre and postnatal growth failure, mental retardation, sensorineural deafness, and microcephaly (76-78). The differences in the clinical presentation are most likely due to the fact that some degree of IGF-I production is present in patients with GH deficiency, GHR, and GHRH defects. More detailed studies on transgenic mice have clearly demonstrated this fact with selective deletion of IGF-I gene expression only in the liver, showing low serum IGF-I concentrations with only 6-8% postnatal growth retardation. In contrast, animals with total IGF-I deletion or only peripherally produced IGF-I deletion showed marked growth retardation (79).

Both elevated and reduced levels of serum IGF-I are associated with excess mortality in human adults (80). In addition, it is well recognized in many species including worms, flies, rodents, and primates that a reciprocal relationship exists between longevity and activation of the insulin/IGF axis (80). The underlying mechanisms are subject to continued scrutiny and are likely to be complex. In this regard, it is noteworthy that calorie restriction is associated with increased longevity and reduced insulin/IGF activity in many species (81) albeit GH levels are increased by calorie restriction and fasting (82).

In the context of GH and IGF-I physiology it can be concluded that 1) during childhood and adolescence the combined actions of GH and IGF-I in the presence of sufficient nutrition promote longitudinal growth and somatic maturation, 2) continued excess IGF-I activity in adulthood increases the risk for cardiovascular and neoplastic diseases and hence reduces longevity, 3) calorie restriction, which suppresses IGF-I activity and stimulates GH secretion, may promote longevity in human adults (82).

METABOLIC EFFECTS OF GROWTH HORMONE

Nutritional status dictates GH effects. In the state of feast and sufficient nutrient intake where insulin is increased in the liver and IGF-I production is stimulated, GH promotes protein anabolism. Whereas, in the state with decreased nutrient intake and during sleep and exercise, the direct effect of GH are more predominant and this is mainly characterized by stimulation of lipolysis.

Glucose Homeostasis and Lipid Metabolism

The involvement of the pituitary gland in the regulation of substrate metabolism was originally detailed in the classic dog studies by Houssay (83). Fasting hypoglycemia and pronounced sensitivity to insulin were distinct features of hypophysectomized animals. These symptoms were readily corrected by the administration of anterior pituitary extracts. It was also noted that pancreatic diabetes was alleviated by hypophysectomy. Finally, excess of anterior pituitary lobe extracts aggravated or induced diabetes in hypophysectomized dogs. Furthermore glycemic control deteriorates following exposure to a single supraphysiological dose of human GH in hypophysectomized adults with type 1 diabetes mellitus (84). Somewhat surprisingly, only modest effects of GH on glucose metabolism were recorded in the first metabolic balance studies involving adult hypopituitary patients (85,86).

More recent studies on glucose homeostasis in GH deficient adults have generated results, which at first glance may appear contradictory. Insulin resistance may be more prevalent in untreated GH deficient adults, whereas the impact of GH replacement on this feature seems to depend on the duration and the dose (87).

Below, some of the metabolic effects of GH in human subjects, with special reference to the interaction between glucose and lipid metabolism, will be reviewed.

STUDIES IN NORMAL ADULTS

More than fifty years ago, it was shown that infusion of high dose GH into the brachial artery of healthy adults reduced forearm glucose uptake in both muscle and adipose tissue, which was paralleled by increased uptake and oxidation of FFA (88). This pattern was opposite to that of insulin, and GH in the same model abrogated the metabolic actions of insulin.

Administration of a GH bolus in the post absorptive state stimulates lipolysis following a lag time of 2-3 hours (89). Plasma levels of glucose and insulin, on the other hand, change very little. This is associated with small reductions in muscular glucose uptake and oxidation, which could reflect substrate competition between glucose and fatty acids (i.e., the glucose/fatty acid cycle) (Figure 7). In line with this, sustained exposure to high GH levels induces both hepatic and peripheral (muscular) resistance to the actions of insulin on glucose metabolism together with increased (or inadequately suppressed) lipid oxidation. However, GH excess reduces intrahepatic lipid content suggesting that GH-induced insulin resistance is not associated with hepatic lipid accumulation (90). Apart from enhanced glucose/fatty acid cycling, it has been shown that GH induced insulin resistance is accompanied by reduced muscle glycogen synthase activity (91) and diminished glucose dependent glucose disposal (92). Bak et al. also demonstrated insulin binding and insulin receptor kinase activity from muscle biopsies to be unaffected by GH (91).

Undoubtedly, a causal link exists between GH-induced lipolysis and insulin resistance (93). Acute GH exposure in healthy individuals downregulates important suppressors of lipolysis, the G0/G1 switch gene (G0S2) and fat specific protein 27 (FSP27), in addition to regulating the suppressor of the insulin signaling, phosphatase and tensin homolog (PTEN) (94).

LESSONS FROM ACROMEGALY

Active acromegaly clearly unmasks the diabetogenic properties of GH. In the basal state plasma glucose is elevated despite compensatory hyperinsulinemia. In the basal and insulin-stimulated state (euglycemic glucose clamp) hepatic and peripheral insulin resistance is associated with enhanced lipid oxidation and energy expenditure (95). There is evidence to suggest that this hyper-metabolic state ultimately leads to beta cell exhaustion and overt diabetes mellitus (96), but it is also demonstrated that the abnormalities are completely reversed after successful surgery (95). Conversely, it has been shown that only two weeks of the administration of GH in supraphysiological doses induces comparable acromegaloid, and reversible abnormalities in substrate metabolism and insulin sensitivity (97).

Interaction of Glucose and Lipid Metabolism

Relatively few studies have scrutinized the exact modes of action of GH on glucose metabolism. There is no evidence of a GH effect on insulin binding to the receptor (91,98), which obviously implies post receptor metabolic effects. The effect of FFA on the partitioning of intracellular glucose fluxes was originally described by Randle et al. (99). According to his hypothesis (the glucose/fatty acid cycle), oxidation of FFA initiates an upstream, chain-reaction-like inhibition of glycolytic enzymes, which ultimately inhibits glucose uptake (Figure 7).

Figure 7. The glucose fatty-acid cycle.

Randle proposed in 1963 that increased FFA compete with and displace glucose utilization leading to a decreased glucose uptake. The hypothesis stated that an increase in fatty acid oxidation in muscle and fat results in higher acetyl CoA in mitochondria leading to inactivation of two rate-limiting enzymes of glycolysis (i.e., phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH) complex). A subsequent increase in intracellular glucose-6-phosphate (glucose 6-P) results in high intracellular glucose concentrations and decreased glucose uptake by muscle and fat.

However, in contrast to the proposed hypothesis by Randle, studies using MR spectroscopy have shown reductions in intramyocellular glucose 6-P and glucose concentrations and have led to an alternative hypothesis. The new hypothesis proposes that a transient increase of intracellular diacylglycerol (DAG) activates theta isoform of protein kinase C (PKCθ) that causes increased serine phosphorylation of IRS-1/2 and consecutively decrease PI3K activation and glucose-transport activity leading to decrease intracellular glucose concentrations

When considering the pronounced lipolytic effects of GH the Randle hypothesis remains an appealing model to explain the insulin-antagonistic effects of GH. In support of this, experiments have shown that co-administration of anti-lipolytic agents and GH reverses GH-induced insulin resistance. Similar conclusions were drawn from a recent study in GH deficient adults, which showed that insulin sensitivity was restored when acipimox (a nicotinic acid derivative) was co-administered with GH (100). We have also shown that GH-induced insulin resistance is associated with suppressed pyruvate dehydrogenase activity in skeletal muscle (101). It has, however, also been reported that GH-induced insulin resistance precedes the increase in circulating levels of fatty acids and forearm uptake of lipid intermediates (102). This early effect of GH on muscular glucose uptake could reflect intramyocytic FFA release and oxidation and thus be compatible with the Randle hypothesis. According to the Randle hypothesis the fatty acid-induced insulin resistance will result in elevated intracellular levels of both glucose and glucose-6-phosphate (Figure 7). By contrast, muscle biopsies from GH deficient adults after GH treatment have revealed increased glucose but low-normal glucose-6-phosphate levels (103). Moreover, NMR spectroscopy studies in healthy adults indicate that FFA infusion results in a drop in the levels of both glucose and glucose-6-phosphate (104). The latter study, which did not involve GH administration, reported that FFA suppressed the activity of PI-3 kinase, an enzyme stimulated by insulin, which is considered essential for glucose transportation into skeletal muscle via translocation of glucose transporter activity (GLUT 4). A more recent study showed that GH infusion does not impact insulin-stimulated PI-3 kinase activity (65).

IMPLICATIONS FOR GH REPLACEMENT

Regardless of the exact mechanisms, the insulin antagonistic effects may cause concern when replacing adult GH deficient patients with GH, since some of these patients are insulin resistant in the untreated state. There is evidence to suggest that the direct metabolic effects on GH may be balanced by long-term beneficial effects on body composition and physical fitness, but some studies report impaired insulin sensitivity in spite of favorable changes in body composition. There is little doubt that these effects of GH are dose-dependent and may be minimized or avoided if an appropriately low replacement dose is used. Still, the pharmacokinetics of subcutaneous (s.c.) GH administration is unable to mimic the endogenous GH pattern with suppressed levels after meals and elevations only during post absorptive periods, such as during the night. This may be considered the natural domain of GH action, which coincides with minimal beta-cell challenge. This reciprocal association between insulin and GH and its potential implications for normal substrate metabolism was initially described by Rabinowitz & Zierler (105). The problem arises when GH levels are elevated during repeated prandial periods. The classic example is active acromegaly, but prolonged high dose s.c. GH administration may cause similar effects. Administration of GH in the evening probably remains the best compromise between effects and side effects (106), but it is far from physiological.

Long-acting GH analogues have been developed to improve adherence and compliance. The clinical experience is limited now but seem not to impact adversely the glucose metabolism compared with daily GH (107). However, long-term surveillance data are required to consolidate its safety profile (108).

Effects of GH on Muscle Mass and Function

The anabolic nature of GH is clearly evident in patients with acromegaly and vice versa in patients with GH deficiency. A large number of in vitro and animal studies throughout several decades have documented stimulating effects of GH on skeletal muscle growth. The methods employed to document in vivo effects of GH on muscle mass in humans have been exhaustive including whole body retention of nitrogen and potassium, total and regional muscle protein metabolism using labeled amino acids, estimation of lean body mass by total body potassium or dual x-ray absorptiometry, and direct calculation of muscle area or volume by computerized tomography (CT) and magnetic resonance imaging.

EFFECT OF GH ON SKELETAL MUSCLE METABOLISM IN VITRO AND IN VIVO

The clinical picture of acromegaly and gigantism includes increased lean body mass of which skeletal muscle mass accounts for approximately 50%. Moreover, retention of nitrogen was one of the earliest observed and most reproducible effects of GH administration in humans (1). Thoroughly conducted studies with GH administration in GH deficient children using a variety of classic anthropometric techniques strongly suggested that skeletal muscle mass increased significantly during treatment (109,110). Indirect evidence of an increase in muscle cell number following GH treatment was also presented (110).

These early clinical studies were paralleled by experimental studies in rodent models. GH administration in hypophysectomized rats increased not only muscle mass, but also muscle cell number (i.e., muscle DNA content) (110). Interestingly, the same series of experiments revealed that work-induced muscle hypertrophy could occur in the absence of GH. The ability of GH to stimulate RNA synthesis and amino acid incorporation into protein of isolated rat diaphragm suggested direct mechanisms of actions, whereas direct effects of GH on protein synthesis could not be induced in liver cell cultures (111). Another important observation of that period was made by Goldberg, who studied protein turnover in skeletal muscle of hypophysectomized rats with 3H-leucine tracer techniques. In these studies it was convincingly demonstrated that GH directly increased the synthesis of both sarcoplasmic and myofibrillar protein without affecting proteolysis (112).

The most substantial recent contributions within the field derive from human in vivo studies of the effects of systemic and local GH and IGF-I administration on total and regional protein metabolism by means of amino acid isotope dilution techniques. Systemic GH administration for 7 days in normal adults increased whole body protein synthesis without affecting proteolysis (113), and similar data were subsequently obtained in GH deficient adults (114). Systemically infused GH for 8 hours in normal adults lead to an acute stimulation of forearm (muscle) protein synthesis without any effects on whole body protein synthesis (115). By contrast in a design that also included co-administration of somatostatin to suppress insulin, an acute stimulatory effect of GH on whole body protein synthesis was observed, but no stimulatory effect on leg protein synthesis (116), Finally, infusion of GH into the brachial artery was accompanied by a local increase in forearm muscle protein synthesis (117).

Based on these studies it seems that the nitrogen retaining properties of GH predominantly involve stimulation of protein synthesis without affecting (lowering) proteolysis. Theoretically, the protein anabolic effects of GH could be either direct or mediated through IGF-I, insulin, or lipid intermediates. GHR are present in skeletal muscle (52), which combined with Fryburg’s intra-arterial GH studies, makes a direct GH effect conceivable. An alternative interpretation could be that GH stimulates local muscle IGF-I release, which subsequently acts in an autocrine/paracrine manner. The effects of systemic IGF-I administration on whole body protein metabolism seem to depend on ambient amino acid levels in the sense that IGF-I administered alone suppresses proteolysis (118) whereas IGF-I in combination with an amino acid infusion increase protein synthesis (119). Moreover, intra-arterial IGF-I in combination with systemic amino acid infusion increased protein synthesis (120). It is therefore likely that the muscle anabolic effects of GH, at least to some extent, are mediated by IGF-I. By contrast, it is repeatedly shown that insulin predominantly acts through suppression of proteolysis and this effect(s) appears to be blunted by co-administration of GH (121). The degree to which mobilization of lipids contributes to the muscle anabolic actions of GH has so far not been specifically investigated.

In conclusion several experimental lines of evidence strongly suggest that GH stimulates muscle protein synthesis. This effect is presumably in part mediated through binding of GH to GHR in skeletal muscle. This does not rule out a significant role of IGF-I being produced either systematically or locally.

An interesting discovery has been that infusion of GH and IGF-I into the brachial artery increases forearm blood flow several fold (117,122). This effect appears to be mediated through stimulation of endothelial nitric oxide release leading to local vasodilatation (123,124). Thus, it appears that an IGF-I mediated increase in muscle nitric oxide release accounts for some of the effects of GH on skeletal muscle protein synthesis. These intriguing observations may have many other implications. It is, for instance, tempting to speculate that this increase in skeletal muscle blood flow contributes to the GH induced increase in resting energy expenditure, since skeletal muscle metabolism is a major determinant of resting energy expenditure (24). Moreover, it is plausible that the reduction in total peripheral resistance seen after GH administration in adult growth hormone deficiency is mediated by nitric oxide (124).

EFFECTS OF GH ADMINISTRATION ON MUSCLE MASS AND FUNCTION IN ADULTS WITHOUT GH DEFICIENCY

As previously mentioned, the ability of acute and more prolonged GH administration to retain nitrogen in healthy adults has been known for decades and more recent studies have documented a stimulatory effect on whole body and forearm protein synthesis.

Rudman et al. was the first to suggest that the senescent changes in body composition were causally linked to the concomitant decline in circulation GH and IGF-I levels (24). This concept has been recently reviewed (125) and a number of studies with GH and other anabolic agents for treating the sarcopenia of ageing are currently in progress.

Placebo-controlled GH administration in young healthy adults (21-34 yr) undergoing a resistance exercise program for 12 weeks showed a GH induced increase in lean body mass (LBM), whole body protein balance, and whole body protein synthesis, whereas quadriceps muscle protein synthesis rate and muscle strength increased to the same degree in both groups during training (126). In a similar study in older men (67 yr) GH also increased LBM and whole body protein synthesis, without significantly amplifying the effects of exercise on muscle protein synthesis or muscle strength (127). An increase in LBM but unaltered muscle strength following 10 weeks of GH administration plus resistance exercise training was also recorded (128). A more recent study of 52 older men (70-85 yr) treated with either GH or placebo for 6 months, without concomitant exercise, observed a significant increase (4.4 %) in LBM with GH, but no significant effects on muscle strength (129). A meta-analysis of studies administering GH to healthy adult subjects demonstrate that it increases lean body mass and reduces fat mass without improving muscle strength or aerobic exercise capacity (130).

Numerous studies have evaluated the effects of GH administration in chronic and acute catabolic illness. A comprehensive survey of the prolific literature within this field is beyond the scope of this review, but it is noteworthy, that HIV-associated body wasting is a licensed indication for GH treatment in the USA. In this patient category GH treatment for 12 weeks has been associated with significant increments in LBM and physical fitness (131,132).

CONCLUSIONS

GH/IGF-I axis is specifically regulated and is involved in a multitude of processes during all aspects of life from intrauterine growth, to childhood and puberty, adulthood, and lastly elderly periods. GH actions directly or via its principal metabolite, IGF-I have a wide range of physiological roles being a metabolic active hormone in adulthood. Nutritional status of an organism dictates the effects of GH, either an impairment of insulin action (fasted state) or promoting protein anabolism (feed state). As our knowledge of the GH normal physiology increases, our ability to understand and specifically target the GH/IGF-I pathway for a diverse range of therapeutic purposes should also increase.

REFERENCES

Raben MS. Growth hormone. 1. Physiologic aspects. The New England journal of medicine. 1962;266:31-35.
Petronella N, Drouin G. Gene conversions in the growth hormone gene family of primates: stronger homogenizing effects in the Hominidae lineage. Genomics. 2011;98(3):173-181.
Baumann GP. Growth hormone doping in sports: a critical review of use and detection strategies. Endocrine reviews. 2012;33(2):155-186.
Holt RIG, Ho KKY. The Use and Abuse of Growth Hormone in Sports. Endocrine reviews. 2019;40(4):1163-1185.
Cheung LYM, George AS, McGee SR, et al. Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types. Endocrinology. 2018;159(12):3910-3924.
Willems C, Fu Q, Roose H, et al. Regeneration in the pituitary after cell-ablation injury: time-related aspects and molecular analysis. Endocrinology. 2015:en20151741.
Ribeiro-Oliveira A, Barkan AL. Growth Hormone Pulsatility and its Impact on Growth and Metabolism in Humans. in K. Ho (ed.), Growth Hormone Related Diseases and Therapy: A Molecular and Physiological Perspective for the Clinician, Contemporary Endocrinology. 2011:33-56.
Ho KY, Evans WS, Blizzard RM, et al. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. The Journal of clinical endocrinology and metabolism. 1987;64(1):51-58.
Leung KC, Johannsson G, Leong GM, Ho KK. Estrogen regulation of growth hormone action. Endocrine reviews. 2004;25(5):693-721.
Kargi AY, Merriam GR. Diagnosis and treatment of growth hormone deficiency in adults. Nature reviews. Endocrinology. 2013;9(6):335-345.
Iacovazzo D, Hernandez-Ramirez LC, Korbonits M. Sporadic pituitary adenomas: the role of germline mutations and recommendations for genetic screening. Expert review of endocrinology & metabolism. 2017;12(2):143-153.
Birzniece V, Ho KKY. Sex steroids and the GH axis: Implications for the management of hypopituitarism. Best practice & research. Clinical endocrinology & metabolism. 2017;31(1):59-69.
Veldhuis JD, Evans WS, Bowers CY, Anderson S. Interactive regulation of postmenopausal growth hormone insulin-like growth factor axis by estrogen and growth hormone-releasing peptide-2. Endocrine. 2001;14(1):45-62.
Bray MJ, Vick TM, Shah N, et al. Short-term estradiol replacement in postmenopausal women selectively mutes somatostatin's dose-dependent inhibition of fasting growth hormone secretion. The Journal of clinical endocrinology and metabolism. 2001;86(7):3143-3149.
Anderson SM, Shah N, Evans WS, Patrie JT, Bowers CY, Veldhuis JD. Short-term estradiol supplementation augments growth hormone (GH) secretory responsiveness to dose-varying GH-releasing peptide infusions in healthy postmenopausal women. The Journal of clinical endocrinology and metabolism. 2001;86(2):551-560.
Murray PG, Higham CE, Clayton PE. 60 YEARS OF NEUROENDOCRINOLOGY: The hypothalamo-GH axis: the past 60 years. The Journal of endocrinology. 2015;226(2):T123-140.
Bonnefont X, Lacampagne A, Sanchez-Hormigo A, et al. Revealing the large-scale network organization of growth hormone-secreting cells. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(46):16880-16885.
Le Tissier PR, Carmignac DF, Lilley S, et al. Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene. Molecular endocrinology. 2005;19(5):1251-1262.
Wajnrajch MP, Gertner JM, Harbison MD, Chua SC, Jr., Leibel RL. Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nature genetics. 1996;12(1):88-90.
Alatzoglou KS, Dattani MT. Genetic causes and treatment of isolated growth hormone deficiency-an update. Nature reviews. Endocrinology. 2010;6(10):562-576.
Giustina A, Veldhuis JD. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocrine reviews. 1998;19(6):717-797.
Ren SG, Taylor J, Dong J, Yu R, Culler MD, Melmed S. Functional association of somatostatin receptor subtypes 2 and 5 in inhibiting human growth hormone secretion. The Journal of clinical endocrinology and metabolism. 2003;88(9):4239-4245.
Copinschi G, Wegienka LC, Hane S, Forsham PH. Effect of arginine on serum levels of insulin and growth hormone in obese subjects. Metabolism: clinical and experimental. 1967;16(6):485-491.
Rudman D, Kutner MH, Rogers CM, Lubin MF, Fleming GA, Bain RP. Impaired growth hormone secretion in the adult population: relation to age and adiposity. The Journal of clinical investigation. 1981;67(5):1361-1369.
Rudman D. Growth hormone, body composition, and aging. Journal of the American Geriatrics Society. 1985;33(11):800-807.
Jorgensen JO, Vahl N, Hansen TB, Thuesen L, Hagen C, Christiansen JS. Growth hormone versus placebo treatment for one year in growth hormone deficient adults: increase in exercise capacity and normalization of body composition. Clinical endocrinology. 1996;45(6):681-688.
Williams T, Berelowitz M, Joffe SN, et al. Impaired growth hormone responses to growth hormone-releasing factor in obesity. A pituitary defect reversed with weight reduction. The New England journal of medicine. 1984;311(22):1403-1407.
Vahl N, Jorgensen JO, Jurik AG, Christiansen JS. Abdominal adiposity and physical fitness are major determinants of the age associated decline in stimulated GH secretion in healthy adults. The Journal of clinical endocrinology and metabolism. 1996;81(6):2209-2215.
Vahl N, Jorgensen JO, Skjaerbaek C, Veldhuis JD, Orskov H, Christiansen JS. Abdominal adiposity rather than age and sex predicts mass and regularity of GH secretion in healthy adults. The American journal of physiology. 1997;272(6 Pt 1):E1108-1116.
Papadakis MA, Grady D, Tierney MJ, Black D, Wells L, Grunfeld C. Insulin-like growth factor 1 and functional status in healthy older men. Journal of the American Geriatrics Society. 1995;43(12):1350-1355.
Goodman-Gruen D, Barrett-Connor E. Epidemiology of insulin-like growth factor-I in elderly men and women. The Rancho Bernardo Study. American journal of epidemiology. 1997;145(11):970-976.
Kiel DP, Puhl J, Rosen CJ, Berg K, Murphy JB, MacLean DB. Lack of an association between insulin-like growth factor-I and body composition, muscle strength, physical performance or self-reported mobility among older persons with functional limitations. Journal of the American Geriatrics Society. 1998;46(7):822-828.
Juul A, Bang P, Hertel NT, et al. Serum insulin-like growth factor-I in 1030 healthy children, adolescents, and adults: relation to age, sex, stage of puberty, testicular size, and body mass index. The Journal of clinical endocrinology and metabolism. 1994;78(3):744-752.
Rudman D, Feller AG, Nagraj HS, et al. Effects of human growth hormone in men over 60 years old. The New England journal of medicine. 1990;323(1):1-6.
Jorgensen JO, Flyvbjerg A, Lauritzen T, Alberti KG, Orskov H, Christiansen JS. Dose-response studies with biosynthetic human growth hormone (GH) in GH-deficient patients. The Journal of clinical endocrinology and metabolism. 1988;67(1):36-40.
Moller J, Jorgensen JO, Lauersen T, et al. Growth hormone dose regimens in adult GH deficiency: effects on biochemical growth markers and metabolic parameters. Clinical endocrinology. 1993;39(4):403-408.
Toogood AA, Shalet SM. Growth hormone replacement therapy in the elderly with hypothalamic-pituitary disease: a dose-finding study. The Journal of clinical endocrinology and metabolism. 1999;84(1):131-136.
Burman P, Johansson AG, Siegbahn A, Vessby B, Karlsson FA. Growth hormone (GH)-deficient men are more responsive to GH replacement therapy than women. The Journal of clinical endocrinology and metabolism. 1997;82(2):550-555.
Vahl N, Moller N, Lauritzen T, Christiansen JS, Jorgensen JO. Metabolic effects and pharmacokinetics of a growth hormone pulse in healthy adults: relation to age, sex, and body composition. The Journal of clinical endocrinology and metabolism. 1997;82(11):3612-3618.
Fisker S, Vahl N, Jorgensen JO, Christiansen JS, Orskov H. Abdominal fat determines growth hormone-binding protein levels in healthy nonobese adults. The Journal of clinical endocrinology and metabolism. 1997;82(1):123-128.
Aguiar-Oliveira MH, Bartke A. Growth Hormone Deficiency: Health and Longevity. Endocrine reviews. 2019;40(2):575-601.
Guevara-Aguirre J, Balasubramanian P, Guevara-Aguirre M, et al. Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans. Science translational medicine. 2011;3(70):70ra13.
Chesnokova V, Zonis S, Zhou C, et al. Growth hormone is permissive for neoplastic colon growth. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(23):E3250-3259.
Kleinberg DL, Wood TL, Furth PA, Lee AV. Growth hormone and insulin-like growth factor-I in the transition from normal mammary development to preneoplastic mammary lesions. Endocrine reviews. 2009;30(1):51-74.
Slater MD, Murphy CR. Co-expression of interleukin-6 and human growth hormone in apparently normal prostate biopsies that ultimately progress to prostate cancer using low pH, high temperature antigen retrieval. Journal of molecular histology. 2006;37(1-2):37-41.
Khan J, Pernicova I, Nisar K, Korbonits M. Mechanisms of ageing: growth hormone, dietary restriction, and metformin. The lancet. Diabetes & endocrinology. 2023;11(4):261-281.
Chesnokova V, Zonis S, Apostolou A, et al. Local non-pituitary growth hormone is induced with aging and facilitates epithelial damage. Cell reports. 2021;37(11):110068.
Bodart G, Farhat K, Charlet-Renard C, Salvatori R, Geenen V, Martens H. The Somatotrope Growth Hormone-Releasing Hormone/Growth Hormone/Insulin-Like Growth Factor-1 Axis in Immunoregulation and Immunosenescence. Frontiers of hormone research. 2017;48:147-159.
Villares R, Kakabadse D, Juarranz Y, Gomariz RP, Martinez AC, Mellado M. Growth hormone prevents the development of autoimmune diabetes. Proceedings of the National Academy of Sciences of the United States of America. 2013.
Lanning NJ, Carter-Su C. Recent advances in growth hormone signaling. Rev Endocr Metab Disord. 2006;7(4):225-235.
Dehkhoda F, Lee CMM, Medina J, Brooks AJ. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects. Frontiers in endocrinology. 2018;9:35.
Kelly PA, Djiane J, Postel-Vinay MC, Edery M. The prolactin/growth hormone receptor family. Endocrine reviews. 1991;12(3):235-251.
Brooks AJ, Dai W, O'Mara ML, et al. Mechanism of activation of protein kinase JAK2 by the growth hormone receptor. Science (New York, N.Y.). 2014;344(6185):1249783.
Woelfle J, Chia DJ, Rotwein P. Mechanisms of growth hormone (GH) action. Identification of conserved Stat5 binding sites that mediate GH-induced insulin-like growth factor-I gene activation. The Journal of biological chemistry. 2003;278(51):51261-51266.
Wormald S, Hilton DJ. Inhibitors of cytokine signal transduction. The Journal of biological chemistry. 2004;279(2):821-824.
Leung KC, Doyle N, Ballesteros M, et al. Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(3):1016-1021.
Silva CM, Kloth MT, Whatmore AJ, et al. GH and epidermal growth factor signaling in normal and Laron syndrome fibroblasts. Endocrinology. 2002;143(7):2610-2617.
Hwa V, Little B, Adiyaman P, et al. Severe growth hormone insensitivity resulting from total absence of signal transducer and activator of transcription 5b. The Journal of clinical endocrinology and metabolism. 2005;90(7):4260-4266.
Jorgensen JO, Jessen N, Pedersen SB, et al. GH receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus. American journal of physiology. Endocrinology and metabolism. 2006;291(5):E899-905.
Dominici FP, Argentino DP, Munoz MC, Miquet JG, Sotelo AI, Turyn D. Influence of the crosstalk between growth hormone and insulin signalling on the modulation of insulin sensitivity. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society. 2005;15(5):324-336.
Emanuelli B, Peraldi P, Filloux C, Sawka-Verhelle D, Hilton D, Van Obberghen E. SOCS-3 is an insulin-induced negative regulator of insulin signaling. The Journal of biological chemistry. 2000;275(21):15985-15991.
Ridderstrale M, Degerman E, Tornqvist H. Growth hormone stimulates the tyrosine phosphorylation of the insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase in primary adipocytes. The Journal of biological chemistry. 1995;270(8):3471-3474.
Thirone AC, Carvalho CR, Saad MJ. Growth hormone stimulates the tyrosine kinase activity of JAK2 and induces tyrosine phosphorylation of insulin receptor substrates and Shc in rat tissues. Endocrinology. 1999;140(1):55-62.
del Rincon JP, Iida K, Gaylinn BD, et al. Growth hormone regulation of p85alpha expression and phosphoinositide 3-kinase activity in adipose tissue: mechanism for growth hormone-mediated insulin resistance. Diabetes. 2007;56(6):1638-1646.
Jessen N, Djurhuus CB, Jorgensen JO, et al. Evidence against a role for insulin-signaling proteins PI 3-kinase and Akt in insulin resistance in human skeletal muscle induced by short-term GH infusion. American journal of physiology. Endocrinology and metabolism. 2005;288(1):E194-199.
Nielsen C, Gormsen LC, Jessen N, et al. Growth hormone signaling in vivo in human muscle and adipose tissue: impact of insulin, substrate background, and growth hormone receptor blockade. The Journal of clinical endocrinology and metabolism. 2008;93(7):2842-2850.
Salmon WD, Jr., Daughaday WH. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. The Journal of laboratory and clinical medicine. 1957;49(6):825-836.
Werner H, Weinstein D, Bentov I. Similarities and differences between insulin and IGF-I: structures, receptors, and signalling pathways. Archives of physiology and biochemistry. 2008;114(1):17-22.
Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine & growth factor reviews. 2005;16(4-5):421-439.
Kim JJ, Accili D. Signalling through IGF-I and insulin receptors: where is the specificity? Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society. 2002;12(2):84-90.
Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. Endocrine reviews. 2002;23(6):824-854.
Cabrera-Salcedo C, Mizuno T, Tyzinski L, et al. Pharmacokinetics of IGF-1 in PAPP-A2-Deficient Patients, Growth Response, and Effects on Glucose and Bone Density. The Journal of clinical endocrinology and metabolism. 2017;102(12):4568-4577.
Fujimoto M, Andrew M, Liao L, et al. Low IGF-I Bioavailability Impairs Growth and Glucose Metabolism in a Mouse Model of Human PAPPA2 p.Ala1033Val Mutation. Endocrinology. 2019;160(6):1363-1376.
Behringer RR, Lewin TM, Quaife CJ, Palmiter RD, Brinster RL, D'Ercole AJ. Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology. 1990;127(3):1033-1040.
Powell-Braxton L, Hollingshead P, Giltinan D, Pitts-Meek S, Stewart T. Inactivation of the IGF-I gene in mice results in perinatal lethality. Annals of the New York Academy of Sciences. 1993;692:300-301.
Gluckman PD, Gunn AJ, Wray A, et al. Congenital idiopathic growth hormone deficiency associated with prenatal and early postnatal growth failure. The International Board of the Kabi Pharmacia International Growth Study. The Journal of pediatrics. 1992;121(6):920-923.
Savage MO, Blum WF, Ranke MB, et al. Clinical features and endocrine status in patients with growth hormone insensitivity (Laron syndrome). The Journal of clinical endocrinology and metabolism. 1993;77(6):1465-1471.
Woods KA, Camacho-Hubner C, Savage MO, Clark AJ. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. The New England journal of medicine. 1996;335(18):1363-1367.
Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Developmental biology. 2001;229(1):141-162.
Bartke A, Sun LY, Longo V. Somatotropic signaling: trade-offs between growth, reproductive development, and longevity. Physiological reviews. 2013;93(2):571-598.
Fontana L, Partridge L, Longo VD. Extending healthy life span--from yeast to humans. Science (New York, N.Y.). 2010;328(5976):321-326.
Moller N, Jorgensen JO. Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocrine reviews. 2009;30(2):152-177.
BA H. The hypophysis and metabolism. The New England journal of medicine. 1936;214:961-985.
Luft R, Ikkos D, Gemzell CA, Olivecrona H. Effect of human growth hormone in hypophysectomised diabetic subjects. Lancet. 1958;1(7023):721-722.
Raben MS, Hollenberg CH. Effect of growth hormone on plasma fatty acids. The Journal of clinical investigation. 1959;38(3):484-488.
Henneman DH, Henneman PH. Effects of human growth hormone on levels of blood urinary carbohydrate and fat metabolites in man. The Journal of clinical investigation. 1960;39:1239-1245.
Hew FL, Koschmann M, Christopher M, et al. Insulin resistance in growth hormone-deficient adults: defects in glucose utilization and glycogen synthase activity. The Journal of clinical endocrinology and metabolism. 1996;81(2):555-564.
Rabinowitz D, Klassen GA, Zierler KL. Effect of human growth hormone on muscle and adipose tissue metabolism in the forearm of man. The Journal of clinical investigation. 1965;44:51-61.
Moller N, Jorgensen JO, Schmitz O, et al. Effects of a growth hormone pulse on total and forearm substrate fluxes in humans. The American journal of physiology. 1990;258(1 Pt 1):E86-91.
Arlien-Søborg MC, Madsen MA, Dal J, et al. Ectopic lipid deposition and insulin resistance in patients with GH disorders before and after treatment. European journal of endocrinology / European Federation of Endocrine Societies. 2023;188(1).
Bak JF, Moller N, Schmitz O. Effects of growth hormone on fuel utilization and muscle glycogen synthase activity in normal humans. The American journal of physiology. 1991;260(5 Pt 1):E736-742.
Orskov L, Schmitz O, Jorgensen JO, et al. Influence of growth hormone on glucose-induced glucose uptake in normal men as assessed by the hyperglycemic clamp technique. The Journal of clinical endocrinology and metabolism. 1989;68(2):276-282.
Hjelholt AJ, Charidemou E, Griffin JL, et al. Insulin resistance induced by growth hormone is linked to lipolysis and associated with suppressed pyruvate dehydrogenase activity in skeletal muscle: a 2 × 2 factorial, randomised, crossover study in human individuals. Diabetologia. 2020;63(12):2641-2653.
Hjelholt AJ, Lee KY, Arlien-Søborg MC, et al. Temporal patterns of lipolytic regulators in adipose tissue after acute growth hormone exposure in human subjects: A randomized controlled crossover trial. Molecular metabolism. 2019;29:65-75.
Moller N, Schmitz O, Joorgensen JO, et al. Basal- and insulin-stimulated substrate metabolism in patients with active acromegaly before and after adenomectomy. The Journal of clinical endocrinology and metabolism. 1992;74(5):1012-1019.
Sonksen PH, Greenwood FC, Ellis JP, Lowy C, Rutherford A, Nabarro JD. Changes of carbohydrate tolerance in acromegaly with progress of the disease and in response to treatment. The Journal of clinical endocrinology and metabolism. 1967;27(10):1418-1430.
Moller N, Moller J, Jorgensen JO, et al. Impact of 2 weeks high dose growth hormone treatment on basal and insulin stimulated substrate metabolism in humans. Clinical endocrinology. 1993;39(5):577-581.
Rosenfeld RG, Wilson DM, Dollar LA, Bennett A, Hintz RL. Both human pituitary growth hormone and recombinant DNA-derived human growth hormone cause insulin resistance at a postreceptor site. The Journal of clinical endocrinology and metabolism. 1982;54(5):1033-1038.
Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1963;1(7285):785-789.
Nielsen S, Moller N, Christiansen JS, Jorgensen JO. Pharmacological antilipolysis restores insulin sensitivity during growth hormone exposure. Diabetes. 2001;50(10):2301-2308.
Nellemann B, Vendelbo MH, Nielsen TS, et al. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity. Acta physiologica (Oxford, England). 2014;210(2):392-402.
Jensen RB, Thankamony A, Day F, et al. Genetic markers of insulin sensitivity and insulin secretion are associated with spontaneous postnatal growth and response to growth hormone treatment in short SGA children: the North European SGA Study (NESGAS). The Journal of clinical endocrinology and metabolism. 2015;100(3):E503-507.
Christopher M, Hew FL, Oakley M, Rantzau C, Alford F. Defects of insulin action and skeletal muscle glucose metabolism in growth hormone-deficient adults persist after 24 months of recombinant human growth hormone therapy. The Journal of clinical endocrinology and metabolism. 1998;83(5):1668-1681.
Shulman GI. Cellular mechanisms of insulin resistance. The Journal of clinical investigation. 2000;106(2):171-176.
Rabinowitz D, Zierler KL. A METABOLIC REGULATING DEVICE BASED ON THE ACTIONS OF HUMAN GROWTH HORMONE AND OF INSULIN, SINGLY AND TOGETHER, ON THE HUMAN FOREARM. Nature. 1963;199:913-915.
Jorgensen JO. Human growth hormone replacement therapy: pharmacological and clinical aspects. Endocrine reviews. 1991;12(3):189-207.
Takahashi Y, Biller BMK, Fukuoka H, et al. Weekly somapacitan had no adverse effects on glucose metabolism in adults with growth hormone deficiency. Pituitary. 2023;26(1):57-72.
Ho KK, O'Sullivan AJ, Burt MG. The physiology of growth hormone (GH) in adults: translational journey to GH replacement therapy. The Journal of endocrinology. 2023;257(2).
Tanner JM, Hughes PC, Whitehouse RH. Comparative rapidity of response of height, limb muscle and limb fat to treatment with human growth hormone in patients with and without growth hormone deficiency. Acta endocrinologica. 1977;84(4):681-696.
DB C. Effect of growth hormone on cell and somatic growth. . In: Handbook of physiology (Eds. Knobil and Sawyer)Washington DC. 1974:159-186.
Korner A. Growth hormone control of biosynthesis of protein and ribonucleic acid. Recent progress in hormone research. 1965;21:205-240.
Goldberg AL. Protein turnover in skeletal muscle. I. Protein catabolism during work-induced hypertrophy and growth induced with growth hormone. The Journal of biological chemistry. 1969;244(12):3217-3222.
Horber FF, Haymond MW. Human growth hormone prevents the protein catabolic side effects of prednisone in humans. The Journal of clinical investigation. 1990;86(1):265-272.
Russell-Jones DL, Weissberger AJ, Bowes SB, et al. The effects of growth hormone on protein metabolism in adult growth hormone deficient patients. Clinical endocrinology. 1993;38(4):427-431.
Fryburg DA, Barrett EJ. Growth hormone acutely stimulates skeletal muscle but not whole-body protein synthesis in humans. Metabolism: clinical and experimental. 1993;42(9):1223-1227.
Copeland KC, Nair KS. Acute growth hormone effects on amino acid and lipid metabolism. The Journal of clinical endocrinology and metabolism. 1994;78(5):1040-1047.
Fryburg DA, Gelfand RA, Barrett EJ. Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. The American journal of physiology. 1991;260(3 Pt 1):E499-504.
Turkalj I, Keller U, Ninnis R, Vosmeer S, Stauffacher W. Effect of increasing doses of recombinant human insulin-like growth factor-I on glucose, lipid, and leucine metabolism in man. The Journal of clinical endocrinology and metabolism. 1992;75(5):1186-1191.
Russell-Jones DL, Umpleby AM, Hennessy TR, et al. Use of a leucine clamp to demonstrate that IGF-I actively stimulates protein synthesis in normal humans. The American journal of physiology. 1994;267(4 Pt 1):E591-598.
Fryburg DA, Jahn LA, Hill SA, Oliveras DM, Barrett EJ. Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms. The Journal of clinical investigation. 1995;96(4):1722-1729.
Fryburg DA, Louard RJ, Gerow KE, Gelfand RA, Barrett EJ. Growth hormone stimulates skeletal muscle protein synthesis and antagonizes insulin's antiproteolytic action in humans. Diabetes. 1992;41(4):424-429.
Copeland KC, Nair KS. Recombinant human insulin-like growth factor-I increases forearm blood flow. The Journal of clinical endocrinology and metabolism. 1994;79(1):230-232.
Fryburg DA. NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF- I: possible role of nitric oxide in muscle protein synthesis. The Journal of clinical investigation. 1996;97(5):1319-1328.
Boger RH, Skamira C, Bode-Boger SM, Brabant G, von zur Muhlen A, Frolich JC. Nitric oxide may mediate the hemodynamic effects of recombinant growth hormone in patients with acquired growth hormone deficiency. A double-blind, placebo-controlled study. The Journal of clinical investigation. 1996;98(12):2706-2713.
Bartke A, Darcy J. GH and ageing: Pitfalls and new insights. Best practice & research. Clinical endocrinology & metabolism. 2017;31(1):113-125.
Yarasheski KE, Campbell JA, Smith K, Rennie MJ, Holloszy JO, Bier DM. Effect of growth hormone and resistance exercise on muscle growth in young men. The American journal of physiology. 1992;262(3 Pt 1):E261-267.
Yarasheski KE, Zachwieja JJ, Campbell JA, Bier DM. Effect of growth hormone and resistance exercise on muscle growth and strength in older men. The American journal of physiology. 1995;268(2 Pt 1):E268-276.
Taaffe DR, Pruitt L, Reim J, et al. Effect of recombinant human growth hormone on the muscle strength response to resistance exercise in elderly men. The Journal of clinical endocrinology and metabolism. 1994;79(5):1361-1366.
Papadakis MA, Grady D, Black D, et al. Growth hormone replacement in healthy older men improves body composition but not functional ability. Annals of internal medicine. 1996;124(8):708-716.
Hermansen K, Bengtsen M, Kjaer M, Vestergaard P, Jorgensen JOL. Impact of GH administration on athletic performance in healthy young adults: A systematic review and meta-analysis of placebo-controlled trials. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society. 2017;34:38-44.
Waters D, Danska J, Hardy K, et al. Recombinant human growth hormone, insulin-like growth factor 1, and combination therapy in AIDS-associated wasting. A randomized, double-blind, placebo-controlled trial. Annals of internal medicine. 1996;125(11):865-872.
Schambelan M, Mulligan K, Grunfeld C, et al. Recombinant human growth hormone in patients with HIV-associated wasting. A randomized, placebo-controlled trial. Serostim Study Group. Annals of internal medicine. 1996;125(11):873-882.

Multiple Endocrine Neoplasia Type 4

ABSTRACT

MEN4 (OMIM #610755) has many similarities with MEN1 but is caused by germline mutations in CDKN1B. MEN4 is rarer than MEN1. Clinical manifestations of MEN4 encompass primary hyperparathyroidism, pituitary adenomas, and gastroenteropancreatic neuroendocrine neoplasms. In line with MEN1 other neoplasms may occur.

INTRODUCTION

MEN4 (OMIM #610755) was initially named MENX and was first described in rats (1-3). MEN4 is caused by germline mutations in CDKN1B (Cdkn1b in rats), a tumor suppression gene encoding for the protein p27Kip1 (commonly referred to as p27 or as KIP1) (4). The CDKN1B gene is located on chromosome 12p13.1 (5). p27 is a member of the cyclin-dependent kinase inhibitor (CDKI) family which regulates the cell cycle (6, 7). Germline mutations in CDKN1B lead to reduced expression of p27, thereby resulting in uncontrolled cell cycle progression. To date, most of the reported human mutations were missense. These mutations were deemed pathogenic due to their in vivo or in vitro effects on the function of p27. In humans, two CDKI families have been identified: the INK4a/ARF family and the Cip/Kip family (8). Natalia Pellegata and colleagues reported in 2006 a three-generation family with apparently MEN1-related tumors, but this kindred turned out to become the first reported cases of MEN4 in humans (2). The incidence of CDKN1B mutations in patients with a MEN1-related phenotype is likely to be in the range of 1-4% (9-11). MEN4 screening has been recommended for all patients with a MEN1-related phenotype without the presence of a MEN1 gene mutation, but the yield seems to be extremely low (< 0.1%) (12, 13). All first-degree relatives of patients with MEN4 should be offered genetic testing (14-16). The offspring of an individual with MEN4 has a 50% chance of inheriting the CDKN1B pathogenic variant (17). Possible genotype-phenotype correlations might exist (18).

CLINICAL FEATURES OF MEN4

Primary Hyperparathyroidism

Primary hyperparathyroidism has been reported in up to 80%-90% of cases with MEN4 (3). The indications for parathyroid surgery in MEN4 are the same as for MEN1 and the approach in MEN4-related primary hyperparathyroidism may be similar to that in MEN1 (19-22). It is suggested that screening for hyperparathyroidism with serum calcium measurements (and parathyroid hormone levels (PTH) if indicated) should start at the age of 15 years in MEN4 mutation carriers (23, 24).

Pituitary Adenomas

Pituitary involvement in MEN4 is the second most common manifestation of the disease, affecting approximately 1/3 of the reported cases to date. The types of pituitary disorders in MEN4 include: nonfunctional pituitary adenoma, acromegaly and gigantism, prolactinoma, or Cushing’s disease (16, 22, 25-36). Pituitary tumors in MEN4 generally present with less aggressiveness and smaller size compared to those in MEN1 (28). The management of pituitary tumors in MEN4 is similar to other sporadic or familial cases (19). Routine surveillance for the development of pituitary tumors in patients with MEN4 should be performed on a case-by-case basis and follow the current guidelines for MEN1 (19, 24).

Gastroenteropancreatic Neuroendocrine Neoplasms (GEP NENs)

The prevalence of GEP NENs in MEN4 is approximately 25%. These include gastroduodenal or pancreatic NENs (panNENs), which are either nonfunctioning or secreting several peptides and hormones, including gastrin, insulin, adrenocorticotropic hormone (ACTH), or vasoactive intestinal polypeptide (VIP) (11, 20, 22, 25, 37-39). It appears that there is a decreased penetrance of gastroduodenal NENs or panNENs in MEN4 as compared to MEN1. The clinical syndromes associated with these hormonal overproductions can be found elsewhere in Endotext (40-43). The diagnosis and management of panNENs in MEN4 is similar to that in MEN1 (19). Screening for gastroduodenal NENs and panNENs should be initiated according to MEN1 screening protocols (19).

Other Neoplasms

Cervical neuroendocrine carcinoma (NEC), secreting and nonsecreting adrenal tumors, testicular cancer, breast cancer, papillary and medullary thyroid cancer, colon cancer, thymic and lung carcinoids, and meningioma have been reported incidentally in MEN4 cases (2, 9, 11, 15, 22, 23, 34, 36, 44, 45).

REFERENCES

Fritz A, Walch A, Piotrowska K, Rosemann M, Schäffer E, Weber K, et al. Recessive transmission of a multiple endocrine neoplasia syndrome in the rat. Cancer Res. 2002;62(11):3048-51.
Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson E, Bink K, Höfler H, et al. Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. Proc Natl Acad Sci U S A. 2006;103(42):15558-63.
Lee M, Pellegata NS. Multiple endocrine neoplasia type 4. Front Horm Res. 2013;41:63-78.
Bencivenga D, Stampone E, Azhar J, Parente D, Ali W, Del Vecchio V, et al. p27(Kip1) and Tumors: Characterization of CDKN1B Variants Identified in MEN4 and Breast Cancer. Cells. 2025;14(3).
Philipp-Staheli J, Payne SR, Kemp CJ. p27(Kip1): regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res. 2001;264(1):148-68.
Hengst L, Reed SI. Translational control of p27Kip1 accumulation during the cell cycle. Science. 1996;271(5257):1861-4.
Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994;78(1):59-66.
Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13(12):1501-12.
Georgitsi M, Raitila A, Karhu A, van der Luijt RB, Aalfs CM, Sane T, et al. Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia. J Clin Endocrinol Metab. 2007;92(8):3321-5.
Molatore S, Marinoni I, Lee M, Pulz E, Ambrosio MR, degli Uberti EC, et al. A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization. Hum Mutat. 2010;31(11):E1825-35.
Agarwal SK, Mateo CM, Marx SJ. Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states. J Clin Endocrinol Metab. 2009;94(5):1826-34.
Chevalier B, Coppin L, Romanet P, Cuny T, Maïza JC, Abeillon J, et al. Beyond MEN1, When to Think About MEN4? Retrospective Study on 5600 Patients in the French Population and Literature Review. J Clin Endocrinol Metab. 2024;109(7):e1482-e93.
Faggiano A, Fazzalari B, Mikovic N, Russo F, Zamponi V, Mazzilli R, et al. Clinical Factors Predicting Multiple Endocrine Neoplasia Type 1 and Type 4 in Patients with Neuroendocrine Tumors. Genes (Basel). 2023;14(9).
de Laat JM, van der Luijt RB, Pieterman CR, Oostveen MP, Hermus AR, Dekkers OM, et al. MEN1 redefined, a clinical comparison of mutation-positive and mutation-negative patients. BMC Med. 2016;14(1):182.
Alrezk R, Hannah-Shmouni F, Stratakis CA. MEN4 and CDKN1B mutations: the latest of the MEN syndromes. Endocr Relat Cancer. 2017;24(10):T195-t208.
Schernthaner-Reiter MH, Trivellin G, Stratakis CA. MEN1, MEN4, and Carney Complex: Pathology and Molecular Genetics. Neuroendocrinology. 2016;103(1):18-31.
Brock P, Kirschner L. Multiple Endocrine Neoplasia Type 4. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A, editors. GeneReviews(®). Seattle (WA): University of Washington, Seattle; 1993.
Halperin R, Arnon L, Nasirov S, Friedensohn L, Gershinsky M, Telerman A, et al. Germline CDKN1B variant type and site are associated with phenotype in MEN4. Endocr Relat Cancer. 2023;30(1).
Pieterman CRC, van Leeuwaarde RS, van den Broek MFM, van Nesselrooij BPM, Valk GD. Multiple Endocrine Neoplasia Type 1. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, et al., editors. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000.
Tonelli F, Giudici F, Giusti F, Marini F, Cianferotti L, Nesi G, et al. A heterozygous frameshift mutation in exon 1 of CDKN1B gene in a patient affected by MEN4 syndrome. Eur J Endocrinol. 2014;171(2):K7-k17.
Mazarico-Altisent I, Capel I, Baena N, Bella-Cueto MR, Barcons S, Guirao X, et al. Novel germline variants of CDKN1B and CDKN2C identified during screening for familial primary hyperparathyroidism. J Endocrinol Invest. 2023;46(4):829-40.
Seabrook A, Wijewardene A, De Sousa S, Wong T, Sheriff N, Gill AJ, et al. MEN4, the MEN1 Mimicker: A Case Series of three Phenotypically Heterogenous Patients With Unique CDKN1B Mutations. J Clin Endocrinol Metab. 2022;107(8):2339-49.
Frederiksen A, Rossing M, Hermann P, Ejersted C, Thakker RV, Frost M. Clinical Features of Multiple Endocrine Neoplasia Type 4: Novel Pathogenic Variant and Review of Published Cases. J Clin Endocrinol Metab. 2019;104(9):3637-46.
Thakker RV. Multiple endocrine neoplasia type 1 (MEN1) and type 4 (MEN4). Mol Cell Endocrinol. 2014;386(1-2):2-15.
Occhi G, Regazzo D, Trivellin G, Boaretto F, Ciato D, Bobisse S, et al. A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype. PLoS Genet. 2013;9(3):e1003350.
Crona J, Gustavsson T, Norlén O, Edfeldt K, Åkerström T, Westin G, et al. Somatic Mutations and Genetic Heterogeneity at the CDKN1B Locus in Small Intestinal Neuroendocrine Tumors. Ann Surg Oncol. 2015;22 Suppl 3:S1428-35.
Sambugaro S, Di Ruvo M, Ambrosio MR, Pellegata NS, Bellio M, Guerra A, et al. Early onset acromegaly associated with a novel deletion in CDKN1B 5'UTR region. Endocrine. 2015;49(1):58-64.
Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, et al. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes. Clin Genet. 2010;78(5):457-63.
Ikeda H, Yoshimoto T, Shida N. Molecular analysis of p21 and p27 genes in human pituitary adenomas. Br J Cancer. 1997;76(9):1119-23.
Dahia PL, Aguiar RC, Honegger J, Fahlbush R, Jordan S, Lowe DG, et al. Mutation and expression analysis of the p27/kip1 gene in corticotrophin-secreting tumours. Oncogene. 1998;16(1):69-76.
Lindberg D, Akerström G, Westin G. Mutational analysis of p27 (CDKN1B) and p18 (CDKN2C) in sporadic pancreatic endocrine tumors argues against tumor-suppressor function. Neoplasia. 2007;9(7):533-5.
Takeuchi S, Koeffler HP, Hinton DR, Miyoshi I, Melmed S, Shimon I. Mutation and expression analysis of the cyclin-dependent kinase inhibitor gene p27/Kip1 in pituitary tumors. J Endocrinol. 1998;157(2):337-41.
Chasseloup F, Pankratz N, Lane J, Faucz FR, Keil MF, Chittiboina P, et al. Germline CDKN1B Loss-of-Function Variants Cause Pediatric Cushing's Disease With or Without an MEN4 Phenotype. J Clin Endocrinol Metab. 2020;105(6):1983-2005.
Tichomirowa MA, Lee M, Barlier A, Daly AF, Marinoni I, Jaffrain-Rea ML, et al. Cyclin-dependent kinase inhibitor 1B (CDKN1B) gene variants in AIP mutation-negative familial isolated pituitary adenoma kindreds. Endocr Relat Cancer. 2012;19(3):233-41.
De Sousa SMC. From bench to bedside in the sella: translational developments in pituitary tumour genetics. Endocr Relat Cancer. 2025.
Mercè F, Asla Q, Illana FJ, Victòria F, Javier HL, Marta S, et al. A novel likely pathogenic germline variant in CDKN1B in a patient with MEN4 and medullary thyroid cancer. Fam Cancer. 2025;24(1):24.
Malanga D, De Gisi S, Riccardi M, Scrima M, De Marco C, Robledo M, et al. Functional characterization of a rare germline mutation in the gene encoding the cyclin-dependent kinase inhibitor p27Kip1 (CDKN1B) in a Spanish patient with multiple endocrine neoplasia-like phenotype. Eur J Endocrinol. 2012;166(3):551-60.
Belar O, De La Hoz C, Pérez-Nanclares G, Castaño L, Gaztambide S. Novel mutations in MEN1, CDKN1B and AIP genes in patients with multiple endocrine neoplasia type 1 syndrome in Spain. Clin Endocrinol (Oxf). 2012;76(5):719-24.
Han HJ, Moalem J, Shih AR, Gigliotti BJ. Insulinoma: A Novel Presentation of Multiple Endocrine Neoplasia 4. AACE Clin Case Rep. 2025;11(2):93-7.
Jensen RT, Ito T. Gastrinoma. In: Feingold KR, Ahmed SF, Anawalt B, Blackman MR, Boyce A, Chrousos G, et al., editors. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000.
de Herder WW, Hofland J. Insulinoma. In: Feingold KR, Ahmed SF, Anawalt B, Blackman MR, Boyce A, Chrousos G, et al., editors. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000.
de Herder WW, Hofland J. Vasoactive Intestinal Peptide-Secreting Tumor (VIPoma). In: Feingold KR, Ahmed SF, Anawalt B, Blackman MR, Boyce A, Chrousos G, et al., editors. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000.
Tsoli M, Dimitriadis GK, Androulakis, II, Kaltsas G, Grossman A. Paraneoplastic Syndromes Related to Neuroendocrine Tumors. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, et al., editors. Endotext. South Dartmouth (MA): MDText.com, Inc.; 2000.
Desrosiers-Battu LR, Lee JH, Tarasiewicz I, Gilbert AR, Galvan EM, Singh AK, et al. Anaplastic meningioma in a 6-year-old with somatic YAP1::MAML2 fusion and multiple endocrine neoplasia type 4 (MEN4) syndrome. Cancer Genet. 2025;292-293:106-10.
Singeisen H, Renzulli MM, Pavlicek V, Probst P, Hauswirth F, Muller MK, et al. Multiple endocrine neoplasia type 4: a new member of the MEN family. Endocr Connect. 2023;12(2).