Adrenal Incidentalomas



Wider application and technical improvement of abdominal imaging procedures in recent years, has led to the discovery of unsuspected adrenal tumors in an increasing frequency. These serendipitously detected lesions, also called adrenal incidentalomas, have become a common clinical problem and need to be investigated for evidence of hormonal hypersecretion and/or malignancy. In this chapter, information on the prevalence, etiology, radiological features, and appropriate biochemical evaluation are discussed in order to delineate the nature and hormonal status of adrenal incidentalomas. Despite the flurry of data accumulated, controversies are still present regarding the accuracy of diagnostic tests and cut-offs utilized to establish hormonal hypersecretion, potential long-term sequelae, indications for surgical treatment as well as duration and intensity of conservative management and follow-up. Recently, clinical guidelines proposing a diagnostic and therapeutic algorithm have been published to aid in clinical practice, however several areas are still debatable and require further research.




Abdominal computed tomography (CT), since its introduction in the late 1970’s, has proven to be an excellent tool for identifying pathology in patients with suspected adrenal disease. It was also predicted that the ability of CT to image both adrenal glands could lead to the occasional discovery of asymptomatic adrenal disease (1). Nowadays, further technological advances and broader availability of CT and other imaging modalities such as Ultrasonography (US), Magnetic Resonance Imaging (MRI), and Positron Emission Tomography (PET) have made the detection of unexpected lesions in adrenal and other endocrine glands a common finding (2). Although earlier detection of adrenal disease may be important in certain cases, it is now recognized that diagnostic evaluation and follow-up of clinically inapparent adrenal masses, or so-called “adrenal incidentalomas” may put a significant burden on patient’s anxiety and health and produce increasing financial consequences for the health system (3). It is therefore important to develop cost-effective strategies to diagnose and manage patients with adrenal incidentalomas.




According to the NIH State-of-the-Science Statement (4), adrenal incidentalomas (AIs) are defined as clinically inapparent adrenal masses discovered inadvertently in the course of diagnostic testing or treatment for conditions not related to the adrenals. Although an arbitrary cut-off of 1 cm or more has been employed to define an adrenal lesion as AI (5,6), this cut-off might be challenged following the higher resolution that modern imaging modalities offer, mainly MRI and CT. Nonetheless, in all published guidelines this cut-off is accepted as the minimum size above which additional diagnostic work-up should be performed, unless clinical signs and symptoms suggestive of adrenal hormone excess are present. Patients harboring an AI, by definition, should not have any history, signs or symptoms of adrenal disease prior to the imaging procedure that led to its discovery. This strict definition excludes cases in which symptomatic adrenal-dependent syndromes are “missed” during history taking or physical examination (6), but is also subject to some controversy regarding the ‘‘a priori’’ suspicion (7). In this context, an adrenal tumor detected in a patient undergoing abdominal imaging for staging and work-up of extra-adrenal malignancy should not be considered an AI, since adrenal metastases are a common finding in this setting, with a prevalence ranging from 3 to 40% in autopsy and from 6 to 20% in radiological series (8).




The precise prevalence and incidence of AI is difficult to define since data from population-based studies are lacking. Most data are derived from autopsy or radiological studies that are relatively difficult to interpret due to their retrospective nature, insufficient clinical information provided, referral bias, and different patient selection criteria.


In autopsy studies the prevalence of AIs varies, depending on patient’s age and the size of the tumors. The mean prevalence in a total of 71,206 cases was found to be 2.3%, ranging from 1 to 8.7% (9–21), without any significant gender difference. The prevalence of AIs increases with age, being 0.2% in young subjects compared to 6.9% in subjects older than 70 years of age (22), and is higher in white, obese, diabetic, and hypertensive patients (8). The variability of the reported prevalence in different series also reflects the difficulty in distinguishing small nodules from adenomas, as in some post-mortem series, small nodules (<1 cm) were detected in more than half of the patients examined (13).


In radiological studies, the prevalence of AIs differs depending on the imaging modality used. Transabdominal US during a routine health examination identified AIs in 0.1% of those screened (23), while studies using CT reported a mean prevalence of 0.64% ranging from 0.35 to 1.9% in a total of 82,483 scans published in the literature between 1982 and 1994 (11,24–28). However, two recent studies utilizing high-resolution CT scanning technology, have reported a prevalence of 4.4% and 5% which are similar to that observed in autopsy studies (29,30). This increase in detection frequency paralleled by the technological advances in imaging modalities, can explain why AIs are considered a “disease of modern technology”. Age has also been found to affect AI detection rates, as these lesions are found in 0.2% of individuals younger than 30 years, in 3% in the age of 50 years and up to 10% in individuals above 70 years of age (22,29,31). The prevalence of AIs is very low in childhood and adolescence accounting for 0.3-0.4% of all tumors (32). Adrenal incidentalomas appear to be slightly more frequent in women in radiological series, in discordance with autopsy studies, probably because women undergo abdominal imaging more frequently than men (31). Adrenal masses are located bilaterally in 10-15% of cases (33), while distribution between the two adrenals appears to be similar in post-mortem and CT studies (8,31).




Adrenal Incidentalomas are not a single pathological entity, but rather comprise a spectrum of different pathologies that share the same path of discovery and include both benign and malignant lesions arising from the adrenal cortex, the medulla or being of extra-adrenal origin (Table 1).


In general, the vast majority (80-90%) of AIs are benign adrenal adenomas, as shown by accumulated follow-up data from their natural history, even in the absence of pathological confirmation, since adrenal adenomas are rarely excised (5). However, a number of these lesions may still be malignant and/or related to autonomous hormonal secretion that is not clinically detected due to subtle secretory pattern or periodical secretion. Therefore, the question a physician faces when dealing with an AI is to exclude pathology other than an adrenal adenoma, particularly an adrenocortical carcinoma (ACC), and evaluate its secretory potential.


Table 1: The Spectrum of Lesions Presenting as AIs (modified from (34))

Adrenal Cortex lesions

·    Adenoma (non-functioning)

·    Adenoma (functioning)- Cortisol-secreting, Aldosterone-secreting

·    Nodular hyperplasia (primary bilateral macronodular adrenal hyperplasia)*

·    Adrenocortical Carcinoma (secreting or non-secreting)

Adrenal Medulla lesions

·    Pheochromocytoma (benign or malignant)*

·    Ganglioneuroma

·   Neuroblastoma, ganglioneuroblastoma

Other adrenal lesions

·    Myelolipoma, lipoma

·    Hemangioma, angiosarcoma

·    Cyst

·    Hamartoma, teratoma

Metastases (lung, breast, kidney, melanoma, lymphoma)*


·    Amyloidosis

·    Sarcoidosis

·    Lymphoma


·    Abscess


·    Fungal/parasitic (histoplasmosis, coccidiomycosis, tuberculosis)

·    Cytomegalovirus

Adrenal hemorrhage or hematomas*

Adrenal pseudotumors

Congenital Adrenal Hyperplasia (CAH)*

* can present with bilateral adrenal lesions


Autonomous cortisol secretion (ACS) is the most frequent endocrine dysfunction detected in patients with AIs, with a prevalence ranging from 5 to 30%, depending on the study design, work-up protocols and mainly diagnostic criteria used (5). This condition exclusively identified in the setting of AIs, also termed subclinical Cushing’s syndrome or subclinical hypercortisolism, is characterized by the absence of the typical clinical phenotype of hypercortisolism and by the presence of subtle alterations of the hypothalamic-pituitary-adrenal (HPA) axis.


Pheochromocytomas (PCCs), albeit rare in the general population, are discovered in approximately 5% of patients with AIs (35), while more than 30% of PCCs are diagnosed as AIs (36). Clinical manifestations are highly variable and the classic clinical triad (headache, palpitations and diaphoresis) is not present in the majority of patients. In addition, several patients harbor ‘‘silent pheochromocytomas’’, being totally asymptomatic or having intermittent and subtle symptoms. In a large multicentric study, approximately half of the patients with PCCs presenting as AIs were normotensive, whereas the remaining had mild to moderate hypertension (31).


Primary aldosteronism (PA) has a median prevalence of 2% (range 1.1-10%) among patients with AIs (37). After excluding cases with severe hypertension and hypokalaemia a retrospective study found that 16 out of 1004 subjects with AIs (1.5%) had PA (31). This figure is relatively low when compared to the prevalence of PA in unselected hypertensive populations which ranges from 4.6 to 16.6% (38) and may be related to the different investigational protocols and cut-offs indicative of autonomous aldosterone secretion used. The absence of hypokalaemia does not exclude this condition but absence of hypertension makes PA unlikely, although normotensive patients with PA have occasionally been reported (39). A recent study using a new diagnostic approach, considering the stimulatory effect that adrenocorticotropin (ACTH) could exert on aldosterone secretion, revealed a 12% prevalence of PA in normotensive and normokalaemic patients with AIs (40).


Combining studies that used a broad definition of incidentaloma without clearly stated inclusion criteria and those that reported descriptions of individual cases, Mansmann et al found 41% of AIs to be adenomas, 19% metastases, 10% ACCs, 9% myelolipomas, and 8% PCCs, with other benign lesions, such as adrenal cysts, ganglioneuromas, hematomas and infectious or infiltrative lesions representing rare pathologies (41). However, the relative prevalence of any pathology depends on the inclusion criteria used and is highly influenced by referral bias. Surgical series tend to overestimate the prevalence of malignant and secretory tumors, because the suspicion of a carcinoma and a functioning or large tumor are considered as indications for surgery. The reported prevalence of ACCs in these studies is also misleading, as it is derived from highly selected patient populations and does not reflect the prevalence seen in population-based studies. Presence of primary adrenal malignancy is more related to mass size, as ACCs represent 2% of all tumors ≤4 cm in diameter, 6% of tumors with size 4.1-6 cm and 25% of the tumors >6 cm (4). On the other hand, benign adenomas comprise 65% of masses ≤4 cm, and 18% of masses >6 cm (41). Similarly, metastatic lesions are much more common when patients with known extra-adrenal cancer are included. Among patients with extra-adrenal malignancies (most commonly carcinomas of the lung, breast, kidney, and melanoma), up to 70% of AIs are metastases. In contrast, the probability of a serendipitously discovered adrenal lesion in a patient without a history of cancer to be metastatic is as low as 0.4% (27). Studies applying more strict inclusion criteria may identify a greater number of small masses and biochemically silent tumors. In a comprehensive review, Cawood et al. (3) concluded that the prevalence of malignant and functioning lesions among AIs is likely to be overestimated if strict inclusion and exclusion criteria for the study populations are not used. By analyzing 9 studies that more accurately simulated the clinical scenario of a patient referred for assessment of an AI, they reported a mean prevalence of 88.1% (range 86.4-93%) for non-functioning benign adrenal adenomas (NFAIs), 6% (range 4-8.3%) for ACS, 1.2% for aldosterinomas, 1.4% (range 0.8-3%) for ACCs, 0.2% (range 0-1.4%) for metastases and 3% (range 1.8-4.3%) for PCCs. These low rates for clinically significant tumors compared to those noted from previous studies, that did not use such a narrow definition of AI (6,8,41), highlighted the limitations of epidemiological data due to inherent bias and raised significant questions concerning the appropriate diagnostic and follow-up protocols.


In the case of bilateral AIs, a broader spectrum of diagnoses needs to be considered, particularly in a relevant clinical setting, including metastatic or infiltrative diseases of the adrenals, hemorrhage, congenital adrenal hyperplasia (CAH), bilateral PCCs, bilateral cortical adenomas, and primary bilateral macronodular adrenal hyperplasia (PBMAH) (42). Occasionally, adrenal tumors of different nature may simultaneously be present in the same patient or in the same adrenal gland (43–46). Adrenal pseudotumor is a term used to describe radiological images of masses that seem to be of adrenal origin, but arise from adjacent structures, such as the kidney, spleen, pancreas, vessels and lymph nodes or are results of technical artifacts.




The pathogenesis of AIs is largely unknown. Early observations in autopsy studies which revealed that AIs are more frequent in older patients, led to the notion that these tumors are a manifestation of the ageing adrenal and could represent focal hyperplasia in response to ischemic injury, a concept that was supported by histopathological findings of capsular arteriopathy (47). Clonal analysis of adrenal tumors later revealed that the vast majority are of monoclonal origin and only a few arise from polyclonal focal nodular hyperplasia under the putative effect of local or extra-adrenal growth factors (48,49). In this sense, it has been postulated that hyperinsulinemia associated with the insulin resistance in individuals with the metabolic syndrome, which frequently coexists in patients harboring AIs, could contribute to the development of these tumors, through the mitogenic action of insulin on the adrenal cortex (50,51). However, the opposite causal relationship, that subtle autonomous cortisol production from AIs results in insulin resistance, has also been proposed (52). Another interesting hypothesis involves alterations in the glucocorticoid feedback sensitivity of the HPA axis. In a recent study, unexpected ACTH and cortisol responses to the combined dexamethasone-CRH (corticotropin-releasing hormone) test were found, in about half of the patients with bilateral AIs, when compared to control and unilateral adenoma cases (53). Such a dysregulated ACTH secretion during lifetime may lead to subtle but chronic trophic stimulation of the adrenals by repeatedly inappropriately higher ACTH levels, particularly in response to stress, favoring nodular adrenal hyperplasia.


Although several genetic syndromes are known to be associated with adrenal tumors, germline or somatic genetic alterations are identified only in subgroups of sporadic tumors that are mainly functioning (54–56). Elucidation of specific signaling pathways involved in these familial syndromes has led to the identification of several mutations in genes not previously described in ACCs, cortisol- and aldosterone-secreting adenomas as well as PCCs, creating new insights in adrenal tumorigenesis (Figure 1). However, the genetics of benign non-functional AIs that account for the majority of AIs are poorly understood.

Figure 1. Genes Involved in the Development of Adrenocortical Tumors

MEN: Multiple Endocrine Neoplasia; CTNNB1: catenin beta-1 gene; CYP21A2: 21-hydroxylase gene; CAH: Congenital Adrenal Hyperplasia; APC: Adenomatous polyposis coli; FAP: Familial adenomatous polyposis; KCNJS: gene encoding potassium channel, inwardly rectifying subfamily J, member 5; ATP1A1: gene encoding sodium/potassium-transporting ATPase subunit alpha 1; ATP2B3: plasma membrane calcium-transporting ATPase 3; CACNA1D: gene encoding calcium channel, voltage-dependent, L type, alpha 1D subunit; ARMCS: Armadillo repeat containing 5; ZNRF3: gene encoding Zinc and Ring Finger3; IGF-2: Insulin-like growth factor 2; TP53: tumor protein p53; CDKN2A: cyclin-dependent kinase inhibitor 2A; RB1: retinoblastoma protein; DAXX: death-associated protein 6; GNAS: gene encoding G-protein alpha subunit: PDE8B: phosphodiesterase 8B; PRKACA: gene encoding catalytic subunit alpha of protein kinase A; SDH-A-B-C-D: gene encoding succinate dehydrogenase complex subunit A, B, C, and D; SDHAF2: succinate dehydrogenase complex assembly factor 2; VHL: von-Hippel-Landau; RET: rearranged during transfection proto-oncogene; MAX: myc-associated factor X; TMEM127: gene encoding transmembrane protein 127.




Although the prevalence of potentially life-threatening disorders associated with AIs is relatively low, the question of whether a lesion is malignant (mainly an ACC) or functioning needs to be addressed in patients with an incidentally discovered adrenal mass. A careful clinical examination and a detailed medical history, evaluation of the imaging characteristics of the adrenal tumor(s), and biochemical evaluation to exclude hormonal excess can help clinicians identify the few cases that pose a significant risk to the patient’s health.


Clinical Evaluation


Per definition, patients with AIs should have no signs or symptoms implying adrenal dysfunction before the radiological detection of the adrenal tumor(s). In everyday clinical practice though, physicians who are not familiar with endocrine diseases may overlook mild signs of hormone excess and pursue evaluation of adrenal function following the incidental discovery of an adrenal mass. In this setting, such cases should not be designated as AIs and highlight the need for detailed and careful clinical history and examination (57).


Imaging Evaluation


Current morphological imaging modalities mainly CT and MRI have proven to be reliable means to predict with high diagnostic accuracy the nature of the lesion and its malignant potential. The size of the lesion has been considered as indicative of malignancy as most ACCs are large or significantly larger than adenomas at the time of diagnosis (31). In a meta-analysis, ACCs represented 2% of all tumors ≤4 cm in diameter, but the risk of malignancy increased significantly with tumor size greater than 4 cm, being 6% in tumors with size 4.1-6 cm and 25% in tumors >6 cm (58). However, size alone has low specificity in distinguishing benign from malignant lesions, since ACCs can also be relatively small during early stages of development and exhibit subsequent progressive growth (5). Other than size, findings suggestive of malignancy include irregular shape and borders, tumor heterogeneity with central necrosis or hemorrhage and invasion into surrounding structures. Benign adenomas are usually small (<4 cm), homogenous, with well-defined margins. Slow growth rate or stable size of an adrenal mass have also been proposed as indicators of benign nature (4). However, studies on the natural history of AIs suggest that up to 25% of benign adenomas can display an increase in size by almost 1 cm, while adrenal metastases with no change in CT appearance over a period of 36 months have been described, not allowing for the introduction of a safe cut-off of absolute growth or growth rate to distinguish benign from malignant lesions (59).


Certain imaging properties of AIs, depending on the modality used, can be helpful for the differential diagnosis between benign and malignant adrenal lesions and are summarized in Table 2.




CT has a high spatial and contrast resolution, which allows assessment of tissue density by measuring X-ray absorption compared to water (attenuation, expressed in Hounsfield Units - HU) (Figure 2).  Water and air are conventionally allocated a HU value of 0 and -1000 respectively, while fat is usually characterized by a HU value between -40 and -100. Because there is an inverse linear relation between the fat content of a lesion and attenuation, lipid-rich adenomas express lower HU in unenhanced (without contrast medium) CT images compared to malignant lesions, which are usually lipid-poor (60). A value of 10 HU in unenhanced CT images is the most widely used and accepted attenuation value threshold for the diagnosis of a lipid-rich, benign adrenal adenoma (61,62). In several studies a density of ≤10 HU was found to be superior to size in differentiating benign from malignant masses, displaying a sensitivity of 96-100% and a specificity of 50-100% (63). In this context, the risk of malignancy in a homogeneous 5 cm adrenal mass with a CT attenuation value of 10 HU is close to 0% (42). On the other hand, up to 30-40% of benign adenomas are considered lipid-poor and have an attenuation value of >10 HU on non-contrast CT, which is considered indeterminate since it overlaps with those found in malignant lesions and PCCs. Hence, unenhanced CT attenuation is a useful screening tool to identify a lesion as benign and exclude malignancy but is less reliable in diagnosing a malignant mass with certainty. When considering patients with a history of extra-adrenal malignancy though, several studies evaluating the >10 HU cut-off as indicative of malignancy showed high sensitivity (93%) for the detection of malignancy but variable specificity, meaning that 7% of adrenal metastases were found to have a tumor density of ≤10 HU (62). Attenuation values in non-contrast CT can also reliably identify typical myelolipomas that have a density lower than minus 40 HU (42).


For these indeterminate adrenal lesions (>10HU) intravenous contrast administration reveals their hemodynamic and perfusion properties that can be utilized to distinguish benign from malignant lesions (Figure 2). The attenuation on delayed images (10-15 min post contrast administration) decreases more quickly in adenomas because they exhibit rapid uptake and clearance compared to malignant lesions that usually enhance rapidly but demonstrate a slower washout of contrast medium (64). There are two methods of estimating contrast medium washout: absolute percentage washout (APW) and relative percentage washout (RPW) and can be calculated from values of pre-contrast (PA), enhanced (EA, 60-70 seconds after contrast medium administration) and delayed (DA, 10-15 mins after contrast medium administration) attenuation values according to the formulas below:

APW=100 x (EA-DA) / (EA-PA)

RPW=100 x (EA-DA) / EA


Lipid-poor adenomas demonstrate rapid washout with APW >60% (sensitivity of 86-100%, specificity 83-92%) and a RPW >40% (sensitivity of 82-97%, specificity 92-100%) (65). The use of APW and RPW criteria can effectively discriminate benign from malignant adrenal masses. Metastases usually demonstrate slower washout on delayed images (APW<60%, RPW<40%) than adenomas and ACCs typically have an RPW of <40%. Furthermore, contrast-enhanced washout CT studies may not suffice for characterization of lesions such as PCCs, cysts, and myelolipomas; in these cases, further biochemical, anatomical and/or functional imaging may be required. Findings consistent, but not diagnostic, of PCC on CT include high attenuation values, prominent vascularity, and delayed washout of contrast medium (66).

Figure 2: CT images of adrenal pathologies presenting as adrenal incidentalomas. a,b,c: A patient with a benign (lipid-rich) adrenal adenoma with unenhanced attenuation value - 3 HU (a), early attenuation (60 seconds after i.v. contrast medium administration) 35 HU (b) and delayed attenuation (10 min post-contrast administration) 18 HU. ARW = 45% and RPW=49%. Absolute washout (APW) less than 60% is indeterminate. However, the low pre-contrast attenuation is suggestive of an adenoma. Relative washout (RPW) of 40% or higher is consistent with an adenoma; d,e,f: Biochemically and histologically proven pheochromocytoma with unenhanced attenuation of 49 HU (d), early attenuation 90 HU (e) and delayed attenuation 64 HU. ARW = 63% and RPW=29%. Absolute washout >60% is suggestive of an adenoma, however relative washout less than 40% and unenhanced attenuation >10 HU are indeterminate; g,h: A patient with a primary adrenocortical carcinoma characterized by heterogeneity an unenhanced attenuation value >10 HU (g) and inhomogeneous contrast medium uptake due to central areas of necrosis; i: Typical myelolipoma.


It is important to note that the aforementioned figures of sensitivity and specificity were produced in studies with limitations and high risk of bias because of lack of definitive pathological diagnosis, different timing in acquiring post-contrast images and the use of broad inclusion criteria, including not only AIs but also clinically overt adrenal masses. In agreement with this statement, a recent study (67), showed that only a minority (21%) of cortisol-secreting adenomas has the typical unenhanced attenuation value of <10 HU, because cortisol secretion is associated with decreased intra-cytoplasmic lipid droplets containing cholesterol esters which are necessary for cortisol synthesis. Nevertheless, among the adenomas with high pre-contrast density (>10 HU), washout analysis after contrast administration was consistent with the benign nature of the tumor in 60% of the cases.


Another crucial key point in clinical practice is that most abdominal and chest CT scans leading to the unexpected discovery of an adrenal mass are obtained with the use of intravenous contrast that may not fulfill current technical recommendations for an optimal CT study of the adrenal glands, such as analysis on contiguous 3-5 mm-thick CT slices, preferentially on multiple sections using multidetector (MDCT) row protocols (68). In such cases, it may be worthwhile to obtain a new CT scan, specifically aimed for the study of the adrenal glands, including washout protocols in order to avoid the radiation exposure of a subsequent third CT scan in case of indeterminate unenhanced attenuation values.


Finally, the importance of thorough and standardized reporting by radiologists (including common terminology, nodule size and HU) needs to be highlighted, in order to improve the percentage of patients with AIs that receive appropriate diagnostic testing and follow-up. This is a recently raised issue based on evidence that suggests that most of AIs are not adequately investigated according to international guidelines due to inconsistent use of terms and lack of specific details and recommendations in radiology reports (69,70).




Adrenal imaging with MRI can also aid in the differential diagnosis between benign and malignant adrenal pathology (Figure 3). Benign adrenal adenomas appear hypotense or isotense compared to the liver on T1-weighted images and have low signal intensity on T2-weighted images. The majority of PCCs show high signal intensity on T2-weighted imaging (“light bulb sign”) which is a non-specific finding; however, a wide range of imaging features of PCCs mimicking both benign and malignant adrenal lesions have also been described (66). Primary ACCs are characterized by intermediate to high signal intensity on T1- and T2-weighted images and heterogeneity (mainly on T2- sequence due to hemorrhage and/or necrosis) as well as avid enhancement with delayed washout. However, these features are not specific and display significant overlap between benign and malignant lesions. The MRI technique of chemical-shift imaging (CSI) exploits the different resonance frequencies of protons in water and triglyceride molecules oscillating in- or out-of-phase to each other under the effect of specific magnetic field sequences, to identify high lipid content in adrenal lesions (71). Adrenal adenomas with a high content of intracellular lipid usually lose signal intensity on out-of-phase images compared to in-phase images, whereas lipid-poor adrenal adenomas, malignant lesions and PCCs remain unchanged. Signal intensity loss can be assessed qualitatively by simple visual comparison or by quantitative analysis using the adrenal-to-spleen signal ratio and can identify adenomas with a sensitivity of 84-100% and a specificity of 92-100% (72). It must be remembered however, that ACC and clear renal cell cancer metastases may sometimes also show signal loss (73).


Overall, MRI is probably as effective as CT in distinguishing benign from malignant lesions. Although quality of data is poor and there are no randomized studies comparing the two conventional imaging modalities, a few studies have concluded that for lipid-rich adenomas, there is no apparent difference, but MRI with CSI might be superior when evaluating lipid-poor adenomas with an attenuation value up to 30 HU (74). Hence, CT is considered to be the primary radiological procedure for evaluating AIs because it is more easily available and cost-effective, whereas MRI should be employed when a CT is less desirable (as in pregnant women and in children), for lipid-poor adenomas with relatively high attenuation values, and for other suspected lesions such as PCCs (75). When MRI is the examination that revealed the AI, additional imaging with CT (unenhanced and/or PW studies) could be performed if the imaging phenotype is equivocal and following discussion of the individual case in a multidisciplinary team of experts.

Figure 3: MRI images of different adrenal lesions presenting as incidentalomas, using the chemical shift imaging (CSI) technique. The loss of signal in out of phase images is typical in benign lipid-rich adenomas (a, b) in contrast with pheochromocytomas (c, d) and adrenocortical carcinomas (e, f) which do not display any signal loss.




In recent years, positron emission tomography (PET) using 18-fluoro-deoxyglucose (18F-FDG) has emerged as an effective tool in identifying malignant adrenal lesions. By utilizing the increased glucose uptake properties of cancer cells, 18F-FDG-PET combined with a CT scan (18F-FDG-PET/CT) achieves a sensitivity and specificity in identifying malignancy of 93-100% and 80-100% respectively (76,77). Both quantitative analysis of FDG uptake using maximum standardized uptake values (SUVmax) and qualitative assessment using a mass/liver SUV ratio have been used as a criterion, with the latter displaying better performance (78). A SUV ratio <1.45–1.6 between the adrenal and the liver is highly predictive of a benign lesion (79). Caveats in utilizing 18F-FDG-PET/CT include cost and availability, risk of false negative results in case of necrotic or hemorrhagic malignant lesions, size <1cm, extra-adrenal malignancies with low uptake (such as metastases from renal cell cancer or low-grade lymphoma) and false positive results in cases of sarcoidosis, tuberculosis, other inflammatory or infiltrative lesions and some adrenal adenomas and PCCs that show moderate FDG uptake (80). Because of its excellent negative predictive value, 18F-FDG-PET may help in avoiding unnecessary surgery in patients with non-secreting tumors with equivocal features in CT demonstrating low FDG uptake. Moreover, 18F-FDG-PET/CT may favor surgical removal of tumors with elevated uptake and no biochemical evidence of a PCC (76). Newer tracers such as 18F-fluorodihydroxyphenylalanine (F-DOPA) and 18F-fluorodopamine (FDA) for detection of PCC on PET have also been developed but their availability is limited (81).


Conventional adrenal scintigraphy using radiolabeled cholesterol molecules such as 131I-6-b-iodomethyl-norcholesterol (NP-59) and 75Se-selenomethyl-19-norcholesterol has been used in the past to discriminate benign from malignant lesions. These tracers enter adrenal hormone synthetic pathways and act as precursor-like compounds, providing information regarding the function of target tissue. Typically, benign hypersecreting tumors, and non-secreting adenomas, show tracer uptake, whereas primary and secondary adrenal malignancies, space-occupying or infiltrative etiologies of AIs appear as ‘cold’ masses, providing an overall sensitivity of 71-100% and a specificity of 50-100% (82). However, some benign adrenal tumors such as myelolipomas and some functioning ACCs, may also be visualized with these modalities. Several additional limitations of adrenal scintigraphy such as insufficient spatial resolution, lack of widespread expertise, limited availability of the tracer, being a time-consuming procedure (which requires serial scanning over 5-7 days), and high radiation doses received by the patient, have limited its value in routine clinical practice, especially when conventional imaging can provide more reliable information. Recently, 123I-iodometomidate has been introduced as a tracer because it binds specifically to adrenocortical enzymes, but its application is hampered by its limited availability and heterogeneous uptake by ACCs (83). Scintigraphy with 123I-meta-iodo-benzyl-guanidine (MIBG) is the preferred method for identifying PCCs when clinical, biochemical, and imaging features are not conclusive, or when multiple or malignant lesions need to be excluded (35).


Table 2: Imaging Findings Differentiating Common Adrenal Pathologies in AIs


Benign adenoma





Usually <4cm

Usually >4cm



Growth rate

Stable or <0.8cm/year

Significant growth (>1cm/year)

Slow growth

Significant growth (>1cm/year)

Shape & margins

Round or oval with well-defined margins

Irregular shape and margins. Invasion to surrounding tissues





Heterogeneous (hemorrhage, necrosis)

Heterogeneous (necrosis)

Heterogeneous (hemorrhage, necrosis)

CT Unenhanced attenuation

≤10 HU (or >10 HU for lipid-poor adenomas)

>10 HU

>10 HU

>10 HU

CT Percent Washout (PW)

APW >60%


APW<60%, RPW<40%



APW<60%, RPW<40%


(out-of phase)

Signal loss

(except in lipid-poor adenomas)

No change in signal intensity

No change in signal intensity

No change in signal intensity

FDG uptake (PET)

Low (some can have low to moderate uptake)


Low (malignant pheochromocytomas show high uptake)


NP-59 uptake


Absent (except in some secreting tumors)



ACC: Adrenocortical carcinoma; HU: Hounsfield Units; APW: Absolute PW; RPW: Relative PW; CSI: Chemical-shift Imaging; FDG: fluoro-deoxyglucose; NP-59: 131I-6-b-iodomethyl-norcholesterol


Hormonal Evaluation


Patients with AIs should be screened at presentation for evidence of excess catecholamine or cortisol secretion and, if hypertensive and/or hypokalemic, for aldosterone excess. As already discussed, the definition of AI per se implies the absence of clinical symptoms/signs related to these entities, however subtle hormonal hypersecretion not leading to the full clinical phenotype of a related syndrome may be present in patients with an AI (6).




According to the Endocrine Society’s Clinical Practice Guidelines for the diagnosis of Cushing’s syndrome and the AACE/AAES Medical Guidelines for the management of AIs, all patients with an incidentally discovered adrenal mass should be tested for the presence of hypercortisolism (57,84). Signs and symptoms of overt Cushing’s syndrome if present in a thorough clinical evaluation should prompt the physician to proceed with the recommended diagnostic approach described in the relevant Endocrine Society’s Clinical Guidelines (84). In this case, as discussed earlier, the validity of the term “incidentaloma” is debated.


In the absence of overt disease, biochemical investigation frequently reveals subtle cortisol hypersecretion and abnormalities of the HPA axis, a state previously termed as subclinical Cushing’s syndrome (6). Based on the most recent clinical practice guidelines by the European Society of Endocrinology (ESE) and European Network for the Study of Adrenal Tumors (ENSAT) the term “autonomous cortisol secretion” (ACS) is preferred and will also be used throughout this chapter. Although ACS is poorly defined, and its natural history is largely unknown (3), the prevalence of hypertension, diabetes, obesity, other features of the metabolic syndrome, and osteoporosis has been found to be increased in such patients (5,85). Because standard biochemical tests used to screen for Cushing’s syndrome were not designed to reveal the subtle changes encountered in ACS, and since a definitive clinical phenotype to ascertain the presence of this condition is missing, a combination of various parameters used to assess the integrity of the HPA axis have been employed. Alterations of the HPA axis suggestive of ACS in AIs include altered dexamethasone suppression (DST) and response to CRH, increased mean serum cortisol and urinary free cortisol (UFC) levels and reduced ACTH levels (31), although the latter has recently been questioned (86). Reduced dehydroepiandrosterone sulfate (DHEA-S) is considered a less reliable marker since it normally decreases with age and creates diagnostic problems in the elder AI patients, whereas the incorporation of midnight salivary cortisol as a means to diagnose ACS has produced inconsistent results (87,88). Recently, a study utilizing gas chromatography-tandem mass spectrometry (GC-MS/MS) to measure serum levels of several steroids in patients with ACS, non-functioning AIs and controls showed that decreased levels of adrenal androgens, their metabolites, and pregnenolone metabolites displayed sensitivity and specificity that were comparable to that of routine methods in selecting patients with ACS (89). Currently though, the 1 mg overnight DST, remains the most reliable and easily reproducible method and is the recommended test to detect cortisol secretion abnormalities based on pathophysiological reasoning, simplicity and the fact that it was incorporated in the diagnostic algorithms of most studies. (5,90).


Different cortisol cut-off values following the 1 mg DST have been advocated from different authors and were adopted by several authorities, ranging from 50 to 138 nmol/l (1.8 to 5 μg/dl) (57,91). Higher thresholds increase the specificity of the test but lower its sensitivity (92). It is also important to consider drugs or conditions that interfere with this test by altering dexamethasone absorption, metabolism by CYP3A4, or falsely elevate cortisol levels through increased cortisol-binding globulin (CBG) levels (93). The post 1 mg DST cortisol cutoff of >5 μg/dl (138 nmol/l) approach was substantiated by studies showing that all patients with such a cortisol value had uptake only on the side of the adenoma on adrenal scintigraphy (94). On the other hand, studies that used post-surgical hypoadrenalism as indicative of autonomous cortisol secretion suggested that lower cortisol cut-offs may be needed to identify these cases (95–97). Although two or more abnormal tests are usually required to establish the diagnosis of ACS (57), the 1 mg DST should be the initial screening test based on pathophysiological reasoning and the fact that it represents the most common HPA axis abnormality described in the majority of studies (42). The formal low dose dexamethasone suppression test (LDDST) can be used to confirm and quantify the degree of autonomous cortisol secretion or to exclude a false positive test (98,99). A negative DST using a cortisol cut-off value of 1.8 μg/dl (50 nmol/l) virtually excludes ACS. Furthermore, a number of studies have found that patients with post DST cortisol values >1.8 μg/dl (50 nmol/l) have increased morbidity or mortality (100,101). A value of >5 μg/dl (138 nmol/l) on the other hand, is highly suggestive of the presence of ACS (5). In the case of a positive test with intermediate cortisol values (1.8-5 μg/dl) the term “possible ACS” is proposed by recent guidelines (90) and consideration of further parameters, such as the presence of other abnormalities of the HPA axis and/or comorbidities, employment of a higher cortisol cut-off level, and re-testing after 3-6 months, have been suggested (102). In our opinion, the post-LDDST cortisol value should be considered in patients with such intermediate cortisol values following the 1 mg DST because, in addition to its high specificity, it correlates well with other indices of cortisol excess and the size of the adenoma, thus providing a quantitative measure of the degree of cortisol production from the adenoma and a more robust means for further follow-up (98,103).




Because catecholamine secretion can be intermittent, and there are cases of “silent” PCCs, screening should be performed even in normotensive patients with AIs in order to prevent the morbidity and mortality that may accompany this tumor (104). The initial recommended biochemical screening test is measurement of plasma free (from blood drawn in the supine position) or urinary fractionated metanephrines using liquid chromatography with mass spectrometric or electrochemical detection methods (35). This approach has a sensitivity and specificity of 99% and 97% respectively and has proven to be superior to measurement of plasma or urine catecholamines and vanillylmandelic acid (VMA) (105). Some studies have suggested higher specificity of the plasma than the urine test albeit without head-to-head comparisons using mass spectrometric-based methods. Thus, until data directly comparing plasma and urinary measurements are produced, urinary free fractionated metanephrines can be used as an alternative, if plasma free metanephrines measurement is not available (106). Sane et al suggested that routine biochemical screening for PCC in small (<2cm) homogenous AIs characterized by attenuation values <10 HU may not be necessary, since none of the 115 patients in his cohort with lipid-rich tumors (<10 HU) had constantly elevated 24-hour urinary metanephrines or normetanephrines, whereas all 10 histologically proven PCCs were larger than 2cm and were characterized by >10 HU in unenhanced CT scans (107). This was also confirmed from a recent multicenter retrospective study including 376 PCCs with sufficient data from CT imaging. Based on the lack of PCCs with an unenhanced attenuation of <10 HU and the low proportion (0.5%, 2/376) of PCCs with an attenuation of 10 HU, it was suggested that abstaining from biochemical testing for PCC in AIs with an unenhanced attenuation of ≤10 HU is reasonable, whereas contrast washout measurements were unreliable for ruling out PCC (108).


A recent study (109) comparing the clinical, hormonal, histological and molecular features of normotensive incidentally discovered PCCs (previously referred as “silent”) with tumors causing overt symptoms, revealed lower diagnostic sensitivity (75%) for plasma and urinary metanephrines irrespective of tumor size, while genetic and histological studies showed decreased expression of genes and proteins associated with catecholamine production and increased cellularity and mitotic activity in “silent” tumors. It was implied that asymptomatic incidentally discovered PCCs do not represent an early stage of development of PCCs but rather correspond to a distinct entity characterized by cellular defects in chromaffin machinery resulting in lower efficiency to produce or release catecholamines. It is, therefore, crucial to consider that normotensive patients with an AI and normal values of metanephrines, may indeed harbor a PCC. In such instance, the CT and MRI scan features of the tumor if suspicious for PCC, should alert the clinician to perform complementary investigations, such as plasma chromogranin A measurement, MIBG scintigraphy, 18F-FDG-PET/CT, or other alternative functional imaging (F-DOPA/PET or FDA/PET) to rule out this possibility.




According to published guidelines from the Endocrine Society, all patients with an AI and hypertension, irrespective of serum potassium levels, should be tested for PA using the plasma aldosterone/renin ratio (ARR) as a screening test (37). However, the knowledge that PA can be diagnosed in normotensive patients with hypokalemia necessitates testing of all patients with hypertension or hypokalemia (39). Although there is no current consensus regarding the most diagnostic ARR cut-off, values >20-40 (plasma aldosterone expressed as ng/dl and plasma renin activity [PRA] as ng/ml/h) obtained in the morning from a seated patient are highly suggestive. However, the plasma aldosterone level also needs to be considered because extremely low PRA, even in the presence of normal aldosterone levels, will result in a high ARR; an aldosterone level less than 9 ng/dl makes the diagnosis of PA unlikely, whereas a level in excess of 15 ng/dl is suggestive (42). Attention should also be given to certain technical aspects required for the correct interpretation of the ARR such as unrestricted dietary salt intake, corrected potassium levels, and washout of interfering antihypertensive medication. Patients may be treated with a non-dihydropyridine calcium channel blocker (verapamil slow release) as a single agent or in combination with α-adrenergic blockers (such as doxazosin) and hydralazine for blood pressure control during the washout period, if needed. When suspected based on the ARR, PA should be verified with one of the commonly used confirmatory tests (oral sodium loading, saline infusion, fludrocortisone suppression, and captopril challenge). Admittedly, the extent that patients with AI should be investigated to exclude PA is still not known. Although PA has been reported with a low prevalence between patients with AIs (1-10%), substantially higher rates (24%) have recently been described using a recumbent post-low dose dexamethasone suppression (LDDST)-saline infusion test (PD-SIT) (40). Further studies evaluating the optimal biochemical diagnostic approach of PA in patients with AIs are required by comparing established versus evolving investigational protocols.




Measurement of sex hormones is not recommended in patients with an AI on a routine basis (57). Elevated levels of serum DHEA-S, androstenedione, 17-OH progesterone as well as testosterone in women and estradiol in men and postmenopausal women can be found in more than half of patients with ACCs (110). Although cases of androgen or estrogen excess have been rarely described in patients with benign adrenocortical adenomas (111–114), they are usually accompanied by symptoms or signs of virilization in women (acne, hirsutism) or feminization in men (gynecomastia), and therefore such lesions cannot be considered as true AIs. Thus, the usefulness of measuring sex hormones and steroid precursors is limited in cases of adrenal lesions with indeterminate or suspicious for malignancy imaging characteristics, where elevated levels can point towards the adrenocortical origin of the tumor and suggest the presence of an ACC rather than a metastatic lesion. Additionally, increased basal or after consytropin stimulation levels of 17-OH progesterone can also indicate CAH in patients with bilateral AIs (6).




Bilateral AIs caused by metastases of extra-adrenal malignancies or infiltrative diseases can rarely cause adrenal insufficiency (115). Therefore, in all patients with bilateral adrenal masses, adrenal insufficiency should be considered and evaluated clinically and if likely, diagnosis should be established using the standard 250μg consytropin stimulation test according to the Endocrine Society’s recently published clinical guidelines (116). 


Fine-Needle Aspiration Biopsy (FNAB)


The use of percutaneous fine-needle aspiration biopsy (FNAB) as a mean to clarify the nature of an AI has now been surpassed by the non-invasive radiological methods because they have better diagnostic accuracy and are devoid of potential side effects (117,118). It should be noted that FNAB is not considered an accurate method of differentiating benign from malignant primary adrenal tumors but can be helpful in the diagnosis of metastases from extra-adrenal malignancies with a sensitivity of 73-100% and a specificity of 86-100% using variable population inclusion criteria, reference standards, and biopsy techniques (119–121). Therefore, in patients with suspicion of a rare tumor or a history of an underlying extra-adrenal malignancy and/or inconclusive imaging features of an AI (non-contrast CT attenuation value >10 HU and an RPW<40%), FNAB could be performed, but only if management would be altered by the histologic findings. FNAB has significant procedural risk with complications such as pneumothorax, bleeding, infection, pancreatitis, and dissemination of tumor cells along the needle track reported at a rate up to 14% by some, but not all available studies (117). To avoid the risk of a potentially lethal hypertensive crisis, PCC should always be excluded biochemically before FNA of an adrenal mass is attempted (122).




Since AIs do not represent a single clinical entity, their natural history varies depending on the underlying etiology. Primary malignant adrenal tumors typically display rapid growth (>2 cm/year) and a poor outcome with an overall 5-year survival of 47%. It is not known whether prognosis of patients with incidentally discovered ACC is different from symptomatic cases, however detection of the tumor at an early stage provides the possibility of definitive surgical cure (123). Patients with adrenal metastases have a clinical course depending on stage, grade, and site of the primary tumor (4). PCCs grow slowly and are mostly benign, but if untreated are potentially lethal displaying high cardiovascular mortality and morbidity, whereas 10-17% of the cases can be malignant (35). This is further emphasized by the fact that PCCs detected in autopsy series had not been suspected in 75% of the patients while they were alive, although they contributed to their death in approximately 55% of cases (124).


In benign adrenal tumors, which constitute the majority of AIs, the major concerns about their natural history revolve around their progressive growth, the possibility of malignant transformation, and the risk of evolution towards overt hypersecretion. Several cohort studies, despite their limitations, have shown that the majority of benign tumors remain stable in size; only 5-20% show a >1 cm increase in size, mostly within the first three years after prolonged follow-up (125,126), whereas occasional shrinkage, or even complete disappearance, of an adrenal mass have also been reported in about 4% of cases (8,127). Although there is not as yet a specific growth rate cut-off indicative of a benign nature, ACCs initially presenting as AIs, are invariably characterized by a rapid growth within months (at least > 0.8cm/year). The risk of an AI initially considered to be benign to become malignant has been estimated at <1/1000 (3,8) by Cawood et al, who found only two reports of a malignancy detected during the follow-up of AIs presenting as benign at diagnosis; the first was a renal carcinoma metastasis in a patient with a known history of renal carcinoma and the other was a non-Hodgkin’s lymphoma that showed a mass enlargement after 6 months (3). Two case reports of patients with a well-documented history of adrenal incidentalomas with totally benign imaging features on CT, who were diagnosed on follow-up (8 and 14 years later) with a malignant tumor in the same adrenal gland have recently been described (128,129). It is not known whether these cases can be explained by the independent occurrence of two events in a single adrenal (initially a typical benign adenoma and consequently the occurrence of an ACC) or whether a malignant transformation of a benign adenoma to carcinoma was the underlying course of events. Although there are some evidence to suggest the adenoma-carcinoma sequence is possible in the adrenal cortex (130,131), the high prevalence of adenomas contrasting with the extremely low prevalence of ACCs suggest that this process is probably exceptionally rare. These findings highlight the low risk of malignant transformation of AIs and the adequacy of current imaging to ascertain the diagnosis at presentation deterring the need for long-term imaging follow-up.


The appearance of hormonal hypersecretion over time in initially non-functioning AIs varies in different series. New-onset catecholamine or aldosterone overproduction is extremely rare (<0.3%), whereas development of overt hypercortisolism during follow-up is found in <1% (8). The most common disorder observed during follow-up is the occurrence of autonomous cortisol secretion eventually leading to ACS, reported with a frequency of up to 10% (127). This risk is higher for lesions >3 cm in size and during the first 2 years of follow-up but seems to plateau after 3-4 years, even if it does not subside completely (132). On the other hand, subtle hormonal alterations discovered at initial screening may also improve over time, indicating possible cyclical cortisol secretion from AIs and/or highlighting the inherent difficulty in biochemical confirmation of this condition (126).


Another issue of debate regarding the natural history of AIs that has attracted research, producing frequently conflicting data, is the sequelae of ACS on cardiovascular risk and subsequent mortality and morbidity. Several cross-sectional and cohort studies have reported a clustering of unfavorable cardiovascular risk factors in patients with AIs similar to those found in patients with overt Cushing’s syndrome (133,134). It is biologically plausible to anticipate that the presence of even mild to minimal cortisol excess may lead to some extent to the classic long-term consequences of overt hypercortisolism, such as hypertension, obesity, impaired glucose tolerance or frank diabetes, dyslipidemia and osteoporosis (figure 4). Because these metabolic derangements are common in the general and particularly the elderly population, in whom AIs are more frequently found, it is difficult to extrapolate whether there is a causal relationship between them. Whether these metabolic abnormalities in patients with AIs result in increased cardiovascular mortality and morbidity has not as yet been fully clarified. Although, some recent retrospective studies (100,101,135) have shown higher rates of cardiovascular events and mortality in patients with higher cortisol levels after the 1 mg DST, data from patients who underwent adrenalectomy are contradictory, regarding the outcome on metabolic and cardiovascular profile, whereas there are relatively few data on the risk of major cardiovascular events or mortality (97,136–138). Similarly, evidence on the detrimental effects of ACS on bone metabolism, such as lower bone density and high prevalence of vertebral fractures (43-72%) in postmenopausal women and eugonadal male patients with AIs (85,139–142) are conflicting with studies not showing reversal of these effects following surgical treatment (136,143). Additionally most of the detected vertebral fractures were minor and of uncertain clinical impact (85).

Moreover, there is growing evidence that even non-functioning AIs may be associated with similar metabolic disturbances and manifestations of the metabolic syndrome that are considered cardiovascular risk factors (144–146). Compared with controls, patients with non-functioning AIs exhibit subtle indices of atherosclerosis such as increased carotid intima-media thickness (IMT)(147), impaired flow-mediated vasodilatation (FMD) (148) and left ventricular hypertrophy (149). A recent study excluding patients with traditional risk factors (diabetes, hypertension or dyslipidemia) reported similar findings in patients harboring non-functioning AIs, with increased insulin resistance and endothelial dysfunction that correlated with subtle but not autonomous cortisol excess (150). Furthermore, an observational study suggested that patients with non-functioning AIs had a significantly higher risk of developing diabetes compared with control subjects without adrenal tumours prompting a re-assessment of whether the classification of benign adrenal tumors as “non-functional” adequately reflects the continuum of hormone secretion and metabolic risk they may harbor (151).


A recent meta-analysis (152) of 32 studies including patients with non-functioning AIs and adrenal tumors associated with ACS provided important insights on the natural history of such tumors that help in solving controversy and informing practice. First and foremost, it was observed that only a small proportion of patients with non-functioning AI or ACS had tumor growth or changes in hormone production during follow-up. Only 2.5% of adrenal incidentalomas grew by 10 mm or more over a mean follow-up of 41.5 months, whereas the mean difference in adenoma size between follow-up and baseline in all patients was negligible at 2.0 mm. Larger adenomas at diagnosis (≥25 mm) were even less likely than smaller tumors to grow during follow-up, which, according to the authors, suggests attainment of maximum growth potential. More importantly malignant transformation was never observed at the end of follow-up. Similarly, in patients with non-functioning AIs or ACS at diagnosis, the risk of developing clinically overt hormonal hypersecretion syndromes (Cushing’s, PA or catecholamine excess) was negligible (<0,1%), suggesting that these rare cases are probably attributed to the development of subsequent adrenal tumors and that ACS does not represent a preliminary stage of overt Cushing’s. Inapparent cortisol autonomy ensued only in 4,3% of patients with initially nonfunctioning tumours. The third and most novel finding of this thorough meta-analysis pertained to comorbidities, cardiovascular risk and mortality. It was confirmed, like in other similar studies, that patients with ACS had a high prevalence of cardiovascular risk factors (such as hypertension, obesity, dyslipidemia, and type 2 diabetes) and were more likely than those with non-functioning AIs to develop or show worsening of these factors during follow-up. However, the prevalence of such factors in patients with non-functioning AIs was also significant and higher than expected for Western populations. This finding could be explained by subtle degree of glucocorticoid excess not detected by current diagnostic criteria or perhaps by cyclical cortisol secretion or even by excess cortisol secretion in response to stress situations. It could also represent ascertainment bias since patients with diseases are more likely to have imaging tests that may detect an AI or could be a result of the previously theorized reverse causality concept that diabetes or the metabolic syndrome promote adrenal tumor development (153). Interestingly, reported all-cause and cardiovascular mortality in patients with non-functioning AI during follow-up were similar to those in patients with ACS, warranting close clinical follow-up and treatment for both groups of patients.




Management of AIs is currently a debatable work in progress. Although the majority of AIs comprising of benign adenomas without evidence of hormone excess should not pose any compelling challenges, the few cases with equivocal imaging features, subtle hormone hypersecretion, or unusual evolution (i.e. significant tumor growth) should be ideally discussed in a multidisciplinary expert team meeting (90).


All published guidelines and expert reviews agree that patients with unilateral adrenal masses causing unambiguous hormonal overactivity, and those with suspected malignancy (mainly ACC), are candidates for surgical interventions (5,6,35,37,57,90,91,154,155). There is also broad consensus that the majority of AIs with clearly benign imaging phenotype in unenhanced CT and no relevant clinical activity do not require surgery. However, some authors have also advocated considering size as an indication for surgery. The 2002 NIH state-of-the-science report recommended surgical excision of all AIs greater than 6 cm and to use clinical judgment, based on the results of the initial or follow-up evaluations, when assessing masses between 4 and 6 cm for surgery (4). Considering the high prevalence of ACC in tumors >4cm, some have proposed lowering the size cut-off to 4 cm (156). Despite the paucity of data regarding the natural history of such large tumors, an attenuation value of ≤10 HU in unenhanced CT combined with washout properties consistent with a benign tumor and absence of significant growth over time, can be reassuring. Non-functioning lesions <4cm with indeterminate imaging features on unenhanced CT should be investigated further with contrast-washout studies, MRI-CSI, or 18F-FDG-PET/CT. If uncertainty remains, immediate surgery or repeat imaging after 3-6 months could be offered. It would also be prudent to exclude the possibility of a “silent” PCC in patients with an indeterminate lesion, before proceeding to surgery because hemodynamic instability during surgical excision may ensue.


The management of patients harboring AIs who have ACS is debatable and the beneficial effect of adrenalectomy has not been proven adequately in the literature. Some, but not all, predominantly retrospective studies have shown a beneficial effect in hypertension and diabetes mellitus in patients with AIs who underwent an adrenalectomy, compared to those who did not undergo such a procedure (97,136,138). In one prospective study with an 8-year follow-up, operated patients with ACS had an improvement in features of the metabolic syndrome, but not of osteoporosis, compared to those who were conservatively managed; however, no control group was included in the study (136). In a recent retrospective study, an improvement of blood pressure and blood glucose was noted in adrenalectomized patients with ACS, whereas these indices worsened in non-operated patients; even so, some patients apparently with non-functioning AI also showed an improvement in some of these parameters (97). Until results from randomized prospective trials, reporting outcome on metabolic and bone comorbidities as well as overall mortality and major cardiovascular events, become available, adrenalectomy should be considered on an individual basis. Since improvement of comorbidities and clinically relevant endpoints with adrenalectomy is not yet definitively proven, several other factors that are also linked to surgical outcome, such as patient’s age, duration and evolution of comorbidities and their degree of control, and presence and extent of end organ damage, should also be considered. Young patients with ACS and those with new onset and/or rapidly worsening comorbidities resistant to medical treatment (6,157) could thus be candidates for surgical intervention. A proposed algorithm for diagnostic approach and management of AIs is presented in Figure 4.


Before proceeding to surgical therapy, appropriate medical therapy must be given to all functioning lesions, aiming at symptom control. Apart from patients with Cushing’s syndrome, post-surgical adrenal insufficiency may ensue in ACS patients (158,159). Because the need for glucocorticoid coverage cannot be predicted before surgery, patients should be covered by steroids post-operatively until the HPA-axis can be formally assessed (95). Low morning cortisol levels the day after surgery, and before glucocorticoid replacement, provide evidence for post-surgical hypoadrenalism (97). All patients diagnosed with PCC, including normotensive patients with “silent” tumors should receive preoperative α-adrenergic blockade for 7 to 14 days to prevent perioperative cardiovascular complications. Treatment should also include a high-sodium diet and fluid intake to reverse catecholamine-induced blood volume contraction preoperatively and prevent severe hypotension after tumor removal (35). Finally, patients diagnosed with PA and bilateral tumors or a unilateral AI (if older than 40 years of age) who seek a potential surgical cure, should be considered for adrenal venous sampling (AVS) before proceeding to surgery, to confirm lateralization of the source of the excessive aldosterone secretion.


According to AACE/AAES Medical Guidelines for the management of adrenal incidentalomas, patients with AIs not elected for surgery after the initial diagnostic work-up, should undergo re-imaging 3-6 months after the initial diagnosis and then annually for the next 1-2 years, while annual biochemical testing is advised for up to 4-5 years following  the diagnosis (57). As the natural history of AIs remains largely unknown, a widely accepted follow-up imaging and hormonal protocol has not been formulated yet. It has recently been suggested by some authors that given the low probability of the transformation of a benign and non-functioning adrenal mass to a malignant or functioning one, the routine application of the current strategies in all patients with AIs is likely to result in a number of unnecessary biochemical and radiological investigations (3,160,161). Such an approach is costly, and it does not take into account harmful consequences of diagnostic evaluation such as patients’ anxiety associated with repeated clinical visits and a high rate of false positive results leading to further testing or unnecessary adrenalectomy. Moreover, exposure to ionizing radiation from repeated CT scans increases the future cancer risk to the level that is similar to the risk of the adrenal lesion becoming malignant (3,162).


Based on available data, it is safe to conclude that lesions <2 cm in size, and with an attenuation value <10 HU, have the lowest possibility of growth and thus long-term imaging follow-up is probably unnecessary. For larger tumors despite the high sensitivity and adequate specificity of unenhanced CT for identifying adenomas, the lack of prospective studies precludes suggesting stringent recommendations regarding optimal radiological follow-up (5). It is our practice for large lesions, particularly those >4 cm with attenuation values <10 HU, to repeat a CT scan after 6-12 months and if there is no increase in size and the imaging features remain unaltered, to defer further radiological follow-up. It is thought that a one-time follow-up scan in 6-12 months may be reassuring to the physician and the patient (42). An increase of >20% of the largest tumor diameter together with an at least 5 mm increase in this diameter (90), or an absolute increase by >8 mm over 12 months (59), probably warrant further follow-up and re-evaluation of radiological features.


The appropriate hormonal follow-up of patients not elected for surgery is also not established. Patients without any biochemical abnormalities at presentation could be spared the burden of repeated testing, since the risk of developing clinically overt hormonal excess is extremely low. Clinical follow-up with assessment of cardiovascular risk factors that have been associated with the presence of AIs may be adequate to detect the reported ~10% of the cases of new-onset ACS (5). Patients with worsening of their metabolic parameters should be retested with the 1mg DST and be advised to apply lifestyle changes and effective medical treatment to reduce cardiovascular risk. If biochemical abnormalities suggesting ACS are present during the initial screening, annual clinical follow-up including evaluation of potentially cortisol excess-related comorbidities, as well as periodic testing of the HPA axis, is advisable. Patients with ACS who do not reach the treatment goals despite an adequate medical therapy could be offered surgery. Duration of follow-up is also under debate, however based on available data, annual hormonal evaluation may be suggested for up to five years, and especially for lesions >3 cm (57).




AIs are increasingly being recognized, particularly in the aging population. Adrenal CT and MRI can reliably distinguish benign lesions, while 18F-FDG-PET/CT scan can be helpful in identifying tumors with malignant potential. ACS is the most common hyperfunctional state that is best substantiated using the 1 mg DST; urinary/plasma metanephrines and aldosterone/renin ratio are used to screen for PCCs and hyperaldosteronism. Adrenal lesions with suspicious radiological findings, PCCs, and tumors causing overt clinical syndromes, as well as those with considerable growth during follow-up, should be treated with surgical resection. Although there is no consensus, the interval for diagnostic follow-up testing relies on the radiological and hormonal features of the tumors at presentation. The benefit of surgical resection in patients with substantial comorbidities and associated subclinical adrenal hyperfunction, mainly in the form of ACS, is still under investigation.

Figure 4. Proposed Algorithm for Diagnosis and Management of AIs




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Utility of Advanced Lipoprotein Testing in Clinical Practice



A standard lipid panel includes total cholesterol, triglycerides, and HDL-C. LDL-C can then be calculated (LDL-C = total cholesterol – HDL-C – TG/5). In some instances, a direct LDL-C assay is employed because once the triglyceride levels are > 400mg/dl a calculated LDL-C is not valid. Non-HDL-C can also be calculated (non-HDL-C = total cholesterol – HDL-C). Increasing levels of LDL-C and non-HDL-C are associated with an increased risk of atherosclerotic cardiovascular disease (ASCVD). However, numerous studies have demonstrated that the association of non-HDL-C with ASCVD is more robust. It is possible to measure apolipoprotein B and A-I levels, LDL and HDL size, LDL and HDL particle number, and Lp(a). Numerous studies have documented a link between small dense LDL particles and an increased risk of ASCVD; however, the association is markedly reduced or entirely eliminated when the analyses are adjusted for other factors that affect ASCVD risk. Similarly, there is little data demonstrating that HDL subfractions are useful in risk prediction beyond HDL and other traditional risk factors. Apolipoprotein B levels and LDL particle number are more strongly associated with ASCVD than LDL-C, particularly when the levels of LDL-C and apolipoprotein B levels or LDL particle number are discordant. Similarly, while apolipoprotein B levels or LDL particle number are significantly better than non-HDL-C in predicting ASCVD risk when the levels of non-HDL-C and apolipoprotein B levels or LDL particle number are discordant whether this will alter therapy is debated. The guidelines put forth by a variety of different expert panels and organizations do not require apolipoprotein B or LDL particle number. It is also the author’s opinion that at this time the routine measurement of apolipoprotein B and/or LDL particle number is not required. Until data demonstrate the superiority of measuring apolipoprotein B or LDL particle number on clinical outcomes it is hard to recommend the routine use of such testing. Studies have demonstrated an association of Lp(a) with ASCVD. Many experts recommend measuring Lp(a) in a) patients with unexplained premature CHD, b) patients with a strong family history of premature CHD, c) patients with resistance to LDL lowering with statins, d) patients with rapid unexplained progression of atherosclerosis, and e) patients with familial hypercholesterolemia. Elevations in Lp(a) will stimulate more aggressive lowering of LDL and the consideration of adding drugs that lower Lp(a) such as niacin or PCSK9 inhibitors. While routine use of advanced lipoprotein testing is not recommended it should be recognized that in selected patients the additional information provided can be helpful and result in changes in treatment. As additional drugs to treat lipids are developed and our understanding of lipid and lipoprotein metabolism expands in the future the use of advanced lipoprotein analysis may assume a more important role.




A variety of specialized lipid and lipoprotein tests are available and a question that is frequently asked is whether and when to utilize these tests in evaluating and treating patients with lipid disorders. The standard lipid panel includes the measurement of total cholesterol, triglycerides, and HDL cholesterol (HDL-C). The LDL cholesterol (LDL-C) can then be calculated using the Friedewald formula (LDL-C = total cholesterol – HDL-C – TG/5). In some instances, a direct LDL-C assay is employed because as the triglyceride levels increase the accuracy of the calculated LDL-C decreases and once the triglyceride levels are greater than 400mg/dl most laboratories will no longer provide a calculated LDL-C level. In patients with normal triglyceride levels calculated LDL-C and directly measured LDL-C are very strongly correlated and the difference between the levels is relatively small (1,2). However, if the triglyceride levels are greater than 200mg/dl the calculated LDL-C will be lower than the directly measured LDL-C level (1). Additionally, if the LDL-C level is low the calculated LDL-C also tends to underestimate the true LDL-C level (1-4). Because of the inaccuracies of LDL-C levels calculated by the Friedewald formula a new and more accurate formula (Martin Hopkins Formula) has been developed (5). Several studies have demonstrated the increased accuracy of this new formula compared to the Friedewald formula with a particular advantage in settings of low LDL-C and high triglycerides (6-11). Major laboratories such as Quest now calculate LDL-C levels using the Martin Hopkins formula. A disadvantage of the Martin Hopkins formula is that it is more complex than the Friedewald formula and the LDL-C cannot be simply calculated. However, there is free, online access that allows for the automated calculation of LDL cholesterol by the Martin Hopkins formula ( and a smart phone application (LDL cholesterol calculator:

Non-HDL cholesterol (non-HDL-C) levels can also be calculated from a routine lipid panel (non-HDL-C = total cholesterol – HDL-C). “Remnant cholesterol can also be calculated from the routine lipid panel (Remnant cholesterol = total cholesterol – LDL-C – HDL-C) (12). Of note most guidelines and risk calculators do not require lipid and lipoprotein measurements beyond a routine lipid panel. For example, the ACC/AHA, QRISK, Reynolds, SCORE, and Framingham calculators utilize total cholesterol and HDL-C levels in order to calculate the risk of atherosclerotic cardiovascular disease (ASCVD) (13-17).


In the past fasting lipid panels were exclusively recommended but recent guidelines recommend either fasting or non-fasting lipid panels ((18-20). Non-fasting lipid panels will increase the convenience of obtaining lipid studies. Additionally, in patients with diabetes, fasting for the lipid panel increases the risk of hypoglycemia (21). Moreover, studies have shown that the ability of fasting and non-fasting lipid panels to predict ASCVD is similar (22-26). Fasting and non-fasting total cholesterol, HDL-C, and non-HDL-C levels are virtually identical (27,28). Triglyceride levels may increase in the fed state depending upon the amount of fat consumed and the time after consumption and therefore depending upon the circumstances there may be a considerable difference between fasting and non-fasting triglyceride levels in some patients (27,28). LDL-C levels calculated by the Friedewald formula are often decreased in the fed state due to increases in triglyceride levels (27). In the non-fasting state when LDL-C levels were calculated using the Friedewald formula 30% of patients had a ≥ 10 mg/dL difference compared to direct LDL-C measurements (8). In contrast, when LDL-C was calculated using the Hopkins Martin formula the results were very similar to direct measurements. Therefore, if one is using non-fasting LDL-C in decision making one should calculate the LDL-C level using the Hopkins Martin formula to increase accuracy. It should be noted that in patients where a genetic disorder of lipid metabolism is suspected or with previously elevated triglyceride levels a fasting lipid panel is preferred. Similarly, if triglyceride levels are markedly elevated with a non-fasting lipid panel the lipid panel should be repeated while fasting.




LDL-C and non-HDL-C levels are strongly correlated and increasing levels of either parameter are associated with an increased risk of ASCVD. Numerous studies have compared the ability of LDL-C and non-HDL-C to predict ASCVD events (29). In general, while both LDL-C and non-HDL-C predict an increased risk, non-HDL-C levels are a better predictor (29-36). For example, in the Women’s Health Study, a prospective cohort study of 15,632 initially healthy US women aged 45 years or older, the relative risk of a cardiovascular event in the top vs. bottom quintile was 1.62 for LDL-C and 2.51 for non-HDL-C (35). Similarly, in the Health Professionals Follow-up Study, a study of 51,529 US male health professionals between 40 to 75 years of age, the relative risk of a cardiovascular event in the highest quintile compared with the lowest quintile was 1.81 for LDL-C and 2.76 for non-HDL-C (36).


While LDL-C and non-HDL-C are strongly correlated there are some individuals where these measurements are discordant (i.e. a relatively low LDL-C and a relatively high non-HDL-C or conversely a relatively high LDL-C and a relatively low non-HDL-C). In discordant situations the non-HDL-C levels are a much better predictor of cardiovascular events than the LDL levels. For example, in a study by Mora of 27,533 healthy women, 11.6% had discordant levels with discordance defined as an LDL-C above the median and a non-HDL-C below the median or an LDL-C below the median with a non-HDL-C above the median (37). Most significantly, in women with a below-median LDL-C but a non-HDL-C above the median coronary risk was underestimated by almost 3-fold for women when the LDL-C was used to predict events (37). Conversely, in women with above-median LDL-C but a non-HDL-C below the median coronary risk was overestimated by almost 3-fold when their LDL-C was used to predict events (37). Thus, the risk of ASCVD tracks more closely with non-HDL-C levels and these results highlight the advantage of non-HDL-C measurements compared to LDL-C measurements in determining risk of ASCVD.


In addition, this discordance between calculated LDL-C (measured by the Friedewald formula) and non-HDL-C levels can result in the misclassification of patients. For example. in patients with LDL-C levels <70 mg/dl, 15% had a non-HDL-C level ≥ 100 mg/dl and if the triglyceride levels were between 150-199mg/dl 22% had a non-HDL-C ≥ 100 mg/dl (38). Thus, a significant number of patients who have reached their LDL-C goal of < 70mg/dl have not reached their non-HDL-C goal. The method used to determine LDL-C levels influences the rate of discordance between LDL-C and non-HDL-C levels. When the LDL-C levels were measured by the Friedewald formula the discordance was considerable higher than when LDL-C levels were measured using the Hopkins Martin formula (Table 1) (39).


Table 1. Discordance Between LDL-C and Non-HDL-C Levels


Percent with Non-HDL-C > 100mg/dl

LDL-C < 70mg/dL Friedewald Formula


LDL-C < 70mg/dL Hopkins Martin Formula



Percent with Non-HDL-C > 130mg/dl

LDL-C < 100mg/dl Friedewald Formula


LDL-C < 100mg/dl Hopkins Martin Formula

~ 1%


Finally, studies have examined the relative utility of LDL-C and non-HDL-C levels in determining the benefits of statin therapy. A meta-analysis by Boekholdt and colleagues looked at 8 statin trials with 62,154 patients (40). They found that while on treatment levels of both LDL-C and non-HDL-C were associated with the risk of future cardiovascular events the association was more robust for non-HDL-C (40).


Taken together these data indicate that while both LDL-C and non-HDL-C levels are predictive of ASCVD events non-HDL-C is a better predictor. The older NCEP guidelines recommended non-HDL-C as a therapeutic target if the triglyceride levels were greater than 200mg/dl and the newer National Lipid Association and American Association of Clinical Endocrinologists recommendations consider non-HDL-C as a target along with LDL-C (13,20,41). The non-HDL-C targets are 30mg/dl higher than the LDL-C targets (for example if the LDL-C target is 70mg/dl the non-HDL-C target would be 100mg/dl). It is the opinion of this author that clinicians should utilize non-HDL-C levels more frequently in the evaluation and management of patients with hyperlipidemia. Additionally, non-HDL-C levels are easily calculated when one obtains a routine lipid panel.




In addition to a routine lipid panel it is possible for the clinician to measure a number of other parameters including apolipoprotein B and A-I levels, LDL and HDL size, LDL and HDL particle number, and lipoprotein (a) (Lp(a)) levels. A number of different tests are offered by large commercial laboratories. Currently, lipoprotein analysis by Nuclear Magnetic Resonance Spectroscopy (NMR) is offered by LabCorp and Ion-Mobility Analysis is offered by Quest Diagnostics. Density Gradient Ultracentrifugation (VAP) by Atherotec was discontinued (Feb 2016). Both, LabCorp and Quest provide routine lipid panel measurements plus LDL particle number, apolipoprotein B levels, indication of LDL and HDL size, and Lp(a) measurements.


It should be recognized that the standardization of certain of these assays is not as rigorous as the standardization of routine lipid panel assays. The Centers for Disease Control and Prevention (CDC) maintains a Lipid Standardization Program (LSP) that provides standards for measuring total cholesterol, triglycerides, HDL-C, apolipoprotein A-I, and apolipoprotein B. Measurements of LDL and HDL size and particle number are not as standardized and studies have shown differences in results between different methods (42,43). For example, Witte and colleagues compared LDL size using NMR and gradient gel electrophoresis and observed a correlation of only 0.39 between the two methods with an average difference in LDL size of 5.38nm with NMR values being lower (44). When these investigators classified patients according to whether they had small dense LDL (Pattern B) less than 50% of patients classified as pattern B using gradient gel electrophoresis were classified as pattern B using NMR (44). Similarly, Ensign et al., compared VAP, NMR, tube gel electrophoresis, and gradient gel electrophoresis to determine LDL subclasses and found a strong disagreement in patient LDL phenotyping among these four different methods (45). Measurement of LDL and HDL particle number has also shown discrepant results between different methods (46,47). These and other results highlight the lack of rigorous standardization (48).




The size of LDL particles is heterogeneous and there are a number of different methods to determine LDL size (ultracentrifugation, gradient gel electrophoresis, ion mobility, NMR) (49). As noted above, the different methods of LDL subclass analysis may produce different results and significant variations are possible even within one method (42). Studies have shown that small dense LDL is more pro-atherogenic than large LDL particles. Small dense LDL are thought to be more atherogenic because they are better able to penetrate the endothelial cell barrier and enter the intima, are more susceptible to oxidation, bind to proteoglycans in the arterial wall, and have a longer half time in the circulation than large LDL particles (50). It should be noted though that large LDL particles are also pro-atherogenic (51-55). For example, patients with familial hypercholesterolemia tend to have large LDL particles and these patients are at high risk to develop ASCVD (54). Small LDL particles are typically seen in patients with elevated triglyceride levels and decreased HDL-C levels (i.e. patients with the metabolic syndrome, obese patients, patients with diabetes) (56). Numerous studies have documented a link between small dense LDL particles and an increased risk of ASCVD (57,58). However, the association of small dense LDL with ASCVD is markedly reduced or entirely eliminated when the analyses are adjusted for other factors that affect the risk of ASCVD (57,58). The National Lipid Association expert panel was unable to identify any patient subgroups in which measuring LDL size is necessary (59). The authors concur with that viewpoint.




HDL particles are heterogeneous and vary in size (60,61). The metabolism and function of the spectrum of HDL particles is poorly understood. Additionally, there are a number of different methods of measuring HDL size and the comparability of the various methods is uncertain (48,60,61). Finally, and most importantly there is little data demonstrating that measurements of HDL subfractions are useful in risk prediction beyond measuring HDL and other traditional risk factors (58,61,62). Because of these issues the National Lipid Association Expert Panel was unable to find situations where HDL subfraction measurements would be recommended (59).


It should be recognized that the crucial issue with HDL may not be the HDL levels per se but rather the function of the HDL particles (48). Assays have been developed to determine the ability of HDL to facilitate cholesterol efflux from macrophages and these studies have shown that the levels of HDL-C do not necessarily indicate the ability to mediate cholesterol efflux (63). Moreover, cholesterol efflux from macrophages had a strong inverse association with both carotid intima-media thickness and the likelihood of angiographic coronary artery disease, independently of the HDL-C level (64). Additionally cholesterol efflux was also inversely associated with the incidence of cardiovascular events (65,66). These results indicate that it is the functional capability of HDL to facilitate cholesterol efflux that is important rather than simply HDL-C levels (67).


Assays have also been developed to measure the ability of HDL to protect LDL from oxidation (68). The ability of HDL to protect LDL from oxidation is decreased in patients with cardiovascular disease and in patients with inflammatory disorders who are at increased risk of developing cardiovascular disease (68,69). Similar to studies of cholesterol efflux these observations suggest that HDL function is a key variable. Unfortunately assays to measure cholesterol efflux or the ability of HDL to prevent oxidation are not available outside of research laboratories.




All of the pro-atherogenic lipoproteins (chylomicron remnants, VLDL remnants, IDL, LDL, and Lp(a)) carry one apolipoprotein B on their surface such that apolipoprotein B levels reflect the total number of atherogenic particles (70). Most of the circulating apolipoprotein B is associated with LDL particles (70). However, the contribution of very high Lp(a) levels to total Apo B levels can be substantial (Apo B in LDL/VLDL = Apo B mg/dl – (Lp(a) mg/dl x 0.16) (71). Apo B levels measured in the non-fasting state are similar to fasting values.


The levels of apolipoprotein B, LDL-C, and non-HDL-C are strongly correlated. Almost all studies have shown that apolipoprotein B levels are more closely associated with ASCVD than LDL-C levels and the general consensus is that apolipoprotein B levels are a more accurate predictor of ASCVD events than LDL-C (35,36,59,72-79). Whether apolipoprotein B levels are significantly better than non-HDL-C levels in predicting ASCVD is less certain.  


There are two large meta-analyses that have compared the ability of non-HDL-C and apolipoprotein B to predict ASCVD. The Emerging Risks Factor Collaboration examined 22 long term perspective studies with 91,307 subjects with a large number of events (4499) (22). In this study there were no differences in the ability of non-HDL-C or apolipoprotein B to predict ASCVD. The hazard ratio was increased approximately 2-fold in the upper quantile of non-HDL-C and apolipoprotein B compared to the lowest quantile. In contrast, another meta-analysis of 12 studies (not all perspective) with 233,455 subjects and 22,950 events reported slightly different results (80). In this study the relative risk ratio for apolipoprotein B was 1.43 (1.35-1.51) vs. 1.34 (1.24-1.44) for non-HDL-C, indicating a slightly greater predictive ability of apolipoprotein B (80).


A recent very large study has compared the predictive ability of non-HDL-C and apolipoprotein B (81). In the UK Biobank study 346,686 individuals without baseline CVD and not taking statins were followed for a median of 8.9 years. Fatal or nonfatal CVD events occurred in 6216 participants (1656 fatal). The conclusion of this very large study was that measurement of non-HDL-C was sufficient to capture the lipid-associated risk in CVD prediction, with no meaningful improvement from addition of apolipoprotein B.


Studies have also examined the predictive ability of non-HDL cholesterol and apolipoprotein B during treatment of dyslipidemia. In the Heart Protection Study (placebo vs. simvastatin) with over 20,000 participants and over 5,000 events the ability of non-HDL-C and apolipoprotein B to predict cardiovascular events were virtually identical (82). A meta-analysis by Boekholdt and colleagues looked at 8 statin trials with 62,154 patients and the adjusted hazard ratios for major cardiovascular events per 1-SD increase were very similar for apolipoprotein B and non-HDL-C (40). A meta-analysis by Robinson et al of 25 trials (n = 131,134): 12 on statin, 4 on fibrate, 5 on niacin, 2 on simvastatin-ezetimibe, 1 on ileal bypass surgery, and 1 on aggressive versus standard low-density lipoprotein (LDL) cholesterol and blood pressure targets observed that decreases in non-HDL cholesterol levels modestly outperformed apolipoprotein B in predicting cardiovascular events (83). Additionally, apolipoprotein B and non-HDL-C decreases similarly predicted cardiovascular disease risk in the statin trials.


While apolipoprotein B and non-HDL-C are strongly correlated there are some individuals where these measurements are discordant (i.e. a relatively low apolipoprotein B and a relatively high non-HDL-C or conversely a relatively high apolipoprotein B and a relatively low non-HDL-C). An analysis of the Interheart study explored the effect of discordance of apolipoprotein B and non-HDL-C (84). The Interheart study is a case-control study of acute myocardial infarction with blood samples in 9345 cases and 12,120 controls from 52 countries. Concentrations of non-HDL-C and apolipoprotein B were expressed as percentiles within the population. Concordance was defined as percentile non-HDL-C = percentile apolipoprotein B. Discordance was defined as percentile non-HDL-C > percentile apolipoprotein B or percentile non-HDL-C < percentile apolipoprotein B by 5%. The results of this study demonstrated that when apolipoprotein B and non-HDL-C levels were discordant the apolipoprotein B measurement was a significantly better predictor of ASCVD (84). Subjects with a low apolipoprotein B and a high non-HDL-C were at low risk (Odds Ratio 0.72 (0.67-0.77 95% CI) whereas subjects with a high apolipoprotein B and a low non-HDL-C were at a high risk (Odds Ratio 1.58 (1.38-1.58 95% CI). Similar results have recently been reported from the Women’s Health Study (85). Subjects with a high apolipoprotein B level and a discordant lower non-HDL cholesterol level had an increased risk (hazard ratio 1.22 CI 1.07- 139). Of note the subjects with higher apolipoprotein B levels relative to non-HDL-C had an increased prevalence of the metabolic syndrome including higher triglyceride levels and decreased HDL-C levels. Finally, the Cardia study compared the ability of apolipoprotein B and non-HDL-C levels to predict the development of coronary artery calcium, a surrogate marker of cardiovascular events (86). In this study apolipoprotein B levels were superior to non-HDL-C in predicting the development of coronary artery calcium (Table 2) (86). It is worth noting that the number of subjects that are discordant is relatively small (430 discordant/ 2794 total; 15.4% discordant).


Table 2. Cardia Study

Apo B/non-HDL-C (number of subjects)                  

Odds Ratio (CI)

Low/low (1184)


Low/high (213)

1.30 (0.91-1.85)

High/low (217)

1.63 (1.15-2.32)

High/high (1180

2.32 (1.91-2.83)


A key question is whether measuring apolipoprotein B in addition to routine risk factors will significantly affect our ability to decide on whether and how to treat patients. Using data from the Framingham Heart Study it was shown that adding apolipoprotein B to non-HDL-C and standard risk factors increased the C-statistic from 0.723 to 0.730, a very small increase suggesting that routine measurements of apolipoprotein B would not be very helpful (75,87). Similarly, the Emerging Risk Factor Collaboration group and the Women’s Health Study also examined the effect of adding apolipoprotein B results on the C-statistic and found very little change (77,88). Additionally, the Emerging Risk Factor Collaboration modelled the effect of measuring apolipoprotein B levels on patient classification using the NCEP III guidelines. In 15,436 subjects with a cardiovascular risk of 10-20% over the next 10 years the addition of apolipoprotein B measurements would result in a change in classification in only 488 subjects (3.2%) (88). Most subjects would be moved to a lower risk category (334) and a very small number would be reclassified to a higher risk category (154). These results coupled with the C-statistic results noted above suggest that the routine addition of apolipoprotein measurements in primary prevention patients would likely not have a major effect in altering patient management.


In patients treated with statins a meta-analysis has compared the association of apolipoprotein B and non-HDL-C levels on the risk of major cardiovascular events (40). While both on-treatment decreases in apolipoprotein B and non-HDL-C levels were associated with a decrease in cardiovascular events the strength of the association was somewhat greater for non-HDL-C than apolipoprotein B (Table 3) (40). A meta-analysis of seven randomized controlled trials comprising more than 60 000 study participants has also shown that changes in LDL-C, apoB100, and non-HDL-C all predicted similar CVD risk reduction after 1-year of statin therapy (-20, -24, and -20% risk reduction, respectively) (89). Finally, in another meta-analysis of 25 trials (12 statin, 4 fibrate, 5 niacin, 2 simvastatin-ezetimibe, 1 ileal bypass, 1 intensive vs. standard statin) the authors concluded that “across all drug classes, apo B decreases did not consistently improve risk prediction over LDL cholesterol and non-HDL cholesterol decreases” (83). Thus, in patients treated for hyperlipidemia the measurement of apolipoprotein B levels also does not appear to significantly contribute to the management of these patients.


Table 3.  Risk of Cardiovascular Disease in Statin Treated Patients (Hazard Ratios)



Apo B


1 (reference)

1 (reference)











Another approach to addressing the question of the importance of routinely measuring apolipoprotein levels is to determine if measuring apolipoprotein B level will alter our therapeutic approach. While most guidelines have not included apolipoprotein B goals there are guidelines that do recommend apolipoprotein B levels. For example, the National Lipid Association recommends in very high risk patients a LDL-C < 70mg/dL, a non-HDL-C < 100mg/dL, and an apolipoprotein B level < 80mg/dL (90). In an analysis by Sathiyakumar and colleagues if the LDL-C was < 70mg/dL and the non-HDL-C was < 100mg/dL (over 9000 subjects) fewer than 2% of the patients had an apolipoprotein B level > 80mg/dL (39). These results indicate that measuring apolipoprotein B levels will not identify a large number of patients that are not meeting the proposed goals.


In summary while measurement of apolipoprotein B levels is an excellent and likely the best predictor of ASCVD events whether it provides a substantial amount of information above and beyond what is provided by LDL-C and non-HDL-C and standard risk factors to justify routine apolipoprotein B measurement remains to be definitively determined. Whether routinely measuring apolipoprotein B levels will alter management in a sufficient number of patients to justify the extra expense of measuring apolipoprotein B needs to be rigorously studied. As noted earlier many of the patients with elevated apolipoprotein B levels relative to non-HDL-C levels are obese, diabetic, and have the metabolic syndrome and it is likely that clinicians will recognize based on non-lipid risk factors that these individuals are at high risk for ASCVD. There will of course be individual patients where measuring apolipoprotein levels will be helpful in determining treatment. For example, in patients thought to have Familial Dysbetalipoproteinemia (Type 3 disease) the non-HDL-C/apolipoprotein B ratio is a simple test for selecting patients with mixed hyperlipidemia that may have Familial Dysbetalipoproteinemia for additional studies (91).




The cholesterol content of LDL is not constant and can vary greatly between individuals and can change over time in a particular individual. For example, treatments that lower serum triglyceride levels can increase the size and cholesterol content of LDL (92,93). Measuring LDL particle number is an alternative way to quantitate LDL burden. While LDL-C and LDL particle number are strongly correlated there are some individuals who are discordant (relatively high LDL-C and relatively low LDL particle number or relatively low LDL-C and relatively high particle number). In patients with elevated triglycerides and/or low HDL levels the LDL-C levels are relatively low compared to LDL particle number (94,95).  Studies have shown that LDL particle number is more strongly associated with ASCVD than LDL-C, particularly when the levels of LDL-C and LDL particle number are discordant (37,77,96-99). Whether LDL particle number is a better predictor than non-HDL-C is discussed below.


Several studies have compared the ability of LDL particle number and non-HDL-C to predict ASCVD. In the Framingham Offspring Study there were 3,066 subjects with 431 events and LDL particle number was measured by NMR (96). In this study LDL particle number was more strongly associated with ASCVD than non-HDL-C (Hazard ratio 1.28 (CI 1.17-1.39) for LDL particle number vs. 1.21 (CI 1.10-1.33) for non HDL-C) (96). In the Women’s Health Study there were 27,673 subjects with 1015 events and LDL particle number was also measured by NMR (77). In this study the association of LDL particle number and non-HDL-C with ASCVD was very similar with the hazard ratio of 2.51 for LDL particle number and 2.52 for non-HDL-C (77). Finally, in the Multi-Ethnic Study of Atherosclerosis subjects (n = 6693) no benefit of measuring LDL particle number compared to routine lipid measurements on predicting ASCVD could be demonstrated (100).


While there are several studies that have examined patients discordant for apolipoprotein B levels and non-HDL-C levels (see section on apolipoprotein B) only two studies have examined discordance between LDL particle number and non-HDL-C. In the Multi-Ethnic Study of Atherosclerosis there were 6,814 men and women and LDL particle number was measured by NMR (101). The endpoint in this study was carotid intima-media thickness (CIMT) and coronary artery calcium (CAC), surrogate markers for ASCVD events. When there was discordance between LDL particle number and non-HDL-C, LDL particle number was more closely associated with CIMT and CAC but the differences were very modest (101). In the Women’s Health Study subjects with high LDL particle number measured by NMR that was discordant with non-HDL cholesterol levels were at increased risk of CHD (hazard ratio 1.13 CI 0.99-1.29) (85).


In patients on-treatment there is only a single study comparing LDL particle number and non-HDL-C. In the Heart Protection study 20,536 subjects were treated with simvastatin or placebo and LDL particle number was measured by NMR (82). The predictive strength of LDL particle number and non-HDL-C was very similar in both the placebo group and the statin group indicating no advantage of measuring LDL particle number (82).


It should also be noted that while LDL particle number and Apo B levels are highly correlated there are circumstances when they are discordant (102). High LDL particle number relative to Apo B levels was seen with insulin resistance, smaller LDL particle size, increased systemic inflammation, and low circulating LDL-C and HDL-C levels while high Apo B levels relative to LDL particle number was seen with larger LDL particle size and elevated levels of lipoprotein(a) (102).


In summary, while measurement of LDL particle number is an excellent predictor of ASCVD events whether it provides a substantial amount of information beyond what is provided by non-HDL-C and standard risk factors to justify routine LDL particle measurement remains to be definitively determined.




Lp(a) is an LDL particle with a single apolipoprotein B with a plasminogen like protein, apoprotein (a), attached by a disulfide bond (103-105). Apoprotein (a) is genetically very heterogeneous due to variations in molecular weight (from 300-800 kDa) due to differences in the number of Kringle repeats (103-105). The plasma levels of Lp(a) vary greatly with undetectable levels in some individuals (0.1mg/dl) and very high levels in others (>200mg/dl) (106). Individuals with genetically determined small apoprotein (a) have high plasma levels of Lp(a) whereas individuals with genetically determined large apoprotein (a) have low levels (103-105). The size of the apo(a) isoforms is inherited with an individual having two distinct apo(a) isoforms derived from apo(a) genes from their mother and father (106). This results in individuals having two different size Lp(a) particles in the serum. It is estimated that up to 90% of the variation in Lp(a) levels is determined genetically with environment having minimal effects. Lp(a) levels are very stable within an individual over their lifespan. Inflammation and renal disease increase while severe liver disease decrease Lp(a) levels (69,107).


Approximately 20% of subjects have Lp(a) levels greater than 50mg/dL and 30% have Lp(a) greater than 30mg/dL. Ethnicity greatly affects Lp(a) levels (107). The levels of Lp(a) in Blacks are approximately 2-3-fold higher than in Caucasians, Caucasians and Chinese have similar levels, South Asians have levels between Blacks and Caucasians, and Mexicans have levels less than Caucasians (Blacks> South Asians > Caucasians/Chinese > Mexicans) (107). Lp(a) levels do not correlate with LDL-C, non-HDL-C, apolipoprotein B, or LDL particle number.


Several large meta-analyses have demonstrated an association of Lp(a) levels with ASCVD. For example, a meta-analysis by the Emerging Risk Factors Collaboration looked at the individual records of 126,634 participants in 36 prospective studies with 9,336 CHD outcomes, 1,903 ischemic strokes, and 8,114 nonvascular deaths (108). They found a continuous association of Lp(a) with the risk of ASCVD that was not greatly affected by adjustment for other lipid levels or other established risk factors. In an analysis of 31 prospective studies with 9,870 events Bennet et al reported an odds ratio of 1.45 for individuals in the top third of Lp(a) compared with those in the bottom third (109). Of note adjustment for lipid levels and other established risk factors also had little effect on this association indicating that Lp(a) is an independent risk factor (109). Additionally, in patients with familial hypercholesterolemia elevated Lp(a) levels markedly increases the risk of the development of ASCVD (110). Mendelian randomization studies and basic science studies including experiments in animals that overexpress apoprotein (a) have suggested that increases in Lp(a) are not just a risk factor for atherosclerosis but causative for atherosclerosis (104,105,111-113). Finally, elevations in Lp(a) account for a significant proportion of the increased risk of ASCVD that is related to family history (114).


While the above studies clearly indicate that Lp(a) levels are a risk factor for the development of ASCVD the significance of Lp(a) in secondary prevention is not clear (115). Some studies have reported that Lp(a) is a risk factor in the setting of ASCVD (116-120) while other studies have failed to demonstrate a role for Lp(a) (121-124). In a meta-analysis of 11 studies with a total of 18,978 subjects the association between Lp(a) and ASCVD  was significant in studies in which the average LDL cholesterol was ≥130 mg/dl (OR: 1.46, 95% CI: 1.23 to 1.73, p < 0.001), whereas this relationship was attenuated and did not achieve statistical significance for studies with an average LDL cholesterol <130 mg/dl (OR: 1.20, 95% CI: 0.90 to 1.60, p = 0.21) (121). This observation suggests that in individuals with elevated LDL-C levels the impact of elevated Lp(a) levels will be magnified. However, in other studies Lp(a) was a risk factor even though LDL-C levels were relatively low (116,120). Recently Williet and colleagues reported a meta-analysis of patient-level data from seven randomized, placebo-controlled, statin outcomes trials that included 29,069 patients with repeat Lp(a) measurements (125). They found that elevated baseline and on-statin lipoprotein(a) showed an independent approximately linear relation with cardiovascular disease risk. Additionally, studies have shown that genetic variations at the LPA locus (apo(a) gene that effects Lp(a) levels) are associated with ASCVD events during statin therapy in patients (126). Taken together the bulk of the data suggests that elevated Lp(a) levels increase ASCVD risk even in patients with underlying cardiovascular disease.


The Emerging Risk Factor Collaboration modelled the effect of measuring Lp(a) levels on patient classification using the NCEP III guidelines (88). In 15,436 subjects with a cardiovascular risk of 10-20% over the next 10 years the addition of Lp(a) measurements would result in a change in classification in 1,517 subjects (9.8%). Most subjects would be moved to a lower risk category (962) and a number of subjects would be reclassified to a higher risk category (555) (88). These results coupled with the above findings suggest that the addition of Lp(a) measurements in patients might be useful in selected patients.


The potential benefits of measuring Lp(a) levels will become clearer when drugs are developed that specifically lower Lp(a) levels and clinical trials determining the effect of these drugs on ASCVD outcomes are completed. Without definitive data from randomized outcome trials demonstrating that specifically lowering Lp(a) levels results in a reduction in ASCVD events the advantages of measuring and treating Lp(a) will remain uncertain. Anti-sense therapy to specifically lower Lp(a) is under development and hopefully in the near future will provide a clear demonstration of the benefits of monitoring and treating Lp(a) levels (127,128).


In the meantime, many experts would recommend measuring Lp(a) levels in certain patients (Table 4) (59,129,130).  Elevations in Lp(a) will stimulate more aggressive lowering of LDL levels and the consideration of adding drugs that lower Lp(a) such as PCSK9 inhibitors (131).



·       Patients with unexplained premature CHD

·       Patients with a strong family history of premature CHD

·       Patients with a family history of elevated Lp(a) levels (Cascade screening)

·       Patients with resistance to LDL-C lowering with statins

·       Patients with rapid unexplained progression of atherosclerosis

·       Patients with familial hypercholesterolemia

·       Patients with aortic valvular stenosis of uncertain cause

·       Patients with intermediate risk profiles?


Standard measurements of LDL-C (either calculated or measured) include Lp(a) cholesterol (129,132). When Lp(a) levels are very high they can make a significant contribution to LDL-C levels. Similarly, when LDL-C levels are markedly reduced with treatment the LDL-C measured may include a significant contribution from Lp(a). The contribution of Lp(a) cholesterol to calculated LDL-C is approximately mg/dL Lp(a) x 0.3 (when both are expressed in mg/dL) (129,132). For example, if the Lp(a) level is 100mg/dL one can estimate that approximately 30mg/dL of the calculated LDL level is due to Lp(a).


Accurate measurement of Lp(a) represents a formidable technical challenge, unequalled in the world of biochemical diagnostics (129,133). This is due to the extreme length polymorphism of apo(a), whose size can vary over five-fold. Measuring Lp(a) mass (in mg/dL), as it is most often done in commercial clinical labs, will not allow for a reliable and consistent way to convert Lp(a) concentration to nmol/l. For example, 50 mg/dL of Lp(a) with 40 kringle IV type 2 repeats is actually fewer particles than 30 mg/dL of an Lp(a) with 15 kringle IV type repeats. The solution is the adoption of an isoform-independent method that equally identifies each Lp(a) particle (129). Such a method is currently approximated by the use of a spectrum of isoform-specific calibrators, and providers should, if possible, have Lp(a) measured using this method and reported as concentration in nmol/l.




While advanced lipoprotein measurements can provide additional insights and information it is not clear that for the evaluation and treatment of the vast majority of our patients that these measurements are necessary. Notably, the guidelines on the evaluation and treatment of hyperlipidemia put forth by a variety of different expert panels and organizations do not require advanced lipoprotein measurements. It is also the author’s opinion that at this time the routine use of advanced lipoprotein testing in clinical practice is not required and that LDL-C and non-HDL-C levels provide sufficient information to guide evaluation and treatment for most patients. Until clinical trial data demonstrate the superiority of utilizing advanced lipoprotein testing on clinical outcomes it is hard to recommend the routine use of such testing. However, it should be recognized that in selected patients the additional information provided can be helpful and result in changes in treatment. It is hoped that as additional drugs to treat lipids are developed and our understanding of lipid and lipoprotein metabolism expands that in the future the use of advanced lipoprotein analysis will assume a more important role in the evaluation and treatment of patients to prevent ASCVD.




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Growth hormone Stimulation Tests in Assessing Adult Growth Hormone Deficiency



The clinical features of adult GH deficiency (GHD) are nonspecific, and GH stimulation testing is often required to arrive at a correct diagnosis. However, diagnosing adult GHD can be challenging due to the episodic and pulsatile endogenous GH secretion, concurrently modified by age, gender, and body mass index. Hence, a GH stimulation test is often required to establish the diagnosis, and should only be considered if there is a clinical suspicion of GHD and the intention to treat if the diagnosis is confirmed. Currently, there is no ideal stimulation test and the decision to consider performing a GH stimulation test to diagnose adult GHD must factor in the validity of the chosen test, the appropriate GH cut-points, and the availability of local resources and expertise. For now, the insulin tolerance test should still be considered as the gold standard GH stimulation test, and the glucagon stimulation test and macimorelin test are reasonable alternatives to the insulin tolerance test, whereas the arginine test is no longer recommended by the 2019 AACE Clinical Practice Guidelines because this test has insufficient diagnostic accuracy and requires a very low peak GH cut-point of 0.4 μg/L to make the diagnosis. In this chapter, we discuss recently published evidence of the GH stimulation tests used in the United States and the inherent caveats and limitations of each individual test. We propose utilizing the lower GH cut-point to 1mg/L for the glucagon stimulation test to improve its diagnostic accuracy in some overweight and all obese patients, and summarize current knowledge of the new oral macimorelin test as the recently and only approved diagnostic test for adult GHD by the United States Food and Drug Administration and the European Medicines Agency.




Physiological growth hormone (GH) secretion from the anterior pituitary gland is episodic, pulsatile, and accounts for > 85% of total daily GH secretion (1). Due to its pulsatility, serum GH levels vary between peaks and troughs, with very low levels between pulses. Hypothalamic growth hormone–releasing hormone (GHRH) and somatostatin traverse the hypothalamic–pituitary portal system to stimulate and suppress GH production, respectively, by signaling through specific somatotroph cell-surface G protein-coupled receptors (2), while gastric-derived ghrelin also stimulates GH secretion and synergizes the action of GHRH (3). Additionally, other factors such as gender, nutritional status, sleep patterns, physical activity, and metabolic and hormonal signals from other endocrine glands, including glucocorticoids, thyroid hormones, and sex steroids, also play an important role in modulating day-to-day GH secretion (1). Growth hormone regulates its own secretion by a feedback mechanism that involves other peripheral mediators, such as insulin-like growth factor-I (IGF-I), free fatty acids, glucose, and insulin (4). Peripheral GH actions are primarily mediated through IGF-I synthesized mainly by the liver. Because IGF-I has a longer half-life in the circulation than GH, it is considered to provide an integrated measure of GH secretion. Like GH, serum IGF-I levels decline with aging (5), and tend to be low in obesity (6) and in patients with non-alcoholic fatty liver disease (7) and the levels may overlap with the levels observed in younger GH–deficient patients. Hence, for these reasons, the diagnosis of adult GH deficiency (GHD) cannot be established in most patients by a random single measurement of serum GH or IGF-I level.




Adult GHD is a heterogeneous disorder that commonly results from a variety of organic causes, including hypothalamic-pituitary tumors and/or their treatment, head trauma, and infiltrative diseases (8). This condition is characterized by decreased lean body mass and increased fat mass, dyslipidemia, cardiac dysfunction, decreased fibrinolysis and premature atherosclerosis, decreased muscle strength and exercise capacity, decreased bone mineral density, increased insulin resistance, and impaired quality of life (9). Treatment with GH replacement improves most of these abnormalities (10, 11). However, due to the high cost of GH replacement (GH costs approximately $18,000 to $30,000 per year depending on the dose and brand used) (12) and concerns of potential long-term risks, particularly the development of diabetes mellitus, cancer and tumor recurrence, it is imperative that the correct diagnosis is established so that appropriate GH replacement is offered to adults who are truly GH-deficient, and not for anti-aging and sporting enhancement (13, 14).


The diagnosis of adult GHD is challenging for the clinician because of the lack of a single biological end-point, such as growth failure seen in children with GHD. Other biochemical measurements like IGF-I, IGF-binding protein-3, or GH secretion over a 24-hour period have shown poor diagnostic value as there is an overlap between healthy and adults with GHD, particularly in adults > 40 years of age (5, 15). Hence, a GH stimulation test is often required to establish the diagnosis, and should only be considered if there is a clinical suspicion of GHD and the intention to treat if the diagnosis is confirmed. Currently, there is no ideal stimulation test and the decision to consider performing a GH stimulation test to diagnose adult GHD must factor in the validity of the chosen test and its GH cut-points, and the availability of local resources and expertise.


Clinical practice guidelines recommend the evaluation of adult GHD to be based on medical history, clinical findings, and utilizing the appropriate GH stimulation test for biochemical confirmation (8, 16-18). The exception when GH stimulation testing is exempted include those with organic hypothalamic-pituitary disease with ≥ 3 pituitary hormone deficiencies and low serum IGF-I levels [< -2.0 standard deviation scores (SDS)] (19), patients with genetic defects affecting the hypothalamic-pituitary axes, and those with hypothalamic-pituitary structural brain defects (8, 16, 18). Evaluation for adult GHD should not be performed in patients with no evidence of a suggestive history, e.g., sellar/parasellar mass lesion or a history of a hypothalamic–pituitary insult, such as surgery, radiation therapy, head trauma, or brain tumor. Additionally, GH stimulation testing should not be performed in patients with commonly encountered, generalized, nonspecific symptoms of weakness, frailty, or obesity, since a misleading diagnosis of adult GHD can be made in some patients with these symptoms whose underlying pituitary function is otherwise intact (8, 16, 18).


All GH stimulation tests are based on the concept that a pharmacological agent acutely stimulates pituitary GH secretion, and peak serum GH levels detected by sequential blood sampling of serum GH levels after administration of the GH provocative agent. The desired criteria of an ideal GH stimulation test should include the following: the ability to accurately and reliably differentiate adults with GHD from GH-sufficient individuals, high reproducibility, safe with minimal side-effects, affordable, short test duration, and simple to perform.


The insulin tolerance test (ITT) has historically been accepted as the gold-standard test for the assessment of adult GHD provided adequate hypoglycemia (blood glucose <40 mg/dL) is achieved (8, 16, 17). However, multiple drawbacks associated with the ITT hamper its wider use (20), and they include the requirement of close medical supervision by a physician throughout the test, the possibility of inducing severe life-threatening hypoglycemia, and the potential of causing seizures and altered consciousness resulting from neuroglycopenia in certain susceptible sub-populations. This test is also contraindicated in the elderly (> 65 years of age) and in patients at risk of and/or with a history of cardio-/cerebrovascular disease and seizures.


In recent years, finding a reliable alternative to the ITT for the diagnosis of adult GHD has been challenging. When the GHRH-arginine test was available in the United States before EMD Serono discontinued manufacturing the GHRH analog (Geref@) in November 2008 (8, 16, 17), it was considered the most acceptable alternative to the ITT. Since then, the glucagon stimulation test (GST) has grown in popularity replacing the GHRH-arginine test as the test of choice if the ITT cannot be performed or is contraindicated (21). Previous studies have examined the diagnostic utility of the GST for adult GHD, but these studies have either not taken body mass index (BMI) into consideration (22, 23)or included only controls with normal BMIs (24, 25). Several recent retrospective studies have questioned the diagnostic accuracy of the GST when the GH cut-point of 3mg/L is applied to overweight/obese adults (26-29) and in those with glucose intolerance (28, 29), while Hamrahian et al. (30) demonstrated in a prospective study of 28 patients by comparing the GST to the ITT that a lower GH cut-point of 1 mg/L improved its diagnostic accuracy with a 92% sensitivity and 100% specificity.


In this chapter, we will discuss recently published evidence of the GH stimulation tests used in the United States and the inherent caveats and limitations of each individual test. We propose utilizing the lower GH cut-point to 1mg/L for the GST to improve its diagnostic accuracy in some overweight and all obese patients, and will summarize current knowledge of the new oral macimorelin test as the recently and only approved diagnostic test for adult GHD by the United States Food and Drug Administration (FDA) and the European Medicines Agency.




The responses to all GH stimulation tests show intra-individual variability, and the GH cut-points vary depending on the test used. For the ITT and GST, the cut-points advocated by previous consensus guidelines were 3-5 μg/L and 2.5-3 μg/L, respectively (8, 16). Other GH stimulatory agents such as clonidine, L-DOPA, and arginine are weaker GH secretagogues, and would require very low GH cut-points with utilization of sensitive GH assays to achieve adequate specificity (e.g., arginine of 0.4 μg/L) (31). Hence, these tests are generally not recommended in the United States (8, 16). Other limitations include the relative lack of validated normative data based on age, gender, BMI, glycemic status, and the paucity of data for specific etiologies of adult GHD that have recently been described, such as traumatic brain injury, subarachnoid hemorrhage, ischemic stroke, and central nervous system infections (32, 33).


One of the caveats in interpreting the results of GH stimulation tests is that adult GHD itself is complicated by an increased susceptibility to central obesity (34). Obesity per se is a state of relative GHD (35-39)(36-40), and earlier physiologic studies in obese individuals have shown that spontaneous GH secretion is reduced, GH clearance is enhanced, and stimulated GH secretion is reduced (40-42). Conversely, serum IGF-I levels are unaffected, or even increased, and this discordance is related to the increased hepatic GH responsiveness (43). The decreased serum GH levels in obesity results in up-regulation of GH receptor and sensitivity. Furthermore, non-alcoholic fatty liver disease is increasingly recognized in overweight and obese adults with GHD (44), with lower serum IGF-I levels being associated with increasing severity of the disease (7). Thus, these data suggest that BMI-specific cut-points should be considered when testing patients for adult GHD. Table 1 summarizes the accepted GH cut-points for the GH stimulation tests used in the United States, as recommended by different consensus guidelines.


Table 1   GH cut-points (µg/L) for GH stimulation tests by different consensus guidelines for diagnosis of adult GHD.



GRS 2007




AACE 2009



ES 2011



AACE 2019






< 3.0


≤ 5.0


< 3.0 to 5.0


≤ 5.0



- BMI < 25 kg/m2

- BMI 25-30 kg/m2

- BMI ≥ 30 kg/m2




< 11.0

< 8.0

< 4.0



≤ 11.0

≤ 8.0

≤ 4.0



< 11.0

< 8.0

< 4.0



No recommendation as not commercially available in the United States



- BMI < 25 kg/m2

- BMI 25-30 kg/m2

- BMI ≥ 30 kg/m2




< 3.0

< 3.0

< 3.0



≤ 3.0

≤ 3.0

≤ 3.0



< 3.0

< 3.0

< 3.0



≤ 3.0

≤ 3.01 or ≤ 1.02

≤ 1.0





Not commercially available in 2007



Not commercially available in 2009



Not commercially available in 2011


≤ 2.8





Not recommended to be used


≤ 0.4



Not recommended to be used


No longer recommended to be used


1GH cut-point of ≤ 3.0 µg/L for patients with a high pre-test probability; 2GH cut-point of ≤ 1.0 µg/L for patients with a low pre-test probability. AACE, American Association of Clinical Endocrinologists; BMI, body mass index; ES, Endocrine Society; GHRH, growth hormone releasing hormone; GRS, Growth Hormone Research Society; ITT, insulin tolerance test.




Insulin Tolerance Test


The ITT has traditionally been accepted as the gold standard test for the assessment of adult GHD, with a GH cut-point of 3-5 mg/L when adequate hypoglycemia (blood glucose < 40 mg/dL) is achieved (8, 16, 17). This GH cut-point was originally proposed by Hoffman et al. (45) in 1994 based on GH responses to insulin-induced hypoglycemia, mean 24-hour GH levels derived from 20-min sampling, and serum IGF-I and IGFBP-3 levels in 23 patients considered GH-deficient due to organic pituitary disease, and in 35 sex-matched normal subjects of similar age and BMI. The ranges of stimulated peak GH responses separated GH-deficient (0.2-3.1 mg/L) from GH-sufficient (5.3-42.5 mg/L) patients. However, an overlap in mean 24-hour GH, IGF-I, and IGFBP-3 levels was observed, demonstrating the challenge in utilizing random single serum GH, IGF-I and IGFBP-3 levels to accurately differentiate GH-sufficiency from GHD.


Disadvantages of the ITT include the requirement of close medical supervision, may be unpleasant and cautioned in some patients because of potential adverse effects (e.g., seizures or loss of consciousness resulting from neuroglycopenia), and contraindicated in elderly patients and in patients at risk of and/or with a history of cardio-/cerebrovascular disease and seizures. Furthermore, normoglycemic and/or hyperglycemic obese patients with insulin resistance may fail to achieve adequate hypoglycemia (46), necessitating the use of higher insulin doses (0.15-0.2 IU/kg), thus increasing the risk of delayed hypoglycemia. Although the ITT demonstrates good sensitivity, its reproducibility is another major limitation. Differences in peak GH responses have been demonstrated in healthy subjects undergoing ITT at varying times (47) and in women at different times of their menstrual cycle (48).


Table  2. Protocol for the Insulin Tolerance Test


History of epileptic seizures, coronary artery disease, pregnancy, or age > 55 years.


Patients commonly develop neuroglycopenic symptoms during the test and should be encouraged to report these symptoms (administration of IV anti-emetics can be considered).

Late hypoglycemia may occur (patients should be advised to eat small and frequent meals after completion of the test).


Fast from midnight for 8-10 hours.

All morning medications can be taken with water (if the HPA axis is simultaneously assessed, then glucocorticoids should be withheld ≥ 12 hours before testing).

Weigh patient.

1Place IV cannula for IV access in both forearms.

2Administer IV human Regular insulin (standard dose: 0.05-0.1 units/kg for non-diabetic subjects with a BMI < 30 kg/m2 and high dose: 0.15-0.3 units/kg for subjects with a BMI ≥ 30 kg/m2).


Blood is drawn for glucose measurement with a glucometer.

Blood draw for baseline glucose, GH and IGF-I levels (cortisol and ACTH, if HPA axis is assessed simultaneously).


Blood samples are drawn from the IV line every 5-10 mins for measurement of glucose levels using a glucometer.

Signs and symptoms of neuroglycopenia are recorded.

When blood glucose levels from the glucometer approaches 45 mg/dL (2.5 mmol/L), blood samples are sent to the laboratory for measurements of blood glucose levels.

When symptomatic hypoglycemia is achieved (laboratory blood glucose < 40 mg/dL or 2.2 mmol/L), additional blood samples are collected to measure glucose and GH levels (+/- cortisol if the HPA axis is assessed simultaneously) at 20, 25, 30, 35, 40, 60 and 90 min.

The patient can begin drinking orange juice and eat to raise his/her blood glucose levels (IV 100 ml of 5% Dextrose can be administered if the patient cannot tolerate oral intake due to nausea or vomiting).


Blood glucose levels measured from the glucometer should increase to levels > 70 mg/dL (3.9 mmol/L) before the patient is discharged from the testing unit.


If adequate (symptomatic) hypoglycemia is not achieved (< 40 mg/dL or 2.2 mmol/L), then adult GHD cannot be diagnosed.

Peak serum GH levels ≤ 5 µg/L at any time point during the hypoglycemic phase of the test is diagnostic of adult GHD.


If adequate (symptomatic) hypoglycemia is not achieved (< 40 mg/dL or 2.2 mmol/L), then adult GHD cannot be diagnosed.

HPA: hypothalamic-pituitary-adrenal, 1Two IV lines are placed, one IV line is used for the administration of insulin bolus and possibly for administration of IV 5% Dextrose administration if the patient requires resuscitation from hypoglycemia, while the other IV line is used for repeated blood draws. 2In certain patients with BMIs > 30 kg/m2 who appear muscular with increased insulin sensitivity, clinical discretion is required in deciding the insulin dose for these patients. A dose of 0.05-0.1 units/kg may be more appropriate to prevent severe or delayed hypoglycemia.   


Glucagon Stimulation Test


Glucagon is reportedly to be more potent than arginine or clonidine in stimulating GH secretion (24, 25). It has also been shown that glucagon stimulates GH secretion more effectively when administered intramuscularly or subcutaneously compared to the intravenous route (49). However, the mechanism(s) by which glucagon-induced GH stimulation occurs is unclear, although it is known that glucagon decreases ghrelin-independent effects of glucose or insulin variations (50).


There have been three earlier studies that have assessed the GST in identifying adult GHD in patients with pituitary disorders (22, 23, 51). Gomez et al. (51) and Conceicao et al. (23) compared the diagnostic characteristics of GST to ITT and included a control group matched for age and sex in both studies, and for BMI in one study (51). Using receiver operating characteristic (ROC) analysis, both studies proposed that a GH cut-point of 3 mg/L provided optimal sensitivity and specificity (51, 52). Gomez et al. (51) also demonstrated an inverse correlation between age (R = - 0.389, P = 0.0075) and BMI (R = - 0.329, P = 0.025) with peak GH levels in healthy controls. These data suggest that there is a potential association between relative, but not organic, GHD in aging and obesity. However, this study was conducted in a European cohort, where the frequency and severity of obesity is generally to a lesser degree than in the United States (53). Conversely, Conceicao et al. (23) demonstrated that peak GH levels were unaffected by age in either the control or patient group, and neither were there any gender differences. Additionally, Gomez et al. (51)used intramuscular glucagon doses of 1 mg and 1.5 mg for body weights ≤ 90 kg and > 90 kg respectively, whereas Conceicao et al. (23) used intramuscular glucagon of 1 mg for all subjects. In another study, Berg et al. (22)demonstrated an optimal peak GH cut-point of 2.5 mg/L with 95% sensitivity and 79% specificity using ROC analysis. This study also reported lower peak GH levels with GST compared to ITT (5.1 vs 6.7 mg/L, P < 0.01) and a positive correlation between peak GH levels during ITT and GST (R = 0.88, P < 0.0001), but no correlation between BMI or age to peak GH responses (54, 55). However, these (22, 23, 51) and other earlier studies (24, 25, 49, 56) did not specifically evaluate patients with glucose intolerance; hence, the diagnostic accuracy of the GST in testing for GHD in this population remains unclear.


Advantages of the GST is its reproducibility, safety, and lack of influence by gender and hypothalamic GHD (21), whereas disadvantages include the lengthy test duration (3-4 hours), and the need for an intramuscular injection that might not appeal to some patients. Side-effects frequently reported include nausea, vomiting, and headaches ranging from < 10% (22) to 34% (54), mainly occur between 60-210 min and tend to resolve by 240 min into the test, and seem to be more pronounced in elderly subjects, where severe symptomatic hypotension, hypoglycemia, and seizures have been observed (57).    


However, since the publication of the 2009 American Association of Clinical Endocrinologists (AACE) (16) and 2011 Endocrine Society (8) Clinical Practice Guidelines, there have been several studies that have suggested that the fixed-dose GST using a GH cut-point of 3 mg/L may potentially over-diagnose adult GHD in a substantial number of overweight/obese subjects and in those with glucose intolerance. In two large retrospective studies, Toogood et al.(58) and Yuen et al. (29) found an inverse correlation between BMI and peak GH during the GST, and that this relationship appeared to be strongest with BMIs between 30 and 40 kg/m2 and seemed to plateau for those with BMIs > 40 kg/m2 (58). Alternatively, a negative correlation between BMI and peak GH following glucagon stimulation has been reported by Gomez et al. (51) in healthy subjects but not in patients with underlying pituitary disease. Dichtel et al. (26) evaluated 3 groups of overweight/obese men, i.e., controls who were younger than the patients, patients with 3-4 pituitary hormone deficits, and patients with 1-2 pituitary hormone deficits. Using ROC analysis, the GH cut-point of 0.94 mg/L provided the optimal sensitivity (90%) and specificity (94%), whereas BMI and amount of visceral adipose tissue inversely correlated with peak GH levels in controls. Almost half of the healthy overweight/obese individuals (45%) failed the GST using the 3 mg/L GH cut-point. Diri et al. (27) evaluated 216 patients with pituitary disease and 26 healthy controls and compared the GST to the ITT. These investigators used a GH cut-point of 3.0 mg/L for the ITT and two GH cut-points of 3.0 mg/L and 1.07 mg/L for the GST, yielding the diagnosis of adult GHD in 86.1%, 74.5%, and 54.2 % patients, respectively. Additionally, patient age, BMI, and number of pituitary hormone deficits correlated with IGF-I and peak GH levels. Twelve out of 26 (46.2 %) healthy subjects failed the GST using a GH cut-point of 3.0 mg/L, but none when the cut-point was lowered to 1.07 mg/L. Wilson et al. (28) studied 42 patients with a high pre-test probability of adult GHD. After excluding 10 patients with severe GHD based on peak GH levels ≤ 0.1 mg/L, these investigators found that body weight negatively correlated with GH area under the curve (AUC) (R = -0.45; P = 0.01) and peak GH response (R = -0.42; P = 0.02) and positively correlated with nadir blood glucose levels (R = 0.48; P < 0.01). Conversely, nadir blood glucose levels during GSTs inversely correlated with GH AUC (r= -0.38; p=0.03) and peak GH (r= -0.37; p=0.04), implying that patients with higher nadir blood glucose levels tended to have a lesser glucagon-induced GH response. Recently, Hamrahian et al. (30) compared the fixed-dose GST (1 mg or 1.5 mg in patients > 90 kg body weight) and weight-based GST (WB-GST: 0.03 mg/kg) with the ITT using a GH cut-point of 3.0 mg/L. Patients with hypothalamic-pituitary disease and 1-2 (n = 14) or ≥ 3 (n = 14) pituitary hormone deficiencies, and control subjects (n = 14) matched for age, sex, estrogen status and BMI undertook the ITT, GST and WB-GST in random order. Using ROC analyses, the optimal GH cut-point was 1.0 (92% sensitivity, 100% specificity) for fixed-dose GST and 2.0 mg/L (96% sensitivity and 100% specificity) for WB-GST. Therefore, lowering the GH cut-point from 3 mg/L to 1 mg/L is important to reduce misclassifying adult GHD in overweight (BMI 25-30 kg/m2) patients with a low pre-test probability and in obese (BMI > 30 kg/m2) patients.


It remains unclear whether hyperglycemia influences peak GH responses to glucagon stimulation, independent of central adiposity. No peak GH responses have been studied using the GST in normal controls > 70 years of age, and none of the previous studies included patients with poorly controlled diabetes mellitus. Studies by Yuen et al. (29) and Wilson et al. (28) demonstrated that higher fasting (range 90-316 mg/dL), peak (range 156-336 mg/dL), and nadir (range 52-200 mg/dL) blood glucose levels during the GST were associated with lower peak GH responses. Therefore, stratification of GH responsiveness by the degree of glycemia will be helpful to clinicians in interpreting the GST results in patients with impaired glucose tolerance and diabetes mellitus. Because these data are currently unavailable, caution should be exercised when interpreting abnormal GST results in these patients. Further larger prospective studies are needed to address the effects of varying degrees of hyperglycemia on the ability of glucagon to stimulate GH secretion.


Table  3. Protocol for the Glucagon Stimulation Test.


Malnourished patients or patients who have not eaten for > 48 hours.

Severe fasting hyperglycemia > 180 mg/dL.


Patients may feel nauseous during the test (administration of IV anti-emetics may be considered).

Late hypoglycemia may occur (patients should be advised to eat small and frequent meals after completion of the test).


Fast from midnight for 8-10 hours.

All morning medications can be taken with water.

Weigh patient.

Place IV cannula for IV access in one forearm.

Administer IM glucagon (1.0 mg if patient body weight ≤ 90 kg and 1.5 mg if patient body weight > 90 kg).


Blood is drawn for serum GH and blood glucose levels at 0, 30, 60, 90, 120, 150, 180, 210 and 240 mins.


Peak GH levels ≤ 3.0 µg/L in normal-weight (BMI < 25 kg/m2) patients and in

overweight (BMI 25-30 kg/m2) patients with a high pre-test probability, and ≤ 1.0 ug/L in

overweight (BMI 25-30 kg/m2) patients with a low pre-test probability and in

obese (BMI > 30 kg/m2) patients at any time point during testing are diagnostic of adult



Clinical suspicion of pre-test probability should be taken into consideration when interpreting GST results in patients > 70 years of age and in patients with impaired glucose tolerance and poorly controlled diabetes mellitus, as no peak GH responses have been studied in these patients.

Serum GH: peak GH levels tend to occur between 120-180 mins; blood glucose: usually peaks around 90 mins and then gradually declines (not a requirement to interpret the test).


Macimorelin Test


Growth hormone secretagogues (GHSs) are peptidyl (GH-releasing peptide [GHRP]) and nonpeptidyl molecules that exert strong dose-dependent and specific stimulatory effects on the animal and human somatotrope secretion (59). These agents act as functional somatostatin antagonists by binding to their specific GH secretagogue receptor-1a in the hypothalamus and pituitary. The natural ligand for this receptor is the gut peptide ghrelin (60). Growth hormone secretagogues are now considered as ghrelin mimetic agents and can be administered parenterally (e.g., GHRP-2, GHRP-6, hexarelin) or orally (e.g., MK-677 and macimorelin).


Macimorelin (formerly known as AEZS-130, ARD-07, and EP-01572) is a novel GH secretagogue that binds the GHS-R1a receptor and to pituitary and hypothalamic extracts with a similar affinity to ghrelin (61). In healthy volunteers, it is readily absorbed with good stability and oral bioavailability, and effectively stimulates endogenous GH secretion (61). An open-label, crossover, multicenter trial examined the diagnostic accuracy of a single oral dose of macimorelin (0.5 mg/kg) compared to GHRH plus arginine in adults with GHD and healthy matched controls (62). Peak GH levels were 2.36 ± 5.69 and 17.71 ± 19.11 mg/L in adults with GHD and healthy controls, respectively, with optimal GH cut-points ranging between 2.7 and 5.2 mg/L (62). Macimorelin showed good discrimination comparable to GHRH plus arginine, with peak GH levels that were inversely associated with BMI in controls. In a recent multicenter, open-label, randomized, two-way crossover study, oral macimorelin was compared to the ITT to validate its use for the diagnosis of adult GHD (63). The GH cut-point levels of 2.8 mg/L for macimorelin and 5.1 mg/L for ITT provided 95.4% (95% CI, 87% to 99%) negative agreement, 74.3% (95% CI, 63% to 84%) positive agreement, 87% sensitivity, and 96% specificity. In both studies (62, 63), macimorelin was well-tolerated, reproducible, and safe. In December 2017, the United States FDA approved macimorelin for use as a diagnostic test for adult GHD and mandated the GH cut-point of2.8 mg/L to be used to differentiate patients with normal GH secretion from those with GHD. However, in the study by Garcia et al. (63), when the GH cut-point was increased to 5.1 mg/L for both macimorelin and ITT, negative agreement and specificity was unchanged at 94% (95% CI, 85% to 98%) and 96%, respectively, but interestingly, positive agreement and sensitivity was higher at 82% (95% CI, 72% to 90%) and 92%. Because measured serum GH levels are dependent on the GH assays used, using the GH cut-point of 5.1 mg/L for macrimorelin that is identical to the cut-point accepted for the ITT could be considered in patients with peak serum GH levels between 2.8 mg/L to 5.1 mg/L, especially if the patient has a high pre-test probability, e.g. history of surgery on a sellar/parasellar mass with 1-2 other pituitary hormone deficiencies. However, it remains to be determined whether BMI-adjusted peak GH cut-points for this test are needed for overweight and obese patients.


Main advantages of macimorelin is that the drug is orally administered, unlike the ITT, GHRH plus arginine or GST, that requires intravenous or intramuscular administration, and no risk of causing hypoglycemia. In addition, the test only lasts 90 minutes with 3-4 blood sample collections required, in contrast to more blood sample collections over 2 hours for the ITT and 3-4 hours for the GST. The most commonly reported side effect was mild dysgeusia, which did not require any intervention and resolved spontaneously (63). One drug-related serious adverse event was reported; that was in a subject with an asymptomatic QT interval prolongation on the electrocardiogram that resolved spontaneously within 24 h (62). However, it is important to note that this subject was taking citalopram, a serotonin selective uptake inhibitor known to be associated with QT prolongation (64). Careful assessment of the patient’s concurrent medications is recommended as well as discontinuation of strong CYP3A4 inducers, provided this is considered safe by the prescribing physician and with sufficient washout time prior to testing.


Table  4. Protocol for the Macimorelin Test.


Drugs that may increase its plasma levels and prolong QT.




Fast from midnight for 8-10 hours.

All morning medications can be taken with water.

Weigh patient.

Place IV cannula for IV access in one forearm.

Dissolve in water 1 (120 ml) or 2 pouches (240 ml) of macimorelin (≤ 120 kg = 1 pouch; > 120 kg = 2 pouches)

Calculate macimorelin dose (0.5 mg/kg as a single oral dose) and volume of water required to reconstitute macimorelin solution (patient body weight X kg = X ml macimorelin solution, e.g., patient with a body weight of 70 kg would require 70 mL of reconstituted macimorelin solution)

After volume of macimorelin is calculated, stir the solution gently and thoroughly for 2-3 min, and use within 30 min of preparation.

Draw the exact macimorelin volume of solution into a needleless syringe, transfer the exact volume of into a drinking glass, and instruct the patient drink the entire volume of solution within 30 seconds.


Blood is drawn for measurements of serum GH levels at 30, 45, 60 and 90 min.


Peak serum GH levels tend to occur between 45-60 mins.

When used according to prescribing package label, peak GH levels ≤ 2.8 µg/L at any time point is diagnostic of adult GHD.


Peak GH levels ≤ 5.1 µg/L at any time point may be considered in patients with a high-pre-test probability to diagnose adult GHD, as this higher GH cut-point limits the risk of a false-positive diagnosis and maintains a high detection rate for GH-deficient patients because of the more potent GH stimulatory effect of macimorelin compared with the ITT.

Safety and diagnostic performance in patients < 18 and > 65 years of age, and in patients with impaired glucose tolerance and poorly controlled diabetes mellitus, and BMI-adjusted peak GH cut-points for overweight and obese patients is not established.


Summary of Tests


Table 5 displays a summary of the desirable test characteristics of GH stimulation tests currently available in the United States.


Table 5. Summary of Characteristics of GH Stimulation Tests










Gold standard























1if appropriate BMI-specific GH cut-points are used; 2contraindicated in patients with a history hypoglycemia, history of previous seizures, in the elderly (> 65 years of age), and in patients at risk of and/or with a history of cardio-/cerebrovascular disease; 3caution in patients with propensity for nausea and vomiting, and elderly patients who may be at risk of developing symptomatic hypotension, and dizziness (57); 4patients may not tolerate severe symptomatic hypoglycemia. GST, glucagon stimulation test; ITT, insulin tolerance test.




Accurate measurement of GH levels is critical for establishing the diagnosis of adult GHD because the analytical method influences the results of GH stimulation tests, which is dependent on specific GH cut-point levels. However, circulating GH is present in several different isoforms and isomers, including the most common variant of 22 kDa, and other smaller molecules, such as the 20 kDa GH variant. Monoclonal antibodies binding to a specific molecular form of GH are used to limit detection to the 22 kDa GH, but will not detect other GH isoforms. Other molecules similar to GH (e.g., placental GH and prolactin) could potentially cross-react and affect the measurement of GH levels. Growth hormone binding protein, to which approximately 50% of circulating GH is bound, can also cause interference in a GH assay. Furthermore, substantial heterogeneity exists among currently utilized assays due to the use of different standard preparations for calibration of GH immunoassays, and lack of harmonization between various GH assays makes it difficult to directly compare diagnostic cut-points across different published studies. Another source of confusion when interpreting data of GH stimulation tests was that some laboratories reported GH levels in activity (mU/L), whereas others used mass units (mg/L) (65).


Due to the heterogeneity of GH assays, it is important that GH assays utilize a universal GH calibration standard 98/574 (National Institute for Biological Standards and Control), a recombinant pituitary GH preparation of high purity (66). All assay manufacturers should also specify the validation of their assay, which should include specification of the GH isoforms detected (20 kDa GH, 22 kDa GH, and other isoforms), the analyte being measured, the specificities of the antibodies used, and the presence or absence of growth hormone binding protein interference.




The decision to perform GH stimulation tests should be based on the clinical suspicion of the treating endocrinologist. If the clinical suspicion is high, such as in a patient with history of surgery on a sellar mass, concurrent 1-2 other pituitary hormone deficiencies and a low (< -2 SDS) or low-normal (< 0 SDS) serum IGF-I level, then performing one GH stimulation test is sufficient. If the clinical suspicion is low, such as in cases where there is no suggestive history, such as hypothalamic-pituitary disease, surgery or radiation therapy, head trauma, or childhood-onset GHD, then the diagnosis of adult GHD should not be pursued and GH stimulation testing should not be performed. For now, the ITT should still be considered as the gold standard GH stimulation test, and the GST and macimorelin test are reasonable alternatives to the ITT, whereas the arginine test is no longer recommended by the 2019 AACE Clinical Practice Guidelines because the test has insufficient diagnostic accuracy due to the weak GH-stimulating effects of arginine that consequently requires a very low peak GH cut-point of 0.4 μg/L to make the diagnosis (18). As the reliability of the GST GH cut-point of 3 mg/L in overweight/obese subjects and in those with glucose intolerance has been shown to misclassify some patients, the utilization of GH cut-points of the GST is now based on the clinician’s level of suspicion of the patient’s pre-test probability and underlying BMI. Macimorelin, a drug administered orally that was approved by the United States FDA in December 2017, appears to be a very promising and attractive test because it is easy to conduct with high reproducibility, is safe, and has a diagnostic accuracy comparable to the ITT and GHRH plus arginine test. The only factor that may hinder its wider use over time is its cost (one 60 mg macimorelin packet costs approximately $4,500) (67) and the potential of drug-to-drug interactions that may cause QT prolongation. Further studies with larger numbers of patients including children, adolescents, elderly (> 70 years of age), and those with obesity, diabetes mellitus, and traumatic brain injury are needed to determine the sensitivity and specificity of this agent in these cohorts of patients. Additionally, more studies are also needed to improve the palatability of the drug, and to help outline any potential safety issues associated with this test (i.e., concomitant use with drugs that may induce QT prolongation).




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Approach To Hypercalcemia



A reduction in serum calcium can stimulate parathyroid hormone (PTH) release which may then increase bone resorption, enhance renal calcium reabsorption, and stimulate renal conversion of 25-hydroxyvitamin D, to the active moiety 1,25-dihydroxyvitamin D [1,25(OH)2D] which then will enhance intestinal calcium absorption. These mechanisms restore the serum calcium to normal and inhibit further production of PTH and 1,25(OH)2D. Normal serum concentrations of total calcium generally range between 8.5 and 10.5 mg/dL (2.12 to 2.62 mM) and ionized calcium between 4.65-5.30 mg/dL (1.16-1.31 mM). Decreased PTH and decreased 1,25(OH)2D should accompany hypercalcemia unless PTH or 1,25(OH)2D is causal. Hypercalcemia may be caused by: Endocrine Disorders with Excess PTH including primary sporadic and familial hyperparathyroidism, and tertiary hyperparathyroidism; Endocrine Disorders Without Excess PTH including hyperthyroidism, pheochromocytoma, VIPoma, hypoadrenalism, and Jansen's Metaphyseal Chondrodysplasia; Malignancy-Associated Hypercalcemia, which can be caused by elevated PTH-related protein (PTHrP), or other factors (e.g. increased 1,25(OH)2D in lymphomas); Inflammatory Disorders including Granulomatous Diseases, where excess 1,25(OH)2D production may be causal, and HIV/AIDS; Pediatric Syndromes including Williams Syndrome and Idiopathic Infantile Hypercalcemia, where inappropriate levels of 1,25(OH)2D may occur due to a mutation in the 25-hydroxyvitamin D-24-hydroxylase gene (CYP24A1); Medication, including thiazide diuretics, lithium, vitamin D, vitamin A, estrogens and antiestrogens, theophylline, and prolonged immobilization, particularly in states of high bone turnover. Treatment should be aimed at the underlying disorder, however, if serum calcium exceeds 12 to 14mg/dL (3 to 3.5mM), acute hydration and agents that inhibit bone resorption are required. Under selected conditions, calcimimetics, calciuresis, glucocorticoids, or dialysis may be needed.    



Hypercalcemia can be defined as a serum calcium greater than 2 standard deviations above the normal mean in a reference laboratory. Calcium in the blood is normally transported partly bound to plasma proteins (about 45%), notably albumin, partly bound to small anions such as phosphate and citrate (about 10%) and partly in the free or ionized state (about 45%) (1). Although only the ionized calcium is metabolically active i.e. subject to transport into cells and capable of activating cellular processes, most laboratories report total serum calcium concentrations. Concentrations of total calcium in normal serum generally range between 8.5 and 10.5 mg/dL (2.12 to 2.62 mM) and levels above this are considered to be consistent with hypercalcemia. Nevertheless, reference ranges may vary between laboratories. The normal range of ionized calcium is generally 4.65-5.25 mg/dL (1.16-1.31 mM), but again values may vary slightly between laboratories. When protein concentrations, and especially albumin concentrations, fluctuate substantially, total calcium levels may vary, whereas the ionized calcium may remain relatively stable. Thus, dehydration or hemoconcentration during venipuncture may elevate serum albumin and a falsely elevated total serum calcium may be reported. Such elevations in total calcium, when albumin levels are increased, can be "corrected" by subtracting 0.8 mg/dL from the total calcium for every 1.0 g/dL by which the serum albumin concentration is >4 g/dL. Conversely when albumin levels are low, total calcium can be corrected by adding 0.8 mg/dL for every 1.0 g/dL by which the albumin is <4 g/dL. Thus, to correct for an abnormally high or low serum albumin the following formula can be used: Corrected calcium (mg/dL) = measured total serum calcium (mg/dL) + [4.0- serum albumin (g/dL) X 0.8] or Corrected calcium (mM) = measured total Ca (mM) + [40 - serum albumin (g/L)] X 0.02. Even in the presence of a normal serum albumin, changes in blood pH can alter the equilibrium constant of the albumin-Ca++ complex, with acidosis reducing the binding and alkalosis enhancing it. Consequently, when major shifts in serum protein or pH are present it is most prudent to directly measure the ionized calcium level in order to determine the presence of hypercalcemia.


The extracellular fluid (ECF) concentration of calcium is tightly maintained within a rather narrow range because of the importance of the calcium ion to numerous cellular functions including cell division, cell adhesion and plasma membrane integrity, protein secretion, muscle contraction, neuronal excitability, glycogen metabolism and coagulation.


The skeleton, the gut and the kidney play a major role in assuring calcium homeostasis. Overall, in a typical individual, if 1000 mg of calcium are ingested in the diet per day, approximately 200 mg will be absorbed. Approximately 10 g of calcium will be filtered daily through the kidney and most will be reabsorbed with about 200 mg being excreted in the urine. The normal 24-hour excretion of calcium may however vary between 100 and 300 mg per day (2.5 to 7.5 mmoles per day). The skeleton, a storage site of about 1 kg of calcium, is the major calcium reservoir in the body and bone turnover (bone formation coupled with bone resorption) will determine the net entry of calcium into or egress of calcium out of the skeleton. When bone turnover is balanced, approximately 500 mg of calcium is released from bone per day and the equivalent amount is accreted per day (Fig. 1).

Figure 1. Calcium balance. On average, if, in a typical adult approximately 1g of elemental calcium (Ca+2) is ingested per day, about 200mg/day will be absorbed and 800mg/day excreted. Approximately 1kg of Ca+2 is stored in bone and about 500mg/day is released by resorption or deposited during bone formation. Of the 10g of Ca+2 filtered through the kidney per day only about 200mg appears in the urine, the remainder being reabsorbed.

Tight regulation of the ECF calcium concentration is maintained through the action of calcium-sensitive cells which modulate the production of hormones (2-5). These hormones act on specific cells in bone, gut and kidney which can respond by altering fluxes of calcium to maintain ECF calcium. The parathyroid glands detect ECF calcium via a calcium sensing receptor (CaSR) (6). Thus, a reduction in ECF calcium can reduce stimulation of the parathyroid CaSR and facilitate release of parathyroid hormone (PTH) from the parathyroid glands in the neck. PTH can then act to enhance calcium reabsorption in the kidney while at the same time inhibit phosphate reabsorption producing phosphaturia. Reduced ECF calcium can also act via a CaSR in the loop of Henle to allow renal calcium reabsorption.


PTH and hypocalcemia per se can both stimulate the conversion of the inert metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], to the active moiety 1,25-dihydroxyvitamin D [1,25(OH)2D] (7), which in turn will enhance intestinal calcium absorption, and to a lesser extent phosphate reabsorption. 1,25(OH)2D can stimulate the production of the hormone fibroblast growth factor 23 (FGF23) from osteocytes in bone which can inhibit phosphate transport in the renal proximal tubule and therefore cause phosphaturia and hypophosphatemia. PTH can also increase bone resorption and liberate both calcium and phosphate from the skeleton. The net effect of the increased reabsorption of renal calcium, the increased absorption of calcium from the gut, and the mobilization of calcium from bone, is to restore the ECF calcium to normal and to inhibit further production of PTH and 1,25(OH)2D. FGF23 elevation will also reduce 1,25(OH)2D production. The opposite sequence of events i.e. diminished PTH and 1,25(OH)2D secretion should occur when the ECF calcium is raised above the normal range and the effect of suppressing the release of these hormones should diminish skeletal calcium release, intestinal calcium absorption, and renal calcium reabsorption and restore the elevated ECF calcium to normal. Consequently, decreased levels of PTH and decreased levels of 1,25(OH)2D should accompany hypercalcemia unless the PTH or 1,25(OH)2D is the cause of the hypercalcemia.



Regulation of Parathyroid Hormone Production

PTH is an 84 amino acid peptide whose known bioactivity resides within the NH2-terminal 34 residues. Consequently, a synthetic peptide, PTH (1-34), can mimic many of its actions. The major regulator of PTH secretion from the parathyroid glands is the ECF calcium acting via CaSR. The relationship between ECF calcium and PTH secretion is governed by a steep inverse sigmoidal curve which is characterized by a maximal secretory rate at low ECF calcium, a midpoint or "set point" which is the level of ECF calcium which half-maximally suppresses PTH, and a minimal secretory rate at high ECF calcium (8). The rate at which ECF calcium falls may also dictate the magnitude of the secretory response with a rapid fall in ECF calcium stimulating a more robust secretory response. As well higher levels of PTH are observed at the same ECF calcium when calcium is falling rather than rising, producing a hysteresis response (9).


CaSR has a large NH2-terminal extracellular domain which binds ECF calcium, seven plasma membrane-spanning helices and a cytoplasmic COOH-terminal domain. It is a member of the superfamily of G protein coupled receptors and in the parathyroid chief cells is linked to various intracellular second-messenger systems. Transduction of the ECF calcium signal via this molecule leads to alterations in PTH secretion.


A change in ECF calcium will also produce a change in PTH metabolism in the parathyroid cell however this response is somewhat slower than the secretory response. Thus, a rise in calcium will promote enhanced PTH degradation and the release of bioinert mid-region and COOH fragments and a fall in calcium will decrease intracellular degradation so that more intact bioactive PTH is secreted (10-12). Bioinactive PTH fragments, which can also be generated in the liver, are cleared by the kidney (13). With sustained low ECF calcium there is a change in PTH biosynthesis which represents an even slower response. Thus, low ECF calcium, acting via CaSR leads to increased transcription of the gene encoding PTH and enhanced stability of PTH mRNA (14,15). Finally, sustained hypocalcemia can eventually lead to parathyroid cell proliferation (16) and an increased total secretory capacity of the parathyroid gland. Although sustained hypercalcemia can conversely reduce parathyroid gland size, hypocalcemia appears less effective in diminishing parathyroid chief cells once a prolonged stimulus to hyperplasia has occurred.


Additional factors including catecholamines and other biogenic amines, prostaglandins (17), cations (e.g. lithium and magnesium), phosphate per se (5) and transforming growth factor alpha (TGFa) (18) have been implicated in the regulation of PTH secretion (5). The phosphaturic factor, FGF23, also suppresses PTH gene expression and secretion (19). One of the most important regulators appears to be 1,25(OH)2D which may tonically reduce PTH release (20), decrease PTH gene expression (15) and inhibit parathyroid cell proliferation (16, 21).

PTH Actions


The kidney is a central organ in ensuring calcium balance and PTH has a major role in fine-tuning this renal function (22-24). PTH has little effect on modulating calcium fluxes in the proximal tubule where 65% of the filtered calcium is reabsorbed, coupled to the bulk transport of solutes such as sodium and water (23). Nevertheless, in this region PTH binds (25) to its cognate receptor, the type I PTH/PTHrP receptor (PTHR1) a 7-transmembrane-spanning G protein-coupled protein which is linked to both the adenylate cyclase system and the phospholipase C system (26-28). Stimulation of adenylate cyclase is believed to be the major mechanism whereby PTH causes internalization of the type II Na+/Pi (inorganic phosphate) co-transporters, NaPi-IIa and NaPi-IIc, in the proximal tubule, leading to decreased phosphate reabsorption and phosphaturia (29).


In this nephron region, PTH can, after binding to the PTHR1, also stimulate CYP27B1, the 25(OH)D-1a hydroxylase [1a(OH)ase], leading to increased synthesis of 1,25(OH)2D (30). A reduction in ECF calcium can itself stimulate 1,25(OH)2D production but the precise mechanism of this action is presently unknown. Finally, PTH can also inhibit Na+ and HCO3- reabsorption in the proximal tubule by inhibiting the apical type 3 Na+/H+ exchanger (31), and the basolateral Na+/K+-ATPase (32) as well as by inhibiting apical Na+/Pi cotransport.


About 20% of filtered calcium is reabsorbed in the cortical thick ascending limb of the loop of Henle (CTAL) and 15% in the distal convoluted tubule (DCT) and it is here that PTH also binds to PTHR1 (27) and again by a cyclic AMP-mediated mechanism (33), enhances calcium reabsorption. In the CTAL, at least, this appears to occur by increasing the activity of the Na/K/2Cl cotransporter that drives NaCl reabsorption and also stimulates paracellular calcium and magnesium reabsorption (34). The CaSR is also resident in the CTAL (35) and can respond to an increased ECF calcium by activating phospholipase A2, reducing the activity of the Na/K/2Cl cotransporter and of an apical K channel, and diminishing paracellular calcium and magnesium reabsorption. Consequently, a raised ECF calcium antagonizes the effect of PTH in this nephron segment and ECF calcium can in fact participate in this way in the regulation of its own homeostasis. Furthermore, the inhibition of NaCl reabsorption and loss of NaCl in the urine that results may contribute to the volume depletion observed in severe hypercalcemia. ECF calcium may therefore act in a manner similar to "loop" diuretics such as furosemide.


In the DCT, PTH can also influence transcellular calcium transport (36). This is a multistep process involving transfer of luminal Ca2+ into the renal tubule cell via the transient receptor potential channel (TRPV5), translocation of Ca2+across the cell from apical to basolateral surface a process involving proteins such as calbindin-D28K, and finally active extrusion of Ca2+ from the cell into the blood via a Na+/Ca2+ exchanger, designated NCX1. PTH markedly stimulates Ca2+ reabsorption in the DCT primarily by augmenting NCX1 activity via a cyclic AMP-mediated mechanism.


In bone, the PTHR1 is localized on cells of the osteoblast lineage which are of mesenchymal origin (37) but not on osteoclasts which are of hematogenous origin. Nevertheless, in the postnatal state the major physiologic role of PTH appears to be to maintain normal calcium homeostasis by enhancing osteoclastic bone resorption, notably cortical bone resorption, and liberating calcium into the ECF. This effect of PTH on increasing osteoclast stimulation is indirect, with PTH binding to the PTHR1 on pre-osteoblastic stromal cells (38) and other cells of the osteoblast lineage including osteocytes (39) and enhancing the production of the cytokine RANKL (receptor activator of NFkappaB ligand), a member of the tumor necrosis factor (TNF) family (40). Simultaneously, levels of a soluble decoy receptor for RANKL, termed osteoprotegerin, are diminished facilitating the capacity for increased cell-bound RANKL to interact with its cognate receptor, RANK, on cells of the osteoclast series. Multinucleated osteoclasts are derived from hematogenous precursors which commit to the monocyte/macrophage lineage, and then proliferate and differentiate as mononuclear precursors, eventually fusing to form multinucleated osteoclasts (41). These can then be activated to form bone-resorbing osteoclasts. RANKL can drive many of these proliferation/differentiation/fusion/activation steps although other cytokines, notably monocyte-colony stimulating factor (M-CSF) may participate in this process (41).


Endogenous PTH has also been shown to exert a physiologic anabolic effect on trabecular bone formation in both the fetus and neonate (42,43). PTH has been reported to increase growth factor production, notably insulin-like growth factor-1 (IGF-1) production, which may contribute to this (44). In addition, the anabolic effect of PTH in part lies via activation of the canonical Wnt growth factor signaling pathway, a critical pathway for bone formation. One mechanism of this activation is via inhibition of sclerostin (39), an osteocyte-derived antagonist. PTH has been suggested to elicit increases in production and activity of cells of the osteoblast pathway and to decrease osteoblast apoptosis (45). It is conceivable that different modes of anabolic action occur depending on the stage of development of the organism and environmental stimuli.


It has been noted that although increased PTH activity increases coupled bone turnover i.e. both osteoblastic bone formation and osteoclastic bone resorption, continuous exogenous administration of PTH in vivo can lead to net enhanced bone resorption and hypercalcemia whereas intermittent exogenous administration can lead to net increasing bone formation and therefore an anabolic effect (46).

 Regulation of Vitamin D Production

Vitamin D3 (cholecalciferol) is a biologically inert secosteroid that is made in the skin (47). After exposure to sunlight 7-dehydrocholesterol is transformed by UVB radiation to previtamin D3 which undergoes isomerization into vitamin D3. Vitamin D3 is then translocated into the circulation where it is bound to the vitamin D-binding protein (DBP). There are no documented cases of vitamin D intoxication occurring due to excessive sunlight exposure most likely due to the fact that prolonged UVB exposure transforms both previtamin D3 and vitamin D3 to biologically inactive metabolites. Vitamin D3 (and vitamin D2 or ergocalciferol) can also enter the circulation after absorption from food in the gut notably fatty foods, fish oils, and foods fortified with vitamin D. In the liver, vitamin D can be converted to 25(OH)D by a cytochrome P450-vitamin D 25-hydroxylase (CYP2R1), which generally converts vitamin D to 25(OH)D almost constitutively (48).


Consequently serum 25(OH)D is the most abundant circulating metabolite of vitamin D, reflects the integrated levels of vitamin D from both cutaneous and dietary sources, and can be used as an index of vitamin D deficiency, sufficiency, or intoxication. However, 25(OH)D is also biologically inert except when present in very high concentrations, and is transported, bound to DBP, to the kidney where it is converted by the cytochrome P450- monooxygenase, 25(OH)D-1a hydroxylase (CYP27B1) to the active moiety, 1,25(OH)2D (49). Although the kidney is the major source of circulating hormonal 1,25(OH)2D, a variety of extra-renal cells have been reported to synthesize 1,25(OH)2D, notably skin cells, monocytes/macrophages, bone cells (50), and the placenta during pregnancy (51). The 1,25(OH)2D produced by many of these non-renal tissues may act in a paracrine/autocrine fashion to regulate cell growth, differentiation, and local function. The renal production of 1,25(OH)2D is stimulated by hypocalcemia, hypophosphatemia, and elevated PTH levels. The renal 1a(OH)ase is potently inhibited by the phosphaturic hormone, fibroblast growth factor (FGF) 23 and also by 1,25(OH)2D per se in a negative feedback loop. As well, FGF23 and 1,25(OH)2D can both stimulate a 24-hydroxylase enzyme (CYP24A1). This cytochrome P450 monooxygenase produces 24,25(OH)2D and 1,24,25(OH)3D from 25(OH)D and 1,25(OH)2D respectively (52). These metabolites are generally believed to be biologically inert and represent the first step in biodegradation. After several further hydroxylations, cleavage of the secosteroid side chain occurs between carbons 23 and 24 leading to the production of the inert, water soluble end product calcitroic acid. This metabolism may occur in kidney, liver and target tissues such as intestine and bone.

Vitamin D Actions

The unbound active form of vitamin D, 1,25(OH)2D can enter target cells and interact with the ligand-binding domain of a specific nuclear receptor (VDR) (53). The 1,25(OH)2D-VDR complex heterodimerizes with the retinoid X receptor (RXR) and then interacts with a vitamin D-responsive element (VDRE) on a target gene to enhance or inhibit the transcription of such target genes. The activity of the VDR is enhanced by co-activator proteins that can also bind to discrete regions of the VDR and remodel chromatin, acetylate nucleosomal histones and contact the basal transcriptional machinery. Co-repressors can bind to the VDR in the absence of ligand and also modify its activity. Although ligand-independent VDR activation and non-genomic actions of 1,25(OH)2D have been reported their physiologic significance is currently unclear.


A major biologic function of circulating 1,25(OH)2D is to increase the efficiency of the small intestine to absorb dietary calcium. Intestinal absorption of calcium occurs by an active transcellular path and by a non-saturable paracellular path. Active calcium absorption accounts for 10-15% of a dietary load (54). Active transcellular intestinal absorption involves (as does Ca+2 reabsorption in the kidney), three sequential cellular steps, a rate-limiting step involving transfer of luminal Ca+2 into the intestinal cell via the epithelial Ca+2  channel TRPV6, or via other calcium channels, intracellular diffusion, mediated by the Ca+2 -binding protein, calbindin-D9K or by other calcium binding proteins such as calmodulin, and extrusion at the basolateral surface into the blood predominantly through the activity of the Ca+2 -ATPase, PMCA 1b (55). 1,25(OH)2D, by interacting with the VDR (56) mainly, but not exclusively, in the duodenum, appears to increase all 3 steps by increasing gene expression of TRPV6, a channel-associated protein, annexin2 calbindin-D9K and to a lesser extent, the basolateral extrusion system PMCA1b (36,55). Calcium within the cell may also be sequestered by intracellular organelles such as the endoplasmic reticulum and mitochondria which could also contribute to the protection of the cell against excessively high calcium. Increasing evidence now supports regulation by 1,25(OH)2D of active transport of calcium by distal as well as proximal segments of the intestine as well as paracellular calcium transport (57). Reductions in dietary intake of calcium can lead to increased PTH secretion and increased 1,25(OH)2D production which can enhance fractional calcium absorption and compensate for the dietary reduction. Although 1,25(OH)2D also increases phosphate absorption, mainly in the jejunum and ileum, nearly 50% of dietary phosphorus can be absorbed even in the absence of 1,25(OH)2D.


Although vitamin D is known to be essential for normal mineralization of bone, its major role in this respect appears to be largely indirect i.e. by enhancing intestinal absorption of calcium and phosphate in the small intestine, maintaining these ions in the normal range and thereby facilitating hydroxyapatite deposition in bone matrix. The major direct function of 1,25(OH)2D on bone appears to be to enhance mobilization of calcium stores when dietary calcium is insufficient to maintain a normal ECF calcium (58). As with PTH, 1,25(OH)2D enhances osteoclastic bone resorption by binding to receptors in cells of the osteoblast lineage and stimulating the RANK/RANK system to enhance the proliferation, differentiation and activation of the osteoclastic system from its monocytic precursors (41), but high concentrations may also inhibit calcium deposition in bone (59). Endogenous 1,25(OH)2D has also been reported to have an anabolic role in vivo (55,60).


Although effects of 1,25(OH)2D on both calcium and phosphorus handling in the kidney have been reported, it remains controversial whether 1,25(OH)2D plays a major role in altering renal tubular reabsorption or excretion of these ions in humans.

Parathyroid Hormone Relation Peptide (PTHrP)

PTHrP was discovered as the mediator of the syndrome of "humoral hypercalcemia of malignancy" (HHM) (61). In this syndrome a variety of cancers, essentially in the absence of skeletal metastases, produce a PTH-like substance which can cause a constellation of biochemical abnormalities including hypercalcemia, hypophosphatemia, and increased urinary cyclic AMP excretion. These mimic the biochemical effects of PTH but occur in the absence of detectable circulating levels of this hormone.


PTHrP is encoded by a single-copy gene located on chromosome 12 whereas the gene encoding PTH is found on chromosome 11. Nevertheless, these two chromosomes encode many similar genes and are believed to have arisen by an ancient duplication event. Consequently, PTHrP and PTH may be members of a single gene family (62,63). The human PTHrP gene which is driven by at least three promoters, contains at least seven exons, shows several patterns of alternative splicing, and is considerably more complex than the PTH gene. Each gene encodes a leader or "pre" sequence, a "pro" sequence and a mature form. In the case of human PTH, the mature form is 84 amino acids. In the case of human PTHrP, 3 isoforms of 139, 141 and 173 residues can occur by alternate splicing. Several common structural features of these genes however suggest that they are related. Thus, the major coding exon of both genes starts precisely at the same nucleotide, one base before the codons encoding the Lys-Arg residues of the prohormone sequences of each hormone. In the NH2 terminus of both peptides, 8 of the first 13 amino acid residues are identical. These identities although limited are believed to be responsible for the similar bioactivities of the NH2terminal domains of the peptides (64), such that synthetic PTH (1-34) and synthetic PTHrP (1-34) interact with a common receptor (PTHR1) (26,27) and have similar effects on calcium and phosphate homeostasis. Thus, PTHrP is the second member of the PTH family to have been discovered. A hypothalamic peptide called tuberoinfundibular peptide of 39 residues (TIP 39) appears to represent a third member of the PTH gene family (65) and can interact at a second PTH receptor termed the type II receptor (66) to which PTHrP does not bind (Fig. 2). The precise physiologic role of TIP 39 and of the type II receptor remain to be elucidated.

Figure 2. PTH and PTHR gene families: PTHrP, PTH and TIP39 appear to be members of a single gene family. Although only nine amino acids in the NH2-terminal domains of these three peptides are conserved these are functionally important residues. The receptors for these peptides, PTHR1 and PTHR2, are both 7 transmembrane-spanning G protein-coupled receptors which seem to be members of a single gene family. PTHrP binds and activates PTHR1; it binds weakly to PTHR2 and does not activate it. PTH can bind and activate both PTHR1 and PTHR2. TIP39 can bind to and activate PTHR2 but not PTHR1.

Regulation of PTHrP Production

In contrast to PTH, whose expression is limited mainly to parathyroid cells, PTHrP is widely expressed in many fetal and adult tissues (67). This is compatible with its primary role as a modulator of cell growth and differentiation. A major locus of regulation of PTHrP production is at the level of gene transcription although both regulated and constitutive secretion of the hormone have been described in various cell types (68,69).


Key stimulators of gene transcription are a variety of growth factors and cytokines (70) including epidermal growth factor (EGF) (71), IGF-1 (72), transforming growth factor b (TGFb) (73). Inhibition of growth factor action, by employing a farnesyl transferase inhibitor to decrease ras-mediated cell signaling, has proved effective in inhibiting PTHrP production in vitro and in studies in vivo using an animal model of malignancy which overproduced PTHrP (74). Hypercalcemia associated with these tumors was also diminished.


Several steroidal hormones including 1,25(OH)2D (75), glucocorticoids (76), and androgens (77) have been reported to be potent inhibitors of PTHrP gene expression. This prompted the use of 1,25(OH)2D (78) and of low calcemic analogues of vitamin D (79) in studies with tumor cells, both in vitro and in animals in vivo, to determine if overproduction of PTHrP by these tumors could be inhibited. Indeed, PTHrP production was inhibited, the associated hypercalcemia was reduced, and survival of the animals was increased.


PTHrP is biosynthesized as a precursor form, proPTHrP and the propeptide must be cleaved to the mature peptide in order to achieve optimal bioactivity. This occurs by prohormone convertase activity (80). This processing locus was attacked using a furin antisense approach to block prohormone convertase activity in an animal tumor model which overproduces PTHrP (81). Bioactive PTHrP production was diminished with this intervention, and, in vivo, hypercalcemia associated with the control tumor was not observed.


PTHrP is considerably longer than PTH with three isoforms of 139, 141 and 173 amino acids whose sequences are identical through residue 139. Serine proteases may also act internally in various cell types to cleave an NH2 terminal fragment, a midregion fragment (82) and carboxyl terminal fragments (83) from the mature forms, each with apparently distinct bioactivities. The in vivo significance of this processing remains to be determined. Nevertheless, PTHrP has been described as a polyhormone.


PTHrP Actions

The major effects of PTHrP appear to be mediated by binding of an NH2 terminal domain, PTHrP (1-36), to the PTHR1 linked to adenylate cyclase, or phospholipase C. In some developing tissues, e.g. teeth. PTHrP is expressed in epithelial cells whereas the PTHR1 is in adjacent mesenchymal cells facilitating epithelial-mesenchymal interactions (84).


A mid-region domain of PTHrP (37-86) has been implicated in placental calcium transport (82) and a COOH terminal region (107-139) has been reported to inhibit osteoclasts (83). Nevertheless, distinct receptors for these putative bioactive regions have not been described.

A bipartite nuclear localization sequence (NLS) has been discovered in PTHrP at sequence positions 87 to 106 and has been shown to be capable, in vitro, of directing PTHrP to the nucleus and, in fact, to the nucleolus (85). Translocation from the cytoplasm to the nucleus is facilitated by binding to importin beta and seems cell cycle dependent. Although cyclin-dependent (cdc2) kinase can phosphorylate PTHrP this may not be the sole regulator of PTHrP nuclear import (86). Inasmuch as PTHrP contains a presequence or leader sequence which directs it to the secretory pathway, 3 pathways have been postulated which could lead it to access to the cytoplasm and thence the nucleus. Thus, PTHrP has been shown in some studies to be internalized after secretion and to access the cytoplasm by this route (87). Reverse transport of PTHrP from the endoplasmic reticulum to the cytoplasm has been reported in other studies (88). Finally, alternate initiation of translation at downstream non-AUG codons that allowed nascent PTHrP to bypass ER transit and localize to the nucleus and nucleolus has also been reported (89). In vitro studies have suggested that nuclear localization of PTHrP may be involved in its proliferative activity and/or in inhibition of apoptosis (84), and in vivo, PTHrP “knockin” mice have been reported which express truncated forms of PTHrP that lack the NLS and the carboxyl -terminus but retain the amino terminus and the capacity to bind to PTHR1. The resulting mutants show growth retardation, defects in multiple organs and early lethality. Consequently, these studies indicate a functional in vivo role for the nuclear localization of this protein (90,91).


Overall, reported physiologic effects of PTHrP can be grouped into those relating to ion homeostasis; those relating to smooth muscle relaxation; and those associated with cell growth, differentiation and apoptosis. The majority of the physiological effects of PTHrP appear to occur by short-range i.e. paracrine/autocrine and intracrine mechanisms rather than long-range i.e. endocrine mechanisms.


With respect to ion homeostasis PTHrP can modulate placental calcium transport and appears necessary for normal fetal calcium homeostasis (92). In the adult, however the major role in calcium and phosphorus homeostasis appears to be carried out by PTH rather than by PTHrP in view of the fact that PTHrP concentrations in normal adults are either very low or undetectable. This situation reverses when neoplasms constitutively hypersecrete PTHrP in which case PTHrP mimics the effects of PTH on bone and kidney and the resultant hypercalcemia suppresses endogenous PTH secretion.


PTHrP has been shown to cause smooth muscle relaxation in a variety of tissues including blood vessels (93) (leading to dilatation), uterus (94), and bladder (95). The physiologic significance of these effects however remains to be determined.


Finally, PTHrP has been shown to modify cell growth, differentiated function and programmed cell death in a variety of different fetal and adult tissues. Most notable have been breast (96), skin (97), nervous tissue (98) and pancreatic islets (99) where PTHrP appears to function to assure normal development. The most striking developmental effects of PTHrP however have been in the skeleton. Targeted deletion of the PTHrP gene in mice produces a lethal chondrodysplasia (100,101), demonstrating the important and non-redundant role of PTHrP in endochondral bone formation. Animals die at birth, although the cause of death is uncertain. A major alteration appears to occur in the cartilaginous growth plate where, in the absence of PTHrP, chondrocyte proliferation is reduced and accelerated chondrocyte differentiation and apoptosis occurs. Increased bone formation occurs, apparently due to secondary hyperparathyroidism (42) and the overall effect is a severely deformed skeleton. Even more severe skeletal dysplasia occurs when either the gene encoding the PTHR1 itself (102) or the genes encoding both PTH and PTHrP are deleted (42). Both models produce similar phenotypes in mice. In the PTHrP knock-in mice that express PTHrP (1-84) but not the NLS or carboxyl terminus, the epiphyseal growth plate was markedly abnormal in this model, but the abnormality consisted of a reduced proliferative zone but normal hypertrophic zone architecture, suggesting that secreted and intracellular PTHrP may act synergistically to regulate the growth plate. In humans, an inactivating mutation of the PTHR1 produces a similar lethal chondro-osseous dysplasia termed Blomstrand's Syndrome (103,104). Consequently these in vivo observations demonstrate that PTHrP is essential, at least for normal development of the cartilaginous growth plate and endochondral bone formation. Interestingly mice that are heterozygous for PTHrP ablation appear normal at birth but develop reduced trabecular bone as they age demonstrating an osseous phenotype due to haploinsufficiency (37). This has been shown to be via a paracrine effect of PTHrP located in osteoblastic cells (105). Furthermore, hypoparathyroid mice that have PTHrP haploinsufficiency do not develop the increased trabecular bone mass that is a characteristic of hypoparathyroidism (106). PTHrP knock-in mice that express PTHrP (1-84) but lack the NLS and carboxyl terminus also appear to develop reductions in osteoblastic activity again suggesting synergy between the extra-cellular and intracellular actions of PTHrP (107). In humans, variants of the gene PTHLP that encodes PTHrP have been associated with achievement of peak bone mass and in genome wide association studies have been associated with reduced bone mineral density. Overall therefore the two ligands of PTHR1 i.e. PTH and PTHrP appear to have differing roles in utero and post-natally. In the fetus PTH appears to exert anabolic activity in trabecular bone whereas PTHrP regulates the orderly development of the growth plate. In contrast, in postnatal life, PTHrP acting as a paracrine/autocrine modulator assumes an anabolic role for bone whereas PTH predominantly defends against a decrease in extracellular fluid calcium by resorbing bone. 


Normal adult bone is constantly undergoing "turnover" or remodeling (108). This is characterized by sequences of activation of osteoclasts followed by osteoclastic bone resorption followed by osteoblastic bone formation. These sequential cellular activities occur in focal and discrete packets in both trabecular and cortical bone and are termed bone remodeling units or bone multicellular units (BMUs). This coupling of osteoblastic bone formation to bone resorption may occur via the action of growth factors released by resorbed bone e.g. TGFb, IGF-1 and fibroblast growth factor (FGF) which can induce osteoclast apoptosis and also induce osteoblast chemotaxis proliferation and differentiation at the site of repair. In addition, direct activation of cells of the osteoblast phenotype by osteoclast family members appears to occur, although the molecular signals regulating this direct interaction remain elusive. A number of systemic and local factors regulate the process of bone remodeling. In general, those factors which enhance bone resorption may do so by creating an imbalance between the depth of osteoclastic bone erosion and the extent of osteoblastic repair or by increasing the numbers of remodeling units which are active at any given time i.e. by increasing the activation frequency of bone remodeling. These latter processes can also result in thinning and ultimately in perforation of trabecular bone and in increased porosity of cortical bone. One predominant example in which osteoblastic activity does not completely repair and replace the defect left by previous resorption is in multiple myeloma; in this case it has been reported that myeloma cells may release inhibitors of the Wnt signaling pathway such as the protein Dickoff (Dkk) which inhibit osteoblast production (109), while stimulation of osteoclastic resorption continues. Such an imbalance can occasionally also occur in association with some advanced solid malignancies.


Systemic hormones such as PTH, PTHrP and 1,25(OH)2D can all initiate osteoclastic bone resorption and increase the activation frequency of bone remodeling. Thyroid hormone receptors are present in osteoblastic cells and triiodothyronine can stimulate osteoclastic bone resorption and produce a high turnover state in bone (110). Vitamin A has a direct stimulatory effect on osteoclasts and can induce bone resorption as well (111).


A variety of local factors are critical for physiologic bone resorption and regulation of the normal bone-remodeling sequence and can be produced by osteoblastic, osteoclastic and immune cells. Thus, for example, interleukin-1 (IL-1) and M-CSF can be produced by both osteoblastic cells and by cells of the osteoclastic lineage. TNFa is released by monocytic cells, TNFb (lymphotoxin) by activated T lymphocytes, and interleukin-6 (IL-6) by osteoclastic cells (112). All can enhance osteoclastic bone resorption. Leukotrienes are eicosanoids that are produced from arachidonic acid via a 5-lipoxygenase enzyme and can also induce osteoclastic bone resorption. Prostaglandins, particularly of the E series, may also stimulate bone resorption in vitro but appear to predominantly increase formation in vivo (113). Consequently, a variety of cytokines, growth factors, and eicosanoids may be produced in the bone environment and act to regulate the bone remodeling sequence. The inappropriate production of these regulators in pathologic conditions such as cancer (Fig. 3) may therefore contribute to altered bone dynamics, altered calcium fluxes through bone, and ultimately in altered ECF calcium homeostasis.

Figure 3. Production of bone resorbing substances by neoplasms. Tumor cells may release proteases which can facilitate tumor cell progression through unmineralized matrix. Tumors cells can also release PTHrP, cytokines, eicosanoids and growth factors (eg EGF) which can act on cells of the osteoblastic lineage to increase production of cytokines such as M-CSF and RANKL and to decrease production of OPG. RANKL can bind to its cognate receptor RANK in osteoclastic cells, which are of hepatopoietic origin, and increase production and activation of multinucleated osteoclasts which can resorb mineralized bone.


Hypercalcemic disorders can be broadly grouped into Endocrine Disorders, Malignant Disorders, Inflammatory Disorders, Pediatric Syndromes, Medication-Induced Hypercalcemia, and Immobilization (Table 1).


Table 1. Hypercalcemic Disorders

  A. Endocrine Disorders Associated with Hypercalcemia

       1.  Endocrine Disorders with Excess PTH Production

       2.  Endocrine Disorders without Excess PTH Production

 B. Malignancy-Associated Hypercalcemia (MAH)

      1. MAH with Elevated PTHrP

      2. MAH with Elevation of Other Systemic Factors

C. Inflammatory Disorders Causing Hypercalcemia

      1. Granulomatous Disorders

      2. AIDS

D. Pediatric Syndromes

      1.Williams Syndrome

      2. Idiopathic Infantile Hypercalcemia

E. Medication-Induced

      1. Thiazides

      2. Lithium

      3. Vitamin D

      4. Vitamin A

      5. Estrogens and Antiestrogens


      7. Aluminium Intoxication

      8. Milk-Alkali Syndrome

F. Immobilization



Endocrine Disorders with Excess PTH Production

A detailed discussion of primary hyperparathyroidism appears in an associated Endotext chapter. Consequently, only selected issues will be addressed here.


Sporadic primary hyperparathyroidism (PHPT) is generally (at least 85-90% of cases) associated with a single parathyroid adenoma which overproduces PTH. Although 10-15% of cases may be associated with multigland hyperplasia, it seems prudent to consider that at least some if not most of these cases represent familial rather than sporadic disease. The presence of multiple adenomas should also suggest the possibility that all glands are involved as part of a familial syndrome. Malignant sporadic PHPT may occur as a consequence of parathyroid carcinoma, but is a relatively rare event (about 1% of cases).


To date, the only genes definitively implicated in sporadic benign PHPT are an oncogene Cyclin D1, that encodes a key regulator of the cell cycle and MEN1, a tumor suppressor gene, also implicated in familial multiple endocrine neoplasia type I (114). HRPT2, also called CDC73, a tumor suppressor gene associated with the Hyperparathyroidism-Jaw Tumor syndrome (115), has also been implicated in most sporadic parathyroid carcinomas (116). Other important parathyroid regulatory pathways that may play a role in the pathogenesis of hyperparathyroidism are those related to the principal regulators or parathyroid cell proliferation and PTH secretion i.e. 1,25(OH)2D, Ca+2 and phosphate. Rarely, sporadic hyperparathyroidism with hypocalciuria may occur, caused by inhibitory antibodies to the calcium-sensing receptor. This syndrome has been termed Autoimmune Hypocalciuric Hypercalcemia (117). The clinical manifestations of these disorders are caused by the overproduction of PTH and its effect on bone resorption, on its capacity to stimulate renal 1,25(OH)2D production and renal calcium reabsorption, and on the resultant effect on ECF calcium which can increase the filtered renal load of calcium and itself increase calcium excretion by stimulating the renal tubular CaSR and inhibiting tubular calcium reabsorption (Fig. 4).


About 80% of cases of the most common form of PHPT i.e. benign sporadic PHPT present as mild or “asymptomatic” hyperparathyroidism in which hypercalcemia is generally less than 1mg/dL (0.25 mM) above the upper limit of normal and may be normal intermittently (118). However significant increases in serum calcium may occur even after 13 years of follow up. Excess PTH production can produce significant bone loss. Classically this is manifested by discrete lesions including subperiosteal bone resorption of the distal phalanges, osteitis fibrosa cystica characterized by bone cysts and "brown tumors" (i.e. collections of osteoclasts intermixed with poorly mineralized woven bone), and ultimately fractures. However, these manifestations are rarely seen in Western nations (2% of cases) but were common manifestations in the past and may still be common in some areas of the East (119-122). Whether this severe bone disease reflects a delay in detecting primary hyperparathyroidism early, or as seems equally plausible, is a manifestation of excess PTH action in the face of marginal or deficient vitamin D and calcium intake (123), remains to be determined. The more common skeletal manifestation of excess circulating PTH in the West now appears to be resorption primarily of cortical bone, reflecting the "catabolic bone activity" of PTH, with relative preservation of trabecular bone, reflecting its "anabolic activity" (124) and production of an osteoporosis clinical picture. Consequently, the severity of bone disease in the West appears considerably diminished. Possibly as a consequence of less severe bone disease, hypercalcemia is also less marked, the filtered load of renal calcium is lower and the incidence of kidney stones and particularly of nephrocalcinosis has declined as well. Nevertheless, hypercalciuria still occurs in 35-40% of patients with primary benign sporadic hyperparathyroidism and kidney stones occur in 15-20% (125). About 25% of patients with mild (“asymptomatic”) sporadic PHPT have been reported to develop renal manifestations within 10 years, including renal concentrating defects or kidney stones. In other regions of the globe, where relative or absolute vitamin D deficiency may limit the severity of hypercalcemia and therefore the filtered load of calcium, the incidence of nephrolithiasis (10-40%) does not appear to be as different as is the incidence of bone disease. The higher incidence in the West of benign sporadic PHPT in women and in an older age group (126) also appears to distinguish the presentation of this disorder in the West relative to the East.

Figure 4. Disordered mineral homeostasis in hyperparathyroidism. In primary sporadic hyperparathyroidism PTH is generally overproduced by a single parathyroid adenoma. Increased PTH secretion leads to a net increase in skeletal resorption with release of Ca+2 and Pi (inorganic phosphate) from bone. PTH also increases renal 1α (OH)ase activity leading to increased production of 1,25(OH)2D from 25(OH)D and increased Ca+2 and Pi absorption from the small intestine. PTH also enhances renal Ca+2 reabsorption and inhibits Pi reabsorption resulting in increased urine Pi excretion. The net result is an increase in ECF calcium and a decrease in ECF phosphate.

Abnormalities other than skeletal and renal have been associated with benign sporadic PHPT. These include gastrointestinal manifestations such as peptic ulcer and acute pancreatitis. The incidence of peptic ulcer disease in sporadic PHPT is currently estimated to be about 10%, the same as in the general population but, the presence of multiple peptic ulcers may suggest the presence of multiple endocrine neoplasia type I (MENI). Acute pancreatitis. may be a manifestation of hypercalcemia per se but is estimated to occur in only 1.5% of those with sporadic PHPT. Neuromuscular abnormalities manifested by weakness and fatigue and accompanied by EMG changes may occur although the pathophysiology is uncertain. The relationship of hypertension and other cardiovascular manifestations as well as neuropsychiatric symptoms to the hyperparathyroidism remains unclear inasmuch as the former is generally not reversible when the hyperparathyroidism is treated and the latter is quite common in the population at large and difficult to ascribe to hyperparathyroidism. Rarely, primary sporadic PHPT may present with severe acute hypercalcemia (parathyroid crisis) (127).


Although estrogen therapy has been advocated for the treatment of PHPT in postmenopausal women (128) potential adverse effects of estrogen therapy, including breast cancer and cardiovascular complications, make this option unattractive. Although selective estrogen receptor modulators may be an alternative, few long-term studies have been done to assess this. Bisphosphonates (129) may increase bone mineral density (BMD) at the lumbar spine and hip regions but generally do not substantially reduce the hypercalcemia. Calcimimetic agents (those that mimic or potentiate the action of calcium at the CaSR) (130) can effectively reduce hypercalcemia and have been approved for use in parathyroid carcinomas but may not significantly improve the skeletal abnormalities. Calcimimetic agents may also be useful for the treatment of severe hypercalcemia in patients with PHPT who are unable to undergo parathyroidectomy.


Surgical removal of the parathyroid adenoma currently remains the treatment of choice if the ECF calcium is greater than 1mg/dL (0.25mM) above normal, if there is evidence of bone disease [i.e. a BMD T-score of <−2.5 at the lumbar spine, total hip, femoral neck, or 33% radius (1/3 site) and/or a previous fracture fragility], if creatinine clearance (calculated) is reduced to <60 ml/min or if the patient is less than age 50. Surgery is also indicated in patients for whom medical surveillance is either not desired or not possible (131). In addition, although hypercalciuria is only one of several risk factors affecting the development of kidney stones, some physicians still regard 24-hour urinary calcium excretion of greater than 400 mg as an indication for surgery.


Imaging is not recommended to establish or confirm the diagnosis of PHPT, but has become routine for preoperative localization of the abnormal parathyroid tissue. The most commonly employed preoperative parathyroid imaging techniques are radionuclide imaging (i.e. sestamibi scanning) and ultrasound. Computed tomography, magnetic resonance imaging, and positron emission tomography scanning, arteriography, and selective venous sampling for PTH are usually reserved for patients who have not been cured by previous explorations or for whom other localization techniques are not informative or are discordant.


The type of surgical procedure i.e. noninvasive or standard, and the use of operative adjuncts (e.g. rapid PTH assay) is institution specific and should be based on the expertise and resource availability of the surgeon and institution. Where more than one gland is enlarged it is reasonable to assume that this is multiple glandular disease and removal of 3½ glands or total parathyroidectomy with or without a parathyroid autograft is indicated. Severe, chronic hypercalcemia is more commonly associated with parathyroid carcinoma. Complete resection of the primary lesion is urgent in this case.


Multiple Endocrine Neoplasia, Type 1 (MEN1)

MEN1 is a familial disorder with an autosomal dominant pattern of inheritance which is characterized by tumors in pituitary, parathyroid, and enteropancreatic endocrine cells (although tumors in several other endocrine and non-endocrine tissues may also be associated with the syndrome) (132) (Fig. 5). Patients exhibit loss-of-function germline mutations in the tumor suppressor gene, MEN1, which encodes the nuclear protein, menin (133). Probands or kindreds with MEN1-like features with germline autosomal dominant mutation of the cyclin dependent kinase inhibitor CDKN1B, encoding P27(Kip1) have been described and the syndrome named MEN4. The prevalence of MEN4 among all MEN1 probands plus MEN1-like probands is approximately 1% (134). Tumors in a proband in at least 2 MEN1 site (pituitary, parathyroid, or enteropancreatic endocrine cells) and in at least one of these sites in a first-degree relative confirms the clinical phenotype. The most common and the earliest endocrinopathy is PHPT (80-100% of cases) (135). In contrast to sporadic PHPT however MEN1 occurs in both sexes equally and patients are younger at the time of diagnosis. Furthermore, in contrast to the frequent occurrence of a single adenoma in sporadic disease, multigland involvement in an asymmetric fashion is the norm in MEN1. Enteropancreatic tumors are usually multiple and gastrinomas are the most common. These may produce the Zollinger-Ellison Syndrome, and occur in the duodenum as well as in the pancreatic islets. Gastrinomas can potentially produce considerable morbidity due to the potential for ulcers and the possibility of metastatic disease. Insulinomas, glucagonomas, VIPomas, and other islet tumors can occur as well. A variety of functioning anterior pituitary tumors can occur although prolactinomas are most frequent and anterior pituitary tumors may also be non-functioning. Finally, foregut carcinoids and other endocrine tumors have been described with lesser frequency and skin tumors such as facial angiofibromas and truncal collagenomas may occur and appear specific for MEN1.

Figure 5. Familial hyperparathyroidism. Familial hyperparathyroidism (FHPT) can occur in the MENI Syndrome, in which MEN1 is mutated; in the MEN2A Syndrome, in which RET is mutated; in FHH and NSHPT in which CaSR, GNA11 or AP2S1 is mutated; and in the Hyperparathyroidism-Jaw Tumor (HPT-JT) Syndrome in which CDC73 is mutated. Familial isolated hyperparathyroidism (FIH) refers to familial hyperparathyroidism in the absence of the specific features of the other documented syndromes and suggests that other genes relevant to parathyroid neoplasia await identification.

Patients with hyperparathyroidism due to MEN1 have multiglandular disease and surgical resection of fewer than 3 glands leads to high rates of recurrence. Consequently, either subtotal parathyroidectomy with 3½ gland removal or total parathyroidectomy is recommended. The latter may be accompanied by autotransplantation of resected parathyroid gland fragments.

Multiple Endocrine Neoplasia Type 2A (MEN2A)

MEN2A is an autosomal dominant familial syndrome characterized by medullary thyroid carcinoma (MTC), bilateral pheochromocytomas, and hyperparathyroidism (136,137). This syndrome results from activating germline mutations in the rearranged during transfection (RET) protooncogene which is a receptor tyrosine kinase (138). Two variants of this disorder are MEN2B which includes familial MTC, familial pheochromocytomas, mucosal and intestinal neuromas, and a Marfanoid habitus, but no hyperparathyroidism, and familial MTC alone (139). Two other variants of MEN2 include MEN2 with cutaneous lichen amyloidosis, and MEN2 with Hirschsprung's disease. Analyses of RET mutations in these syndromes have provided good genotype-phenotype correlations (140).


The dominant feature of the MEN2A syndrome is MTC, a calcitonin-secreting neoplasm of thyroid C cells (141). Genetic testing for mutations in the RET oncogene is of value in considering prophylactic thyroidectomy to prevent MTC. Another major feature is pheochromocytomas which are frequently bilateral but which generally have low malignant potential.


Hyperparathyroidism is milder and less frequent (5-20%) in MEN2A than in MEN1 but is also associated with multigland involvement in which gland enlargement may be asymmetric. The treatment of the hyperparathyroidism is as for MEN1.

Hyperparathyroidism-Jaw Tumor Syndrome  

Hyperparathyroidism - Jaw Tumor Syndrome (HPT-JT) is an autosomal-dominant syndrome with incomplete penetrance and variable expression, caused by inactivating germline mutations of the tumor suppressor gene, Cell Division Cycle 73 (CDC73) (formerly called HRPT2), which encodes a protein termed parafibromin. Patients may present with early onset of single or multiple cystic parathyroid adenomas which may develop asynchronously, and ossifying fibromas of the mandible and maxilla. These jaw tumors lack osteoclasts and therefore differ from "brown tumors" (142,143). Affected individuals also have an increased risk (15–38%) of developing parathyroid carcinoma. Surgical removal of the affected parathyroid tissue is clearly indicated in this disorder. A variety of renal tumors have been described in some kindreds and other e.g. uterine tumors have been described in others. Mutations in CDC73, have been implicated in this syndrome (115), in sporadic parathyroid cancer (116), and in a minority of families with isolated hyperparathyroidism (144). Genetic testing in relatives can result in identification of individuals at risk for parathyroid carcinoma, enabling preventative or curative parathyroidectomy.   


Familial Hypocalciuric hypercalcemia (FHH) and Neonatal Severe Primary hyperparathyroidism (NSHPT)

Familial hypocalciuric hypercalcemia (FHH) (145), also called Familial Benign Hypercalcemia (FBH) (146) is an autosomal dominant trait characterized by moderate hypercalcemia and relative hypocalciuria i.e. urine calcium that is low in relationship to the prevailing hypercalcemia. The molecular basis, in most cases, is a loss-of-function mutation in the calcium-sensing receptor (CaSR) gene (147) in which case the syndrome is now called FHH1. The protein product, CaSR, is a G-protein coupled receptor that predominantly signals via the G-protein subunit alpha-11 (Ga11) to regulate calcium homeostasis. As a consequence, in heterozygotes, diminished ability of the CaSR in the parathyroid cell and in the CTAL of the kidney to detect ECF calcium occurs leading to increased PTH secretion and enhanced renal tubular reabsorption of calcium and magnesium respectively, to hypercalcemia, and often hypermagnesemia. Loss-of-function mutations in GNA11, the gene encoding Ga11, have also been reported to cause the syndrome, now termed FHH2. Hypercalcemia may be milder in FHH2 than in FHH1 (139). Loss-of-function mutations of the gene AP2S1, encoding adaptor protein-2 δ-subunit (AP2 δ), which plays a pivotal role in clathrin-mediated endocytosis of CaSR, may also lead to FHH and this variant is now termed FHH3. Hypercalcemia may also be more severe in this variant. The calcium to creatinine clearance ratio is usually low i.e. below 0.01, in patients with all forms of FHH, but above this level in sporadic PHPT. This increased ECF calcium is also inadequately sensed by altered CaSR function in the parathyroid gland so that mild hyperplasia may occur and "normal" levels or elevated levels of PTH are secreted despite the hypercalcemia. Patients are generally asymptomatic. Testing serum and urine calcium in three relatives may be of value in establishing the diagnosis in a proband, however genetic testing may be of value when hypocalciuric hypercalcemia occurs in an isolated patient without access to additional family members or familial isolated hyperparathyroidism (FIH) occurs in the absence of classical features of FHH.


In view of the fact that the renal lesion related to loss of CaSR function, and therefore hypercalcemia persists after parathyroidectomy, and that the patients are generally asymptomatic, it is important to identify these patients to ensure that they are not subjected to parathyroidectomy.


Individuals who are homozygous for the mutated genes, or who are compound heterozygotes and therefore have little functional CaSR, can develop Neonatal Severe Hyperparathyroidism (NSHPT) (148). This disorder generally presents within a week of birth and is characterized by severe life-threatening hypercalcemia, hypermagnesemia, increased circulating PTH concentrations, massive hyperplasia of the parathyroid glands and relative hypocalciuria. Skeletal abnormalities include demineralization, widening of the metaphyses, osteitis fibrosa and fractures. Inasmuch as the course of NSHPT can be self-limited, aggressive medical management is first indicated in all case of NSHPT, but prompt surgical intervention including total parathyroidectomy with immediate or delayed parathyroid autotransplantation should be performed in patients who deteriorate.

Familial Isolated Hyperparathyroidism (FIH)

Finally, a number of cases of Familial Isolated Hyperparathyroidism (FIH) have been reported in which familial PHPT occurs in the absence of any other manifestation of the familial disorders described. GCM2 mutations have been described in some cases but this remains to be confirmed. Undoubtedly additional research will uncover new genetic mutations which contribute to the pathogenesis of these cases.


Tertiary hyperparathyroidism appears to represent the autonomous function of parathyroid tissue that develops in the face-of-long-standing secondary hyperparathyroidism (149). This may occur with monoclonal expansion of nodular areas of the parathyroid gland. These in turn can be associated with decreased VDR and decreased CaSR expression which may lead to an increased set point for PTH secretion. The most common circumstance in which this occurs is in chronic renal failure where 1,25(OH)2D deficiency, hyperphosphatemia and hypocalcemia produce chronic stimulation of the parathyroid glands. However, hypercalcemic hyperparathyroidism has also been described in some cases of X-linked hypophosphatemic rickets, or other hypophosphatemic osteomalacias, after long-term treatment with supplemental phosphate which is believed to induce intermittent slight decreases in ECF calcium and stimulation of PTH secretion. In symptomatic patients, the use of the calcimimetic agent, cinacalcet, which enhances the capacity of ECF calcium to stimulate the CaSR, may be tried or surgical treatment may be required, i.e. either sub-total removal of the parathyroid mass or total parathyroidectomy possibly with autografting of parathyroid tissue.


After renal transplantation for end-stage kidney disease, pre-existing parathyroid gland hyperplasia associated with pre-transplant chronic kidney disease may persist over a period of months to years. This persistent hyperparathyroidism, in the presence of restored renal calcitriol production and normalized phosphate balance may lead to transient or prolonged hypercalcemia.

Endocrine Disorders Without Excess PTH Production


Hypercalcemia has been reported in as many as 50% of patients with thyrotoxicosis (150)., Bone turnover and resorption are increased due to direct effects of increased triiodothyronine on bone (151,152). The liberated calcium appears to suppress PTH release so that 1,25(OH)2D levels are reduced and renal calcium reabsorption is diminished. Treatment with a beta-adrenergic antagonist may reduce the hypercalcemia and therapy of the hyperthyroidism reverses the hypercalcemia (153,154).


Hypercalcemia has been reported with pheochromocytomas and may be due to excess PTHrP production (155).Serum PTHrP concentrations may be reduced by alpha-adrenergic blockers in these patients (156).


Hypercalcemia has frequently been reported in association with the neuroendocrine tumor VIPoma but whether the hypercalcemia is due to the overproduction of vasoactive intestinal polypeptide (VIP) per se causing bone resorption or to other co-secreted peptides such as PTHrP is uncertain (157).  


Although both primary and secondary hypoadrenalism have been associated with hypercalcemia (158,159), the underlying etiology is unclear. Ionized calcium appears to be elevated and PTH and 1,25(OH)2D are suppressed. The hypercalcemia is reversed by volume expansion and glucocorticoids.


Jansen's Syndrome is a rare autosomal dominant form of short-limbed dwarfism in which neonates presents with metaphyseal chondro-dysplasia, hypercalcemia and hypophosphatemia which is lifelong (160). PTH and PTHrP levels in serum are undetectable but renal tubular reabsorption of phosphate is decreased and urinary cyclic AMP is increased. An activating mutation of the PTHR1 has been described in such patients. A variety of skeletal abnormalities have been noted which reflect the overactivity of PTH and PTHrP during development, growth and in the adult skeleton.


It has been estimated that hypercalcemia can occur in up to 10% of malignancies. Malignancy-associated hypercalcemia (MAH) can occur in the presence or the absence of elevated PTHrP production. Using two-site immunoradiometric assays for PTHrP several groups have confirmed that 50-90% of patients with solid tumors and hypercalcemia and 20-60% of patients with hematologic malignancies and hypercalcemia have elevated circulating PTHrP. MAH both with and without elevated serum PTHrP concentrations will therefore be discussed.

MAH With Elevated PTHrP


The association between hypercalcemia and neoplastic disease was first reported in the 1920's and the suggestion was made that the direct osteolytic action of malignant cells was responsible for the release of calcium from bone, resulting in hypercalcemia (161). An association between neoplasia and hypercalcemia was later reported in the English medical literature (162). In 1941 Fuller Albright, while discussing a case of a patient with a renal cell carcinoma, who in fact had a bone metastasis, noted that hypophosphatemia should not have accompanied the hypercalcemia if the tumor was simply producing hypercalcemia by causing osteolysis (163). He suggested that the tumor might be secreting a hypercalcemic substance which was also phosphaturic. Consequently, the concept of "ectopic" PTH production by tumors arose and lead to the term’s ectopic hyperparathyroidism (164) or pseudohyperparathyroidism. Nevertheless, immunoreactive PTH could not be detected in the circulation of these patients (165) and PTH mRNA could not be detected in their tumors (166). To circumvent these issues, bioassays sensitive to PTH were employed to identify PTH-like bioactivity in blood and tumor extracts (167,168) and eventually to identify a novel protein (169), PTHrP. Despite the limited homology of PTH and PTHrP within the NH2-terminal 13 amino acids, PTH (1-34) and PTHrP (1-34) exhibit similar effects on raising blood calcium and lowering blood phosphorus, reducing renal calcium clearance, and inhibiting the renal tubular reabsorption of phosphate. The molecular basis of these effects was shown to be cross-reactivity at the PTHR1. In animal models of MAH associated with high PTHrP secretion, passive immunization with PTHrP antiserum reduced hypercalcemia (170,171). Initially after passive immunization, urine calcium increased reflecting reduction in PTHrP induced renal calcium reabsorption; only subsequently did urine calcium decline as bone resorption was neutralized and the filtered load of calcium fell (171). Consequently, the hypercalcemia induced by PTHrP involved a renal mechanism as well as an osseous one. Other similarities were noted between PTHrP and PTH including similar capacities to raise 1,25(OH)2D levels (172) and effects on bone characterized, for both PTHrP and PTH, by enhanced bone turnover and increased bone formation as well as resorption (173).


The classic neoplasms associated with hypercalcemia and excess PTHrP have few or no skeletal metastases, and are solid tumors (Fig. 6). This constellation has been termed humoral hypercalcemia of malignancy (HHM). The availability of assays to detect PTHrP demonstrated a broad spectrum of tumors that produce the peptide (174-178). Hypercalcemia is notably associated with squamous cell tumors arising in most sites including esophagus, cervix, vulva, skin, and head and neck, but especially in lung. Renal cell carcinomas are also commonly associated with the syndrome as are bladder and ovarian carcinomas. On the other hand, patients with colon, gastric, prostate, thyroid, and non-squamous cell lung cancers manifest hypercalcemia much less commonly.

Figure 6. Growth factor-regulated PTHrP production in tumor states. Tumor cells at a distance from bone may be stimulated by autocrine growth factors (GF) to increase production of PTHrP which can then travel to bone via the circulation and enhance bone resorption. Tumor cells metastatic to bone (inset) may secrete PTHrP which can resorb bone and release growth factors which in turn can act in a paracrine manner to further enhance PTHrP production. PTHrP may itself promote tumor growth and progression.

Inasmuch as PTHrP is broadly distributed in normal tissues, PTHrP secretion by tumors likely represents eutopic overproduction rather than ectopic PTHrP production. Although demethylation of the PTHrP promoter (179) and gene amplification (180) have been involved as mechanisms responsible for PTHrP overproduction by malignancies, it seems likely that in most cases overproduction of PTHrP is driven by enhanced gene transcription of tumor growth factors and by oncogenes which are signaling molecules in the growth factor pathways.


Patients who manifest hypercalcemia usually present with advanced disease. These tumors are generally obvious clinically, and carry a poor prognosis. Elevated PTHrP per se may be an independent prognostic factor signaling a poor prognosis (181).This appears to be because in addition to its role in hypercalcemia, (182) PTHrP produced by tumor cells acts in an intracrine manner, increasing cell survival, apoptosis resistance, and anoikis evasion, and in autocrine manner via the PTHR1 to increase tumor cell proliferation, survival, apoptosis resistance. PTHrP is also a potential candidate for premetastatic niche formation in bone marrow, causing expansion of myeloid cells required for forming a conducive niche for metastatic growth in bone. Thus, PTHrP participates in all steps of the metastatic process, including tumor growth, progression, invasion, migration and survival in bone in order to skeletal support metastases (183).


Hypercalcemia in association with malignancy is generally more acute and severe than in association with primary hyperparathyroidism. Nevertheless, as in primary hyperparathyroidism hypercalcemia is accompanied by hypophosphatemia, reduced tubular reabsorption of phosphorus, enhanced tubular reabsorption of calcium, and increased excretion of nephrogenous cyclic AMP, reflecting the PTH-like actions of PTHrP. Nevertheless, serum 1,25(OH)2D concentrations which are generally high or high normal in hyperparathyroidism are frequently low or low normal in HHM (184). This may reflect the higher levels of ambient calcium observed in HHM which may directly inhibit the renal 1a(OH)ase enzyme. In hyperparathyroidism, bone resorption is increased but osteoblastic bone formation is also accelerated reflecting a relatively balanced increase in turnover. However, in HHM, osteoclastic bone resorption is markedly increased and osteoblastic bone formation may be reduced (185). The reasons for this uncoupling are unclear and could reflect the action of osteoblast inhibitory factors co-released from the tumor or in the bone microenvironment or perhaps the effect of the very high levels of calcium per se.


Several studies have indicated that elevated PTHrP correlates better with hypercalcemia than does the presence or absence of skeletal metastases (174,176,178). This appears particularly relevant to certain neoplasms such as breast cancer which is commonly-associated with hypercalcemia but is even more commonly associated with osteolytic skeletal metastases. Elevated circulating PTHrP concentrations (176,177) may contribute to the development of hypercalcemia in these cases in part through augmented bone resorption and in part through increased renal calcium reabsorption. PTHrP may also contribute to the pathogenesis of local osteolytic lesions (186,187). PTHrP per se may be a supportive factor for the growth and progression of cancers by acting in paracrine, autocrine and intracrine modes to modulate tumor cell proliferation, apoptosis, survival, and anoikis, and can therefore influence cell processes which enhance the capacity for tumor dissemination and metastasis (182). In addition, tumors, such as breast tumors that are metastatic to bone, may release PTHrP in the bone microenvironment which will bind to cells of the osteoblastic lineage (including stromal cells, osteoblasts and likely osteocytes), release RANKL ligand (RANKL) and decrease osteoprotegerin (OPG), stimulate osteoclasts  to resorb bone and release, in addition to calcium, growth factors such as TGFb (188), IGF-1 FGF, PDGF and BMP; released growth factors can then stimulate further PTHrP release from the tumor, thus setting up a positive feedback loop (Fig. 6, see above). PTHrP released from cancers such as osteoblasts may also release the chemokine and angiogenic factor CCL2/MCP-1 from osteoblasts which may also increase osteoclastic activity and potentially angiogenesis, and further enhance tumor proliferation (189). Consequently, the presence of skeletal metastases in association with a malignancy is not mutually exclusive with high circulating PTHrP, which can contribute to the hypercalcemia, through both osseous and renal mechanisms; at the same time, locally released PTHrP may contribute to the focal osteolysis. It is in fact uncertain whether local osteolysis per se ever effectively raises ECF calcium in the absence of some cause of reduced renal calcium excretion.


Hematologic malignancies that may cause hypercalcemia (190,191) include non-Hodgkin's lymphoma, chronic myeloid and lymphoblastic leukemia, adult T cell leukemia/lymphoma (ATL) and multiple myeloma.


ATL is an aggressive malignancy that develops after 20-30 years of latency in about 5% of individuals infected with human T-cell lymphotrophic virus type I (HTLV-I). This tumor can be associated with hypercalcemia and increased PTHrP production (192). The mechanism of PTHrP production appears to be stimulation of the PTHrP promoter by the viral protein TAX in the infected lymphoid cells, causing increased PTHrP gene transcription.


Non-Hodgkin's lymphoma may also be associated with increased PTHrP secretion and hypercalcemia and this appears to occur predominantly in patients with late-stage disease and high-grade pathology (191). Multiple Myeloma, although frequently associated with hypercalcemia (about 30% of cases) is rarely associated with increased PTHrP production.


PTHrP assays that recognize NH2-terminal regions or mid-regions of the molecule, and two-site assays detecting two molecular epitopes have been developed. These assays have generally been quite sensitive and specific and successful in detecting PTHrP in MAH where PTHrP overproduction occurs. Circulating levels in normal individuals are generally low or undetectable. Studies have also shown that PTHrP levels do not fall after treatment of the hypercalcemia of MAH but do fall after reducing the tumor burden (176,193). Consequently, the assays may prove useful to track PTHrP as a tumor marker to monitor the course of therapy. Detection of elevated serum PTHrP concentrations in malignancy may, however, predict a poor prognosis (194). Nevertheless, further work is necessary to understand the identity of PTHrP fragments which circulate in order to produce even more useful assays. In most reported NH2-terminal or mid-region assays, PTHrP levels may be elevated in some normocalcemic cancer patients. Whether this represents the detection of bioinert fragments which might be useful as tumor markers or the detection of bioactive PTHrP which presages the development of hypercalcemia and therefore also has predictive value needs to be clarified.

MAH with Elevation of Other Systemic Factors

Although PTHrP is the principal mediator of MAH, and elevated circulating PTHrP levels correlate strongly with hypercalcemia in patients with common tumors of widely diverse histological origin, other systemic factors have been described which may act with PTHrP or in the absence of PTHrP.


Although concentrations of 1,25(OH)2D are generally normal or low in most patients with MAH, elevated serum concentrations have been reported in some cases of non-Hodgkin's and Hodgkin's lymphoma in association with hypercalcemia (195-197). In view of the fact that extra-renal production of 1,25(OH)2D has been shown in various cell types and that renal impairment accompanied several of the reported lymphoma cases it seems likely that synthesis was occurring in the tumor tissue. This would be analogous to expression of a 1a(OH)ase enzyme in granulomatous tissue. Although it is likely that elevated 1,25(OH)2D contributed to the hypercalcemia, co-production of PTHrP has also been reported in some cases (190). Consequently, production of 1,25(OH)2D, lymphoid cytokines and PTHrP individually or in concert might all contribute to disordered skeletal and calcium homeostasis in these tumor states.


A variety of manifestations of malignancy including anorexia, cachexia, and dehydration may be due to tumor-produced circulating proinflammatory cytokines. Cytokines such as Il-1, IL-6, IL-8, IL-11, TNF, and RANKL which are produced in the bone microenvironment have been identified as regulators of bone turnover. PTHrP released from tumors may increase the local production of several of these cytokines however animal studies have reported that tumor activity can increase systemic levels of certain cytokines such as IL-6 and IL-1 which may contribute along with PTHrP to skeletal lysis and hypercalcemia. Some studies of tumor models have implicated a soluble form of RANKL as contributing to MAH (198). Overall therefore it seems likely that other modulators of skeletal and calcium metabolism may be secreted by malignancies and, generally in the presence but occasionally in the absence of PTHrP, may contribute to the dysregulation of bone and mineral homeostasis occurring with MAH.


Inasmuch as PTH per se is expressed mainly in the parathyroid gland, the secretion of PTH by non-parathyroid tumors constitutes true ectopic hyperparathyroidism. A number of such cases of MAH with true PTH production have now been well documented by immunological and molecular biological techniques (199,200). These tumors include ovarian, lung, thyroid, thymus and gastric malignancies (201). Consequently, true ectopic hyperparathyroidism may occur as a cause of MAH but is rare.


Unlike other hematologic malignancies, multiple myeloma appears to have a special predilection to grow in bone (202). This may be related to production of growth factors, notably IL-6, by osteoblastic and osteoclastic cells, which facilitate its growth and factors such as MIP-1a which may promote its adherence to bone. The annexin AXII axis also appears to play a critical role in supporting multiple myeloma cell growth and adhesion to stromal cells/osteoblasts in the bone marrow (203). In order to grow in bone, myeloma cells must secrete bone-resorbing factors. A number of such factors have been implicated including MIP-1a, IL-1, IL-6, TNF-b (lymphotoxin) and hepatocyte growth factor (HGF). Increased RANKL expression by stromal cells with decreased OPG expression also occurs in multiple myeloma and this correlates with the extent of the bone resorption (204). Although bone resorption is stimulated there is little active new bone formation. Consequently, the serum alkaline phosphatase, a marker of osteoblast function is usually normal and bone scans may be negative. Production by myeloma cells of Dickkopf-1 (DKK-1) protein, a soluble inhibitor of signaling via the Wnt pathway, an important growth factor pathway for osteoblasts, has been implicated in the suppression of osteoblast differentiation (205). Other Wnt signaling antagonists, such as soluble-frizzled-related protein (206) and sclerostin (207) have also been implicated in inhibition of osteoblast differentiation in myeloma. All patients with myeloma therefore have extensive bone destruction which may be discrete and focal or diffuse throughout the axial skeleton. Consequently, bone pain is a frequent complaint (80% of cases) and pathological fractures are a disabling consequence.


Although all patients develop osteolysis, hypercalcemia occurs in only about 30% of patients. It is likely that as long as renal function is intact and no circulating factor is produced which enhances renal calcium re-absorption (PTHrP is rarely produced by myeloma cells), increased renal excretion of calcium can accommodate the increased filtered load consequent to bone resorption. Impairment of renal function can occur however due to Bence Jones nephropathy or "myeloma kidney" (in which free light chain fragments of immunoglobulins are filtered and damage glomerular and tubular function), or due to amyloidosis, uric acid nephropathy, or recurrent infections. At this time hypercalcemia may become evident and be associated, because of the renal damage, with hyperphosphatemia rather than the hypophosphatemia occurring in other disorders causing MAH. Therapy aimed at inhibiting bone resorption (e.g. bisphosphonates) may therefore have a special effect in Myeloma, not only in reducing hypercalcemia but also in limiting tumor growth.

Therapeutic Considerations for MAH

Therapy of MAH should be directed primarily at treating the hypercalcemia, which may be of acute onset and considerable magnitude, and at treating the underlying tumor burden. Several approaches have been directed at reducing PTHrP production by those tumors in which PTHrP hypersecretion occurs. These include immunoneutralization (171), antisense inhibition, inhibition of growth factor stimulation through farnesyl transferase inhibition (74), inhibition of gene transcription with low calcemic vitamin D analogs (79), and convertase inhibition (81). To date these remain experimental but if PTHrP contributes to the local growth of the tumor, which some studies have reported, reduction of PTHrP levels may contribute not only to the long-term amelioration of skeletal and calcium homeostasis but also to a reduction in tumor burden.


Granulomatous Disorders

Both infectious and non-infectious granuloma-forming disorders have been associated with 1,25(OH)2D-mediated hypercalcemia (208). Noninfectious disorders include sarcoidosis, silicone-induced granulomatosis, paraffin-induced granulomatosis, berylliosis, Wegener's granulomatosis, eosinophilic granuloma, and infantile fat necrosis. Infectious disorders include tuberculosis, candidiasis, leprosy, histoplasmosis, coccidiomycosis, and Bartonella Hensalae infection (cat-scratch disease). The disorder in which the hypercalcemia was first noted, is perhaps best documented, and has best been studied is sarcoidosis. Consequently, this will be discussed as a prototype of granulomatous diseases.


Up to 50% of patients with sarcoidosis will become hypercalciuric during the course of their disease and mild to severe hypercalcemia will be detected in 10% (294). Hypercalciuria and hypercalcemia generally occur in patients who have widespread disease and high serum angiotensin-converting enzyme activity. Normocalcemic patients with sarcoidosis are prone to the development of hypercalciuria and hypercalcemia after receiving small amounts of dietary vitamin D or after exposure to UV light (210). This is due to the fact the serum 1,25(OH)2D levels in active sarcoidosis are exquisitely sensitive to increases in the 25(OH)D substrate levels. This leads to inappropriately elevated serum 1,25(OH)2D concentrations and absorption of high fractions of dietary calcium (Fig. 7). PTH is suppressed and its calcium reabsorptive effect in the kidney is lost leading to hypercalciuria. However urinary calcium often exceeds dietary calcium intake suggesting a role for 1,25(OH)2D-mediated bone resorption as an alternate or additional source of filtered calcium and indeed accelerated trabecular bone loss and decreased bone mass has been documented in patients with active sarcoidosis (211,212). The source of the inappropriate 1,25(OH)2D is believed to be an extra-renal 1a(OH)ase (as in malignant lymphoproliferative disease) produced by macrophages which are prominent components of the sarcoid granuloma (213). This enzyme exhibits similar kinetics and substrate specificity as the renal 1a(OH)ase but is clearly not regulated as is the renal 1a(OH)ase by PTH, 1,25(OH)2D, calcium, or phosphorus. This extra-renal 1a(OH)ase does however appear to be suppressible by glucocorticoids (214), chloroquine (215) analogs, and cytochrome P-450 inhibitors such as ketoconazole (216).

Figure 7. Disordered calcium homeostasis in granulomatous disease. Production of an extra-renal 1α(OH)ase by macrophages in a granuloma can increase conversion of circulating 25(OH)D to 1,25(OH)2D. This secosteroid will increase Ca+2 absorption from the gut and Ca+2 resorption from bone resulting in an increased ECF Ca+2. The increased ECF Ca+2 and 1,25(OH)2D will inhibit PTH production by the parathyroid glands. The increased filtered load of Ca+2 through the kidney and suppressed PTH will contribute to hypercalciuria.

Therapy of hypercalcemia associated with granulomatous disease is therefore aimed at reducing dietary intake of calcium and vitamin D, limiting sunlight exposure, and treating the underlying disease. Glucocorticoid therapy, if not already indicated for treating the underlying disease, or chloroquine analogs or ketoconazole should be considered to specifically decrease 1,25(OH)2D concentrations.

Viral Syndromes. Autoimmune Deficiency Syndrome (AIDS), and HIV and Cytomegalovirus(CMV) infections

A number of mechanisms may contribute to causing hypercalcemia in AIDS however direct skeletal resorption has been described due to human immunodeficiency virus (HIV), HTLV-III, or cytomegalovirus (CMV) infections of the skeleton (217). Use of foscarnet as an antiviral agent has also been associated with hypercalcemia (218).


Williams Syndrome

Williams Syndrome (William-Beuren Syndrome) is a sporadic disorder characterized by dysmorphic features including an "elfin facies", cardiac abnormalities, the most typical of which is supravalvular aortic stenosis, neurologic deficits, musculoskeletal abnormalities, and hypercalcemia (219). Hypercalcemia occurs in about 15% of cases and may be associated with increased sensitivity to vitamin D (220). Williams Syndrome has been associated with loss of genetic material at 7qll.23 which likely represents a continuous gene deletion that includes the elastin gene (ELN) (221). The hypercalcemia typically occurs during infancy and resolves between 2 and 4 years of age. The pathophysiology is not well understood.

Idiopathic Infantile Hypercalcemia

Idiopathic Infantile Hypercalcemia is a disorder in which patients lack phenotypic features of Williams Syndrome and do not have a 7q11.13 deletion. However, they also manifest hypercalcemia in infancy which is associated with apparent vitamin D sensitivity and which resolves in the first few years of life (222). Loss-of-function mutations in CYP 24A1, the gene encoding the 24-hydroxylase appear to occur. Consequently, inactivation of the active form of vitamin D, 1,25(OH)2D is impaired. This results in increased 1,25(OH)2D levels and enhanced calcium absorption and hypercalcemia (223). Loss-of-function mutations of SLC34A1, encoding the renal proximal tubular sodium-phosphate cotransporter, Na/Pi-IIa result in phosphaturia, hypophosphatemia, stimulation of CYP27B1 and inhibition of CYP24A1, causing increased 1,25(OH)2D and hypercalcemia which may also manifest as Idiopathic Infantile Hypercalcemia.

Congenital Lactase Deficiency

Infants with congenital lactase deficiency may develop increased calcium absorption in the ileum in the presence of nonhydrolyzed lactose, and hypercalcemia and medullary nephrocalcinosis may ensue. The hypercalcemia generally resolves quickly after a lactose-free diet is initiated but nephrocalcinosis may persist (224).


Thiazide Diuretics

Thiazides reduce renal calcium clearance, however in the presence of a normal calcium homeostatic system (e.g. in the absence of primary hyperparathyroidism) this should not produce sustained hypercalcemia (225). Thiazides have however been reported to produce hypercalcemia in anephric individuals. Overall therefore the mechanism is unknown although "unmasking" of mild underlying primary hyperparathyroidism has been suggested as a mechanism. Hypercalcemia is however a rare event in thiazide use and is rapidly reversed by discontinuing the medication.


Lithium carbonate, at 900 to 1500 mg/day, has occasionally (5% of cases) been reported to cause hypercalcemia. Lithium may reduce renal calcium clearance and may also alter the set-point for PTH secretion such that higher ECF calcium levels than normal are required to suppress PTH (226). The hypercalcemia is generally reversible with discontinuation of therapy.

Vitamin D and Analogues

Excessive intake of vitamin D per se, of dihydrotachysterol, of 25(OH)D3, or of 1,25(OH)2D3 can all cause hypercalciuria and hypercalcemia by increasing gut absorption of calcium and bone resorption (227). Only vitamin D, (vitamin D2 or vitamin D3) is available without prescription. Vitamin D per se, is more lipid soluble and has a much longer retention time in the body (weeks to months) than the hydroxylated analogues (hours to days). Therapy consists of hydration, calciuresis, and if needed glucocorticoids and an anti-resorptive agent (bisphosphonate or denosumab).

Vitamin A and Analogues

Vitamin A, in greater than 50,000 IU/day and its analogues cis-retinoic acid and all-trans-retinoic acid (used for the treatment of dermatologic and neoplastic disorders) have been associated with hypercalcemia (228-230). This appears to be due to enhanced bone resorption. Discontinuation of the medication, hydration and administration of an anti-resorptive agent would appear to be the treatments of choice.

Estrogens and Antiestrogens (Tamoxifen)

In up to 30% of patients, hypercalcemia may occur when estrogens or antiestrogens (231) are used to treat breast cancer metastatic to the skeleton. Increased bone resorption appears to be the major mechanism possibly induced by cytokines and growth factors released when the tumor undergoes lysis. The hypercalcemia may require acute treatment but is usually self-limiting.


Hypercalcemia has been reported with theophylline usage for chronic obstructive pulmonary disease or asthma and appears reversible with cessation of therapy or amenable to treatment with beta – adrenergic antagonists (232).

Aluminum Intoxication

Aluminum intoxication was observed when large amounts of aluminum-containing phosphate-binding agents were prescribed to patients with chronic renal failure to control hyperphosphatemia. Alternatively, clustered outbreaks of aluminum intoxication occurred when inadequately purified water was employed for dialysis or for total parenteral nutrition (233). Aluminum intoxication can cause adynamic bone disease in patients with renal failure, and hypercalcemia possibly due to inadequate deposition of calcium in bone. In chronic kidney disease, removal of aluminum by treating with the chelating agent desferioxamine is effective in reducing serum calcium levels and improving mineralization. Less frequent use of aluminum-containing medications has considerably diminished the frequency of this disorder.

Milk-Alkali Syndrome

The classic milk-alkali syndrome causing hypercalcemia occurred in the past when large quantities of milk and bicarbonate were ingested together to treat peptic ulcers. The modern-day equivalent appears to be consumption of large quantities of milk or other dairy products with calcium carbonate (234). Quantities of calcium that must be ingested to cause the syndrome are at least 3 g per day or more. Classically hypercalcemia is accompanied by alkalosis, nephrocalcinosis, and ultimately by renal failure. The alkali may enhance precipitation of calcium in renal tissue. Discontinuation of the calcium and antacid, rehydration and rarely, hemodialysis, can be useful for treatment.


Immobilized patients, in association with reduced mechanical load on the skeleton, continue to resorb bone whereas bone formation is inhibited. Thus, high bone resorption with negative calcium balance leading to osteopenia, osteoporosis, and hypercalcemia may occur from prolonged immobilization after burns, spinal injury, major stroke, hip fracture, and bariatric surgery (235). More severe hypercalcemia and hypercalciuria may occur in immobilized individuals with already high bone turnover such as growing children, patients with Paget’s Disease, or patients with primary hyperparathyroidism or MAH (236).


This discussion of the clinical assessment of the hypercalcemic patient will focus primarily on adult patients. Although many of the approaches are relevant to childhood and even neonates, detailed discussion of the issues relevant exclusively to the pediatric age group is beyond the scope of this chapter.

History and Physical Examination

The approach to the history and physical examination of the hypercalcemic patient should focus on the signs and symptoms which are relevant to hypercalcemia, and the signs and symptoms which are relevant to the causal disorder.


Hypercalcemic manifestations will vary depending on whether the hypercalcemia is of acute onset and severe (greater than 12 mg/dL or 3 mM) or whether it is chronic and relatively mild (Table 2). Patients may also tolerate higher serum calcium levels more readily if the onset is relatively gradual, but at concentrations above 14 mg/dL (3.5 mM) most patients are symptomatic. In both acute and chronic cases, the major manifestations affect gastrointestinal, renal, and neuromuscular function. Patients with acute hypercalcemia commonly present with anorexia, nausea, vomiting, polyuria, polydipsia, dehydration, weakness, and depression and confusion which may proceed to stupor and coma. As well the QT interval on EKG may be shortened by hypercalcemia due to the increased rate of cardiac repolarization. Arrhythmias such as bradycardia and first-degree atrioventricular block, as well as digitalis sensitivity may occur. Acute hypercalcemia, therefore, can represent a life-threatening medical emergency. Patients with chronic hypercalcemia may have a history of constipation, dyspepsia (generally not due to a true ulcer), pancreatitis, and nephrolithiasis but few other signs or symptoms.


Table 2. Manifestations of Hypercalcemia












Anorexia, nausea, vomiting



Dyspepsia, constipation, pancreatitis





Polyuria, polydipsia



Nephrolithiasis, nephrocalcinosis





Depression, confusion, stupor, coma








Bradycardia, first degree atrio-ventricular, digitalis sensitivity




The most frequent underlying causes (over 90%) of hypercalcemia are primary sporadic hyperparathyroidism and malignancy-associated hypercalcemia (MAH). In the West, the most frequent presentation of primary sporadic hyperparathyroidism is that of relatively "asymptomatic" disease with only intermittently or mildly (<12 mg/dL or 3 mM) elevated serum calcium concentrations (126). Occasionally a history is obtained of having passed a kidney stone either recently or in the remote past. Neck masses are unusual in primary hyperparathyroidism unless the patient has a particularly large adenoma or a parathyroid carcinoma. In contrast, the most frequent presentation of MAH is of acute, severe hypercalcemia with some or all of the manifestations of this mineral ion abnormality that are noted above. In view of the fact that hypercalcemia is generally a manifestation of advanced disease, tumors causing hypercalcemia are rarely occult. Consequently, evidence for an underlying malignancy may be obtained or suspected on history or physical examination. Endocrine disorders such as hyperthyroidism or hypoadrenalism should be suspected from a careful history and physical examination as should a history of ingestion of medication which have been reported to cause hypercalcemia. The presence of chronic granulomatous disease could be suspected on the basis of an accurate history and physical examination targeted to the known granulomatous diseases that cause hypercalcemia. Finally, a careful family history should provide clues as to whether the patient manifests any of the variants of familial hyperparathyroidism.

Laboratory Examination

Laboratory testing should be guided by the results of a careful history and a detailed physical examination and should be geared toward assessing the extent of the alteration in calcium homeostasis and toward establishing the underlying diagnosis and determining its severity. Useful laboratory screening may include a complete blood count (CBC), serum total and ionized calcium, PTH, 25(OH)D, 1,25(OH)2D, phosphorus, serum creatinine and calculation of estimated glomerular filtration rate (GFR), urinalysis and 24-hour urine collection for calcium and creatinine.


To establish the diagnosis of PHPT the most common cause of hypercalcemia in the clinic, documentation of at least two elevated corrected (or ionized) serum calcium levels with concomitant elevated (or at least normal) serum PTH levels is required (Figure1). Two site assays for PTH are currently the method of choice (237). If mild hyperparathyroidism is documented, then in addition to the level of urine calcium, bone densitometry (BMD), calculation of estimated GFR, and a renal ultrasound or renal CT scan for evidence of nephrolithiasis may help determine the extent of the disease. For severe hyperparathyroidism, appropriate skeletal X-rays would be indicated to provide a baseline of disease extent before parathyroidectomy. Pre-operative localization of a parathyroid adenoma, generally by nuclear imaging (MIBI scans) or ultrasound has been helpful (238). Ultimately an experienced surgeon is the best guarantee for a successful neck exploration.


The presence of a family history of hypercalcemia or of kidney stones should raise suspicion of MEN1 or MEN2a. If, in addition to HPT in the proband, one or more first-degree relatives are found have at least one of the three tumors characterizing MEN1 (parathyroid, pituitary, pancreas) or MEN2a (parathyroid, medullary thyroid carcinoma, pheochromocytoma) then it is highly likely that the disease is familial. Documentation of familial HPT should be transmitted to the surgeon so that multigland disease can be dealt with. The presence of ossifying fibromas of the mandible and maxilla, and renal lesions such as cysts and hamartomas in addition to HPT would suggest HPT-jaw tumor syndrome. In all patients with documented HPT, a 24-hour urine calcium and creatinine level should be obtained to exclude FHH. If the urine calcium to creatinine ratio is less than 0.01 and if testing serum and urine calcium in three relatives discloses hypercalcemia and relative hypocalciuria in other family members, then this diagnosis is likely and parathyroid surgery is to be avoided. If the urine calcium to creatinine ratio is greater than 0.01 then a BMD test should be performed and guidelines for treatment of primary HPTH should be considered (see below).


If hypercalcemia is associated with very low or suppressed serum PTH levels, then malignancy would be an important consideration, either in association with elevated serum PTHrP or in its absence, in which case it is generally as a result of the production of other cytokines. Hypercalcemia is however frequently a late manifestation of malignancy and the presentation of hypercalcemia is often acute and severe. When malignancy-associated hypercalcemia is suspected then an appropriate malignancy screen should be done including skeletal imaging to identify skeletal metastases. As well appropriate biochemical assessment such as a complete blood count, serum creatinine, and serum and urine protein electrophoresis to exclude multiple myeloma would be appropriate. Detection of elevated serum 1,25(OH)2D levels may point toward the need for a search for lymphoma or for infectious or non-infectious granulomatous disease. Other testing (e.g. a TSH level) could be done for specific clinical disorders based on the findings on the history and physical examination. Although increased PTHrP may be associated with pheochromocytoma, serum PTH levels are suppressed in hypercalcemia in association with thyrotoxicosis, pheochromocytoma, VIPoma, and hypoadrenalism. Although these disorders may be suspected from clinical examination, detailed biochemical evaluation is required for confirmation.


An approach to laboratory assessment of the hypercalcemic patient is shown in Figure 8.

Figure 8. Laboratory approach to the diagnosis of hypercalcemia. Abbreviations used are: BMD= bone mineral density, eGFR=estimated glomerular filtration rate,Li=lithium therapy MAH=mailgnancy-associated hypercalcemia, PHPT=primary hyperparathyroidism, SPEP=serum protein electrophoresis, UPEP=urine protein electrophoresis


If the patient's serum calcium concentration is less than 12 mg/dL (3 mM) then treatment of the hypercalcemia should be aimed solely at treatment of the underlying disorder. If the patient has symptoms and signs of acute hypercalcemia as described above and serum calcium is greater than 12 mg/dL (3mM) then a series of urgent measures should be instituted (Table 3). These measures are almost always required with a serum calcium above 14 mg/dL (3.5 mM)


Table 3. Management of Acute Hypercalcemia

      1. Hydration

      2. Inhibition of Bone Resorption

      3. Calciuresis

      4.Glucocorticoids (when indicated)

      5. Dialysis (in renal failure)

      6. Calcimimetics

      7. Mobilization


Hydration to Restore Euvolemia  

Hydration with normal saline is necessary in every patient with acute, severe hypercalcemia to correct the ECF deficit due to nausea, vomiting, and polyuria (239). This may require infusion of 3 to 4 L of 0.9% sodium chloride over 24 to 48 hours (e.g. an initial rate of 200-300 mL/h subsequently adjusted to maintain a urine output at 100-150 mL/h). Hydration can enhance urinary calcium excretion by increasing the glomerular filtration of calcium and decreasing tubular reabsorption of sodium and calcium. This form of therapy although always required should however be used cautiously in patients with compromised cardiovascular or renal function.

Inhibition of Bone Resorption

Accelerated bone resorption is an important factor in the pathogenesis of hypercalcemia in the majority of patients with acute hypercalcemia and a bisphosphonate is the treatment of choice for inhibition of bone resorption. Consequently, after the patient is rehydrated, zoledronic acid 4 mg intravenously in 5 ml over 15 min (240) or pamidronate, 90 mg, intravenously in 500 ml of 0.9% saline or 5% dextrose in water over 4 hours (241) may be administered. These agents may cause transient fevers, flu-like symptoms, or myalgias for a day or two and transient hypocalcemia and/or hypophosphatemia may result. After a single dose both agents may only reduce serum calcium to normal levels after 4 days but the duration of the effect may last from days to 8 weeks. A second treatment is not recommended for at least 8 days. Denosumab (initial dose 60 mg subcutaneously, with repeat dosing based upon response) is an alternative option and in contrast to bisphosphonates is not cleared by the kidney, and therefore can be used in patients with severe or chronic kidney disease. A second dose may be administered if the calcium is not lowered within approximately one week. Low serum 25(OH)D, if present, should be corrected before administering denosumab. Calcitonin is a peptide hormone which is a safe therapeutic agent when acutely administered. Calcitonin can inhibit osteoclastic resorption and also increase calcium excretion (242). It has a rapid onset of action, causing serum calcium to fall generally by 2 mg/dL within 2 to 6 hours of administration. Consequently, it may be used in concert with a bisphosphonate or denosumab to more rapidly reduce the hypercalcemia (within 2-6 hours) (243). It is usually given intramuscularly or subcutaneously at a dose of 4 to 8 IU/kg. Unfortunately, this agent is not as potent as the most potent bisphosphonates and tachyphylaxis may occur after 24-48 hours. 


If PTHrP or PTH is suspected to be a pathogenetic mediator of the presenting hypercalcemia then renal calcium retention may contribute to the maintenance of the hypercalcemia and inhibition of bone resorption alone may be insufficient to normalize serum calcium (244). In this case, a loop diuretic i.e. furosemide may be added, but also only after rehydration. Loop diuretics inhibit both sodium and calcium reabsorption at the CTAL of the kidney. Small doses of furosemide may be administered (10 to 20 mg) intravenously both to control clinical manifestations of volume excess and to promote calciuria.


Glucocorticoids (e.g. hydrocortisone 200 to 300 mg intravenously over 24 hours for 3 to 5 days) may be administered, particularly if the underlying disorder is known to be responsive to this agent. Thus, patients with hypercalcemia due to hematologic malignancies such as lymphoma or myeloma may benefit (245) as may patients with vitamin D intoxication or granulomatous disease (214) where 1,25(OH)2D production and action may be inhibited.


Dialysis is usually reserved for severely hypercalcemic patient’s refractory to other therapies or who have renal insufficiency.  Both peritoneal dialysis (246) and hemodialysis (247) can be effective.


The calcimimetic, cinacalcet, may be used in doses starting from 30 mg twice daily orally to as high as 90 mg 4 times daily for the treatment of hypercalcemia due to severe primary HPT (especially if caused by a parathyroid carcinoma).


Finally, the patient should be mobilized as rapidly as possible (248). If mobilization is not possible then continued treatment with antiresorptive agents may be necessary (249).

Once the acute episode of hypercalcemia has been managed, careful attention must be paid to addressing the underlying hypercalcemic disorder per se.


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Ovarian Reserve Testing



The ovaries affect far more than reproductive health. Estrogen affects cardiovascular, skeletal, mental health, and numerous other aspects of wellness. Additionally, ovarian dysfunction can reflect disequilibrium relating to multiple conditions. Efficient and effective ovarian testing can give women valuable answers about their fertility, time to menopause, and other conditions and symptoms they may face. Though no test is perfect, antral follicle count (AFC) and anti- Müllerian hormone (AMH) provide more sensitive and specific results that allow for the continuum of ovarian function, and have advantages over classic tests such as follicle stimulating hormone (FSH), estradiol, the clomiphene citrate challenge test (CCCT), and others. This chapter explores these and additional ovarian assays, their underlying mechanisms, and limitations that may favor one test over another depending on circumstances. Particular emphasis is given to evaluating perimenopausal status, procreation, and etiologies for amenorrhea.




Ovarian endocrinology is dynamic. Years of quiescence are followed by oscillating secretion until near burnout, but some function remains even after menopause. “Ovarian reserve testing” assesses where the ovaries are within this spectrum. These measures seem to most clearly relate to oocyte quantity, as multiple other factors (especially age) meaningfully affect oocyte quality and fecundability. However, quantity and quality are not completely independent, as abnormal ovarian reserve testing has been linked to increased blastocyst aneuploidy (1).


This chapter will characterize the main biochemical and sonographic approaches used in both classic and modern testing. Moreover, an assay, like any tool, has value relative to the task to which it is applied. Accordingly, this chapter will also discuss application of ovarian reserve tests to several common areas: assessing perimenopausal status, evaluating ovarian reserve for fertility, and addressing primary and secondary amenorrhea. Use of these markers in assessing the male is covered elsewhere (


Because consensus can be difficult, the following summaries reflect trends, though different perspectives exist and the literature continues to evolve. Existing research on ovarian reserve testing is often confusing because of heterogeneity among tested populations (the general population, infertility patients of all ages, infertility patients more than 35 years old, etc., see also data from the Society for Assisted Reproductive Technology (SART), Figure 1, (2)). Additionally, one must always keep in mind that as with all screening tests, no single result is definitive, since findings must be interpreted in context and should be repeated or supplemented as appropriate.

Figure 1. The relative effect of age on fecundity through in vitro fertilization (IVF) in 2017 according to the Society for Assisted Reproductive Technology (SART). (2)



Follicle Stimulating Hormone (FSH)


FSH was the first hormone directly linked to ovarian aging (3). It is secreted by the anterior pituitary and promotes the progression of antral follicles into dominant follicles. Feedback from estrogen, inhibin, and activin influence hypothalamic GnRH pulsatility, which determines pituitary FSH expression. Elevated FSH levels can be seen with dwindling reserve, where a greater FSH stimulus is required to drive folliculogenesis, but elevated levels also can be found in normal ovarian reserve if measured at the time of the LH surge. Low FSH levels are seen prior to puberty or with hypogonadotropic hypogonadism. In addition to medical conditions that shift pituitary FSH expression, exogenous hormones and their modulators (clomiphene, letrozole, etc.), cimetidine, phenothiazines, and other medications can also shift levels.




Many non-FSH substrates can induce an FSH-like effect. Without describing in detail the spectrum of FSH assays that bypass this challenge, for which an excellent review is available (4), in the clinical setting FSH is typically measured by immunoassay. The sample is usually acquired by phlebotomy (24-hour urine collections are rarely used) on menstrual cycle day three for ovulatory patients, with day one being the first full day of flow.


Testing on cycle days two, four, or five is not unreasonable, but if a normal result would prompt retesting, a day three measurement or a different assay is preferred. Multiple cutoffs are used, with FSH levels of >16.7, >11.4, and <10 mIU/mL reflecting high, moderately high, and normal levels based on the World Health Organization (WHO) Second International Standard (5).

Because ovarian reserve is on a continuum, any cutoff selected should relate to goals of balancing positive and negative predictive values, and this is an issue that applies to other measures of ovarian reserve as well. In amenorrheic patients, a random sample is preferred to testing after hormonally induced menses. In the setting of amenorrhea, a concurrent progesterone level (<2 ng/mL) is a reasonable control to ensure that one is in the follicular phase.



Interpersonal and intercycle variation can be meaningful in patients at risk for moderately elevated FSH, which is why it has been called “Fluctuating Severely Hormone.” The problems with FSH’s sensitivity in part stem from it being a late marker of dwindling ovarian function, as summarized in Stages of Reproductive Aging Workshop + 10 conclusions (6). This limited predictive value is reflected in the NHANES III data, which showed 75% of women aged 40 to 44 years having normal levels at less than 10 mIU/mL, even though ovarian factor is typically the rate limiting step at this age, and half of women aged 45 to 49 years had levels less than 11 mIU/mL (7). Sensitivity for FSH is often worse than specificity, with findings ranging from 11-86% and 45-100%, respectively (8). With anti-muellerian hormone (AMH) and antral follicle count (AFC) demonstrating better predictive value for ovarian response than FSH, these are more likely to be the tests of choice (9). Accordingly, relative to emerging alternatives, FSH testing increasingly is seen as less valuable than it used to be for procreative testing and more useful for evaluating perimenopausal status, hypergonadotropic and hypogonadotropic hypogonadism, and central precocious puberty.





As with FSH, estradiol levels vacillate over the course of a menstrual cycle, peaking in both the late follicular and mid luteal phases. As ovarian reserve declines, the follicular phase shortens because of decreasing feedback inhibition by follicles recruited during the previous cycle. (This is why the first clinical sign of decreasing ovarian reserve is shortening menstrual cycle length.) With the follicular phase starting earlier, estradiol levels start rising closer to menses (and the classic day three FSH peak actually can occur prior to menses). As a result, an elevated day three estradiol level could reflect diminishing ovarian reserve.


Elevated estradiol (>60-80 pg/mL) may also lead to an artificially normal FSH, where higher estradiol levels lead to feedback suppression of FSH. Conversely, estradiol levels <20 pg/mL on day three depending on the circumstances can be consistent with normal ovarian function, hypogonadotropic hypogonadism, or ovarian failure.




Estradiol is also typically measured by immunoassay after phlebotomy. The sample is usually drawn at the same time as FSH levels or randomly when assessing amenorrhea. Estrone, the primary postmenopausal estrogen, and estriol, the primary pregnancy estrogen, are not typically tested when evaluating ovarian function. Also, because oral estrogens are typically metabolized into many byproducts (with varying activity), serum estradiol levels often won’t reflect exogenous exposure. (However, transdermal estrogen administration can be monitored through serum levels.) Medical conditions, glucocorticosteroids, sex steroids, clomiphene, letrozole, GnRH agonists and antagonists, and other medications can alter estradiol levels, just as they could shift FSH levels.




For many conditions, an estradiol level is a reasonable proxy for ovarian inactivity. However, for assessing decreasing ovarian reserve, estradiol is neither a sensitive nor specific assay (9). Accordingly, when used for measuring ovarian reserve, estradiol has its greatest value as an internal control to ensure that one is testing at the expected portion of the menstrual cycle (Figure 2).

Figure 2. Ovarian, hormonal, and endometrial changes over the menstrual cycle. Adapted from Hall, et al., Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal follicular transition. (10).


Clomiphene Citrate Challenge Test (CCCT)



The clomiphene citrate challenge test combines measurement of FSH and estradiol levels prior to clomiphene exposure and FSH levels after clomiphene exposure. Clomiphene is a selective estrogen receptor modulator (SERM) that inhibits negative feedback inhibition by estradiol on the hypothalamus. Normally, increased estrogen levels decrease GnRH pulsatility, resulting in lower FSH levels through negative feedback. By using clomiphene to block feedback inhibition by estradiol, there is an increase in FSH, which enhances follicular recruitment, and which is why clomiphene can be used for ovulation induction and superovulation.




FSH and estradiol levels are assessed through immunoassay, as previously described. The CCCT is performed by having an FSH level drawn on the third day of the menstrual cycle, taking 100 mg of clomiphene orally cycle days five to nine, and then repeating the FSH level on cycle day number ten (11,12). An estradiol level is also frequently drawn on the third day and sometimes on the tenth day as well.




When assessing ovarian reserve for fertility, FSH is a limited measure of ovarian response and a poor predictor of pregnancy and estradiol is predictive of neither (9). When combining the two through the CCCT, it is difficult to assess the degree of benefit through receiver operator curves (9). If benefit is unclear, cost-effectiveness is even less so. Accordingly, other measures of ovarian reserve are increasingly used instead of the CCCT, although this assay is still more commonly used than other provocative tests, such as the exogenous FSH ovarian reserve test (EFORT) and the GnRH agonist stimulation test (GAST). The CCCT has particularly suboptimal value in anovulatory patients. The reason is that the CCCT is primarily used to help discriminate normal ovarian reserve from poor reserve in patients with potentially borderline function. However, the typical anovulatory patient tends to have robust reserve (PCOS, hypogonadotropic hypogonadism) or poor reserve (primary ovarian insufficiency), so relative to alternative assays, a test designed to elicit subtleties is typically less important in this population.


Antral Follicle Count (AFC)



Follicular recruitment is in constant flux during the reproductive years, with less than 0.1% of oogonia present at birth ever making it to ovulation. Fluid surrounding numerous oocytes not selected to be the dominant follicle can be seen sonographically prior to regression. The more follicles visualized within the ovary, the greater the probable ovarian reserve, and AFC has been shown to correlate closely with the primordial follicular pool on histologic analysis. (13,14). Though it remains for debate as to how much a dwindling follicular pool reflects oocyte quality as well as quantity, women with infertility are more likely to have lower antral follicle counts than those without infertility (15). Similarly, women with low antral follicle counts are much more likely to have cancellation for under response with IVF than those with normal counts (16). However, though low quantity in younger women may reflect fewer oocytes with which blastocysts can form, it does not clearly seem associated with higher rates of aneuploidy or miscarriage (17).




Antral follicle count can be measured at any time during the menstrual cycle, as well as when a woman is on hormonal contraceptives or is pregnant. Classically, a woman’s AFC is the total number of ovarian follicles measuring between two and nine millimeters, though many studies count follicles up to and including ten millimeters in size (Figure 3).

Figure 3. Ovarian sonographic imaging of women in their mid-30’s. Figure 3a is from a woman with premature ovarian failure and there are no visualized antral follicles (the sonographically anechoic regions measuring approximately two to nine millimeters within the ovary). Figure 3b is from a woman with tubal factor infertility, and for whom seeing a few follicles within a single plane of the ovary would be normal. Figure 3c is from a woman with polycystic ovarian syndrome. Though her ovary is arguably more multicystic than polycystic (which would typically have follicles concentrated on the periphery of the ovary), she met the criteria for PCOS and her ovary is clearly distinct from those shown in 3a and 3b. Of note, all three ultimately conceived with their own oocytes, so it should be remembered that the absence of visualized antral follicles makes conception far less probable, but not impossible.


Multiple cutoffs are used for what constitutes normal and poor ovarian reserve. Given that antral follicle count varies among cycles, it is reasonable to view the AFC as a continuum, with four total antral follicles reflecting limited reserve, but five antral follicles not being entirely reassuring. Additionally, what constitutes normal is age dependent, where ten total antral follicles may be common for women in their 30’s, but not their teens. Though many measures have been used to define polycystic ovarian morphology, the most accepted standard is that used in the Rotterdam criteria of, “12 or more follicles in each ovary measuring 2 to 9 mm in diameter, and/or increased ovarian volume (>10 ml).” This cutoff was chosen, as it was associated with 75% sensitivity and 98% specificity for distinguishing PCOM from normal ovaries (18). Another frequently used definition comes from Adams, who considered an ovary polycystic if there were ≥ 10 follicles measuring <9 mm (19). Of note, in the development of guidelines for the WHO on PCOS, sonography was deemed preferable to AMH levels from a pragmatic standpoint. (20)




There is debate as to how much moving outside of the early follicular phase or hormonal modulation such as pregnancy and oral contraceptives will shift the measurement of antral follicle count. Both central and paracrine effects can occur and these are more likely to be meaningful in patients with suboptimal ovarian reserve. However, patients with reassuring ovarian reserve are unlikely to move into a non-reassuring category through these conditions if the ultrasound resolution allows for early antral follicle visualization and measurement.


Patient dependent and observer dependent limitations should also be considered. Patients with elevated BMI (particularly with increased vaginal adiposity) and/or scarring of the pelvis may be more likely to have ovaries with limited resolution for assessment, which could potentially underestimate ovarian reserve. Similarly, large cysts or endometriomas could exert a temporary paracrine effect underestimating reserve. Patients with previous ovarian surgery could also have inclusion cysts appearing similar to antral follicles, but these won’t develop with stimulation or have oocytes at follicular aspiration for IVF. For observer dependent limitations, it should be noted that in some multi-center studies where anti-Müllerian hormone (AMH) is found superior to antral follicle count, one can find most institutions having AMH and AFC equally predictive, but one site has an observer where there is a meaningful difference. This has led some to conclude AMH superior to AFC, but failure to properly train observers prior to research is a limitation to study design and may not necessarily reflect true diminished value in utilizing AFC for assessing ovarian reserve.


Anti-Müllerian Hormone (AMH, Müllerian Inhibiting Substance, MIS)




AMH is a homodimeric glycopeptide that in reproductive aged women is predominantly granulosa cell derived. The role of systemic AMH is not clear, but at the level of the ovary, it is believed to downregulate FSH mediated folliculogenesis. AMH expression is highest in secondary, preantral, and small antral follicles up until approximately 4 mm in size, and it stops being expressed by granulosa cells when the follicle measures in the 4 to 8 mm range (Figure 4).


Figure 4. The interplay of follicular development and hormonal secretion and responsiveness.


AMH seems to have a role in selecting the dominant follicle in addition to generally mediating preantral follicular recruitment. AMH levels start undergoing a log-linear decline approximately fifteen years prior to menopause and drop to very low levels approximately five years before menopause (21).


The AMH level associated with diminished ovarian reserve is assay specific and depends on the desired balance of sensitivity and specificity, but is typically below 1 ng/mL. The threshold for menopause is typically lower than the lower detectable limit for many assays, being slightly below 0.1 ng/mL (22). AMH <0.5 ng/mL seems associated with fewer than three follicles available at retrieval, 0.5-1 ng/mL with reduced response, 1-3.5 ng/mL with normal response, and >3.5 ng/mL with overresponse, reflecting greater risk for ovarian hyperstimulation syndrome. (23) Normal AMH values often exceed 2 ng/mL at 30, 1.5 ng/mL at 35, and 1 ng/mL at 40 as a quick reference for expected reserve at a given age.




AMH levels are measured through immunoassay on a sample obtained through phlebotomy. Values obtained have the distinct advantage of being equally valid at any point in the menstrual cycle. Because AMH is expressed primarily before FSH responsiveness occurs, it is believed that AMH remains a valid assay even when ovarian suppression occurs through smoking, oral contraceptives, GnRH agonists, and pregnancy (24). Though these factors can lead to transient ovarian suppression, they are unlikely to change levels so much as to meaningfully underestimate true reserve. The magnitude of effect through these reversible factors seems to be low, with age-specific AMH percentiles decreasing by 11% with oral contraceptives and 17% with pregnancy (25). Additionally, AMH levels drawn on day seven of the pill free interval seem to closely correlate with levels seen after oral contraceptive discontinuation (26).


A popular misconception is that just because it is valid to assess AMH throughout the menstrual cycle and under a variety of inhibitory conditions, this should not be mistaken as meaning that AMH levels are static. Though levels of AMH tend to be steady state in perimenopausal patients, for those with higher ovarian reserve, AMH levels fluctuate significantly. This fluctuation, however, is not to the point where a person with robust ovarian reserve is likely to be categorized as having limited reserve (21).




AMH seems to have fewer limitations than most other assays. In fact, it seems to have the advantage that it not only is useful in predicting ovarian response to gonadotropin stimulation, but may even have limited value in predicting pregnancy rates (27). However, like other ovarian reserve assays, it does not appear particularly valuable in predicting viability once pregnancy has already been established. When there is discordance between AFC and AMH levels (e.g. low AFC but normal AMH or vice versa), ovarian response is often a hybrid of the two findings (above those with diminished reserve but less than that of those with normal reserve) (28).


Inhibin B




Inhibin B is similar to AMH in that it is a glycoprotein secreted by preantral follicles, with levels declining with age. Both inhibin A and B downregulate pituitary FSH secretion. However, Inhibin A levels are not used to predict ovarian reserve because they arise primarily from the dominant follicle rather than an earlier follicular cohort and therefore are less predictive. Inhibin B levels are relatively more useful, but overall remain suboptimally predictive, as they are a late finding for diminished ovarian reserve and typically start falling around four years prior to menopause (21).




Inhibin levels are measured by immunoassay after phlebotomy. Inhibin B levels fluctuate over the menstrual cycle, with peaks in the early to mid-follicular phase, as well as during ovulation. Accordingly, inhibin B is typically measured on the third day of the menstrual cycle in ovulatory women. Outside of ovarian reserve testing, in postmenopausal women, where inhibin B levels should be consistently low, a random level is particularly good for following granulosa cell tumors (>89% have elevated inhibin B) and also can be useful for following some epithelial cell ovarian tumors.




In addition to significant variation within the cycle, there is also meaningful variation among cycles. Because of limited sensitivity and specificity, this assay has greater value in those far more likely to have diminished reserve. Some have proposed using inhibin B in combination with other assays, but it is the opinion of the American Society for Reproductive Medicine that “combined ovarian reserve tests models do not consistently improve predictive ability over that of single ovarian reserve tests.” (29).



Ovarian Volume



Follicles, stroma, and vasculature all contribute to ovarian volume. The percentage that each contributes depends on the individual, her age, underlying gynecologic conditions, and where she is during the menstrual cycle.




Typically, ultrasound is used to measure the ovary in all 3 dimensions. These measurements are then applied in the formula for calculating the volume of an ellipse (D1 x D2 x D3 x 0.523). An ovarian volume of >10 cm3 is considered consistent with PCOS. Although increased ovarian stromal volume distinguishes polycystic ovarian morphology from the multicystic ovary, stromal volume is not routinely measured. Alternative approaches that may improve the effectiveness of ovarian volume include the use of trapezoidal volume (30), 3D ultrasound (31), and color Doppler (32).




Ovarian volume shifts in response to normal physiologic changes (such as the presence of a dominant follicle) and coexisting medical conditions (such as endometriomas). Exogenous hormones can decrease ovarian volume (33), even though ovarian reserve itself has not changed. For these reasons, if evaluating the ovaries by ultrasound, antral follicle count is believed to be a better proxy for ovarian reserve.




Assessing Perimenopausal Status


Classically, ovarian insufficiency and failure have been defined as present when persistent FSH levels >40 µIU/mL are found with at least two radioimmunoassays more than a month apart. No detectable antral follicles in a patient without ovarian suppression is consistent with a perimenopausal state and fewer than two antral follicles have been deemed a more sensitive cutoff (29). The reason to not require the complete absence of follicles is that minimal follicular development is not unusual in postmenopausal women, as there can be a 14% prevalence and an 8% incidence of simple cysts in a given year (34). Similarly, though an undetectable AMH level would be consistent with menopause, in women with primary ovarian insufficiency, approximately a quarter of them will have below normal but detectable AMH levels and a sixth will have normal AMH levels (35). Though women with advancing age will have higher FSH levels, it remains unclear if women with elevated FSH earlier in their reproductive life will go through menopause earlier (36). Finally, it should be remembered that confirmation of primary ovarian insufficiency does not automatically mean completion of testing, as fragile X carrier screening and other evaluations may be appropriate.

Evaluating Ovarian Reserve for Fertility in Ovulatory Patients


For ovarian reserve testing prior to fertility therapy, there is more data on FSH than other measures. Generally, women of the same age with higher FSH levels seem to have lower fecundability (37). However, younger women with elevated FSH levels often have much better fecundability than older women with comparably elevated FSH (38) and age can be a better predictor of outcome than FSH (39). Though differences in pregnancy rates can be shown between those with high and low FSH, the assay in general has suboptimal sensitivity for both ovarian response and pregnancy rates, as reflected by receiver-operator curves (9).


A rarely addressed caveat is that though it is true that multiple studies are showing AMH and AFC to have a better balance of sensitivity and specificity than FSH, meta-analyses regarding the predictive value of FSH run the risk of being biased towards the null. The reason is that the earliest ovarian reserve testing research (using FSH) was done at a time when IVF success rates were lower. This caveat won’t apply to modern studies were FSH is directly compared with AMH or AFC, but one should account for temporal bias in meta-analyses if studies from the 1990s are included.


Anti-Müllerian hormone levels and antral follicle count seem to be emerging as the best approaches to procreative testing. After accounting for age, AFC and AMH seem highly accurate in predicting poor response with IVF, while FSH does so only moderately (40). Not only are these measures commonly used for predicting under response, but they can also be used to predict hyperstimulation (41). Ovarian reserve assessment for reproductive purposes is fraught with controversy because different practitioners prefer different balances of sensitivity and specificity. At the minimum it should be recognized that this type of testing is meant to be screening for women who are more likely to have a poor response to ovarian stimulation, and findings are not necessarily diagnostic of ovarian failure or the degree of risk for premature menopause. However, results consistent with perimenopausal findings should be confirmed and appropriate counseling given. As stated by ASRM, “There is fair evidence against the recommendation that any ovarian reserve test should be used as a sole criterion for the use of ART” (29). The American College of Obstetricians and Gynecologists (ACOG) draws similar conclusions (42).


Since combined tests do not consistently improve the ability to predict ovarian response, many clinicians are simply using either AMH or AFC in the context of the patient’s age and reserve additional testing for atypical clinical pictures or to confirm significant ovarian insufficiency. In spite of this ASRM recommended approach, some argue that combined testing improves sensitivity in detecting suboptimal ovarian reserve. Whether or not the literature ultimately demonstrates this, a way of side-stepping this debate is by noting that combined testing is unlikely to be cost-effective. The reason is that if additional testing is unlikely to change management (especially when the vast majority of patients have normal results), it is very hard to show cost-effectiveness when doubling or tripling costs without clear benefit. Accordingly, if using combined testing, it should be selective rather than universal.


A final note on ovarian reserve tests in procreation relates to their limitations. Though some appear better than others in predicting ovarian response to stimulation, most are limited at best in predicting pregnancy, and this predictive value is highly dependent on patient demographics within a study. This is not inherently a flaw in the assays; rather, infertility is often multifactorial, so when ovarian reserve testing is a subset of factors, this tends to bias its relevance towards the null. Studies showing an ovarian reserve test to be predictive of pregnancy in general tend to have older populations. (It has been argued as to whether this constitutes enrollment bias or limits external validity; however, it is reasonable to find a test having greater value when applied to a population at risk.) Another limitation is that abnormal ovarian reserve testing does not seem clearly related to miscarriage, (43) despite an association between abnormal testing and blastocyst aneuploidy (1). Follicular quantity is more valuable in identifying ovarian factors in subfertility patients and does not seem predictive of outcomes in patients who have suboptimal reserve, but have never tried to conceive. (44)

Evaluating Amenorrhea in the Post-menarche, Pre-menopausal Patient

Numerous conditions can cause amenorrhea in reproductive aged women. Testing falls into two categories: diagnosing etiology and reassuring the patient that she is not in ovarian failure. For a more comprehensive discussion of how to evaluate etiology, please see . In general, and in addition to remembering to exclude pregnancy as a cause for amenorrhea, ASRM Practice Committee guidelines recommend FSH, TSH, and prolactin levels in addition to the usual history and physical exam (45) (An important contextual caveat is that AMH and AFC were not as well established when these guidelines came out in 2008). Though increasingly AFC is used in place of FSH, especially for evaluating hyperandrogenic women since AFC is part of the Rotterdam criteria (46), FSH still has a role in differentiating PCOS and forms of functional hypothalamic amenorrhea (47).


Figure 5. Overall direction of laboratory testing in women with amenorrhea. (43) Reproduced with permission of the author.


Regarding reassuring the patient, for gynecologists a transvaginal ultrasound to assess antral follicle count is relatively easy to perform, can often be performed promptly at the initial office visit, and can have a reassuring tangibility to patients when antral follicles are identified and their importance is explained. When sonographic evaluation of ovarian reserve is less available, a normal AMH level should be reassuring. Additionally, for patients who have been placed on oral contraceptives or other hormonal therapy without a diagnosis of etiology for amenorrhea, AFC and AMH levels may be lowered, but are still likely to remain within the normal range if the patient truly has normal reserve.

Figure 6. Relative strengths of assays in clarifying issues of ovarian reserve (balancing positive and negative predictive values, typical costs, and available alternatives). AFC = Antral Follicle Count; AMH =Anti-Mullerian Hormone; FSH = Follicle Stimulating Hormone; CCCT = Clomiphene Challenge Test. * When combined with LH




It has been said that a Rolex keeps time well, but makes for a lousy hammer. All ovarian reserve tests are merely tools and their value relate to the task to which they are applied. Even as we see increased use of AFC and AMH (48), we have to remember that ideal testing is “systematic, expeditious, and cost-effective” (49). In other words, when evaluating ovarian reserve, one should account for not only the symptoms and probable diagnosis, but also the turnaround time for results, and how to maximize value in testing. These latter two factors vary by site, so clinicians will have to find the right balance for their practice. Finally, one of the most important and cost-effective predictors is age (see Figure 1). In the procreative setting, after age is combined with another ovarian reserve test, the marginal benefit from further assays tends to be less (8). When 796 fertility centers were surveyed on preferred tests, 51% thought AMH best, while 40% preferred AFC; however, 80% thought age to be the best predictor if that were given as an option (50). Accordingly, and with the exception of premature ovarian failure where independent confirmation is appropriate (due to discordance between age and the assay), until further studies justify effectiveness and cost-effectiveness, simultaneously using multiple ovarian reserve tests should be for selected patients rather than universal.




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Normal Physiology of Growth Hormone in Adults


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 the 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 synergistic (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 several IGF-I binding proteins. The mechanisms underlying the insulin antagonist effect of GH in humans are causally linked to lipolysis and the ensuing elevated levels of circulating free fatty acids. 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 the present chapter, the normal physiology of GH secretion and the effects of GH on intermediary metabolism throughout adulthood, focusing on human studies, are presented.


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 a 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.


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 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 the pituitary and blood is the 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 thus 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 the adult pituitary gland (5). The differentiation of somatotroph cell is governed by the pituitary transcription factor 1 (Pit-1). Data in mice suggest that the pituitary holds regenerative competence, the GH-producing cells being regenerated form 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 their 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 the 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 and Figure 4) (8). Integrated 24h GH concentration is significantly greater in women than in men and greater in the young than in older 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 in girls and 1 year later in boys (9).

Figure 1 The secretory pattern of GH in young and old female and male. In young individuals the GH pulses are larger and more frequent and that female 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 amplify hypothalamic GHRH secretion and synergize with its pituitary GH-stimulating effects (Figure 2) (10). Interestingly, recently germline or somatic duplication of GPR101 has been shown to constitutively activate the cAMP pathway in the absence of a ligand, leading to GH release. Although the precise physiology of GPR101 is unclear, it is worth mentioning it since it clearly has an effect on GH pathophysiology (11).

In addition, a multitude of other factors may impact the GH axis, most probably due to interaction with GRHR, 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 mute inhibition by exogenous SST (14). Also, exogenous estrogen potentiates ghrelin’s action (15).

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

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


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 up-regulates 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 SST, 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 lead to profound GH deficiency with anterior pituitary hypoplasia. Subsequent 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 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).


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 paraventricular nucleus and arcuate nucleus, produce SST. These neurons secrete SST into the adenohypophyseal portal venous system, via the median eminence, to exert effects on the anterior pituitary. SST has a short half-life of approximately 2 minutes as it is rapidly inactivated by tissue peptidase in humans.

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, the adult pituitary only expresses 4 subtypes (SSTR1, SSTR2, SSTR3, SSTR5). Of these four subtypes, somatotrophs exhibit more sensitivity to SSTR2 and SSTR5 ligands in inhibiting the secretion of GH in a synergistic manner (21). Somatostatin inhibits GH release but not GH synthesis.


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 the stomach and may be involved in the GH response to fasting and food intake.

Clinical Implications

GH levels – influence of body composition, 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 (22,23). It has been hypothesized that many of the senescent changes in body composition and organ function are related to or caused by decreased GH (24), also known as "the somatopause".

Studies carried out 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) (25). It is also known that normal GH levels can be restored in obese subjects following massive weight loss (26), 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 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. life style, 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 performed 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), while 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 (27). In the same population, 24-h spontaneous GH levels also predominantly correlated inversely with intra-abdominal fat mass (Figure 3) (28).

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 (29-31). 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 (32).

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 (33). Whether this reflects an unfavorable balance between effects and side effects in older people or the 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 (34,35), 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 years are highly responsive to even a small dose of GH (36). Interestingly, there is a gender difference response to GH treatment with men being more responsive in terms of IGF-I generation and fat loss during therapy, most probably due to lower estrogen levels that negatively impact the GH effect on IGF-I generation in the liver (37).

The pharmacokinetics and short-term metabolic effects of a near physiological intravenous GH bolus (200μg) were compared in a group of young (30 year) and older (50 year) healthy adults (38). 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 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 proteins correlated strongly and positively with abdominal fat mass (39).

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, leads to increased FFA availability, and induces insulin resistance and hyperinsulinemia; 3. High insulin levels suppress IGF binding protein (IGFBP)-1 resulting in a relative increase in free IGF-I levels; 4. Systemic elevations of FFA, insulin and free IGF-I suppress pituitary GH release, which further increases fat mass; 5. Endogenous GH is cleared more rapidly in subjects with a 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 especially due to concern that the ensuing elevation of IGF-I levels may increase the risk for the development of neoplastic disease (For an extensive discussion of GH in the elderly see the chapter on this topic in the Endocrinology of Aging section of Endotext).

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

Life-long GH deficiency

A real-life model for GH effects in human physiology is represented by patients 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, and central obesity, and are fertile (40). 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 (41). The decrease of cancer risk in life-long GH deficiency together with reports on the permissive role of GH for neoplastic colon growth (42), pre-neoplastic mammary lesions (43), and progression of prostate cancer (44) demands, at least, a careful tailoring of GH substitution dosage in the GH deficient patients.

GH and the 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 possess immunomodulatory actions. Immune cells, including several lymphocyte subpopulations, express receptors for GH, and respond to its stimulation (45). GH stimulates in vitro T and B-cell proliferation and immunoglobulin synthesis, enhances human myeloid progenitor cell maturation, and modulates in vivo Th1/Th2 (8) and humoral immune responses (46). 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 (46). JAK/STAT signaling, the principal mediator of GHR activation, is well-known to be involved in the modulation of the immune system, so is tempting to assume that GH may have a role too, but clear data in humans are needed.

Growth Hormone Signaling in Humans

Growth hormone RECEPTOR (GHR) activation

GHR signaling is a separate and prolific research field by itself (47), so this section will focus on recent data obtained in human models.

GHRs have been identified in many tissues including fat, lymphocytes, liver, muscle, heart, kidney, brain and pancreas (48,49). 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 them binding 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 (48,50). 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 allowing dimerization and translocation to the nucleus, where they bind to DNA and activate gene transcription. A STAT5b binding site has been characterized in the IGF-I gene promoter region (51). Attenuation of JAK2-associated GH signaling is mediated by a family of cytokine-inducible suppressors of cytokine signaling (SOCS) (52). SOCS proteins bind to phosphotyrosine residues on the GHR or JAK2 and suppress GH signaling by inhibiting JAK2 activity and competing with STATs. For 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 (53).

Data on GHR signaling derive mainly from rodent models and experimental cell lines, although GH-induced activation of the JAK2/STAT5b and the mitogen activated protein kinase (MAPK) pathways have been recorded in cultured human fibroblasts from healthy human subjects (54). 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 a normal GHR (55). Activation of GHR signaling in vivo has been reported in healthy young male subjects exposed to an intravenous GH bolus vs. saline (56). Significant tyrosine phosphorylation of STAT5b was recorded after GH exposure at 30-60 minutes in muscle and fat biopsies, but there was no evidence of GH-induced activation of PI 3-kinase, Akt/PKB, or MAPK (56).

GH and insulin signaling

GH impairs the insulin mechanism but the exact mechanisms in humans are still a matter of debate. There is no evidence of a negative effect of GH on insulin binding to the receptor (57,58), which obviously implies post-receptor metabolic effects.

There is animal and in vitro evidence to suggest that insulin and GH share post-receptor signaling pathways (59). Convergence has been reported at the levels of STAT5 and SOCS3 (60) as well as on the major insulin signaling pathway: insulin receptor substrates (IRS) 1 and 2, PI 3-kinase (PI3K), Akt, and extracellular regulated kinases (ERK) 1 and 2 (61-63). Studies in rodent models suggest that the insulin-antagonistic effects of GH in adipose involve suppression of insulin-stimulated PI3-kinase activity (59,64). In 2001 it was demonstrated that GH induces cellular insulin resistance by uncoupling PI3K and its downstream signals in 3T3-L1 adipocytes (65)]. A follow up study has shown that GH increased p85α expression and decreased PI3K activity in adipose tissue of mice, supporting the previous report of a direct inhibitory effect of GH on PI3K activity (64). However, a study performed in healthy human skeletal muscle showed, as expected, that the infusion of GH induced a sustained increase in FFA levels and subsequently insulin resistance as assessed by the euglycemic clamp technique, but was not associated with any change in the insulin-stimulated increase in either IRS-1/PI3K or PKB/Akt activity (66). It was subsequently showed that insulin had no impact on GH-induced STAT5b activation or SOCS3 mRNA expression (67).

Because GH and insulin share some common intracellular substrates, a hypothesis arose claiming that competition for intracellular substrates explains the negative effect of GH on insulin signaling (59). Furthermore, studies have shown that SOCS proteins negatively regulate the insulin signaling pathway (68). Therefore, another possible mechanism by which GH alters the action of insulin is by increasing the expression of SOCS genes.


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 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 (IGFBP) in the circulation.

Following the initial discovery of IGF-I, it was thought that GH governs somatic growth only by IGF-I produced by the liver (69). However, in the 1980s this hypothesis was challenged by the identification of IGF-I production in numerous tissues. 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 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 tetrameric structure, with 2 alpha and 2 beta subunits (70). 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 is more involved 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 IRS-2, which in-turn activates the RAS activating protein SOS. This complex activates the MAPK pathway leading to the stimulation of cell growth (71,72).

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 (73). 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 become 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 (74). 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 (74,75).

Effects of IGF-I

Studies on hypophysectomized animals overexpressing IGF-I demonstrate the independent anabolic effects of IGF-I (76). 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 (77). 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 (78-80). 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, and GHR and GHRH defects. The important growth promoting role of IGF-I is further demonstrated by studies on transgenic mice. Only 6-8% postnatal growth retardation is presented in mice with liver-selective deletion of IGF-I gene showing low serum IGF-I concentrations, whereas animals with total IGF-I deletion or those with only peripherally produced IGF-I deletion showed marked growth retardation (81).

Both elevated and reduced levels of serum IGF-I are associated with excess mortality in human adults (82). 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 (82). In this regard, it is noteworthy that calorie restriction is associated with increased longevity and reduced insulin/IGF activity in many species (83), albeit GH levels being increased by calorie restriction and fasting (84).

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, and 3) calorie restriction, which suppresses IGF-I activity and stimulates GH secretion, may promote longevity also in human adults (84).


The nutritional status dictates the effects of GH. 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 a state with decreased nutrient intake and during the sleep and exercise, the direct effects 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 (85). Fasting hypoglycemia and pronounced sensitivity to insulin were distinct features of hypophysectomized animals. These symptoms were readily corrected by 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 deteriorated following exposure to a single supraphysiological dose of human GH in hypophysectomized adults with type 1 diabetes mellitus (86). Somewhat surprisingly, only modest effects of GH on glucose metabolism were recorded in the first metabolic balance studies involving adult hypopituitary patients (87,88).

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 (89).

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 (90). 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 (91). 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. Apart from enhanced glucose/fatty acid cycling, it has been shown that GH-induced insulin resistance is accompanied by reduced muscle glycogen synthase activity (57) and diminished glucose dependent glucose disposal (92). However, insulin binding and insulin receptor kinase activity from muscle biopsies is not affected by GH (57).

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 (93). There is evidence to suggest that this hyper-metabolic state ultimately leads to beta cell exhaustion and overt diabetes mellitus (94), but it is also shown that the abnormalities are completely reversed after successful surgery (93). Conversely, it has been shown that administration of GH in supraphysiological doses for only two weeks induces comparable acromegaloid - and reversible - abnormalities in substrate metabolism and insulin sensitivity (95).

Interaction of Glucose and Lipid Metabolism

The effect of FFA on the partitioning of intracellular glucose fluxes was originally described by Randle et al. (96). According to this 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. A. 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. B. 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 the 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.

The Randle hypothesis remains an appealing model to explain the insulin-antagonistic effects of GH when considering its pronounced lipolytic effects. To support this, experiments have shown that co-administration of anti-lipolytic agents and GH reverses GH-induced insulin resistance (97). Moreover it has been shown that GH-induced insulin resistance is associated with suppressed pyruvate dehydrogenase activity in skeletal muscle (98). However, 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), whereas the muscle biopsies from GH deficient adults after GH treatment have revealed increased glucose but low-normal glucose-6-phosphate levels (99).

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 (100). 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 (101), but it is far from physiological.

We know and understand that hypoglycemia is a serious and challenging side effect of insulin therapy as a consequence of inappropriately high insulin levels (during fasting). As a corollary, we must realize that hyperglycemia may result from GH therapy. It is therefore important to carefully monitor glucose metabolism and to use the lowest effective dose when replacing adults with GH.

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 LBM by total body potassium or dual x-ray absorptiometry, and direct calculation of muscle area or volume by computerized tomography and magnetic resonance imaging.

Effects of GH on Skeletal Muscle Metabolism in Vitro and in Vivo

The clinical picture of acromegaly and gigantism includes increased LBM 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 (102,103). Indirect evidence of an increase in muscle cell number following GH treatment was also presented (103).

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) (103). 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 (104). Another important observation of that period was that GH directly increases the synthesis of both sarcoplasmic and myofibrillar protein without affecting proteolysis in a rat model (105).

In a human study, the in vivo effects of systemic and local GH and IGF-I administration on total and regional protein metabolism revealed that GH administration for 7 days in normal adults increased whole body protein synthesis without affecting proteolysis (106), and comparable results have been obtained in other human studies (107-110).

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 (49), which allows for direct GH effects; alternatively, GH may stimulate 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 (111) whereas IGF-I in combination with an amino-acid infusion increase protein synthesis (112). 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 (113). The degree to which mobilization of lipids contributes to the muscle anabolic actions of GH has so far not been specifically investigated.

An interesting discovery has been that infusion of GH and IGF-I into the brachial artery increases forearm blood flow several fold (110,114). This effect appears to be mediated through stimulation of endothelial nitric oxide release leading to local vasodilatation (115,116). 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. This increase in muscle blood flow may also contribute to the GH-induced increase in resting energy expenditure, since skeletal muscle metabolism is a major determinant of resting energy expenditure (23). 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 (116).

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. were the first to suggest that the senescent changes in body composition were causally linked to the concomitant decline in circulating GH and IGF-I levels (23). This concept has been recently reviewed (117), 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 undergoing a resistance exercise program for 12 weeks showed a GH induced increase in 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 (118). In a similar study in older men, GH also increased LBM and whole body protein synthesis, without significantly amplifying the effects of exercise on muscle protein synthesis or muscle strength (119). An increase in LBM but unaltered muscle strength following 10 weeks of GH administration plus resistance exercise training was also recorded (120). A more recent study in older men observed a significant increase (4.4 %) in LBM with GH, but no significant effects on muscle strength (121). Finally, a meta-analysis of studies administering GH to healthy adult subjects showed that it increases LBM and reduces fat mass without improving muscle strength or aerobic exercise capacity (122).

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 these patients, GH treatment for 12 weeks has been associated with significant increments in LBM and physical fitness (123,124).


The GH/IGF-I axis is specifically regulated and is involved in a multitude of processes during all the aspects of life from intrauterine growth, to childhood and puberty, adulthood and lastly elderly stages of life. GH acts directly or via its principal metabolite, IGF-I, and has a wide range of physiological roles being a metabolic active hormone in adulthood. The nutritional status of an organism dictates the effects of GH, either an impairment of insulin action (fasting state) or promoting protein anabolism (fed state). As our knowledge of 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. Normal aging is associated with a gradual decline in serum IGF-I levels that run in parallel with reductions in muscle mass and function and other senescent changes in organ function. The cause-effect relationship is uncertain, but GH administration to elderly people without pituitary disease has not proven beneficial and sustained supra-physiological IGF levels and actions are likely to be harmful. On the other hand, a stimulation of endogenous GH secretion induced by exercise and calorie restriction may contribute to healthy aging.  


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  95. Moller N, Moller J, Jorgensen JO, Ovesen P, Schmitz O, Alberti KG, Christiansen JS. Impact of 2 weeks high dose growth hormone treatment on basal and insulin stimulated substrate metabolism in humans. Clin Endocrinol (Oxf). 1993;39(5):577-581.
  96. 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.
  97. Nielsen S, Moller N, Christiansen JS, Jorgensen JO. Pharmacological antilipolysis restores insulin sensitivity during growth hormone exposure. Diabetes. 2001;50(10):2301-2308.
  98. Nellemann B, Vendelbo MH, Nielsen TS, Bak AM, Hogild M, Pedersen SB, Bienso RS, Pilegaard H, Moller N, Jessen N, Jorgensen JO. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity. Acta Physiol (Oxf). 2014;210(2):392-402.
  99. 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. J Clin Endocrinol Metab. 1998;83(5):1668-1681.
  101. Jorgensen JO. Human growth hormone replacement therapy: pharmacological and clinical aspects. Endocr Rev. 1991;12(3):189-207.
  102. 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 Endocrinol (Copenh). 1977;84(4):681-696.
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  108. Fryburg DA, Barrett EJ. Growth hormone acutely stimulates skeletal muscle but not whole-body protein synthesis in humans. Metabolism. 1993;42(9):1223-1227.
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  110. Fryburg DA, Gelfand RA, Barrett EJ. Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. Am J Physiol. 1991;260(3 Pt 1):E499-504.
  111. 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. J Clin Endocrinol Metab. 1992;75(5):1186-1191.
  112. Russell-Jones DL, Umpleby AM, Hennessy TR, Bowes SB, Shojaee-Moradie F, Hopkins KD, Jackson NC, Kelly JM, Jones RH, Sonksen PH. Use of a leucine clamp to demonstrate that IGF-I actively stimulates protein synthesis in normal humans. Am J Physiol. 1994;267(4 Pt 1):E591-598.
  113. 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.
  114. Copeland KC, Nair KS. Recombinant human insulin-like growth factor-I increases forearm blood flow. J Clin Endocrinol Metab. 1994;79(1):230-232.
  115. 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. J Clin Invest. 1996;97(5):1319-1328.
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Nutritional Recommendations for Individuals with Diabetes

The authors are employees of Eli Lilly & Company, and the content of this chapter reflects their own views and training as Registered Dietitian Nutritionists not that of the company. 



The chapter summarizes current information available from a variety of scientifically based guidelines and resources on nutritional recommendations for adult people with diabetes (PWD). It is designed to take these guidelines and provide an overview of practical applications and tips in one place for health care practitioners who treat PWD.  The sections are divided into components of nutritional content, with associated goals for PWD, as well as reviews of present nutritional topics of interest, including weight loss diets in the current press. The information also includes sources for further review, and resources that can be utilized for PWD.  A main message is that nutrition plans should be individualized and flexible to meet the specific needs of the PWD, in consideration of their ability to implement the changes.  Education is best given by a team approach and should not simply be delivered by giving a person a one-size-fits all diet sheet. Referral to a diabetes self-management education (DSMES) program that includes counseling and instruction on nutrition therapy by a Registered Dietitian Nutritionist (RDN) is highlighted.



This chapter will summarize current information available from a variety of scientifically based guidelines and resources on nutritional recommendations for persons with diabetes (PWD) for health care practitioners who treat them. The provided information provides sources for further review and study.  The key take home message is that nutrition plans should be individualized to meet the needs of the PWD, in consideration of their lifestyle, socioeconomic factors, cultural background, and motivation. The modern diet for the individual with diabetes is based on concepts from clinical research, portion control, and individualized lifestyle changes. It cannot simply be delivered by giving a person a diet sheet in a one-size-fits-all approach. The lifestyle modification guidance and support needed requires a team effort, best led by an expert in this area; a registered dietitian (RD) or registered dietitian nutritionist (RDN), or a referral to a diabetes self-management education (DSMES) program that includes instruction on nutrition therapy. Dietary recommendations need to be individualized for and accepted by the given PWD. It’s important to note that the nutrition goals for diabetes are similar to those that healthy individuals should strive to incorporate into their lifestyle.


Leading authorities and professional organizations have concluded that proper nutrition therapy is an important part of the foundation for the treatment of diabetes. However, appropriate nutritional intervention, implementation, and ultimate compliance with the plan remain some of the most vexing problems in diabetes management for three major reasons: First, there are some differences in the dietary structure to consider, depending on the type of diabetes and medication the PWD is taking. Second, a plethora of dietary information is available from many sources to the PWD and healthcare provider. Nutritional science is constantly evolving, so that what may be considered true today may be outdated in the near future. Nutritional intervention may vary based on the type of diabetes; however, many of the basic dietary principles are similar for all PWD, prediabetes, metabolic syndrome or who are overweight or obese.  Lastly, there is not perfect agreement among professionals as to the best nutritional therapy for individuals with diabetes, and ongoing scientific debate reported in the popular press may confuse PWD and health care providers.


The following recommendations are consensus-based, and they emphasize practical suggestions for implementing nutritional advice for most individuals with diabetes.

Ali et al, reported that although there have been improvements in risk factor control and adherence to preventative practices, almost half of U.S. adults with diabetes did not meet the recommended goals for diabetes care from 1999-2010. (1) Thus, still more needs to be done to improve overall care of PWD.




The nutrition therapy goals for the individual with diabetes have evolved and have become more flexible and patient centered. The goals from the American Diabetes Association (ADA) 2019 include the following: (2)


  1. To promote and support healthful eating patterns, emphasizing a variety of nutrient dense foods in appropriate portion sizes in order to improve overall health and:
  • Achieve and maintain body weight goals
  • Attain individualized glycemic, blood pressure, and lipid goals
  • Delay or prevent complications of diabetes
  1. To address individual nutrition needs based on personal and cultural preferences, health literacy and numeracy, access to healthful food choices, willingness and ability to make behavioral changes, as well as barriers to change
  2. To maintain the pleasure of eating by providing nonjudgmental messages about food choices
  3. To provide an individual with diabetes the practical tools for day-to-day meal planning rather than focusing on individual macronutrients, micronutrients or single foods


The American Association of Clinical Endocrinologists (AACE) guidelines have similar goals for people with type 2 diabetes. (3)




How should these goals best be put into practice? The following guidelines summarized from the ADA Standards of Care will address the above goals and provide guidance on nutrition therapy based on numerous scientific resources. The Diabetes Control and Complications Trial (DCCT) and other studies demonstrated the added value individualized consultation with a registered dietitian familiar with diabetes treatments, along with regular follow-up, has on long-term outcomes and is highly recommended to aid in lifestyle compliance. (4) Medical nutrition therapy (MNT) implemented by a registered dietitian is associated with A1C reductions of 1.0–1.9% for people with type 1 diabetes and 0.3–2% for people with type 2 diabetes (2)




Many studies have been completed to attempt to determine the optimal combination of macronutrients. Based on available data, the best mix of carbohydrate, protein, and fat depends on the individual metabolic goals and preferences of the person with diabetes. It’s most important to ensure that total calories are kept in mind for weight loss or maintenance. (2)




The primary goal in the management of diabetes is to achieve as near normal regulation of blood glucose (postprandial and fasting) as possible. The total amount of carbohydrate (CHO) consumed has the strongest influence on glycemic response (2). Yet the ideal amount of CHO in the diet is unclear. The majority of persons with type 1 or type 2 diabetes in the U.S. report eating moderate amounts of carbohydrate (~45% of total energy intake). (5) There are differing opinions in the literature regarding recommendations for low CHO diets in the treatment of diabetes. There have been many studies over the years looking at use of diets with lower CHO content and improvement in blood glucose without detrimental effects. (6) In a major change, the ADA in their 2019 position statement now states “research indicates that low carbohydrate eating plans may result in improved glycemia and have the potential to reduce antihyperglycemic medications for individuals with type 2 diabetes”.  Further, low CHO diets are not recommended for pregnant and lactating women, those who have or are at risk for disordered eating, and those with renal disease. The ADA recommends caution in people taking sodium–glucose cotransporter 2 (SGLT2) inhibitors due to the potential risk of ketoacidosis. (2) The most compelling reasons limiting adoption may be that the definitions of low CHO diets vary and that diets lower in CHO are difficult to maintain in the long term with few longer-term studies to support extended benefits. The ADA recommends the following: (2)


  • Carbohydrate intake should emphasize nutrient-dense carbohydrate sources that are high in fiber, including vegetables, fruits, legumes, whole grains, as well as dairy products.
  • For people with type 1 diabetes and those with type 2 diabetes who are prescribed a flexible insulin therapy program, education on how to use carbohydrate counting, and in some cases how to consider fat and protein content to determine mealtime insulin dosing is recommended to improve glycemic control.
  • For individuals whose daily insulin dosing is fixed, a consistent pattern of carbohydrate intake with respect to time and amount may be recommended to improve glycemic control and reduce the risk of hypoglycemia.
  • People with diabetes and those at risk are advised to avoid sugar-sweetened beverages (including fruit juices) in order to control glycemia and weight and reduce their risk for cardiovascular disease and fatty liver and should minimize the consumption of foods with added sugar that have the capacity to displace healthier, more nutrient-dense food choices


Nutritive Sweeteners


Sucrose, also known as “table sugar,” is a disaccharide composed of one glucose and one fructose molecule and provides 4 kcals/gm. Available evidence from clinical studies shows dietary sucrose has no more effect on glycemia than equivalent caloric amounts of starch. It’s important to note that excess energy intake from nutritive sweeteners or foods and beverages containing high amounts of nutritive sweeteners should be avoided, since they provide “empty” calories and can lead to weight gain. (7)


Fructose is a common naturally occurring monosaccharide found in fruits, some vegetables and honey. High fructose corn syrup is high in processed fructose and is used abundantly in processed foods as a less expensive alternative to sucrose.


  • Fructose consumed as “free fructose” (i.e., naturally occurring in foods such as fruit, (that also contain fiber) may result in better glycemic control compared with isocaloric intake of sucrose or starch, and free fructose is not likely to have detrimental effects on triglycerides as long as intake is not excessive (12% energy).
  • People with diabetes should limit or avoid intake of sugar-sweetened beverages (SSBs) (from any caloric sweetener including high-fructose corn syrup and sucrose) to reduce risk for weight gain and worsening of cardio metabolic risk profile. (7)


A recent meta-analysis of 18 controlled feeding trials in people with diabetes compared the impact of fructose with other sources of carbohydrate on glycemic control. The analysis found that an isocaloric exchange of fructose for carbohydrates did not significantly affect fasting glucose or insulin and reduced glycated blood proteins in these trials of less than 12 weeks duration.  The short duration is a potential limitation of the studies. (8) Strong evidence exists that consuming high levels of fructose-containing beverages may have particularly adverse effects on selective deposition of ectopic and visceral fat, lipid metabolism, blood pressure, and insulin sensitivity compared with glucose-sweetened beverages. (7) Thus, recommendations about the optimal amount of dietary fructose remain controversial due to potential metabolic consequences that could lead to further insulin resistance and obesity.


Non-Nutritive Sweeteners


Non-nutritive sweeteners provide insignificant amounts of energy and elicit a sweet sensation without increasing blood glucose or insulin concentrations. There are currently seven non-nutritive, FDA-approved sweeteners found to be safe when consumed within FDA acceptable daily intake amounts (ADI): (9)


  1. Sucralose (Splenda®) is synthesized from regular sucrose, but altered such that it is not absorbed. Sucralose is 600 times sweeter than sucrose. It is heat stable and can be used in cooking. It was approved for use by the FDA in 1999
  2. Saccharine (Sugar Twin®, Sweet ‘N Low®) is 200 to 700 times sweeter than sugar. A cancer-related warning label was removed in 2000 after the FDA determined that it was generally safe.
  3. Acesulfame K (Ace K, Sunette) is 200 times sweeter than sucrose. It can be used in cooking. The bitter aftertaste of acesulfame can be greatly decreased or eliminated by combining acesulfame with another sweetener. [
  4. Neotame is a derivative of the dipeptide phenylalanine and aspartic acid. It is 7,000-13,000 times sweeter than sucrose and does not have a significant effect on fasting glucose or insulin levels in persons with type 2 diabetes.
  5. Aspartame (Equal®, NutraSweet®) is a methyl ester of aspartic acid and phenylalanine dipeptide, both amino acids. The FDA approved it in 1981 for use in certain foods, in 1983 for use in soft drinks, and in 1996 as a general use sweetener. Although aspartame provides 4 kcal/g, the intensity of the sweet taste means that very small amounts are required to achieve desired sweetness levels, as it is 200x sweeter than sucrose. Aspartame yields phenylalanine, aspartic acid and methanol when hydrolyzed in the intestine. These breakdown products naturally occur in much higher levels in many foods. The FDA requires any foods containing aspartame to have an informational label statement: “Phenylketonurics: contains phenylalanine.” Patients with phenylketonuria should avoid products containing Aspartame.


      With close to 200 studies conducted in humans and animals on the use of common levels of aspartame in food, the safety is considered to be established, and does not suggest any long-term adverse effects.  The European Food Safety Authority (EFSA) recently conducted the most comprehensive review of available animal and human data, both published and unpublished and concluded that in current levels of exposure, no safety issues were noted leading to increased risk of cancer, gene or neurologic damage. (109) An adult weighing 60 kg (132 lb.) would have to consume over 12 cans of soda containing aspartame daily to reach the Acceptable Daily Intake (ADI) of 40 mg/kg/day, lower than the ADI recommended by the FDA of 50 mg/kg/day.  The American Cancer Society and the National Cancer Institute in the U.S. agree that no excessive link to increased cancer risk exists with use of aspartame in food products at current levels. (110) Some controversy has existed for many years around safety of the sweetener, but not from any major organizations.  The most prudent advice to give to patients is to use non-nutritive sweeteners sparingly.

  1. Stevia (Truvia®) derived from the plant stevia rebaudiana, is a non-caloric, natural sweetener. Stevia has been used as a sweetener and as a medicinal herb since ancient times and appears to be well-tolerated. It has an intensely sweet taste. Five randomized controlled trials showed minimal effects on blood glucose, insulin, blood pressure or weight.
  2. Luo han guo is the most recently approved GRAS (generally recognized as safe) sweetener. It is also known as monk fruit or Swingle fruit extract. It is 150-300 times sweeter than sucrose, and may have an aftertaste at high levels.


A recent review of 29 randomized controlled trials which included 741 people, 69 of which have type 2 diabetes, showed that artificial sweeteners on their own do not raise blood glucose levels, but the content of the food or drink containing the artificial sweetener must be considered, especially among PWD. (10)


Sugar Alcohols (Polyols)

Polyols are hydrogenated monosaccharides, and include such sugars as sorbitol, mannitol, erythritol, xylitol and D-tagatose as well as the hydrogenated disaccharides isomalt, maltitol, lactitol and trehalose. The polysaccharide derived hydrogenated starch hydrolysates [HSH] are also included in this category. Polyols are used as sweeteners and bulking agents, and designated GRAS by the FDA. Polyols are only partially absorbed from the small intestine, allowing for the claim of reduced energy per gram. Polyols contain, on average, 2 kcals/gm, or 1/2 the calories of other nutritive sweeteners. Studies of subjects with and without diabetes have shown that sugar alcohols cause less of a postprandial glucose response than sucrose or glucose. (7) However, polyols can cause diarrhea at ≥20 grams, especially in children. Although a diet high in polyols could reduce overall energy intake or provide long-term improvement in glucose control in diabetes, such studies have yet to be conducted.




Patients with DM should consume 20 to 35 g of fiber from raw vegetables and unprocessed grains (or about 14 g of fiber per 1,000 kcal ingested) per day (the same as the general population) (2). The definition and understanding of fiber have changed in past years. Dietary fiber is defined as the carbohydrate and lignin found in plants that is not digested by the stomach or absorbed in the GI tract. Functional fiber is the portion of fiber attributed to have beneficial physiologic effects in humans. Total fiber is the sum of both dietary and functional fiber. Although solubility of fiber was thought to determine physiological effect, more recent studies suggest that other properties of fiber, such as fermentability or viscosity may be more important. (11) Intake of dietary fiber is associated with lower all-cause mortality in people with diabetes. (12) A fiber rich meal is processed more slowly, which promotes satiety, may be less caloric, and lower in added sugars, which can help combat obesity and also may prevent risk of heart disease, type 2 diabetes, and colon cancer. (11) The FDA advocates consumption of 25 g dietary fiber per 2,000 calories consumed. (13) This recommendation is based on epidemiologic studies showing protection against cardiovascular disease. According to the latest NHANES survey, intake of dietary fiber in individuals in the United States from 2009-10 averaged 16 gms/day. (14) Fiber supplements and bulk laxatives are used frequently as additional dietary fiber sources, but since few fiber supplements have been studied for physiological effectiveness, the best advice is to consume foods that are high in fiber. (11) A recently published systematic review of the literature concluded that the consumption of whole grains was not associated with significant improvements in glycemic control in individuals with type 2 diabetes; however, it may have other benefits, such as reductions in systemic inflammation. (15)

As with the general population, individuals with diabetes should consume at least half of all grains as whole grains. High fiber containing carbohydrate sources (>5g/serving) include legumes, whole grain breads and cereals, whole fruits and vegetables and should be included as part of the daily carbohydrate intake. The goal of 25 gms or greater of daily fiber intake may be difficult to achieve for some people, as large amounts of fiber can cause negative GI effects, such as bloating and gas. If the person is not accustomed to larger amounts of fiber in their diet, it should be added slowly. 


The ADA has a website featuring recipes high in fiber to help meet fiber goals.  (16)

The website below contains links to a comprehensive table listing fiber content of foods, and a calculator to help select foods with higher fiber content to help reach daily fiber goals. (17)


Resistant Starches and Fructans


Resistant starches are starch enclosed within intact cell walls. These include some legumes, starch granules in raw potato, retrograde amylose from plants modified to increase amylose content, or high-amylose containing foods, such as specially formulated cornstarch, which are not digested and absorbed as glucose. Resistant starches are completely fermented in the colon. It has been proposed that resistant starches may affect postprandial glucose response, reducing hypo and hyperglycemia.  However, there are no published long-term studies in subjects with diabetes to prove benefit from the use of resistant starch. (7)


Fructans are an indigestible fiber that has been suggested to have a glucose-lowering effect. Inulin is a fructan commonly added to many processed foods in the form of chicory root. A recent review and meta-analysis of 20 randomized clinical trials revealed that the use of inulin-type fructans demonstrated HDL-c improvement and glucose control in the T2DM subgroup. More well-powered, long-term, randomized clinical trials are required for a definitive conclusion on inulin-type fructan supplementation in improving lipid profile and glucose metabolism. (18)


Gluten Free


Gluten is a protein commonly found in wheat, barley, rye and other grains. A gluten free diet is used to treat people with celiac disease, an inflammatory condition in persons who are intolerant to gluten and suffer inflammatory and gastrointestinal side effects when gluten is consumed, leading to damage of the small intestine. It is noted that approximately 10% of people with type 1 diabetes also have celiac disease, which is significantly higher than the population in general. There seems to be no connection with Celiac disease and type 2 diabetes. (19) There is some data in the literature describing benefit of a gluten free diet in preventing T1D in animals and newly diagnosed children with T1D. (20)


The gluten free diet has recently grown in popularity in persons who may be gluten sensitive, but don’t have celiac disease. Gluten in sensitive individuals, causes inflammation, leading to depression and other symptoms. More data is needed in this area in people with diabetes.

According to the ADA, the person with T1D can follow a gluten free diet, but it may provide additional challenges. Some common CHO containing foods that do not contain gluten include:

White and sweet potatoes, brown and wild rice, corn, buckwheat, soy, quinoa, sorghum and legumes. These foods can be used in place of other CHO containing grains.


Practical Tips on CHO Intake


  • Include a good source of fiber containing food with every meal or snack.
  • Add some whole grain to the morning meal. Hot cereals - Old-fashioned or steel-cut oats. Cold cereals - Look for those that list whole wheat, whole oats, or other whole grain first on the ingredient list without added sugars
  • Use whole grain breads for lunch or snacks. Check the label to make sure that whole wheat or another whole grain is the first ingredient listed.
  • Eat less potatoes. Instead, try brown rice or less well-known grains like bulgur, wheat berries, millet, hulled barley, faro, or quinoa.
  • Switch to whole grain pasta. If the whole grain products are too chewy, look for those that are made with half whole wheat or brown rice or other whole grain flour.  Newer pasta products made from legumes such as chickpeas are now available.
  • Include beans/legumes which are an excellent source of slowly digested carbohydrate as well as a great source of lean protein. Substitute for meat as a protein and fiber source.
  • Strive to include a variety of fresh fruits and vegetables in meals every day.




Evidence is inconclusive for an ideal amount of total fat intake for people with diabetes; therefore, goals should be individualized; fat quality appears to be far more important than quantity.


Due to the high risk of CVD (cardiovascular disease) in individuals diagnosed with diabetes, the goal for dietary fat intake (amount and type) for PWD is similar to that of people with CVD but without diabetes. Recent studies have found that decreasing the amount of saturated fatty acids and trans fatty acids, the principal dietary fatty acids linked to elevating LDL cholesterol, reduces the risk of CVD. (7) The American Heart Association, and American College of Cardiology currently recommend limiting the amount of dietary saturated and trans-fat intake. (2,21) Recommendations from the Institute of Medicine and the Academy of Nutrition and Dietetics for healthy individuals are that 20% to 35% of total calories should come from fat. (22,23) Currently, limited research on recommendations on percent of total calories coming from fat exists for individuals with diabetes. An individualized approach to fat content is the current guidance. (2)


The 2019 Lifestyle Management: Standards of Medical Care for Diabetes from the American Diabetes Association recommends: (2)


  • Data on the ideal total dietary fat content for people with diabetes are inconclusive, so an eating plan emphasizing elements of a Mediterranean-style diet rich in monounsaturated and polyunsaturated fats may be considered to improve glucose metabolism and lower CVD risk and can be an effective alternative to a diet low in total fat but relatively high in carbohydrates.
  • Eating foods rich in long-chain n-3 fatty acids, such as fatty fish (EPA and DHA) and nuts and seeds (ALA), is recommended to prevent or treat CVD; however, evidence does not support a beneficial role for the routine use of n-3 dietary supplements.


The American Heart Association has developed the Fat Facts to help individuals learn more about healthy vs. unhealthy fats. Among the campaign's top priorities is to encourage replacing high trans-fat partially hydrogenated vegetable oils, animal fats and tropical oils with healthier oils and foods higher in unsaturated fats — monounsaturated and polyunsaturated. (24)


Monounsaturated Fatty Acids


Monounsaturated fats (MUFA) are typically found in vegetable oils such as olive, peanut, avocado, and canola oil and remain liquid at low temperatures. Foods high in MUFA include avocado, some fatty fish, and nuts and nut butters. Several large prospective observational studies have documented that diets rich in MUFA or PUFA and lower in saturated fat are associated with a reduced risk of CVD. (25) A recent meta-analysis of RCTs comparing diets higher in MUFA vs CHO or PUFA demonstrated that high MUFA containing diets can improve metabolic parameters in people with T2D. (26)


Polyunsaturated Fatty Acids


Polyunsaturated fats (PUFAs) are usually liquid at room temperature, and are found in vegetable oils such as corn oil, safflower oil, and soybean oil. Controversy exists on the best ratio of omega-6 to omega-3 fatty acids. A recent meta-analysis of RC feeding trials provided some evidence that dietary macronutrients have diverse effects on glucose-insulin homeostasis. Most consistent positive effects were seen with PUFA compared to CHO, MUFA, or saturated fat. Replacement with PUFA was linked to improved glycemia, insulin resistance, and insulin secretion capacity. (27)


Omega-3 Fatty Acids


Eating foods rich in long-chain n-3 fatty acids, such as fatty fish (EPA and DHA) and nuts and seeds (ALA), is recommended to prevent or treat CVD; however, evidence does not support a beneficial role for the routine use of n-3 dietary supplements. (2) There are two kinds of omega-3 fatty acids in fish — eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The form of omega-3 in plants is called alpha-linolenic acid (ALA). Some types of fatty fish and certain nuts/seeds contain these unique polyunsaturated fats, one of the most studied areas in nutrition science. The consumption of 2 servings (8 ounces) per week of fish high in EPA and DHA is associated with a reduced risk of both sudden death and death from coronary artery disease in healthy adults. (28) Studies on the effect of omega-3 fatty acids (both from food and supplements) in persons with diabetes are limited and have been inconclusive. (7)  In addition to providing EPA and DHA, regular fish consumption may help reduce triglycerides by replacing other foods higher in saturated and trans fats from the diet, such as fatty meats and full-fat dairy products. Preparing fish without frying or adding cream-based sauces is recommended. Fish with high amounts of omega-3 include salmon, albacore tuna, mackerel, sardines, herring and lake trout. Nuts and seeds high in ALA include walnuts, flax seeds, chia seeds and soybeans. (29)


Saturated Fats


Saturated fats are usually solid or almost solid at room temperature. All animal fats, such as those in meat, poultry, and dairy products, are saturated. Processed and fast foods contain high amounts of saturated fats. Some vegetable oils also can be saturated, including palm, palm kernel, and coconut oils. Oil such as coconut and palm (sometimes referred to as tropical oils) are touted as healthful saturated fats since they are derived from plants however this is not accurate.  The American Heart Association recommends limited consumption of saturated fats and when cooking with oil, to choose nontropical vegetable oils such as canola, corn, olive, peanut, safflower, soybean, and sunflower oils. (101)


Few research studies have been undertaken to look at the difference between the amount of saturated fatty acids (SFA) in the diet and glycemic control and CVD risk in people with diabetes. The ADA nutrition position paper recommends people with diabetes follow the guidelines for the general population. (7) The Dietary Guidelines for Americans, 2015-2020 recommends consuming less than 10% of calories from SFAs to reduce CVD risk. (30)

In general, saturated fats are discouraged because they increase LDL-cholesterol and total cholesterol concentrations. Diets high in saturated fats have been implicated in an increased risk of cardiovascular disease. Three randomized controlled trials found that diets containing ≤7% SFA and ≤200 mg/day cholesterol reduced LDL cholesterol level from 9% to 12% compared to baseline values or to a more standard Western-type diet. (31) As saturated fats are progressively decreased in the diet, they should be replaced with unsaturated fats and not with refined carbohydrates. PWD should strive to limit saturated fat intake to less than10% of total calories.


Trans Fats


Trans fatty acids (TFA) are also called hydrogenated fats, which are fats created when oils are "partially hydrogenated". The process of hydrogenation changes the chemical structure of unsaturated fats by adding hydrogen atoms, or “saturating” the fat. Hydrogenation converts liquid oil into stick margarine or shortening. Manufacturers use hydrogenation to increase product stability and shelf-life. Thus, a larger quantity can be produced at one time, saving manufacturing costs. Research studies show that synthetic TFA can increase LDL cholesterol and lower HDL cholesterol. With the mandatory TFA labeling in 2006, a big push has been made by food manufacturers to remove it from processed and baked goods. Although the TFA content in foods has decreased recently (through food reformulation), it is important to monitor the type of fat used to replace TFA, as it might be saturated fat. Also, the FDA recently determined that trans fats are no longer considered generally recognized as safe (GRAS).  For the majority of uses of TFAs, June 18, 2018, was the date after which manufacturers cannot add TFAs to foods. However, to allow for an orderly transition in the marketplace, FDA is allowing more time for products produced prior to June 18, 2018 to work their way through distribution. FDA is extending the compliance date for these foods to January 1, 2020. (32)




The body makes enough cholesterol for physiological functions, so it is not needed through foods. The most recent Dietary Guidelines for Americans states that available evidence does not support a recommendation to limit cholesterol for the general population, and recommendations for dietary cholesterol for other populations, such as PWD, are not clear. Cholesterol intake correlates with serum cholesterol levels but it has not been well correlated with CVD events. Therefore, additional research is needed regarding the relationship between dietary cholesterol, blood cholesterol, and CVD events in PWD. (108) The most current practical advice to give to PWD regarding dietary cholesterol is to follow the guidelines for limiting saturated fat intake, as these foods are usually highest in dietary cholesterol content.



Type of Fat

Main Source


Canola, peanut, and olive oils; avocados; nuts such as almonds, hazelnuts, and pecans; and seeds such as pumpkin and sesame seeds.


Sunflower, corn, soybean, and flaxseed oils, and also in foods such as walnuts, flax seeds, and fish.


Whole milk, butter, cheese, and ice cream; red meat; chocolate; coconuts, coconut milk, coconut oil and palm oil


Some margarines; vegetable shortening; partially hydrogenated vegetable oil; deep-fried foods; many fast foods; some commercial baked goods (check labels)


Stanols and Sterols


Individuals with diabetes and dyslipidemia may be able to modestly reduce total and LDL cholesterol by consuming 1.6–3 g/day of plant stanols or sterols typically found in enriched foods. (7) Plant sterols are naturally occurring cholesterol derivatives from vegetable oils, nuts, corn, woods and beans. Hydrogenation of sterols produces stanols. The generic term to describe both sterols, stanols and their esters is phytosterols. An important role of phytosterols is their ability to block absorption of dietary and biliary cholesterol from the gastrointestinal tract. The LDL lowering property of both sterols and stanols is considered equivalent in short term studies. (33) The amounts of sterols and stanol esters found naturally in a normal diet are insufficient to have a therapeutic effect. Thus, many manufacturers add them to various foods for their LDL cholesterol lowering effects. You can find added phytosterols in margarine spreads, juices, yogurts, cereals, and even granola bars. 


A recent meta-analysis reviewing well controlled studies found that the short-term use of food supplements high in plant sterols is a safe and effective strategy to help maximize the benefits of dietary and lifestyle treatment, either with or without statin therapy, among the majority of dyslipidemic individuals with a need for further lipid-lowering. Products that contain plant sterols can help reduce LDL cholesterol by more than 10 percent. The amount of daily plant sterols needed for results is at least 2 grams — which equals about two 8-ounce (237-milliliter) servings of plant sterol-fortified orange juice a day. (33) The evidence on long term use and in people with diabetes is less substantiated, as not many studies have been completed. (33,34) The Evidence Analysis Library (EAL) from the Academy of Nutrition and Dietetics advocates use of plant sterol/stanol esters in amounts of 2 g/day, which equates to approximately 2 tablespoons/day as part of a cardioprotective diet. (35) The taste of these fortified margarines is comparable to regular margarine, but they cost 3-4 times more than regular spreads. It is also important to keep in mind that these fortified foods should be used as a substitute for regular foods, not as an additive, as more is not better and will provide extra calories which can lead to weight gain.


Practical Tips on Fat Intake


  • Try to eliminate trans fats from partially hydrogenated oils. Check food labels for trans fats; limit fried fast foods.
  • Limit intake of saturated fats by cutting back on processed and fast foods, red meat, and full-fat dairy foods. Try replacing red meat with beans, nuts, skinless poultry, and fish whenever possible, and switching from whole milk and other full-fat dairy foods to lower fat versions.
  • In place of butter or margarine, use liquid vegetable oils rich in polyunsaturated and monounsaturated fats in cooking and at the table.
  • Eat one or more good sources of omega-3 fats every day—fatty fish, walnuts, soybean oil, ground flax seeds or flaxseed oil




In individuals with type 2 diabetes, ingested protein can increase insulin response without increasing plasma glucose concentrations. Therefore, carbohydrate sources high in protein should not be used to treat or prevent hypoglycemia. (2)


The ADA Standards of Medical Care in Diabetes-2019 states that there is no evidence that adjusting the daily level of protein intake (typically1–1.5 g/kg body weight/day or 15–20% total calories) will improve health in individuals without diabetic kidney disease, and research is inconclusive regarding the ideal amount of dietary protein to optimize either glycemic control or cardiovascular disease (CVD) risk. Therefore, protein intake goals should be individualized based on current eating patterns. Some research has found successful management of type 2 diabetes with meal plans including slightly higher levels of protein (20–30%), which may contribute to increased satiety. Those with diabetic kidney disease (with albuminuria and/or reduced estimated glomerular filtration rate) should aim to maintain dietary protein at the recommended daily allowance of no more than 0.8g/kg desirable body weight/day. (35) Reducing the amount of dietary protein below the recommended daily allowance is not recommended because it does not alter glycemic measures, cardiovascular risk measures, or the rate at which glomerular filtration rate declines. (2)


The National Kidney Foundation recommends 0.8 g protein/kg desirable body weight for people with diabetes and stages 1–4 chronic kidney disease as a means of reducing albuminuria and stabilizing kidney function (36). The Joslin Diabetes Center, advocates a protein intake of 20–30% of total energy intake (for those without kidney disease). (37)


Two misconceptions about dietary protein in diabetes management are that a certain amount of protein consumed is converted into blood glucose and that consuming too much protein can lead to diabetic kidney disease. Also, limited evidence exists to suggest a difference in animal or plant protein sources and diabetes outcomes. Although several prospective cohort studies suggested differences between protein source and T2D-related outcome, there is limited evidence from randomized controlled trials to suggest that protein source is important. (38)


Further research is still needed to define the optimal macronutrient content for fat (SFA, MUFA, PUFA), protein, and carbohydrate to attain the most beneficial lipid and lipoprotein profile in the general population and in those with diabetes at increased risk for CVD. 


Practical Tips for Protein Intake


  • Include a source of lean protein with each meal (8-12 oz/day)
  • Good sources of lean animal protein, such as skinless poultry, lower fat cuts of beef or pork, fish or egg (1 egg =1 oz protein), and reduced fat dairy products (1 c low fat or skim milk/yogurt, 1 oz cheese = 1 oz protein
  • Plant protein sources such as tofu, tempeh, legumes, (1/2c = 2 oz protein) or meat alternative products are options but be aware of possible higher sodium content
  • Nuts or seeds: 1 oz equals 24 almonds, 18 medium cashews, 12 hazelnuts or filberts, 8 medium Brazil nuts, 12 macadamia nuts, 35 peanuts, 15 pecan halves and 14 English walnut halves
  • Nut butters 2 Tbsps. equals 1oz protein
  • Protein should be a supplement to vegetables, fruits and whole grains in a meal, not the entire meal




There is no clear evidence that dietary supplementation with vitamins, minerals (such as chromium and vitamin D), herbs, or spices (such as cinnamon or aloe vera) can improve outcomes in PWD who do not have underlying deficiencies and they are not generally recommended for glycemic control (2)


In PWD who have no underlying deficiencies, there is no clear scientific evidence of benefit from vitamin or mineral supplements, either in preventing or treating progression or complications. It is, however, important to establish that no deficiencies exist. People with diabetes should be aware of the necessity for meeting vitamin and mineral needs from natural food sources through intake of a balanced diet. Specific populations, such as older adults, pregnant or lactating women, strict vegetarians or vegans, and individuals on very low calorie or very low carbohydrate diets may benefit from a multivitamin mineral supplement. (2) Excessive doses of certain vitamin or mineral supplements when there is no deficiency has been shown to be of no benefit and may even be harmful. There is some evidence that those on metformin therapy are at higher risk of B12 deficiency, and may need Vitamin B12 supplementation if tests indicate a deficiency. (2, 39)




Since type 2 diabetes is a state of increased oxidative stress, interest in recommending large doses of antioxidant vitamins has been high. Current studies demonstrate no benefit of carotene and Vitamins E, and C in respect to improved glycemic control or treatment of complications.

Routinely supplementing the diet with antioxidant supplements is not recommended due to lack of evidence showing benefit in large, placebo-controlled clinical trials and concerns regarding potential long-term safety. (2,39) There is also not adequate evidence to recommend routine Vitamin D supplementation without deficiency (2,40)






As for the general population, PWD should limit sodium consumption to 2,300 mg/day. (2)

Since few studies have been undertaken on sodium restriction in PWD, the 2019 ADA standards of medical care recommendation is to follow the guidelines for sodium intake for the general population, which is to limit sodium intake to 2300 mg/day. (2) Food manufacturers and restaurants will need to provide additional reduced sodium alternatives to help accomplish this goal. It requires not adding salt to foods during cooking or at the table, as well as decreasing consumption of most pre-prepared and pre-packaged foods. Some studies in people with type 1 and type 2 diabetes measuring urine sodium excretion have actually shown increased mortality associated with very low sodium intakes, potentially requiring caution for universal sodium restriction to 1,500 mg in the diabetes population without hypertension (7) Other lifestyle modifications, including loss of excess body weight; increasing consumption of fruits and vegetables (8 –10 servings/day), and low-fat dairy products (2–3 servings/day); avoiding excessive alcohol consumption (no more than 2 servings/day in men and no more than 1 serving/day in women); and increasing activity levels can be helpful in people with hypertension and diabetes. These nonpharmacological strategies may also positively affect glycemia and lipid control. (7) The DASH (Dietary Approaches to Stop Hypertension) diet, which is high in fruit and vegetables, low-fat dairy products, and low in saturated and total fat; has been shown in large, randomized, controlled trials to significantly reduce blood pressure. (41) The DASH diet was rated second overall and tied for best diet for diabetes in a recent report published in US News and World Report. (42) The report was based on scores of 41 diets rated by nutrition and diet, diabetes and cardiac experts. (2)




Studies in support of magnesium supplementation to improve glycemic control are unclear and complicated by differences in study designs as well as baseline characteristics. There is some evidence that higher dietary intake of magnesium may help prevent type 2 diabetes in both middle aged men and women at higher risk for developing the disease. (43) Additional long-term studies are needed to determine the best way to assess magnesium status and how magnesium deficiency impacts PWD. (100) Dietary sources of magnesium include nuts, whole grains, and green leafy vegetables.




Several studies have demonstrated a potential role for chromium supplementation in the management of insulin resistance and type 2 diabetes. According to the ADA position statement, the findings with more significant effects were mainly found in poorer quality studies, limiting transferability of the results. Routine supplementation of chromium is therefore currently not recommended for treating diabetes or obesity. (2)




There has been interest in the past several years on the effect of cinnamon, curcumin, and other herbs and spices in individuals with diabetes. The most recent ADA Lifestyle Management recommendations conclude that after a review of the evidence, there is not enough clear data to substantiate recommending the use of herbs or spices as treatment for T2D. (2) The ADA also states that the use of any herbal supplements, which are not regulated and vary in content, may provide more risk than benefit, in that herbs may interact with other medications that are taken to control diabetes. (7)


A good resource to determine general calorie, macro and micronutrient needs based on the DRI is: Dietary Reference Intake Calculator for HCPs: (44)




Probiotics (from pro and biota, meaning "for life"), are certain kinds of “good” bacteria found in fermented foods, such as yogurt, kefir, and kimchi and are available as supplements. They are naturally found in the gut and may be depleted due to poor diet, use of antibiotics, stress, etc. Probiotics have been studied extensively to improve gut flora for use in treatment and possibly prevention of various disorders, including irritable bowel syndrome, diarrhea, constipation, and genitourinary infections, to name a few. Different strains and amounts may work better for some conditions over others, but the FDA does not oversee the supplements, so content and effectiveness are not regulated. They are generally considered safe, as they are found naturally in the digestive tract. (105)


Some research has been done in people with gestational and type 2 diabetes using probiotic supplements and foods to determine if chronic inflammatory and glycemic markers can be improved. The premise is that the microbiome flora may be connected to glucose metabolism by altering insulin sensitivity and inflammation. The micropia in the gut of those with and without T2DM is different and altering the gut flora with certain probiotic strains may be helpful.  A recent review of 12 randomized controlled studies of probiotic supplements in people with T2DM demonstrated a moderate improvement in glycemic and lipid parameters in the majority of the trials. The authors noted that lactobacillus and Bifidobacterium species were most commonly used in the studies and that more studies of longer duration, exact strain and therapeutic dose should be pursued. (106)  


A recent meta-analysis of probiotic yogurt as a method to improve glycemic control in type 2 and obesity showed that compared to conventional yogurt, the probiotic containing yogurt did not yield improvement in glycemic markers. The authors comment that larger randomized trials longer than 12 weeks should be undertaken. (107)




Chocolate is a favorite food of many people.  Chocolate and cocoa are often touted as having healthful benefits.  Some studies in healthy individuals and in hypertensive patients with impaired glucose tolerance have shown an improvement in endothelial function with consumption of dark chocolate compared to white chocolate.  The improvement in endothelial function is thought to be due to the flavonoids in cocoa and dark chocolate.  The improvement in endothelial function may alter glucose metabolism and increase insulin sensitivity.


There has not been any long term randomized controlled trials in people with diabetes to determine the long-term effects of a diet rich in dark chocolate or cocoa. (102) Chocolate is often combined with sugar, fat, and other ingredients to create a snack or candy that is energy dense with low nutritional value.  This may cause an increase in weight that would be detrimental to a person with diabetes.  The overall nutrient content of the chocolate containing food or product, daily calorie intake and energy balance should be considered and if incorporated into their diet, should be offset by adjustments of other energy dense, low nutritional value foods to maintain a healthful diet and energy balance.




Adults with diabetes who drink alcohol should do so in moderation (no more than one drink per day for adult women and no more than two drinks per day for adult men). Alcohol consumption may place people with diabetes at increased risk for hypoglycemia, especially if taking insulin or insulin secretagogues. Education and awareness regarding the recognition and management of delayed hypoglycemia due to alcohol with or without a meal are warranted


The ADA position paper states that moderate alcohol consumption has minimal detrimental short- or long-term effects on blood glucose in people with diabetes, with some epidemiologic data showing improved glycemic control with moderate intake. Moderate intake may also contribute to cardiovascular risk reduction and mortality benefits in people with diabetes, no matter the type of alcohol. Thus, the recommendations for alcohol consumption for people with diabetes are the same as for the general population (2)


Risks of excessive alcohol intake include hypoglycemia (particularly for those using insulin or insulin secretagogue therapies), weight gain, and hyperglycemia (for those consuming excessive amounts). Hypoglycemia can occur through several mechanisms, including the inability of alcohol to be converted into glucose, the inhibitory effect of alcohol on gluconeogenesis, and its interference in normal counter regulatory hormonal responses to impending hypoglycemia. However, one drink for women and two drinks for men per day can usually be incorporated into the diet for individuals with type 1 diabetes with no major effect on blood glucose. One drink is defined as 12 oz beer, 5 oz wine or 1.5 oz of hard liquor. To decrease the risk of hypoglycemia, it is best to have the alcohol with food. Consuming alcohol in a fasting state may contribute to hypoglycemia in people with type 1 diabetes. Symptoms of hypoglycemia can be similar to drunkenness, so advise others that the person has diabetes so proper treatment for hypoglycemia can be undertaken. When calculating the need for meal related boluses of insulin, one should account for the carbohydrate content of the alcohol if drinking sweet wines, liqueurs, or drinks made with regular juice or soda. Selecting dry wine, light beer or hard liquor made with noncaloric mixers is preferable. (45)




People taking insulin should be counseled on the importance of balancing food and beverage intake with timing and dosing of insulin.  This is especially important for individuals with varied or hectic schedules such as shift workers, people that travel frequently, or anyone who has a schedule in which timing of meals and access to food is irregular. (2)  Numerous materials and resources are available that can be provided to PWD to help them consider portion control, consistency in food intake and medication dosing, as well as planning to allow some flexibility in their daily self-care regimen. (46) The health care provider should provide individualized guidelines for a target blood glucose range, considering safety and health. For motivated people, teaching an insulin to CHO ratio, and blood glucose correction factor may assist them with achieving blood glucose targets and achieving better glycemic control.  (2,47)


Carbohydrate Counting


Carbohydrate (CHO) from any food affects blood glucose levels.  Monitoring carbohydrate, whether by carbohydrate counting, using the exchange method, or experienced-based estimation, remain an important strategy used in timing of medication administration and improving glycemic control. (7) CHO counting methodology is based on the concept that each serving of CHO equals approximately 15 gms of CHO.  Generally, blood glucose response to carbohydrate is similar for most foods, however PWD should be educated on more healthful carbohydrate sources including legumes, whole grain or multi-grain foods and whole fruits rather than highly processed foods, fruit juices, and sweetened beverages.  The average woman needs about 3 to 4 choices (45-60 gms), while men may need 4-5 choices (60-75 grams) of CHO at each meal. (46) This number could vary more or less depending on individual calorie needs (i.e., pregnant/nursing, ill, etc.), medication, and level of physical activity.

Carbohydrate counting is a tool that can be taught to motivated PWD, so that they can more easily estimate the amount (grams) of CHO in a particular food. Furthermore, setting a target CHO intake for each meal allows the PWD to more easily match their CHO intake to the appropriate mealtime insulin dose. Potential advantages of CHO counting include improved glucose control, flexibility in food choices, a better understanding of how much insulin to take, and simplification of meal planning. Review with the PWD the understanding of CHO counting and reinforce the importance of choosing foods that are less processed, and contain whole grains and fiber on a regular basis. There are no evidence-based studies showing superiority of different approaches to dietary management methods. CHO counting requires motivation on the PWD’s part. (48) Approaches that are individualized based on the PWD’s capacity and resources are recommended to develop an individualized eating plan (2)


A good online resource for basic carbohydrate counting can be found on the UCSF website: (49)


Glycemic Index (GI) and Load (GL) 


Substituting low–glycemic load foods for higher–glycemic load foods may modestly improve glycemic control


The use of the glycemic index (a scale that ranks carbohydrate rich foods by how much they raise blood glucose levels) has been developed to identify and classify over 600 foods and their blood glucose raising potential. It has been demonstrated that high fiber, low GI foods can help delay the absorption of glucose into the bloodstream, consequently helping to control blood glucose levels. As a rule, refined grain products and potatoes have a higher GI, legumes and whole grains have a moderate GI, and non-starchy fruits and vegetables have a low GI. Many factors can influence the GI of a food, such as methods of cooking, physical state of a food, and how much fat and protein are consumed in conjunction with that food. (50) The ADA states use of the glycemic index and glycemic load may provide a modest additional benefit for glycemic control over that observed when total carbohydrate is considered alone. The reasoning behind a less than robust recommendation is that the literature on GI and GL in individuals with diabetes is complex, and it is often difficult to separate the independent effect of fiber compared with that of the GI on glycemic control and other outcomes. Other organizations more highly advocate its use, including the Diabetes and Nutrition Study Group (DNSG) of the European Association and the Diabetes UK Nutrition Working Group. (7) It is important that persons with diabetes who want to use the GI to better manage their glucose control are taught how specific foods and meals affect their own blood glucose levels, rather than adhering only to the existing GI. For example, a person could compare a low GI food, such as oatmeal (GI = 50) with cornflakes (GI = 84) to determine the relative effect of each on their own blood glucose.


The basic technique for following low GI guidelines is simply a "this for that” approach – i.e.: replacing high GI foods with low GI foods. One need not count numbers or do any sort of mental arithmetic to make sure they are eating a healthy, low GI diet. Some tips include:


  • Increasing the consumption of whole grains, nuts, legumes, fruit, and non-starchy vegetables
  • Decreasing the consumption of starchy high-glycemic index foods like potatoes, white rice, and white bread
  • Decreasing the consumption of sugary foods like cookies, cakes, candy, and soft-drinks


The glycemic load (GL) combines the GI and the total CHO content of an average serving of a food. It is defined as the GI multiplied by the amount of carbohydrate per serving of food in grams and dividing the total by 100. It was introduced as a measure of the overall effect of a food on blood glucose and insulin levels. Lowering the GL of the diet may be an effective method to improve glycemic control in individuals with type 2 diabetes. This approach is not currently included in the overall strategy of diabetes management in the US. (51)


A 2011 review article on GI and GL in the diabetes diet by Marsh, et al concludes that both the amount and type of carbohydrate are important in predicting glycemic response to a meal. Diets based on low GI carbohydrate containing foods have been associated with a reduced risk of type 2 and CVD, and intervention studies have shown improvements in insulin sensitivity and A1C in those with diabetes. Low GI diets may also assist with weight management through effects on satiety and fuel partitioning. Since no demonstrated negative effects of a low GI diet have been demonstrated, the GI can be an important consideration in the dietary management of diabetes. (52)


Special Considerations for PWD Treated with Intensive Insulin Regimens


The following guidelines are the starting point for the nutritional component of PWD on intensified insulin management regimens, regardless of what meal plan approach is chosen: (2,53)


  1. The initial diabetes meal plan should be based on the PWD normal intake with respect to calories, food choices, and times meals eaten.
  2. Choose an insulin regimen that is compatible with the PWD normal pattern of meals, sleep and physical activity.
  3. Synchronize insulin with meal times based on the action time of the insulin(s) used.
  4. PWD should measure blood glucose levels prior to meals and snacks and at bedtime and adjust the insulin doses as needed based on intake.
  5. Monitor A1C, weight, lipids, blood pressure, and other clinical parameters, modifying the initial meal plan as necessary to meet goals
  6. It is also important to educate the PWD on adjustment of prandial insulin considering premeal glucose levels, carbohydrate intake, and anticipated physical activity.
  7. For PWD who are overweight on insulin, counseling on nutrition, weight management and monitoring blood glucose continue to be important components of treatment. Medical nutrition therapy (MNT) for PWD is recommended with continued emphasis on making lifestyle changes to achieve a weight loss of 5% or more to reduce the risk of chronic complications associated with diabetes, CVD and other risk factors that contribute to early mortality.


Children and Adolescents


While medical nutrition therapy provided by registered dietitians resulted in better glycemic control in children with newly diagnosed type 1 diabetes, a survey of 45 pediatric clinics revealed that only 25 clinics had an experienced pediatric/adolescent dietitian available for children with diabetes. (54) Registered Dietitian Nutritionists who are trained and experienced with children and adolescent diabetes management should be involved in the multidisciplinary care team (55). The goals of nutrition therapy for children and adolescents with diabetes include the following (2,55):


  1. Provide individualized nutrition therapy with guidance on appropriate energy and nutrient intake to ensure optimal growth and development.
  2. Assess and consider changes in food preferences over time and incorporate changes into recommendations.
  3. Promote healthy lifestyle habits while considering and preserving social, cultural, and physiological well being
  4. Achieve and maintain the best possible glycemic control
  5. Achieve and maintain appropriate body weight and promote regular exercise


Dietary advice should start gradually:


  1. Emphasis should initially be on establishing supportive rapport with the child and family with simple instructions. More detailed guidelines should be administered later by the entire team, with focus on consistency in message and should include dietary guidelines to avoid hypoglycemia. Instruction on carbohydrate counting should be provided as soon as possible after diagnosis. (55) Nutritional advice needs to be given to all caregivers; babysitters, and extended family who care for the child.
  2. Nutrition guidelines should be based on dietary history of the family and child’s meal pattern and habits prior to the diagnosis of diabetes and focus on nutritional recommendations for reducing risk of associated complications and cardiovascular risk that are applicable to the entire family.
  3. Activity/exercise schedules need to be assessed, along with 24-hour recall and 3-day food diary to determine energy intake. Growth patterns and weight gain need to be assessed every 3-6 months and recommended dietary advice adjusted accordingly. (54)


Dietary recommendations can be illustrated by use of the Plate method. There are numerous resources for visuals and educational materials using the plate method and some are specific to diabetes. Half the plate should consist of fruits and vegetables, while the other half is divided between whole grains and lean sources of protein. The dairy is represented by a glass of nonfat or 1% milk or other nonfat or low-fat dairy source.  The general guidelines for macronutrients are similar to that of the adult population with diabetes. (56)

Figure 1. Video and print materials can be found on the website

Special Considerations  




Hypoglycemia usually occurs more frequently in PWD taking insulin, but can occur in those taking oral antihyperglycemic agents, especially a sulfonylurea. To help prevent hypoglycemia, the following guidelines should be discussed: (57)


  1. Don't skip or delay meals or snacks. If taking insulin or oral diabetes medication, be consistent about the amount eaten and the timing of meals and snacks.
  2. Monitor blood sugar. Depending on treatment plan, check and record blood sugar level several times a week or several times a day. Careful monitoring is the only way to make sure that blood sugar level remains within the individual target range.
  3. Measure medication carefully, and take it on time. Take medication as recommended by the physician coordinating diabetes care.
  4. Adjust medication or eat additional snacks if physical activity increases. The adjustment depends on the blood sugar test results and on the type and length of the activity.
  5. Eat a meal or snack if choosing a drink with alcohol. Drinking alcohol on an empty stomach can contribute to hypoglycemia.
  6. Record low glucose reactions. This can help the health care team identify patterns contributing to hypoglycemia and find ways to prevent them.
  7. Carry some form of diabetes identification so that in an emergency others will know you have diabetes. Use a medical identification necklace or bracelet and wallet card.




Eating and drinking can be a challenge when the PWD is sick. The main rules for sick day management are:


  1. Continue to take diabetes medication (insulin or oral agent)
  2. Self-monitor blood glucose
  3. Test urine ketones
  4. Eat the usual amount of carbohydrate, divided into smaller meals and snacks if necessary; try to take the normal number of calories by eating easy-on-the-stomach foods like regular (non-diet) gelatin, crackers, soups, and applesauce. (if glucose is 250 mg/dL or >, all the usual amount of carbohydrate may not be necessary)
  5. If even these mild foods are too hard to eat, drink liquids that contain carbohydrates. Aim for 50 grams of carbohydrate every three to four hours. This may include regular (not diet) soft drinks. Other high-carbohydrate liquids and almost-liquids are juice, frozen juice bars, sherbet, pudding, creamed soups, and fruit-flavored yogurt. Broth is also a good choice to help stay hydrated, but does not provide a significant amount of CHO
  6. Drink non caloric, caffeine free fluids frequently. Call the diabetes care team
  7. A list of sick foods, including sugar containing items, such as soft drinks and gelatin, should be provided. See more at:




Exercise for individuals with diabetes has many benefits; for most, benefits outweigh risks. Exercise and resistance training may improve glycemic control (2).  PWD should be encouraged to exercise to improve cardiovascular and overall fitness, weight control, and for improved psychological well-being and quality of life. (7) There are several factors that can affect the blood glucose response to exercise: (59)


  • Individual response to exercise varies
  • Type, amount, and intensity of exercise
  • Timing and type of the previous meal
  • Timing and type of the insulin injection or other diabetes agent
  • Pre-exercise blood glucose level
  • Person’s fitness level


In individuals with type 1 diabetes, and PWD with T2 taking insulin, blood glucose monitoring is necessary to adjust insulin dosing and carbohydrate intake to reduce hypoglycemia during exercise. To reduce the risk of hypoglycemia, when exercise is planned, it may be preferable to adjust the dose of insulin before the exercise begins. On the other hand, if the exercise is unplanned, blood glucose should be checked and a carbohydrate snack may be eaten as needed before the exercise begins. If the blood glucose is less than 100mg/dL, a 15- to 30-g carbohydrate snack should be consumed, and glucose should be rechecked in 30 to 60 minutes. If glucose levels are less than 70 mg/dL, exercise should be postponed. Depending on the blood glucose level at the start of exercise, as well as length and intensity of the activity, a snack may need to be consumed before, during and after the exercise. Moderate intensity exercise can increase glucose uptake significantly, which may require an additional 15 gms of carbohydrate for every 30-60 minutes of exercise above the normal routine. (59)


To better help with weight management, and avoid hypoglycemia, exercise should be scheduled post-meals when blood glucose levels are higher. If this is not possible, it may be necessary to decrease medication dose to facilitate exercise without increasing caloric intake. (60)


Exercise can increase the rate of absorption of insulin into exercising limbs, especially when it is started immediately after the insulin injection. Inject insulin into a non-exercising area, such as the abdomen, to minimize the effect of exercise on insulin absorption. The response to exercise varies greatly in every individual, so adjustment in medication and food should be based on individual responses. Blood glucose monitoring is very important in understanding response patterns and tailoring an exercise program. (60)




The greatest risk for hypoglycemia results when the peak insulin action does not coincide with the peak postprandial glucose. For example, the longer duration of action of regular insulin may lead to increased risk of late postprandial hypoglycemia, compared with rapid-acting insulin analogs, which peak closer to meal consumption. In addition, when the pre-meal insulin dose is too large for a particular meal relative to its CHO content, hypoglycemia can result. Such a mismatch may occur due to errors in estimating CHO or food intake in PWD on multiple daily injections (MDI) or on insulin pumps. Insulin calculations can be based on exchanges, carbohydrate counting, or predefined, set menus. If meals and the insulin regimen remain constant, then no problems will usually result. However, any changes in insulin or food intake require adjustment of one or the other, or both. Whatever regimen is employed, it must be individualized to the PWD. Those taking rapid-acting insulin may choose to give their insulin dose after the meal, if unsure of amount of food to be consumed. This approach can be especially helpful in children, nausea related to pregnancy, or other illness. If a smaller than normal meal is eaten, guidelines are available for reducing the insulin dose, or carbohydrate replacement in the form of fruit or fruit juice can be given, depending on the PWD’s particular insulin regimen. (61)




Hypoglycemia is defined as a low blood glucose level ≤70 mg/dL. Symptoms include anxiety, irritability, light-headedness and shakiness. Advanced symptoms include headache, blurred vision, lack of coordination, confusion, anger and numbness in the mouth. Hypoglycemia must be treated immediately with glucose. Follow the 15/15 rule: take 15 gms of simple carbohydrate which should increase blood glucose by 30-45 mg/dL within 15 minutes.  When blood glucose dips below 70 mg/dL, PWD should be advised to have one of the following "quick fix" foods right away to raise the glucose: (2)


  • Glucose tablets (see instructions)
  • Gel tube (see instructions)
  • 4 ounces (1/2 cup) of juice or regular soda (not diet)
  • 1 tablespoon of sugar, honey, or corn syrup
  • Hard candies, jellybeans, or gumdrops—see food label for how many to consume


High-fat foods will delay peak of glucose levels from carbohydrate intake and should be avoided (e.g., treatment of hypoglycemia with chocolate bars). After 15 minutes, blood glucose should be checked again to make sure that it is increasing. If it is still too low, another serving is advised. Repeat these steps until blood glucose is at least 70 mg/dL. Then, a snack should be consumed if it will be an hour or more before the next meal. (62)


Those who take insulin or an oral antidiabetic drug that can cause hypoglycemia, such as a sulfonylurea, should be advised to always carry one of the quick-fix foods with them, when driving, and also available nearby when sleeping. Wearing a medical ID bracelet or necklace is also a good idea, as well as having a glucagon emergency kit handy.



Driven by the explosive increase in the prevalence of obesity, the number of PWD with known diagnosis of type 2 diabetes has reached massive proportions in the U.S. and worldwide. The number of persons worldwide with diabetes has more than tripled since 1980. According to the 2017 National Diabetes Statistic report, diabetes affects 30.3 million people of all ages or 9.4 % of the U.S. population. This includes 23.1 million diagnosed, and an undiagnosed population of 7.2 million people. 90 to 95% of these people have type 2 diabetes. Another estimated 84.1million people, that’s 1 out of every 4 Americans 18 years of age and older have prediabetes. (63)


A lack of physical activity and an overabundance of readily available convenience foods (usually containing too many calories) can lead to obesity and in many cases the metabolic, or insulin-resistance syndrome. The metabolic syndrome combined with insulin resistance increases the chance of developing type 2 diabetes and heart disease. (64)


But adults are not alone in this problem, as there is also an increased rate of the diagnosis of type 2 diabetes in young people. Until 20 years ago, type 2 diabetes accounted for less than 3% of all cases of new-onset diabetes in adolescents, whereas now it has increased to over 45% of cases. (65)


Obesity and insulin resistance are key factors, but not the only variables, that can increase the risk of developing type 2 diabetes. In a study by Van Dam, et al, the Western dietary pattern (high in processed meat, red meat, French fries, refined grains, high-fat dairy products, and sweets), was associated with a 59% greater risk of diabetes in adult men, while a more “whole food” diet, deemphasizing processed foods (high in fruits and vegetables, whole grains, fish, and poultry) was associated with a 16% lower risk of diabetes in adult men. For men who ate a Western diet, the risk for diabetes was even greater if they were also obese or had a low level of physical activity. While these results do not prove that eating a Western diet causes type 2 diabetes, they certainly add to existing evidence that eating these types of food increases the risk for developing type 2 diabetes, and that being overweight, and lack of exercise increases the risk even further. (66)


Other widely publicized studies, the Finland Diabetes Prevention Study (67,68) and the one and two year community implementation results of the Diabetes Prevention Program [69,70,71], confirmed the importance of exercise and nutrition therapy as a preventative measure for development of type 2 diabetes and primary treatment after the initial diagnosis of type 2 diabetes is made. 


What Weight Loss Plan is Best? Keys to Success


While the general principles discussed in the first section apply to all PWD, those people with type 2 diabetes who are overweight or obese (BMI 25.0 and greater) should have a major focus placed on weight loss and increased physical activity. With so many weight loss “diets” available, confusion abounds. Even the scientific literature is inconclusive. Most people are looking for the quickest and easiest way to lose weight, and most have unrealistic expectations. Obesity does not occur overnight, and its treatment requires lifetime adjustments to food (energy) intake and energy expenditure (increased activity). As much as one would like to find the magic bullet that leads to quick and sustained weight loss, the fact remains that there does not appear as yet to be a balance of macronutrients that consistently leads to the loss and maintenance of body weight, other than a reduction of total calories consumed. A study published in Diabetologia reported that a diet of only 600 calories a day for eight weeks may have helped reverse type 2 diabetes in newly diagnosed people. According to the study, the diet helped reduce hepatic and pancreatic lipid levels, which normalized insulin production and blood glucose levels. However, more studies are needed to determine whether the results will be permanent, (72) and maintaining a 600-calorie intake long term is very unrealistic. In Roy Taylor’s 2012 Banting Lecture, the twin cycle hypothesis concept was introduced, which postulates that chronic calorie excess leads to accumulation of liver fat with eventual spill over into the pancreas. He believes that type 2 diabetes is a reversible condition of intra-organ fat excess to which some people are more susceptible than others. This hypothesis is supported by both bariatric surgery and hypocaloric diet evidence demonstrating reversibility of type 2 diabetes. (73)


A plethora of randomized, controlled studies have been undertaken and published to ascertain which macronutrient combination leads to greater weight loss. A two-year head-to-head trial comparing four weight loss diets with differing macronutrient content concluded that all four reduced calorie diets, regardless of macronutrient content, led to comparable modest weight loss with weight regain over time. (74) Another 12-month trial of 259 participants with diabetes compared a low carbohydrate Mediterranean diet, a traditional Mediterranean diet, and a 2003 ADA diet. Greater weight loss, improved glycemic control, and improved HDL levels were demonstrated with the low carb Mediterranean diet. (75) Another study of 115 obese T2D people compared a low carb (LC), healthy fat versus a higher carb, lower fat diet. The outcome of this 52-week trial showed that both diets achieved substantial weight loss and reduced HbA1c and fasting glucose, though the LC diet achieved greater improvements in lipid profile, blood glucose stability, and reductions in diabetes medications. (76)


It seems that in the short term (1-2 years) a lower carb, moderate fat macronutrient intake will lead to weight loss and improved metabolic parameters for many people with T2D, but in the long run, it does not appear to make as much of a difference, leading one to believe that many other factors are in play. A prudent recommendation for losing weight or maintaining a healthy weight is to be aware of the amount of food eaten in relation to the number of calories expended in a day. Keeping a food and activity journal can help keep track and create awareness of the amount of food eaten. A moderate intake of fats, with an emphasis on healthful unsaturated fats, and complex carbohydrates is compatible with a weight-loss or weight-maintaining diet. The most important variable in selecting a weight loss plan is the ability of the individual to follow it and maintain weight loss over the long term.


Weight loss is a major challenge for most people who, in our fast-paced environment, don’t eat properly and fail to establish patterns of regular physical activity. The key to success is having a PWD commit to establishing a healthy lifestyle they can live with that emphasizes and incorporates more healthful food choices on most days and a daily exercise routine, taking into account the presence of possible complications. Developing an individualized weight loss program, preferably guided by a registered dietitian nutritionist familiar with diabetes management, along with regular follow-ups, will help promote and maintain weight loss. Initial physical activity recommendations should be moderate, gradually increasing the duration and frequency to 30-45 min of moderate aerobic activity 3-5 days/week. It is always important that a person checks with their physician before starting an exercise program.


The individualized approach to dieting shows powerful proof through the National Weight Control Registry, a prospective study of successful long-term dieters established in 1994. To be included, members must have maintained a 30-pound weight loss for at least a year. Tracking over 10,000 members, the Registry is the largest collection to date of long-term weight-loss data. Most participants report keys to success are continuing to maintain a lower calorie diet and doing high levels of activity. Other common themes to losing weight and keeping it off, according to data from the registry, include: (77)


  1. 78% eat breakfast every day.
  2. 75% weigh themselves at least once a week.
  3. 62% watch less than 10 hours of TV per week
  4. 90% exercise, on average, about 1hour per day. 


Children and Adolescents


Type 2 diabetes is becoming increasingly prevalent among young people who are driven, as is the case in adults, by lifestyle factors and food choices leading to increased body weight. The diabetogenic process may begin as early as fetal life, with maternal type 2 diabetes, abnormal birth weight and poor nutrition combined with sedentary lifestyle and dietary factors to produce an insulin-resistant phenotype that may accelerate the development of renal pathology and cardiovascular disease. (78) According to a recent paper, the incidence of both T1D and T2D among youths increased significantly in the 2002–2012 period, particularly among those of minority racial and ethnic groups. (79) It is important for children and adolescents to be physically active as well as following healthy eating guidelines to promote normal growth patterns, without exceeding recommended weight ranges for age and/or height.




There is no one “diet” for diabetes. There are, however, many meal planning guidelines available for the PWD. A meal plan should not be thought of as a diet, but more of an individualized guideline for more healthful eating. Listed in the information below are some of the basic guideline and more in-depth approaches. A brief explanation of the approaches, along with the resource list to obtain additional information, is included in this section.




Guideline approaches are less in-depth and complex, but they can offer the foundation for basic nutrition information. In some cases, guidelines alone may be enough to change eating behaviors in some PWD. Guideline approaches focus on making healthy food choices without weighing or measuring foods, using exchanges, or counting calories, fat or carbohydrate. Regardless of whether they are used alone, or in combination with a specific meal plan, guidelines are a good choice for beginning education about nutrition. Recognize that, due to education level, lack of motivation, etc. it may not be appropriate to move some PWD beyond this initial stage.


Choose My Plate


Choose My Plate replaces the retired USDA Food Pyramid (figure 2), and contains general, simple guidelines for healthy eating using a small plate to visually illustrate foods and portion control. An explanation and picture of the guide is listed earlier in this chapter. (56)


Print materials and videos from the USDA are available at

and The Joslin Diabetes Center (80)


Mediterranean-Style Eating


The Mediterranean-style eating pattern derived from the Mediterranean region of the world has been observed to improve glycemic control and cardiovascular disease risk factors. The Mediterranean eating pattern includes: (81,82)


  • Vegetables, fruits, nuts, seeds, legumes, potatoes, whole grains, breads, herbs, spices, fish, seafood and extra virgin olive oil. Emphasis is placed on use of minimally processed foods, seasonal fresh and locally grown foods
  • Olive oil is the primary fat, replacing other fats and oils (including butter and margarine)
  • Total fat ranging from 25% to 35% of total energy, with saturated fat no more than 7% of calories
  • Low-to-moderate amounts of cheese and yogurt
  • Twice-weekly consumption of fish and poultry; approximately seven eggs/week
  • Fresh fruit as daily dessert; sweets only a few times/week
  • Red meat a few times/month (limited to 12 oz to 16 oz per month)
  • Regular physical activity to promote a healthy weight, fitness and well-being
  • Moderate consumption of wine, normally with meals; approximately two glasses/day for men and one glass/day for women  

Figure 2. Mediterranean Food Pyramid. For more information see Mediterranean Diet Plan; Mediterranean Diet Pyramid from Oldways, and Mediterranean Diet Pyramid and the Healthy Eating Plate from Harvard University School of Public Health

What Do I Eat Now?


What Do I Eat Now?  A book primarily used for the initial stage of type 2 diabetes meal planning. It includes an overview of diabetes nutritional management within the framework of basic eating guidelines. Other resources may be added to this tool, as appropriate, to move the PWD toward more in-depth management. (83)


Figure 3. What Do I Eat Now?

Diabetes Place Mat

Figure 4. Nutrition PlaceMat for Diabetes. A sturdy, heavily laminated, 11" by 17" place mat that can be easily used over and over to apply the meal plan.

One side of the Diabetes Place Mat lists food choices and individual portion sizes for each food category of the meal plan. This list replaces easily misplaced or damaged paper lists, which are often given to PWD.


When planning the meal, a wipe-off marker is used to write down the number of servings for each food category, as indicated on the plan. Then circle or tally the food choices in each category to track progress toward the plan’s targets. Carbohydrate categories - starch and bread, fruit, milk and other carbohydrates - which affect blood sugar and which can be exchanged for each other, are color coded in yellow for easy identification and proper selection. Other food categories - vegetables, meat, fat and free foods - are individually color-coded.

Figure 5. Nutrition PlaceMat for Diabetes. A sturdy, heavily laminated, 11" by 17" place mat that can be easily used over and over to apply the meal plan.

The other side of the Diabetes Place Mat illustrates the "Plate Method" of managing a diet for proper nutrition and control of blood sugar and weight. It shows the proportions of each food category that are appropriate for a healthy, balanced diet. The food groups shown on the top half of the Plate Method side are carbohydrates, which affect blood sugar the most - fruit, milk, and starch & bread. These are colored in yellow to distinguish them from the other food groups that don't significantly affect blood sugar (meat, vegetables, fat and free foods). The food categories are shown in proportion to how much of each might be eaten in a healthy, balanced diet. The plate method is a great plan for PWD who have poor math or reading skills, or are non-English speaking. (84)


Create Your Plate: Meal Planning Tool Kit


On line portion control tools to make meal planning easier. (85)


DASH Eating Plan     


Dietary Approaches to Stop Hypertension (DASH) is a flexible and balanced eating plan that is based on research studies sponsored by the National Heart, Lung, and Blood Institute (NHLBI). These studies showed that following the DASH plan lowers high blood pressure and improves levels of blood lipids which reduces the risk of developing cardiovascular disease. (86) The DASH plan was rated #2 by the US World News Report of all healthy dietary plans for 2019.  U.S. News evaluated and ranked 41 popular diet plans with input from a panel of health experts. To be top-rated, a diet had to be relatively easy to follow, nutritious, safe, effective for weight loss and protective against diabetes and heart disease. The government-endorsed Dietary Approaches to Stop Hypertension (DASH) tied for the #2 spot as best food plan for diabetes. (42).  The DASH eating plan:


  • Emphasizes vegetables, fruits, and fat-free or low-fat dairy products
  • Is low in saturated and trans fats
  • Includes whole grains, fish, poultry, beans, seeds, nuts, and vegetable oils
  • Is high in potassium, calcium, magnesium, fiber and protein
  • Limits sodium, sweets, sugary beverages, and red meats
  • Is lower in sodium than the typical American diet. Contains 2,300 mg of sodium per day which has been shown to lower blood pressure. Further lowering to 1,500 mg/day can further reduce blood pressure

For more information go to


Intermittent Fasting


The popularity of intermittent fasting has increased recently as a new way to lose weight and possibly lead to better control of Type 2 diabetes.  There are many suggested types of intermittent fasts; some involve eating only on specific days, or not eating for a specified number of hours, alternated by day or hours in which food consumption is allowed.  Others greatly restrict calories on some days but allow a more normalized diet on other days.  There is no one specific intermittent fasting diet that has been proven to be beneficial.   Since calories are restricted for certain periods of time, an individual with diabetes may lose weight over time if they maintain an overall calorie deficit in relation to energy expenditure as is seen with any successful weight loss method.


For a PWD who is interested in intermittent fasting, their current anti-hyperglycemic medications must be considered.  For those on insulin or taking other anti-hyperglycemia medications, intermittent fasting may lead to frequent hypoglycemia that may become severe and result in death. (103) Careful monitoring of blood glucose is required, and medication adjustment may be necessary.  A more traditional approach to increase physical activity with a lower calorie diet will produce consistent weight loss and may lead to a more realistic long-term weight maintenance plan.


Flexitarian Diet


This eating plan combines the words vegetarian and flexible. It is a mainly plant based plan, but meat can be added occasionally. This plan was tied for #2 spot with the DASH by USWNR. (42)

For more information see: (87)


Ketogenic Diet


The ketogenic diet was originally used in the treatment of epilepsy.  More recently it has been studied for use in patients with T2 diabetes and is also currently being studied as adjunctive treatment for a variety of conditions including pain and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.  The ketogenic diet induces ketosis through high protein, typically high fat, and very low carbohydrate intake.  Studies in people with T2 diabetes have shown a positive benefit on blood glucose, insulin sensitivity, and a lowering of anti-hyperglycemic medications with a sustained very low carbohydrate intake.  There is disagreement on the negative effects of a higher protein intake in patients with T2 diabetes as some studies suggest possible kidney damage due to the high level of nitrogen excreted during protein metabolism.  More studies are needed to determine the long-term effects of a ketogenic diet in PWD.  Nutritional recommendations for a person considering this diet are to limit protein sources that are high in saturated fat and include more plant-based proteins for a more healthful approach. (104)


Volumetrics Plan


This plan uses a food's energy density, and works by cutting the energy density of your meals and making choices that fight hunger. Food is divided into four groups. Category one (very low-density) includes nonstarchy fruits and vegetables, nonfat milk and broth-based soup. Category two (low-density) includes starchy fruits and veggies, grains, breakfast cereal, low-fat meat, legumes and low-fat mixed dishes like chili and spaghetti. Category three (medium-density) includes meat, cheese, pizza, French fries, salad dressing, bread, pretzels, ice cream and cake. And category four (high-density) includes crackers, chips, chocolate candies, cookies, nuts, butter and oil. You'll go heavy on categories one and two, watch your portion sizes with category three, and keep category four choices to a minimum. Each day, you'll eat breakfast, lunch, dinner, a couple snacks and dessert. This plan was also tied for #2 for best diet plan for diabetes by USWNR (42). For more information see: (88)


MAYO Clinic Diet 


Developed by the Mayo Clinic, a two-phase approach to lose and maintain body weight using the Mayo Clinic food pyramid. Learn how to replace bad habits with good. This plan was also tied for #2 for diabetes by USWNR (42) For more information see: (89)


Jenny Craig®


The plan emphasizes restricting calories, fat and portions. Jenny's prepackaged meals and recipes do all three, plus emphasize healthy eating, an active lifestyle and behavior modification. Personal consultants guide members through their journeys from day one. You'll gain support and motivation, and learn how much you should be eating, what a balanced meal looks like and how to use that knowledge once you graduate from the program. By following the plan, you’re expected to drop up to 2 pounds a week.


Jenny Craig offers two programs: its standard program and Jenny Craig for Type 2, which is designed for people with Type 2 diabetes by including a lower-carb menu, reinforcement of self-monitoring of blood sugar levels, consistent meals and snacks, and other self-management strategies for weight loss and support for diabetes control. This plan was voted #6 for best diabetes diet (42) and is good for those that need support from a group and ready-made meals. Because you buy foods, this program can be more expensive, but convenient. For more information see: (90)


Ornish Diet


The diet is low in fat, refined carbohydrates and animal protein. It also emphasizes exercise, stress management and relationships. On nutrition, for instance, Ornish categorizes food into five groups from most (group one) to least (group five) healthful. It tied for #6 for best diabetes diet. (42) The plan has been shown to reverse heart disease. For more information see: (91)


Vegan Diet


Veganism excludes all animal products from the diet – including dairy and eggs. Fruits, vegetables, leafy greens, whole grains, nuts, seeds and legumes are the staples. It is restrictive, but beneficial for the cardiovascular system. It also tied for #6 best diet for diabetes (42)

For more information see: (92)


Weight Watchers®


Its WW Freestyle program, launched in late 2017, builds on its SmartPoints system, which assigns every food and beverage a point value, based on nutritional content. The newest program expands dietary options that are 0 points from only fruits and vegetables to more than 200 foods. A backbone of the plan is multi-model access (via in-person meetings, online chat or phone) to support from people who lost weight using Weight Watchers, kept it off and have been trained in behavioral weight management techniques. It ranked #6 as well for best diet for diabetes. (42) Also good for those that need support group-based approach to losing and maintaining weight loss. For more information see: (93)




For individuals who rely on technology for assistance in managing their daily activities there are several diabetes specific as well as nutrition and physical activity smart phone applications (Apps) available at no charge or for a subscriber’s fee.  A list of some of the more popular are included here and are available either as downloads from the company website or the Apple and Android stores.  There are many other Apps available on the Apple and Android stores.  Each App has different data that is collected, and usefulness will depend on the individual user, their specific needs and interests and the potential to use and interact with their healthcare team.

·                Cronometer

·                Glooko

·                Health2Sync

·                DietSensor

·                Glucose Buddy

·                Nutritionix




The following are approaches that are more in-depth for individuals who are motivated to follow a more structured, focused meal plan and who are able to more actively engage in meal planning and advanced carbohydrate counting.

Individualized Menus Provided by a RD/RDN


Many PWD like to have examples to follow when setting up meal plans. The menu describes in writing what foods and what quantities should be consumed over a period of days. A dietitian creates an individualized menu based on the specific nutritional counseling plan and incorporates the PWD’s unique preferences, schedule, etc. The person then has written examples to follow, and over time may learn how to independently create their own menus and substitutions to fit their individual lifestyle.


Month of Meals


These menus were created by committees of the Council on Nutritional Science and Metabolism of the American Diabetes Association, and staff of the American Diabetes Association National Service Center in response to frequent requests for menus from PWD and their families. The menus are designed to follow the exchange groups and provide 45-50% of calories from CHO, 20% protein, and about 30% fat. The menus provide 1200 or 1800 calories, and instructions are provided on how to adjust caloric levels upward or downward. Each menu provides 28 days of breakfast, lunch, dinner and snacks with a different focus to help make planning meals easier. (94)


Exchange List Approach


The Exchange Lists for Meal Planning were developed by the American Diabetes Association and the Academy of Nutrition and Dietetics, and have been in existence since 1950. It’s now in its seventh edition as Choose Your Foods:  Food Lists for Diabetes. The concept is that foods are grouped according to similar nutritional value, and can be exchanged or substituted in the portion size listed within the same group. In 1995, the exchange lists were revised from 6 food groups to 3. They include:


  • Carbohydrate group – includes starches, fruit, milk and vegetable
  • Meat and Meat Substitutes group – four meat categories based on the amount of fat they contain.
  • Fat group – contains three categories of fats based on the major source of fat contained: saturated, polyunsaturated or monounsaturated.


The exchange lists also give information on fiber and sodium content. They can be utilized for people with type 1 or 2 diabetes. The emphasis for type 1 is on consistency of timing and amount of food eaten, while for type 2, the focus is on controlling the caloric values of food consumed. (95) Use of the exchange list may be helpful for some PWD while others may benefit by learning from other carbohydrate counting resources available online and through numerous publications and resources.


Advanced Carbohydrate Counting


Although CHO counting has been used seemingly effectively for many people with type 1 diabetes, very few clinical trials have been undertaken to report actual outcomes. The results of the first randomized clinical trial designed to test the effects of CHO counting in adults with type 1 diabetes treated with continuous subcutaneous insulin infusion (CSII) concluded that there was an improved Diabetes-Specific Quality-of-Life Scale score related to diet restrictions, and CHO counting was also associated with a modest, although significant, decrease in BMI and waist circumference. When PWD who did not continuously use CHO counting or CSII during the study were excluded from the analyses, CHO counting was also associated with a significant reduction in A1C without an increase in hypoglycemic events. (96) A more recent published meta-analysis on effects of advanced CHO counting revealed a trend toward reduction in AIC, but no significant evidence to definitively determine the effects on glycemic control, weight, psychosocial measures, or hypoglycemic events. (97)


For those PWD managed by insulin and at a more advanced level, the focus is to finely tune food intake, medication and activity based on patterns from daily food intake and blood glucose records. Record keeping is an important first part of advanced CHO counting. The mealtime, amount and type of food eaten, estimates of CHO intake for each food item containing CHO, and total amount of CHO for each meal and snack must be recorded. Also, insulin dose, physical activity, and blood glucose levels must be accurately documented for several weeks. Any unusual circumstances should be noted such as illness, stress, menstrual cycle, etc.


Ratio and correction factor calculation is another aspect of advanced CHO counting. The insulin-to-CHO ratio helps the person with diabetes understand how much rapid or short-acting insulin is needed to metabolize the CHO that is consumed at a meal or snack. It allows greater flexibility in lifestyle and can improve glucose control.  The insulin to carbohydrate ratio (ICR) is the number of grams of carbohydrate that one unit of insulin will “cover”. To calculate the ICR (insulin to carbohydrate ratio), of 1:15, divide the number of grams of CHO for the meal by 15 to determine the number of units of prandial insulin needed to cover the amount of carbohydrate to be consumed. For example, if a person with diabetes plans to eat 75 gms of CHO, they would divide 75 by 15 which equals 5.   Five units of prandial insulin are needed to cover 75 grams of carbohydrate for that individual. The ratio should be individualized for each PWD based on individual responses to carbohydrate and considering possible differences in meals and timeframe. (98) It may be helpful to provide worksheets when a PWD begins to use this formula.  These are available through numerous diabetes education and research organizations.

An "average" ICR can be 1 unit of insulin for every 10 to 15 grams of CHO for an adult or 1 unit for every 20 to 30 grams of carbohydrate for a school-age child, however careful monitoring of blood glucose and individual response should be evaluated to individualize the ratio.

A correction factor is used to correct a high or low blood glucose level before a meal. The correction factor is added or subtracted to the prandial bolus insulin dose. For example, a factor of 1800 is used for rapid-acting insulin, and 1500 for regular insulin. Thus, if a person uses 60 units of total daily insulin and rapid insulin before meals, the correction factor would be 30 (1800 divided by 60). This means that 1 additional unit of insulin will lower blood glucose by approximately 30 mg/dL. Thus, if the pre-meal blood glucose is 169, and the target glucose is 130 or less, 1 extra unit of insulin should be given with the meal (169-130 = 39). (98) The diabetes management team can help establish personal ICRs and help educate on specific amount of CHO in grams that are consumed, and appropriate correction factors.  


Calorie Counting and Fat Counting


These are meal planning methods that can be useful for people with type 2 diabetes who want to lose weight. Knowledge regarding the number of total calories and fat grams in a given food (including pre-prepared and fast foods) and becoming adept at label reading, can help promote weight loss when incorporated into other lifestyle changes. One of the first studies designed to determine empirically if people can learn a calorie counting system and if estimated food intake improves with training demonstrated that use of the Health Management Resources Calorie System tool (HMRe, Boston, MA, USA) helped to teach people how to estimate food intake more accurately. (99)



Ø  Choose My Plate (2019)

Ø  Eat Out, Eat Well

Hope S. Warshaw, MMSc, RD, CDE

Your go-to resource for assembling healthy meals in just about any type of restaurant, from fast food to upscale dining and ethnic cuisines.

Order from:  The American Diabetes Assn.,, 1-800-232-6455

Ø  The CalorieKing Calorie, Fat & Carbohydrate Counter 2018

Find nutrition facts from your favorite brands and chain restaurants

Order from: or

Ø  What Can I Eat? The Diabetes Guide to Healthy Food Choices 2nd Edition

A 28-page guide for planning meals and making the best food choices. Includes carb counting, glycemic index, plate method, eating out, meals/snack ideas, best food choices and more

Order from: The American Diabetes Assn., Inc., 1-800-232-6455

Ø  Eating Healthy with Diabetes, 5th Edition

Picture cues for portion sizes and color codes for food types teach how to put together a healthy diet plan to manage diabetes

Order from:  The Academy of Nutrition and Dietetics. or the American Diabetes Assn., Inc.

Ø  Diabetes Meal Planning Made Easy & Healthy Portions Meal Measure

Meet your health and nutrition goals with healthy diabetes meal plans, shopping strategies and our handy portion control guide.

Order from:  The American Diabetes Association,, 800-232-6455

Ø  Diabetes Place Mat Kit for People with Diabetes

Order from: NCES Health & Nutrition Information Catalog- Available in Spanish OR

School Health Corporation

Ø  The Complete Month of Meals Collection, 2017

Available from: or American Diabetes Association, 1-800-232-6455;

Ø  Choose Your Foods: Food Lists for Diabetes  

Order from: Academy of Nutrition and Dietetics OR American Diabetes Associations; OR or

Available in Spanish

Ø  Diabetes Food Hub

A website available on the American Diabetes Association site that has meal planning, grocery lists, recipes, menus and healthy substitutions. Section in Spanish available.

Ø  The Complete Guide to Carb Counting

American Diabetes Association 4th edition

Has all the expert information you need to practice carb counting, whether you’re learning the basics or trying to master more advanced techniques.

Order from American Diabetes Association, or

Ø  Diabetes and Carb Counting for Dummies 1st Edition

Sherri Shafer, RD, CDE

Provides essential information on how to strike a balance between carb intake, exercise, and diabetes medications while making healthy food choices. — Covering the latest information on why carb counting is important for Type 1 diabetes, Type 2 diabetes, and gestational diabetes.

Available at

Ø  The resources listed above are a sampling of the many available, primarily from the American Academy of Nutrition and Dietetics and the American Diabetes Association. There are several other organizations and websites which have educational ma­terials available for persons with diabetes. A few which should be mentioned with their websites include:

Ø  --------Diabetes Care and Education ( sponsored by the Academy of Nutrition and Dietetics, has a good list of diabetes resource websites

·       --------Joslin Diabetes Center (

·       --------National Diabetes Education Program (;

·       --------Many pharmaceutical companies also have free nutrition education materials which can be obtained for persons with diabetes.




Knowledge and individual application to improve adherence to the core foundational nutrition principles is one of the most important aspects of diabetes lifestyle management. There is no longer such a thing as a 1200 or 1800 calorie ADA diet! The dietary goals covered here, along with other lifestyle changes, if consistently applied, can help to improve metabolic profiles and ultimately help prevent long-term complications associated with diabetes. Motivating the PWD to make changes by working with a diabetes management team to implement an individualized program may help to elicit positive outcomes.




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Social and Environmental Factors Influencing Obesity



The evidence for social and environmental factors that contribute to obesity are often underappreciated. Obesity prevalence is significantly associated with sex, racial ethnic identity, and socioeconomic status, which creates complex relationships between each of these characteristics. Food availability remains an important factor associated with obesity that relates to differences in prevalence seen across geographical areas and higher rates of obesity within low socioeconomic status individuals. Proliferation of high calorie, energy dense food options that are or perceived as more affordable combined with reductions in occupational and transportation related physical activity can contribute to a sustained positive energy balance.  Additionally, environments experiencing deprivation, disorder, or high crime have been shown to be associated with higher odds of obesity, which may appear more frequently in low social status individuals. Both objective and subjective measures of social status and inequality are associated with increased energy intake and decreased energy expenditure, which could place individuals of low social status at greater risk for obesity development. Given the complexity of this multifactorial disease, effective obesity care requires knowledge of these complex relationships and an integration between the health systems and surrounding community. Resources for practicing clinicians regarding methods of screening for social and environmental factors in clinical care are provided in addition to information on a program that has been widely dispersed and made accessible to those who may be the most at risk. 




Many medical providers appreciate the significant social and environmental determinants of obesity but are unsure how to address them. Others consider these factors outside of their control and scope of practice, and are thus hesitant to even broach the topic with their patients. Finally, many medical providers still attribute obesity to causes within a person’s control, such as dietary choices, amount of exercise, or willpower, (1, 2) which perpetuates a stigma that accompanies this disease.  Specifically, the prevailing stigma is that those who suffer from obesity represent a population who lack the willingness to change their poor lifestyle habits or harbor a character flaw that, at its extreme, infers immoral behaviors (e.g., gluttony). In reality, obesity is a multifactorial disease (3) that is caused by a combination of biological, genetic, social, environmental, and behavioral determinants. In order to address this gap in the understanding of the social and environmental determinants of obesity and improve the care of patients with obesity, this chapter will review the evidence for the social and environmental determinants of obesity development. The specific areas to be covered include social identity, social status, societal trends, and influences of the built, industrial, and social environments, all factors that are closely associated with the prevalence or incidence of obesity or that impact efforts to prevent and treat this disease.  Resources for the busy clinician that will support implemental changes in one’s practice to improve the care and management of patients with obesity, as well as evidenced-based opportunities for advocacy in the community, will be included in the final section.


This chapter is divided into three primary sections based on the progression of thought and evidence surrounding the social and environmental determinants of obesity: individual characteristics, environmental characteristics, and social hierarchy influences. Individual characteristics are those that are attributed to the individual with obesity such as their sex, age, race, ethnicity, and socioeconomic status (SES). Environmental characteristics surround the individual, including the physical spaces where people live, work, and play, as well as sociocultural norms. The social hierarchy refers to social status or social rank of individuals within larger society or a local community.




The prevalence of obesity varies according to key individual characteristics such as age, sex, race and ethnicity, and SES. The prevalence of obesity increases cross-sectionally across the lifespan: from 13.9%, in early childhood (2-5 years old)  to 18.4% in childhood (6-11 years old),  20.6% in adolescence (12-19 years old), 35.7%, in young adulthood (20-39 years old),  42.8% in adulthood (40-59 years old), and 41.0% in older adulthood (≥60 years old) (4).  As of 2016, the prevalence of adult obesity in women in the United States was 41.1% and in men was 37.9% (4).  In the decade between 2007-2008 and 2015-2016, obesity significantly increased only in women (4), suggesting a sex-specific vulnerability to expression of this disease. Additionally, when race and ethnicity are considered, significant interactions between race and sex emerge. Non-Hispanic black, non-Hispanic Asian, and Hispanic women all have significantly higher prevalence of obesity than men with the same racial ethnic identity (5). In men and women, non-Hispanic Asians have significantly lower prevalence of obesity compared to all other major races and ethnicities in the United States (Note: not adjusted for ethnic specific cut points for Asians), and Non-Hispanic blacks and Hispanics have significantly higher prevalence of obesity compared to Non-Hispanic whites (5). It is not fully clear why differences in obesity prevalence by race and ethnicity are present, but some evidence points to differences in genetic backgrounds that affect body composition and fat distribution (6, 7), and to differences in cultural body image standards (8). Additionally, in the United States, race and ethnicity are confounded with SES, which is one of the most potent indicators of overall health in the United States (9).


A significantly greater proportion of underrepresented racial ethnic minorities are considered low SES compared to non-Hispanic Asians and non-Hispanic whites in the United States. Socioeconomic status is a composite measure that can be represented by measures of income, educational attainment, or occupational status. In the 2017 Census, 21.2% of non-Hispanic blacks and 18.3% of Hispanics lived below the poverty level compared to 8.7% of non-Hispanic whites and 10% of non-Hispanic Asians (10). Non-Hispanic Asians (53.9%) and non-Hispanic whites (36.2%) are more likely to earn a bachelor’s degree than non-Hispanic blacks (22.5%) and Hispanics (15.5%) (11). In terms of health, low SES in childhood is associated with adult development of cardiovascular risk factors and a 20% increase in the odds of having central obesity (as defined by a waist circumference >102 cm for men or > 88 cm for women) (12). In adult women, obesity prevalence increases with decreasing income and educational attainment; however, in non-Hispanic black women, obesity prevalence differs by education gradients but not by income gradients (13). Conversely, non-Hispanic black men have a higher prevalence of obesity in the highest income group, but all the men’s racial ethnic groups showed similar relationships between obesity rates and education gradients as women (13). Higher SES is also associated with healthy lifestyle behaviors that are often the first line of prevention or treatment for obesity. On the other hand, low SES is associated with less leisure time physical activity (14) and consumption of energy-dense diets that are nutrient poor (15); however, SES is not the only factor that influences these behaviors. Further exploration of how SES affects resources and the ability to practice healthy behaviors is expounded upon in the next section.






Obesity prevalence differs by geographical region in the United States with the South and the Midwest having the highest level of obesity among adults (16). The Midwest and South also have high rates of diabetes and metabolic syndrome, which frequently accompany obesity (16).  Approximately 55% of global increases in BMI can be attributed to rising BMI in rural areas, and this may be as high as 80% in low- and middle-income countries (17). Rural areas are associated with 1.36 higher odds of obesity compared to urban areas; however, mediation analysis shows that individual educational attainment, neighborhood median household income, and neighborhood-built environment features reduce these odds by 94% and render the relationship statistically insignificant (18). Rural areas tend to have farther distances between residences and supermarkets, clinical settings, and recreational opportunities, which may be impacting the ability to practice healthy behaviors that prevent obesity. This is one example of the “built environment”, which alludes to the infrastructure of a geographic area that influences proximity to and types of resources, transportation methods, and neighborhood quality.


Food Availability


The frequency and type of food vendors in a neighborhood determines the types of foods that residents can purchase. Historically, evidence has suggested that fast food restaurant density is associated with obesity prevalence. A state-level analysis of fast food restaurant density and the number of residents per restaurant accounted for 6% of the variance in state obesity prevalence (19). Individual-level factors can interact with built environmental factors (like fast food restaurant density) to increase the odds of obesity. For example, one study in older adults showed that residents who ate 1-2 times per week at a fast food restaurant (odds ratio [OR]: 1.878), did not meet current physical activity guidelines (OR: 1.792), had low self-efficacy for eating healthy food (OR: 1.212), or identified as non-Hispanic black (OR: 8.057) and lived in a high density fast food neighborhood were more likely to have obesity than older adults who lived in a low density fast food neighborhood (20). On the other hand, recent research suggests that fast food restaurant density is not associated with obesity prevalence and the food consumed in these establishments’ accounts for less than 20% of the total energy intake (21). This could reflect the widespread availability of fast food nationally, which weakens the ability to dissect links between its presence and increased consumption specific to obesity.


The term “food desert” is often used to describe areas with limited access to affordable and nutritious food (e.g. supermarkets) and these vary significantly according to neighborhood socioeconomic and racial/ethnic composition (22, 23). Food desert designation has been positively linked to obesity in the United States and simply switching from a non-food desert census tract to a food desert census tract can increase the odds of obesity by 30%, when all other relevant factors are held constant (24). Conversely, access to supermarkets does not automatically result in healthier eating behavior and weight status. A systematic review showed that five out of six studies looking at supermarket access did not find increased fruit and vegetable consumption with greater accessibility; however, four out of five studies looking at changes in weight status found lower BMI and prevalence of obesity in areas with high access to supermarkets compared to low access areas (25). A large natural experiment found that the opening of a new supermarket improved overall diet quality in the neighborhood, but did not affect fruit and vegetable intake or BMI (26). Interestingly, the only positive outcome directly associated with regular use of the new supermarket was higher perceived access to healthy food (26). Although these findings are mixed, it is important to acknowledge that changes in food choices at a neighborhood level might occur too slowly to be captured in these studies.


In addition to food availability and quality, the shift in food type, amount, and pricing is also relevant to the obesity epidemic. For example, available evidence strongly supports a greater risk of weight gain and type 2 diabetes with increased consumption of sugar-sweetened beverages (27). North America still has the highest per capita sales of calorie sugar-sweetened beverages, but is slowly starting to shift to low-calorie sugar sweetened beverages, though sports and energy drink consumption continue to increase (28). Portion sizes in the most popular fast-food, take-out, and family style restaurants exceed current USDA and FDA standard-recommended portion amounts as well as what had been historically served in past decades (29). Increased portion sizes have been robustly linked to increases in energy intake in both adults and children; however, evidence is limited that decreasing portion size results in decreased energy intake (30). In addition, fast foods, snack foods, and foods available through convenience stores are typically ultra-processed (high in processed grains and added sugars; low in fiber and unsaturated fats).  A recent study found that keeping macronutrient content the same, meals that were ultra-processed resulted in greater food intake and weight gain over a two-week follow-up compared to consumption of non-processed foods (31). Contributing to increased intake of fast-foods and ultra-processed foods is the marketing techniques implemented by food industries across multiple mediums. Though adults have shown to be less susceptible to the effects of food advertising, experimental studies with children produce a moderate effect size for increased food consumption after food advertising exposure (32). Food advertising targeted at children is focused on brand building and emotive messages may not be discerned as such by this vulnerable population (33). Another common misconception confronting consumers is that healthy foods are more expensive, but research suggests this perception is based on misleading price metrics as well as changes in fruit and vegetable convenience and level of preparedness (34). Price per calorie metrics show fruits and vegetables to be more expensive than less healthy foods; however, price per average portion and price per edible 100 grams actually shows that fruits and vegetables are less expensive (34). In times of financial constraint, socioeconomically disadvantaged groups maximize energy value for money resulting in energy-dense, nutrient poor diets that contribute to obesity (35).




Infrastructure can dictate means of transportation and neighborhood walkability, which is associated with weight status. High neighborhood walkability has been found to be associated with decreased prevalence of overweight and obesity (36), which can link back to structural differences discussed earlier between urban and rural areas (urban areas having higher walkability). Transport-related physical activity decreased by 17.8% between 1965 and 2009 in the United States, which could be due to growing ubiquity of car ownership and supportive infrastructure for automotive transport in the United States (37). Proximity to recreational facilities, recreational facility density, access to sidewalks and paths that remove pedestrians from traffic hazards, and access to parks, have all been reported to be facilitators of physical activity in qualitative and quantitative research (38, 39).


The quality of infrastructure in a neighborhood and the perceived aesthetics of homes, shops, and recreational facilities can impact the use of these facilities. A study in a high-income neighborhood and a low-income neighborhood showed that even though the number of recreational facilities was equitable in the neighborhoods, the residents of the low-income neighborhood perceived that they had less access to recreational facilities (40).


Additional neighborhood descriptors that are associated with obesity include neighborhood deprivation, disorder, and crime. Neighborhood deprivation, a composite score of socioeconomic position of individuals in a neighborhood that is used to assign a rank to that neighborhood, shows that high levels of deprivation are associated with a 20% increased odds of overweight (41). Neighborhood physical disorder refers to the presence of vandalism, abandoned lots or vehicles, garbage, and quality of building conditions. Women in an urban area with high neighborhood physical disorder have a 1.43 greater odds of obesity (42). Persons living in areas of high crime have a 28% reduced odds of achieving higher levels of physical activity and, conversely, perceived safety increases the odds of achieving higher levels of physical activity by 27% (43). Living in a neighborhood with high crime has been found to be associated with increased weekly snack consumption in women (42). The relevance of the neighborhood environment to obesity is further exemplified in the Moving to Opportunities Study (44). The Department of Housing and Urban Development randomly assigned just under 5000 families in Chicago, Baltimore, Boston, Los Angeles, and New York public housing to 3 possible conditions: receive a housing voucher to move to a low-poverty census track with moving counseling, receive a standard unrestricted housing voucher and no moving counseling, or receive nothing. Despite the fact that this study was not focused on weight or diabetes outcomes, participants that received the voucher to move to a low-poverty census track had 4.61 percentage points lower prevalence of BMI > 35, BMI > 40, and glycated hemoglobin ≥ 6.5% than participants who received nothing (44), showing that a mere change in environment from high- to low-poverty rates was enough to have a significant impact.


Work Environment and Advances in Communication Technology


As the built environment and food environment have changed in the United States, so has the work environment. From 1960 to 2010, jobs in the U.S. private industry shifted from 50% requiring at least moderate to vigorous physical activity to less than 20% requiring this level of activity intensity (45). National Health and Nutrition Examination Survey data has documented an association between decreases in work-related energy expenditure and weight gain over the same time period (45). These changes in occupation related physical activity could be due to improvements in labor-saving technology. Technology advances are not confined to the work environment and have spread into many facets of daily life, such as improvements in smart personal communication devices, internet media platforms, marketing techniques, and enhanced audio-visual media.  Studies show that marketing for unhealthy foods is often targeted at more vulnerable populations such as Non-Hispanic blacks (46) and Hispanics (47). Additionally, the availability of information about healthy weight-loss behaviors on the internet is poor when searched for in Spanish (48).  “Screen time” or the time spent using technology that utilizes a screen interface has been found to be associated with increased risk for obesity (49-51); however, many app companies and academic researchers are now using that same technology to help with obesity prevention and treatment (52-54). 




Animal research consistently shows that animals of subordinate status experience adverse physiological and behavioral changes compared to their high status counterparts: higher levels of cortisol (primates) (55), elevated blood pressure (rats, rabbits, baboons, macaques) (56), elevated heart rate (primates) (56), accumulation of visceral fat (rats) (57), increased ad-libitum energy-dense food consumption (macaques, rats) (57, 58), cardiovascular disease (mice) (59), and shortened lifespan (mice) (59). This implies that social standing, regardless of species, has physiological implications and could be contributing to obesity development and poor health. The findings from animal models thus serve as the basis for parallel outcomes reported in humans of low social status.


Social status can be measured objectively or subjectively. Objective measures typically include socioeconomic status (SES) variables, such as income, education, or occupation, which were discussed as individual level factors at the beginning of this chapter. Social status can also be represented by manifestations of status differentials, including inequality between groups or measurable differences in the ability for someone to obtain basic life necessities, such as food security. High levels of absolute income/wealth may be related to health not only through better material conditions, but also through social position.  However, in an analysis of two nationally representative British panel studies, ranked position of income/wealth, not absolute income/wealth, predicted adverse health outcomes such as obesity, presence of chronic disease, and poor ratings of physical functioning and pain (60). In a worldwide study of physical activity, countries with large activity inequality predicted obesity better than the total volume of physical activity within the country (61). Activity inequality is identified by calculating a Gini coefficient for population step count data from each country, 0 = complete equality, 1= complete inequality. Individuals in the top five countries for physical activity inequality (Saudi Arabia, USA, Egypt, Canada, Australia) were 196% more likely to have obesity than individuals from more equal societies that did not have large disparities in step counts across the population. Gender differences account for 43% of the inequality observed, however, this effect was mitigated in societies that rated higher in walkability (61). Inequality can also drive calorie consumption. Individuals who are experimentally induced to view themselves as poor in reference to others exhibited increased calorie intake (62). Additionally, individuals who believed they were poorer or wealthier than an interaction partner exhibited higher levels of anxiety in regards to that difference in status that, in turn, led to increased calorie consumption (62).


Food insecurity affects approximately 11.8 percent of families in the United States and has been linked to obesity and diabetes. Food insecurity occurs when “the intake of one or more members of a household is reduced and eating patterns are disrupted (sometimes resulting in hunger) because of insufficient money and other resources for food” (63). In women, food insecurity status predicts overweight/obese status differentially across racial ethnic groups. Non-Hispanic white women who are food insecure are 41% more likely to have overweight or obesity whereas Hispanic women who are food insecure are 29% more likely to have overweight and obesity (64). Among non-Hispanic black women and men, food insecurity did not predict overweight or obesity status (64). A population-based study in Canada revealed that persons in food insecure households had double the risk of developing type 2 diabetes compared to persons in food secure households, even after controlling for age, gender, income, race, physical activity, smoking status, alcohol consumption, diet quality, and BMI (65). Reduced food availability is theorized to initiate compensatory biological mechanisms that boost caloric intake, decrease resting metabolic rate, and increase storage of adipose tissue as a protective mechanism for survival (66). Research in youth has provided evidence for a moderating effect of food insecurity on the relationship between income and subjective social status (67). This means that low income is more strongly associated with low subjective social status when the household is also food insecure.


Subjective measures of social status (SSS) are typically measured by asking individuals to place themselves on 10-rung ladders based on where they perceive their rank within society and the community. Experimental evidence demonstrates a relationship between feelings of low social status and increased calorie intake. Cornil and Chandon showed that hometowns of National Football League teams consumed more calories after a team loss than hometowns of winning teams or of hometowns where teams didn’t play (68). Manipulations of social status in an experimental setting show that acute eating behavior post experimental manipulation consists of higher calorie food choices and higher total calorie intake in the low status group (69). Additionally, individuals randomized to a low social status condition, had increased levels of ghrelin, a hormone that stimulates appetite, as compared to the high social status condition, suggesting a physiological hunger response to low perceived social status (70). Studies of physical activity and SSS show that low SSS is associated with significantly lower levels of moderate to vigorous physical activity (71, 72), which could contribute to a lower overall energy expenditure. Closely related to SSS are other perceptive representations of status differentials, such as perceived discrimination, which is associated with increased weight and BMI in women (73) and increased abdominal adiposity in non-Hispanic whites (74).


Researchers have integrated individual and environmental factors into design and development of interventions to improve weight outcomes or weight-related behaviors (healthy eating, physical activity); however, not all of them are successful. For example, a study among low-income women with children in rural Mexico randomly assigned families to cash or in-kind transfers (food baskets) and found that women in the food basket and cash groups actually gained weight compared to women in the control group (75). This study and others that show weight gain occurring in spite of access to resources or poverty relief imply accounting for individual and environmental factors alone may not paint a complete picture of obesity development. Granted, it is important to consider that systemic environmental changes, such as placement of sidewalks or fruits and vegetables in a corner store, may not be adequately captured in a short time frame typical of academic studies. However, the small or nonexistent changes observed when resources are supplied warrants further investigation into deeper realms of social hierarchical constructs, as well as continued study of individual and environmental factors to improve treatment and prevention of obesity.




Given the extent of the information on individual, environmental, and social hierarchy constraints on obesity development, it is important to understand how these can merge with clinical care. It is evident that there is no one simple solution and effective care requires knowledge of these complex relationships and an integration between the health system and the surrounding community. For example, based on the knowledge that the social determinants of health can influence diabetes and its comorbidities, the American Diabetes Association recommends in its clinical guidelines that providers “assess the social context… and apply that information to treatment decisions” (76). In conjunction with recognition of the impact of social and environmental determinants on multiple chronic diseases, some researchers propose that “community vital signs” be integrated into the electronic health record (EHR) (77) and some community health centers have begun pilot testing a social determinants questionnaire in their HER (78). Knowledge provided by these “vital signs” and social determinants could help providers make appropriate lifestyle-tailored recommendations for the patient.


Discussing context surrounding food in a patient’s life can provide insight into the realistic expectations for a patient’s diet.  Food insecurity can be identified with a short two question screener (79) and implementation in clinics has shown that screening improves clinician awareness of food insecurity, helping to better understand the lengths to which it affects patient treatment (80). Positive responses from physicians after pilot testing that incorporates screening into clinical practice mitigates concerns that discussions about food security would be stigmatizing to the patient (80). Patients who identify as food insecure can be referred to local food banks or community programs that will connect patients with resources at a federal and community level.


Patients that are finding it difficult to follow lifestyle modification recommendations to lose weight to prevent diabetes development may benefit from the Diabetes Prevention Program. The Diabetes Prevention Program is a lifestyle program focused on weight loss through dietary change and increased physical activity. While the overall weight loss was modest (~4% after 4 years), participants lowered their chances of developing diabetes by 58% during long-term follow-up (81). This program has been adapted for implementation and dissemination purposes and now the CDC’s National Diabetes Prevention (National DPP) program is available at almost 2,000 sites across the United States including many YMCAs, with a mix of online and in-person options.  This program is covered for eligible individuals by Medicare and many private insurers and cost for non-covered patients is variable and often income-based or free.  Initial evaluation of the real-world evidence for implementation of the National DPP have been promising with 35% achieving 5% weight loss and 42% meeting the activity goal of 150 minutes per week (82). Locations with the best participant retention and attendance share the following qualities: referrals from healthcare providers or health systems, provision of non-monetary incentives for participation, and use of cultural adaptations to address participant needs (83). The National DPP provides an affordable, easy and local referral source so that the provider can be assured their patients are receiving evidence-based lifestyle management in an ongoing program.  




Figure 1 below shows the age-adjusted prevalence of obesity in adults by race and ethnicity, and sex from the Centers for Disease Control 2017 National Center for Health Statistics Data Brief (5).

Figure 1. Prevalence of Obesity by Race/Ethnicity and Sex

Questions to Incorporate into Your EHR About Food Insecurity

  1. “We worried whether (my/our) food would run out before (I/we) got money to buy more” Was that often true, sometimes true, or never true for (you/your household) in the last 12 months?
  2. “The food that (I/we) bought just didn't last and (I/we) didn't have money to get more” Was that often true, sometimes true, or never true for (you/your household) in the last 12 months?

Information on the Diabetes Prevention Program


Opportunities for Advocacy 

The Obesity Action Coalition:

The Obesity Society:

STOP Obesity Alliance:

Rudd Center for Food Policy and Obesity:



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Growth Hormone in Aging



Growth hormone (GH) serves important roles in adult life, including maintenance of lean body mass and bone mass, promoting lipolysis, thereby limiting visceral adiposity, and regulating carbohydrate metabolism, cardiovascular system function, aerobic exercise capacity, and cognitive function. Younger adults with growth hormone deficiency (AGHD) exhibit abnormalities in body composition, physical and cognitive function, and quality of life which are reversed by GH replacement therapy. With advancing age GH production declines, paralleled by physical and functional alterations similar to those of AGHD; however, the degree to which the decrease in GH contributes to these age-related changes is unknown. Seemingly in opposition to the theory that the diminished GH secretion of older age is a net detriment are observations that animal models of congenital GH deficiency have remarkably increased life span and humans with congenital GH deficiency may have decreased rates of age-related diseases such as diabetes and cancer. Several short-term studies aiming to increase GH in older adults by a variety of interventions including exercise, administration of GH, or treatment with GH secretagogues have demonstrated consistent effects to improve body composition, yet inconsistent effects on physical and cognitive function. While side effects of GH administration in older adults include edema, arthralgias, and elevated blood glucose, data regarding the possible long-term effects on “hard end points” such as risk of fractures, cancer, cardiovascular disease, life expectancy, and mortality are lacking.




The decline in growth hormone secretion observed with aging is associated with changes in body composition and physical and psychological function that are similar to those seen in younger adult patients with growth hormone deficiency. These changes include reductions in lean body mass and muscle strength and an increase in body fat, particularly in the visceral compartment. Memory and cognitive function gradually deteriorate with age. Deep (slow-wave) sleep also decreases markedly with age, together with a decrease in nighttime growth hormone secretion, and sleep disorders become a significant clinical problem in old age. Although these changes only show an association, and it is still unknown whether there is any causal link between them, they have led to speculation that replacing or stimulating growth hormone may reverse some of the detrimental features of the aging process (1).



The trophic hormones which rise at puberty, including sex steroids and GH, have dramatic effects on body composition and strength. Their levels plateau in young adult life and then decline progressively, accompanied by a loss of muscle mass and aerobic capacity, and an increase in abdominal fat. These changes resemble some features of hypogonadism and adult GH deficiency (2).


After the third decade of life, there is a progressive decline of GH secretion by approximately 15% for every decade of adult life. Integrated measurements of daily GH secretion demonstrate that secretion peaks at puberty at about 150 µg/kg day, then decreases to approximately 25 µg/kg/day by age 55 (3). The reduction in GH secretion results from a marked reduction in GH pulse amplitude, with only very little change in pulse frequency (4). This process is characterized by lack of day-night GH rhythm resulting from loss of nocturnal sleep-related GH pulses (Figure 1) (4). Growth Hormone Binding Protein decreases from 60 years of age, theoretically increasing the amount of bioavailable growth hormone (5). This decrease is thought to parallel the decrease in growth hormone receptors with age. Though slow-wave sleep (SWS) decreases with age most studies administering GH or GHRH to seniors did not improve SWS. This finding suggests that the age-related decline in GH does not cause reduced SWS, although the reverse may be true.

FIGURE 1. Patterns of GH secretion in younger and older women and men. There is a marked age-related decline in GH secretion in both sexes and a loss of the nighttime enhancement of GH secretion seen during deep (slow-wave) sleep. This decrease is primarily due to a reduction in GH pulse amplitude, with little change in pulse frequency. L = large GH pulses, S = small GH pulses. From Ho et al. 1987 (4).

Circulating levels of IGF-I, the main mediator for the trophic effects of growth hormone, also decline with age (Figure 2). The majority of circulating IGF-I is produced in the liver under the control of GH. It appears that the age-related decline in IGF-I production is a direct result of decreases in GH and there is no evidence to suggest increased “GH resistance”. In fact, studies of GH replacement therapy in patients with pituitary disorders and dose-response studies demonstrate a reduction in GH dose necessary to maintain normal IGF-I concentrations in older subjects, although this is due at least in part to the higher susceptibility to side effects from GH and also that their target IGF-1 is lower (6-7).

FIGURE 2. Changes in serum IGF-I with increasing age. Modified from Juul A et al,1994 (8).



Three hypothalamic factors regulate GH secretion: somatostatin (SRIF), growth-hormone releasing hormone (GHRH) and ghrelin (9) (Figure 3). Somatostatin is a noncompetitive inhibitor of GH secretion as well as of other hormones. It modulates the GH response to GHRH. GHRH is the principal stimulator for GH synthesis and release. Ghrelin is the endogenous ligand to the growth hormone secretagogue receptor-1a (GHSR-1a). Ghrelin is secreted primarily by the stomach and has appetite-stimulating activity separate from its effect on GH secretion. Although recent preclinical data suggest that not all the effects of ghrelin are mediated through GHSR-1a (10), its orexigenic and GH secretagogue effects require the presence of the GHSR-1a (11).Acylated Ghrelin levels decrease with age (12) Growth hormone secretagogues (GHS) are synthetic molecules that stimulate the GHSR-1a exhibiting strong growth hormone-releasing activity.


A variety of stimuli and inhibitors, such as exercise, sleep, food intake, stress and body composition have effects on the hypothalamic factors that regulate GH production (13). All of these factors interact to generate the physiological patterns of pulsatile GH secretion.

FIGURE 3. Major components of the GH neuroregulatory system (3).

There are several mechanisms that could explain the age-related decrease in GH secretion. Possibilities include decreased secretion of GHRH or ghrelin, increase in inhibition by somatostatin, increased sensitivity of somatotrophs to negative feedback inhibition by IGF-I, decline in pituitary responsiveness to GHRH, and pituitary and/or hypothalamic responsiveness to ghrelin.


Whether the aging pituitary responds normally to GHRH and ghrelin is still a matter of debate. Although earlier studies suggest no age–related decline in GH responsiveness to GHRH (13), more recent reports suggest a gender-independent, age-related decline in the GH responsiveness to GHRH and ghrelin (14) (Figure 4).There is no age-related increase in GH sensitivity to IGF-1 (15); however, there may be relative deficiency in GHRH and ghrelin secretion, and an increase in SRIF secretion, in older individuals (16). The density of GHRS-1a receptors in the hypothalamus decreases with aging and this is thought to be responsible for the age-related decreased response to some GHS (17). The aging pituitary is also less responsive to exercise, sleep, and other physiologic stimuli. Based on these observations it is most likely that the age-related change in GH secretion is multifactorial in etiology and is caused by changes above the level of the pituitary.

FIGURE 4. Approximate peak GH relative change in response to I.V. bolus of Ghrelin and GHRH is diminished in older adults compared to young males. Adapted from Broglio F et al. (14).



Although not a perfect parallel with aging, adult growth hormone deficiency (AGHD) is the best documented source of information on signs and symptoms of reduced GH secretion, effects of treatment, dosing strategies appropriate for adults, and side effects and safety of GH replacement.


Several aspects of normal aging resemble features of the AGHD syndrome, including decrease in muscle and bone mass, increased visceral fat, diminishing exercise and cardiac capacity, atherogenic alterations in lipid profile, thinning of skin, and many psychological and cognitive problems (18-19) (Table 1). Although these changes and the GH deficit of aging are milder than seen in AGHD, they remain clinically significant (20).


Table 1. Features of Adult Growth Hormone Deficiency (3)

Increased fat mass especially abdominal fat

Decreased lean body mass

Decreased muscle strength

Decreased cardiac capacity

Decreased exercise performance

Decreased bone mass

Decreased RBC volume

Atherogenic lipid profile

Thin dry skin

Impaired sweating

Poor venous access

Psychosocial problems-

Ø  low self-esteem

Ø  depression

Ø  anxiety

Ø  fatigue and listlessness

Ø  sleep disturbances

Ø  emotional lability and poor self-control

Ø  social isolation

Ø  poor marital and social-economic performance


It is important to distinguish the normal decrease in GH secretion associated with aging from true AGHD. Although aging is a state of relative physiologic GH deficiency, it is not a disease in itself and is clearly a separate entity from AGHD. This is demonstrated by higher GH secretion and physiological responses seen in older adults when compared with AGHD patients of similar age (2, 20). Moreover, aging per-se is not an indication for AGHD diagnosis testing or administration (21, 22).


Biochemical tests for AGHD diagnosis are imperfect, and their accuracy is strongly affected by the pre-test probability of the condition. Therefore, the most important indicator of the likelihood of GHD is the clinical context (21). In the majority of cases this is due to tumors arising in the region of the sella turcica or the treatment for these tumors including surgery and radiation, but there are other etiologies. Traumatic brain injury is an increasingly recognized cause of AGHD and may occur without coexisting deficiencies in other anterior pituitary hormones (22). IGF-1 levels alone are generally not enough for AGHD diagnosis, hence the need for provocative testing with the insulin tolerance test, glucagon stimulation test, or when available the combined GHRH-arginine test (23).


The GHS and ghrelin mimetic, macimorelin, has recently been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for AGHD diagnosis., Macimorelin provides a simple, orally-available, well-tolerated, reproducible, and safe diagnostic test for AGHD with comparable accuracy to the insulin tolerance test in adults (24). Since studies of macimorelin excluded individuals over the age of 65 or with a BMI >40, the safety and efficacy of this test in these populations has not been established.



The age-related increase in body mass index, changes in body composition, and diminished functional capacity parallel the age-associated decline in growth hormone secretion (18, 19). The alterations in body composition that are most pronounced in normal aging include a reduction in bone density and in muscle mass and strength, an increase in body fat, and adverse changes in lipoprotein profiles (2, 16). This decline in GH production is initially clinically silent, but over time may contribute to sarcopenic obesity and frailty.


The decline in GH may also play a role in cognitive changes observed with aging. One of the many systems for classifying different cognitive domains is grouping them as either “crystallized” or “fluid” intelligence. Crystallized intelligence generally refers to vocabulary and long-term memory; whereas the fluid intelligence includes short term memory and active problem-solving and demonstrates a more marked age-related decline. Several studies have shown a correlation between plasma IGF-I concentrations and performance on tests of fluid intelligence (25), suggesting that GH may play a role in maintenance of fluid intelligence.


Mechanistic insights into the role of growth hormone and IGF-I in age-related alterations in cognitive function were assessed in several studies by Sonntag and colleagues demonstrating somatotrophic effects on rodent brain aging (26). These studies suggest that deficiencies in GH and IGF-I contribute to the functional decline in senescent rats whereas augmentation of GH or IGF-I improved cognitive function, increased glucose utilization throughout the brain, increased cortical vascularity, and ameliorated age-related decline in hippocampal neurogenesis. Although some small studies suggest a positive effect of GHRH or GH replacement on cognition (27), there is insufficient data to recommend GH deficiency testing or GH treatment solely for this purpose (22).




Animal studies have called into question the hypothesis that interventions aiming to increase growth hormone secretion and IGF-I should be considered a net benefit (28). In numerous species from nematodes to rodents, caloric restriction, which lowers IGF-1, has been associated with an increased life span. Mice with growth hormone resistance and profoundly reduced IGF-I levels appear healthy and have increased life expectancy, along with reduced fasting glucose and insulin levels (29, 30). In mice with mutations in the gene necessary for the differentiation of pituitary cells to produce GH, life span increases by 42% and tumor development is delayed (30). These findings are accentuated when combined with caloric restriction, while treatment with GH actually reduces lifespan. Mice treated with a metalloproteinase that cleaves an IGF-binding protein to decrease the bioavailability of IGF-1 have a 38% longer lifespan and a lower incidence of tumors. In experiments conducted with a mouse strain prone to developing age-related cognitive decline and decreased life expectancy, treatment with a GHRH-receptor antagonist resulted in increased telomerase activity, improvements in some markers of oxidative stress, improved cognition, and increased mean life expectancy (31) On the other hand, mice treated with a growth hormone antagonist made by a single amino acid substitution in GH do not show increased lifespan.


The mechanism of increased longevity seen in these mice populations is complicated. Some mouse models with increased lifespan have insulin resistance, while those that develop overt diabetes have a shortened lifespan. Long living mice are either leaner than normal or have increased subcutaneous adipose tissue, both of which may have protective aging effects. Caloric restriction and decreased GH/IGF-1 signaling improves resistance to cellular stress, inhibits mTOR by rapamycin, leading to longevity and may be responsible for enhanced tumor resistance.


The parallel between these results and human senescence is not clear. This last assertion is underscored by the recent report that longevity is not increased, but rather reduced in women in a Brazilian population with a GHRH receptor mutation (32) and in a Swiss cohort of patients with isolated GH deficiency from a homozygous mutation spanning the GH1 gene (33). In an Ecuadorian-kindred with GH receptor deficiency and very low levels of IGF-I, however, rates of cancer and diabetes were markedly reduced compared to unaffected people in the same communities (34). A group of Croatian patients with dwarfism and deficiencies in GH, TSH, prolactin, FSH, and LH as a result of a homozygous PROP1 mutation do not die prematurely, do not develop diabetes mellitus, and have delayed appearance of grey hair (30). Ultimately, although size and lifespan appear to have an inverse relationship in some animal studies, no formal longevity studies have been performed in humans with dwarfism.




A large literature of over 2000 published papers has led to a general consensus that GH replacement can reverse many abnormalities in AGHD patients. Recent reviews of this literature report reduced fat mass, increased lean body mass, improved exercise capacity and cardiac function, improved bone mineral density, and enhanced quality of life by subjective and objective measures (35-37).


The similarities between aging and adult GH deficiency, while not exact, have led to interest in administering GH directly or stimulating GH secretion in aging individuals. However, the starting point and the target are not the same in the two conditions, and we cannot assume safety and efficacy will be the same.


To date, studies of interventions to increase GH effect in elderly subjects include administration of GH, IGF-I, GHRH, and ghrelin mimetic (GHS) either alone or in combination with each other, sex steroids, or exercise. The first studies of GH treatment in non-GHD older adults were performed not long after its effects in AGHD were demonstrated. In 1990 Rudman and colleagues reported that healthy men above the age of 60 who were treated with GH for 6 months responded with an 8.8% increase in lean body mass, a 14.4% decrease in adipose tissue mass, and a 1.6% increase bone mineral density (BMD) only at the vertebral spine (38). Although the change in BMD was quite small it was especially remarkable considering that most studies of AGHD have required one year or more of therapy to show an improvement in bone density. The changes in body composition persisted after one year of growth hormone treatment (39).


Although the Rudman study did not include any functional measures, given these results, it was postulated that GH treatment might also improve muscle strength and functional performance. Despite an absence of demonstrated functional efficacy, some clinics began to prescribe GH treatment to healthy older persons. Acknowledging this growing practice and the lack of information on the subject, the NIH National Institute on Aging issued a call for applications in 1991 to study trophic factors in aging. Several studies of GH, either alone or in combination with sex steroids, IGF-I, or exercise conditioning, and one study of GHRH were funded and have since been completed. These reports generally demonstrate that GH replacement in normal seniors can increase levels of IGF-I to the young adult normal range. However, lower doses of GH were used in subsequent studies to maintain IGF-I levels in the normal range for healthy young adults, since attempts to reproduce the doses of the initial study of Rudman and colleagues led to severe side effects. Following is a summary of the findings of these studies.


Effects of GH Treatment on Strength and Functional Performance


Though GH treatment of otherwise healthy seniors has been shown to have potentially beneficial effects on body composition, studies of physical functional effects have been generally disappointing and inconsistent.


In a relatively large study, comparing the effects of 6 months of growth hormone treatment with placebo in men aged 70 to 85, Papadakis and colleagues reported a 13% reduction in fat mass and a 4% increase in lean body mass in the treatment group, effects consistent with earlier studies; however, there was no effect of growth hormone on knee or handgrip strength or endurance (40). It is important to point out that this seemingly negative result is likely undercut and confounded by the excellent baseline functional status of the study subjects, who were close to the top of the range on many of the tests used, even before treatment. It is very likely that such a "ceiling effect" led to difficulties in demonstrating further improvements due to treatment with growth hormone.


In a separate study, Taaffe and co-workers showed that exercise training improved strength and exercise capacity, but growth hormone treatment did not further augment this effect (41). Several other similar studies have since been completed (42). These studies were conducted for 6–12 months, each at a single site; therefore, only short-term outcomes and side effects, not long-term risks, could be observed. Their results do not provide guidance on the effects of GH on long-term clinical outcomes or “hard” endpoints such as falls or fractures, maintenance of functional status, or effects on cardiovascular morbidity and mortality – outcomes that could establish more definitively the rationale for GH treatment in normal aging. Though few long-term risks have been observed, this is mainly indicative of an absence of information rather than a demonstration of safety. In 2004 a review of various interventions for sarcopenia and muscle weakness in the elderly concluded that GH therapy produces a high incidence of side-effects, does not increase strength, and that resistance training is the most effective intervention for increasing muscle mass and strength in the elderly (43).


In 2007 Liu and colleagues published a systematic review of the safety and efficacy of growth hormone in the healthy elderly (42). After a mean treatment duration of 27 weeks, GH treated individuals had decrease in fat mass of 2.1 kg and an equal increase in lean body mass of 2.1 kg, with no change in weight overall. Total cholesterol levels trended downward (0.29 mmol/L), though not significantly after adjustment for the change in body composition. Other outcomes, including bone density and other serum lipid levels, did not change. Despite higher doses of GH per kilogram of body weight, women treated with GH did not increase lean body mass and achieved only borderline significant decreases in fat mass, indicating a difference in response to GH therapy between genders. Persons treated with GH were significantly more likely to experience soft tissue edema, arthralgias, carpal tunnel syndrome, and gynecomastia and were somewhat more likely to experience the onset of diabetes mellitus and impaired fasting glucose (Figure 5).

Figure 5. Adverse events in participants treated with growth hormone versus those not treated. From Liu H et al. (42)

Effects of GH Treatment on Cognition, Sleep, and Mood


As noted above, rodent studies have shown that GH administration increases brain vascularity and improves performance on some cognitive tests, but systematic tests of cognitive effects of GH treatment in humans are lacking (44). A seemingly contradictory finding was reported in 2017 by Basu and colleagues who demonstrated that spatial learning and memory were improved in 12-month-old GH receptor antagonist transgenic mice when compared to their wild type controls, proposing that GH antagonism as well may have cognitive benefits in aging rodents (45). A trial of GH therapy in patients with Down syndrome showed an increase in head circumference but no effect on cognitive performance (46). Early reports suggested that GH increased deep sleep (SWS), but subsequent studies have failed to confirm this and indeed have found increased sleep fragmentation and reduced total deep sleep (27). While GH treatment of adult GH deficiency improves self-rated quality of life (QoL) scores using any of a number of questionnaires, there are no solid comparable data for normal aging.


Combination Interventions with GH and Sex Steroids


In a 6-month study of healthy men and women over the age of 65 using GH alone or in combination with estrogen/progestin in women and testosterone in men, GH administration increased lean body mass (LBM) in women with or without estrogen/progestin (47). In men, GH and testosterone increased LBM when given alone and had an additive effect in combination. In men, total fat mass decreased with either testosterone or GH alone, but the decrease was greatest with the combination, whereas in women GH decreased fat mass while sex steroids did not change fat mass. Body strength did not improve in women and slightly increased in men only in the GH + testosterone arm of the trial. There was no evidence that co-administration of sex steroids altered the frequency or severity of GH-related side effects.


A 2006 British study randomized healthy older men to 6 months treatment with GH, testosterone patch (Te), or combination of both GH and testosterone (GHTe) and compared results to placebo (48). Both GH treated groups experienced similar increases in lean body mass, while this parameter was unchanged by testosterone treatment alone. Fat mass decreased only in the GH/testosterone combination group. Similarly, mid-thigh muscle cross-sectional area and exercise capacity (VO2 max) was increased only in GHTe and not in the GH or Te groups. There was no difference among the groups in 5 of 6 muscle strength measures except for strength of knee flexion that was found to be increased in the GHTe group. Both GH treated groups reported improvement in a quality of life questionnaire. Overall GH treatment was well tolerated in this study, with most GH-related side effects resolving with dose adjustment.


A study published in 2009 randomized men over the age of 65 having IGF-I levels in the bottom tertile of the reference range, and who were treated with testosterone after a “Leydig cell clamp”, to three groups of daily doses of GH either 0, 5 mcg/kg or 10 mcg/kg (49). After 16 weeks the investigators were able to demonstrate significant synergistic effects of GH treatment, when added to testosterone replacement, on all parameters studied including decrease in total fat mass and truncal fat as well as increase in lean body mass and maximal voluntary muscle strength and aerobic endurance. A slight increase in systolic blood pressure was noted in the study, but did not appear to be related to GH therapy.


Growth Hormone and Exercise


Regular exercise has been shown to increase lean body mass, muscle strength, and aerobic capacity in older men (50). Vigorous exercise acutely stimulates growth hormone secretion, a physiologic response that has been utilized as a screening test for growth hormone deficiency in children.


The growth hormone response to exercise decreases with aging (51). This finding led to speculation that some of the effects of exercise might be mediated via effects on growth hormone and IGF-I. Although exercise stimulates an acute rise in growth hormone secretion, subsequent overnight growth hormone secretion is inhibited (52). In older adults, even intensive exercise does not elevate serum IGF-I level (53). Therefore, the effects of exercise on muscle mass and function seem to be separate from those of growth hormone.


Studies assessing the effects of adding GH to progressive resistance training regimens in older adults have found little to no additional benefit of GH therapy on measures of muscle strength or other measures of muscle composition, but did find that GH therapy led to greater reductions in fat mass than resistance training alone (41, 54, 55).


Adverse Effects of GH Treatment


Side effects observed during clinical trials of growth hormone treatment in normal aging must be taken into consideration in a different way from those in patients treated for adult growth hormone deficiency. The possibility that some of the hormonal changes observed with aging could represent adaptive responses must be considered. Whether increasing growth hormone above the age-appropriate normal range may have as many risks, both acute and delayed, as benefits is a worthwhile hypothesis to examine. The acute side effects of growth hormone are largely hormonal. The most worrisome long-term potential side effect, of special importance in the older population where baseline risk is elevated, is the risk of cancer. Though there is no definitive evidence that GH replacement in AGHD increases the risk of de novo or recurrent malignancy, but several case reports note development of cancer after treatment with GH and because it is a mitogen, the use of GH is contraindicated in active malignancy (35, 36). Nevertheless, GH replacement is not associated with tumor regrowth in AGHD patients with pituitary tumors (56).


Older persons are more sensitive to replacement with GH and more susceptible to the side effects of therapy. The acute side effects are due to the hormonal effects of over-replacement, which can be avoided or relieved with careful dose titration. Patients who are older, heavier, or female are more prone to develop complications (22). Common side effects of GH replacement include fluid retention, with peripheral edema, arthralgia, and carpal tunnel syndrome (Figure 5) Although glucose levels often increase with initiation of GH, these levels generally return toward normal with the improvement in body composition and reduced insulin resistance. However, some studies report persistent elevations in fasting glucose and insulin with chronic GH treatment. Other less frequently reported side effects include headache, tinnitus, and benign intracranial hypertension (22). Hypothyroidism is common in the elderly. GH can accelerate both the clearance of thyroxine and promote its conversion to triiodothyronine, and therefore may have variable effects in hypothyroid patients who are on thyroid hormone replacement.




GHRH and GHS stimulate the secretion of GH. Since most AGHD is caused by pituitary lesions, and these patients, unlike healthy seniors, are unresponsive to GHRH or GHS, there are few studies of treatment with these agents.


Theoretically, treatment with GHRH or GHS should lead to more physiologic GH replacement, leading to a pulsatile rather than prolonged elevation in GH and preserving the ability for negative feedback inhibition of GH by increasing IGF-I. GHRH and GHS effects are influenced by the same factors which modulate endogenous GHRH secretion, such as negative feedback by somatostatin. This normal negative feedback regulation would be expected to result in buffering against overdose. The side effects of GHRH treatment are similar in character to GH treatment but are milder and less frequent. Since the GHS are smaller molecules than GH, and generally resistant to digestive enzymes, they can be administered via the oral, transdermal or nasal routes.


Growth Hormone Releasing Hormone (GHRH)


There are several published trials of GHRH treatment in normal aging (57, 58). Once daily GHRH injections can stimulate increases in GH and IGF-I at least to the lower part of the young adult normal range (57). In a study of 6 months treatment with daily bedtime subcutaneous injections of GHRH(1–29)NH2, alone or in combination with formal exercise conditioning, IGF-I levels increased by 35% (56). Participants had an increase in lean body mass and decrease in body fat (mainly abdominal visceral fat). However, there was no improvement in strength or aerobic fitness with GHRH injections. This study confirmed the benefits of exercise but showed no effect upon IGF-I levels; thus, it appears that GH/GHRH and exercise work through different mechanisms. Subjects receiving GHRH also showed no change in scores on an integrated physical functional performance test of activities of daily living, but there was a significant decline in physical function in the placebo group. This finding, suggesting that GHRH can stabilize if not improve physical function, needs confirmation.


Sleep and cognition were also studied in this GHRH trial, with unexpected results. GHRH failed to improve and may even have impaired deep sleep, despite the rise in IGF-I and pulsatile GH. However, GHRH treatment was associated with improved scores in several domains of fluid (but not crystallized) intelligence – those measures previously found to be correlated with circulating IGF-I levels (25).


A 2006 study of the effects of 6-months daily treatment with sermorelin acetate, a GHRH analogue, on cognitive function of 89 elderly adults found significant improvement on several cognitive assessments, particularly those involving problem solving, psychomotor processing speed, and working memory, but no change on tests reflecting crystallized intelligence (27). Higher GH levels were associated with higher Wechsler Adult Intelligence Scale performance IQ scores, and greater increases in IGF-1 were associated with higher verbal fluency test scores, while gender, estrogen status, and initial cognitive function did not interact with the GHRH effect on cognition.


A 2013 pilot study of 30 elderly adults given a stabilized analogue of GHRH, tesamorelin, versus placebo, used magnetic resonance spectroscopy to examine the effects of inhibitory and excitatory neurotransmitters (60). After 20 weeks GABA levels were increased in all brain regions, N-acetylaspartylglutamate levels were increased in the dorsolateral frontal cortex, and myo-inositol (an osmolyte linked to Alzheimer disease) levels were decreased in the posterior cingulate, with similar results across adults with mild cognitive impairment (MCI) and those with normal cognitive function. Treatment related changes in serum IGF-1 were positively correlated with changes in GABA and negatively correlated with myo-inositol. There was a favorable treatment effect on cognition (p = .03), but no significant associations were observed between treatment-related changes in neurochemical and cognitive outcomes.


The follow-up study of 152 elderly patients on tesamorelin versus placebo included those with amnestic MCI (early stage Alzheimer’s disease) and analyzed executive function, episodic memory, mood, sleep, insulin sensitivity, glucose tolerance, body composition, and IGF-1 levels (61). GHRH had a favorable effect on cognition (P = .002) in both groups. Treatment related increases in IGF-I were associated with higher composite change scores in executive function (p = .03). Visual memory, mood, sleep, hemoglobin A1c, and 2-hour OGTT glucose and insulin responses were not affected in either population, though GHRH treatment was associated with increased fasting plasma insulin levels in adults with MCI. Treatment with GHRH reduced body fat by 7.4% (p < .001) and increased lean muscle mass by 3.7% (p < .001), across both populations. Ultimately though, the clinical significance of these results cannot be assessed as no data was collected regarding functional status.


In a non-controlled 3-month trial of GHRH(1-44)amide in 10 postmenopausal women, increases in both GH and IGF-I levels as well as decreased visceral fat were demonstrated (57). This study also reported improvements from baseline in selected measures of functional performance including timed walking and stair climbing.


Thus, as is the case with GH, studies of treatment of healthy seniors with GHRH have arrived at a consensus on hormonal and body composition effects but inconsistent functional effects. There is a very encouraging but still unconfirmed positive effect on some domains of fluid intelligence.


Ghrelin Mimetics and Growth Hormone Secretagogues (GHS)


Ghrelin, a 28 amino-acid octanoylated peptide, is produced in the stomach and increases before meals and during overnight fasting. Ghrelin acts at both hypothalamic and pituitary levels via mechanisms distinct from GHRH. Ghrelin therefore has different effects from GHRH or GH; subjects often gain body weight, lean and fat mass via a number of GH dependent and independent mechanisms (62). The effects of ghrelin on GH secretion depend in part on the presence of GHRH. If GHRH secretion declines with aging, as is thought to be the case, ghrelin’s effects may be blunted. While the effects of these two GHS differ clinically, they have synergistic effects on GH release, and therefore supplementation of both substances may be more effective than either alone. Nevertheless, ghrelin is more potent than GHRH at eliciting GH secretion (14). Additionally, there are other substances which can enhance GH response to GHS by suppressing somatostatin secretion, including arginine and beta-adrenergic antagonists, which could potentially enhance treatment effects (59).


Several studies have shown short-term effects of GHS on GH secretion, but few studies of their chronic effects in normal aging have been reported. Bowers and colleagues showed that chronic repeated injections or subcutaneous infusions of GH-releasing peptide-2 (GHRP-2) could stimulate and maintain increases in episodic GH secretion and raise IGF-I levels (63).

Results of a one-year double-blind, randomized, placebo-controlled, modified-crossover clinical trial of the Merck orally active ghrelin mimetic MK-677 in healthy high functioning older adults were published in 2008 (64). Daily administration of MK-677 significantly increased growth hormone and IGF-I levels to those of healthy young adults without serious adverse effects. Mean fat-free mass decreased in the placebo group but increased in the MK-677 group. No significant differences were observed in abdominal visceral fat or total fat mass. Body weight increased 0.8 kg in the placebo group and 2.7 kg in the MK-677 group (P = 0.003). Fasting blood glucose level increased an average of 0.3 mmol/L (5 mg/dL) in the MK-677 group (P = 0.015), and insulin sensitivity decreased. The most frequent side effects were an increase in appetite that subsided in a few months and transient, mild lower-extremity edema and muscle pain. Low-density lipoprotein cholesterol levels decreased in the MK-677 group relative to baseline values (change, -0.14 mmol/L) (-5.4 mg/dL) P = 0.026); no differences between groups were observed in total or high-density lipoprotein cholesterol levels. Changes in bone mineral density consistent with increased bone remodeling occurred in MK-677 recipients. Increased fat-free mass did not result in changes in strength or function.


A multicenter trial of the Pfizer investigational oral GHS, capromorelin, in pre-frail older men and women recruited over 300 subjects and was initially planned as a two-year intervention (65). The study was stopped, however, after all subjects had been treated for 6 months and many for 12 months, due to failure to see an increase in percent lean body mass, which was a pre-set non-efficacy termination criterion. Absolute lean body mass did increase significantly, but due to the appetite-stimulating and lipogenic/anti-lipolytic effect of ghrelin mimetics – unforeseen in early 1999 when the study was designed and ghrelin was still unknown – subjects also gained weight (about 1.5 Kg) and this washed out the effect on percent lean body mass. However, even this truncated study is currently the largest clinical trial of chronic GHS treatment in aging. It showed the expected increases in IGF-I levels and (as noted) total lean body mass. There were also encouraging effects on physical functional performance. Of seven functional tests, one improved significantly after 6 months of treatment, and another after 12 months. Two other measures showed non-significant trends toward improvement, and the three remaining measures showed no effect. Effects on clinical endpoints such as falls could not be assessed with this relatively brief duration of treatment. Side effects were generally mild, including increases in fasting blood sugar within the normal range. Interestingly, there was a self-reported deterioration of sleep quality, though formal sleep testing was not performed. Cognition was not studied in this trial. The reasons for the difference in functional outcomes between the two trials are not clear, but it is speculated that this may reflect differences in the populations studied. The MK-677 study recruited a robustly healthy population of seniors in whom further improvement in physical function might be difficult to achieve, while the capromorelin trial was limited to participants already manifesting a decline in function.


Thus, as with GH and GHRH, reports of the hormonal and body composition effects of ghrelin mimetic GHS in normal aging are relatively consistent, but there is no consensus on functional effects among these very few studies, and of course none could assess long-term clinical outcomes or risks.


The novel GHS, anamorelin, is currently under clinical development for cancer anorexia and cachexia syndrome (CACS), a syndrome overrepresented in the elderly.  In a phase II randomized, double-blind, placebo-controlled study, 3 days of treatment increased body weight and appetite in these patients when compared to placebo (66). Over 3 months of treatment, anamorelin increased body weight, LBM, hand grip strength, and quality of life (QOL). Anamorelin also increased IGF-1 and IGF binding protein (IGFBP)-3. It was well-tolerated, but it induced a small increase in glucose and insulin concentrations (67). Unfortunately, two large, international, randomized, double-blind, placebo-controlled phase III studies in patients with advanced non-small cell lung cancer and CACS (ROMANA 1 and 2) did not show improvements in handgrip strength with anamorelin, in spite of increased LBM, fat mass, body weight and appetite-related QoL compared to placebo (68). Although these studies were not restricted to the elderly, the mean age of the population was above 60 years of age in all studies.




While aging is not a disease, it results in alterations in body composition and functional decline with subsequent frailty and loss of independence. Interventions that slow this decline could potentially prolong the capacity for independent living and improve quality of life, but this has not yet been demonstrated. It is unknown whether the decrease in trophic hormones including sex steroids and growth hormone that occur with aging represents an adaptive or pathological process. Aging may represent a milder form of adult GHD, and since GH replacement in frank AGHD has met with success, it may be logical to reason that GH replacement or stimulation by GHRH or GHS might be beneficial in aging. However, older persons are more sensitive to GH, and thus more susceptible to the side effects of replacement. To date, definitive conclusions regarding functional effects of treatments in normal aging aimed at increasing GH levels to those of young healthy persons have been elusive. Until more studies are undertaken to determine the long-term effects of GH and GHS supplementation, conclusive statements about the merits of treatment cannot be made. Long term studies on hard clinical endpoints, such as falls and fracture rates, function measures, quality of life, and decreased morbidity and mortality from vascular disease need to be performed in order to establish the role, if any, for GH and GHS treatment in normal aging. In the meantime, GH use for anti-aging purposes is currently prohibited by US federal law (69, 70).




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Surgical Treatment of Pituitary Adenomas


The overwhelming majority of pituitary adenomas are benign and present either with characteristic syndromes of excess hormone secretion or secondary to mass effect by the growing tumor. The common hypersecretory syndromes include Cushing’s disease, acromegaly/gigantism, and hyperprolactinemia. Local mass effects on the pituitary can cause varying degrees of hypopituitarism. As the tumor grows beyond the confines of the sella turcica, the visual pathways are commonly affected and visual field deficits are present. Effective medical therapy is available for prolactin secreting adenomas. With the exception of these tumors, transsphenoidal surgery remains the first-line treatment for most other pituitary adenomas. Medical therapy for growth hormone secreting adenomas and for Cushing’s disease continues to evolve.




Pituitary adenomas may be classified according to their clinical/radiographic characteristics (Table 1) and, more recently, their cell lineage (Table 2). Those tumors that measure less than 10 mm in diameter are considered microadenomas; macroadenomas are those 10 mm or larger (Fig. 1A, B, C, and D). Macroadenomas may also be sub-categorized as "giant" if their extent reaches far beyond the normal confines of the pituitary region or their greatest diameter exceeds 4cm (Fig 1E, F, and G). Pituitary adenomas may also be categorized based on their functional/secretory status. The hypersecretory adenomas cause distinctive clinical syndromes that include acromegaly/gigantism caused by growth hormone (GH) secreting adenomas, the classic Forbes-Albright syndrome (amenorrhea-galactorrhea) caused by prolactin (PRL) secreting adenomas, TSH-secreting adenomas, the occasional hypersecreting FSH/LH adenoma, and Cushing's disease/Nelson’s syndrome caused by corticotropin (ACTH) secreting adenomas. The non-functioning adenomas (NFAs) are “silent” and only perturb the endocrine system due to mass effects on the normal gland causing hypopituitarism (decreased pituitary hormone production) and generally present either incidentally, due to visual loss, or with secondary subtle hormonal abnormalities. The new histopathological classification considers the majority of tumors to be clinically silent gonadotropin tumors staining for SF-1. The next category is the true null cell adenoma which stains for no pituitary hormones with none of the other transcription factors or hormones being detected.


Table 1. Clinical/Radiographic Classification Schemes of Pituitary Adenomas



Microadenoma/ Macroadenoma

 £ 10 mmm/ > 10 mm

Non-Functioning adenoma


Functioning adenoma


 Endocrinologically inactive, patient may present with pituitary deficiency or cranial nerve deficits (CN 1 most commonly)


Excess of pituitary hormone secreting:  GH adenoma; PRL adenoma; ACTH adenoma; TSH adenoma; GH -PRL adenoma; FSH/LH adenoma (rare, most are non-functioning)


Other plurihormonal hypersecretory adenomas

Abbreviations: CN = cranial nerve, GH = growth hormone, PRL = prolactin, ACTH = adrenocorticotropic hormone, TSH = thyroid stimulating hormone, FSH = follicle stimulating hormone, LH = luteinizing hormone

Figure 1. Tumor Classification based on size. Microadenoma: Coronal and sagittal T1 weighted MRIs with contrast with arrow indicating the location of the tumor (A and B). Macroadenoma: Coronal and sagittal T1 weighted MRIs of a typical macroadenoma (C and D). Giant invasive macroadenoma: Coronal and sagittal T1 MRIs with contrast in a patient in whom the tumor compresses the right temporal lobe and invades the sphenoid sinus (E and F). In another patient, the sagittal MRI reveals a tumor that has not only invaded the sphenoid sinus but compresses the brainstem; the tumor is highlighted (G and H).

The new cell lineage classification system of pituitary adenomas is a result of recent studies which have uncovered the shared transcription factor profiles present in adenoma cell lines [1]. For detailed information on the pathology and pathogenesis of pituitary adenomas, see the corresponding Endotext chapter. The most common transcription factor profile is PIT1, which is shared by somatotroph, lactotroph, and thyrotroph adenomas.  PIT1 mediates differentiation, expansion, and survival of these three cell types (Table 2). In adenomas, evidence supports an HMGA mediated upregulation of PIT1 [2]. HMGA genes are usually active during embryogenesis but not in normal adulthood [3]. A new paradigm has evolved, which generally begins with transcription factor mediated monoclonal expansion of a single cell line followed by variable differentiation and retention of secretory capability. Patients harboring multiple pituitary adenomas present a unique scenario in which the true pathogenesis and pathogenetic process underlying neoplastic growth could involve distinct multicentric monoclonal expansion (“Multiple-Hit Theory”) or adenoma transdifferentiation across cell lines (“Transdifferentiation Theory”) [4].


Table 2. Cell Lineage Classification of Pituitary Adenomas [1]


 Cell type



Transcription factor profile



GH ± PRL ± a-subunit




PIT1, ER-a


TSH-b, a-subunit








FSH-b or LH-b or a-subunit



Null cell



Abbreviations: GH= growth hormone, PRL= prolactin, TSH = thyroid stimulating hormone, ACTH = adrenocorticotropic hormone, LMWCK = low molecular weight cytokeratin




Pituitary adenomas account for approximately 10 to 15% of surgically-treated primary tumors of the central nervous system (CNS) [5-9]. The incidence appears higher in African Americans in whom pituitary adenomas account for over 20% of the non-metastatic CNS tumors [10, 11]. The incidence rate of pituitary tumors has increased from 2.5 to 3.1 per 100,000 per year (annual percentage change of 4.25%). Although the incidence varies according to age, sex, and ethnic group, between approximately 0.5 and 8.5 per 100,000 in the population are diagnosed annually with a pituitary adenoma [5, 12-14]. In a large cohort study between 2004 and 2009, the largest incidence peak was 8.5 for males 75-79 years old [14]. Autopsy series indicate that pituitary tumors are quite common, and that nearly 25% of the population may harbor undiagnosed adenomas [15, 16]. The majority of these tumors are less than 3-5 mm in diameter and would not require medical or surgical intervention. More recent series using magnetic resonance imaging (MRI) of healthy subjects indicate that approximately 10% of the population harbors pituitary lesions. Some series report a higher rate of diagnosis among women of childbearing age, despite a similar incidence in women and men  [5, 13]. Because disruption of the hypothalamo-pituitary-gonadal axis in women is more evident in women than men, women with pituitary adenomas may present to clinical attention at a higher rate, and earlier, than men.


Amongst the varying classes of adenomas, prolactinomas and non-functioning adenomas have the highest incidence, and account for nearly two-thirds of all pituitary tumors. Prolactin-secreting adenomas comprise 40 to 60% of functioning adenomas and are the most common subtype of pituitary tumor diagnosed in adolescents [6]. The majority of microadenomas are found in women in their second and third decades. Men generally present later, in their fourth and fifth decades, almost always with macroadenomas.


GH secreting adenomas represent approximately 20-30% of all functioning tumors. Nearly three quarters of GH secreting adenomas are macroadenomas. Approximately 40 to 60 individuals per million have acromegaly [17-19]. Between 3 and 4 new cases per million are diagnosed annually [17-20]. Most present in their 3rd to 5th decades after they have been developing symptoms and signs for many years  [18]. Acromegaly has been associated with an increased incidence of cardiovascular, respiratory, and cerebrovascular disease, as well as an increased risk of colon cancer. Studies have reported an increased risk of mortality compared to the unaffected population [17, 20]. Although some studies report a higher incidence of several cancers, others have only confirmed an increased risk of colon cancer  [21, 22]. There is some evidence that mortality risk may be different between the sexes. Etxabe et al. found a higher mortality rate in men than in women  [18]. Other reports found similarly increased mortality in both sexes  [23]. Still others report increased risks of death in men from cardiovascular, respiratory, cerebrovascular, and malignant disease, but only from cerebrovascular disease in women  [17].


ACTH adenomas account for 15 to 25% of all functioning adenomas and are the most common pituitary tumors diagnosed in pre-pubertal children [6]. The majority of ACTH adenomas, regardless of age, are microadenomas. Approximately 39 individuals per million have Cushing's disease from an ACTH-secreting adenoma and the annual incidence is estimated at 2.4 per million [24]. Cushing's disease is more common in women, most of whom present in their third and fourth decades [24, 25]. There is a high incidence of hypertension and diabetes mellitus as well as higher vascular disease-related mortality [24, 26]. Nelson’s syndrome can develop after adrenalectomy in patients with Cushing’s disease, as negative feedback is then lost to a previously unrecognized intrasellar ACTH adenoma.  These patients may develop hyperpigmentation and the ACTH-secreting pituitary tumors are often aggressive.




Advances in neuroimaging, namely CT, CT angiography and particularly magnetic resonance imaging (MRI) have improved the visualization of the pituitary region. Increasing numbers of adenomas are diagnosed incidentally during the evaluation of sinus disorders (15%), trauma (19%), and stroke (15%), among others. These "incidentalomas" are not necessarily asymptomatic. Visual deficits are present in 5-15% of cases and up to 50% when formal testing is employed [27]. Some degree of pituitary dysfunction is found in up to 15-30% [27, 28]. More than one third are macroadenomas and, of these, approximately 30% will show significant enlargement over time [28-31]. Small asymptomatic incidental microadenomas are less likely to have clinically significant growth and often can be followed over time with repeated MRIs.

Although increasing numbers of tumors are diagnosed incidentally, pituitary adenomas more often present secondary to hypersecretion, hypopituitarism, or mass effect (Table 3).


Table 3. Presenting Features of Pituitary Adenomas


GH-secreting adenoma: Acromegaly

ACTH-secreting adenoma: Cushing's disease/Nelson’s syndrome

Prolactin-secreting adenoma: Amenorrhea-galactorrhea

TSH-secreting adenoma: Secondary hyperthyroidism

Pituitary insufficiency

Symptoms: diminished libido, fatigue, weakness

Gonadal dysfunction, Hypothyroidism, Adrenal Insufficiency, Somatotroph Insufficiency

Mass Effect (symptoms related to compressed adjacent structures)

Optic chiasm: bitemporal visual field deficit and diminished visual acuity

Cavernous sinus: trigeminal nerve, facial pain; cranial nerves III, IV, VI, diplopia, ptosis, mydriasis, anisocoria

Pressure on dura or diaphragma sellae: headache

Hypothalamus: behavior, eating, and vigilance disturbances (somnolence)

Temporal lobe: complex partial seizures, memory and cognitive disturbances


Discovered during the evaluation for headaches, trauma, nasal sinus disorders, dizziness


Hypersecretory Syndromes

(for detailed descriptions see corresponding chapters in Endotext)


Acromegaly induces characteristic growth hormone-induced structural changes in physiognomy. There is an insidious coarsening of facial features with an enlarged forehead, enlarged tongue, malocclusion of the teeth, and prognathism (Fig 2). Patients' hands and feet also enlarge. Many patients also report excessive sweating (hyperhidrosis). The external hypertrophy of tissue is paralleled within the body. Patients suffer enlarged organs (visceromegaly) and overgrowth of joints and cartilage, along with high blood pressure, congestive heart failure, sleep apnea, spinal canal narrowing (facet hypertrophy), and carpal tunnel syndrome. Significant numbers of patients with acromegaly also have impaired glucose metabolism and diabetes mellitus.

Figure 2. Acromegaly. A. Coronal T1 weighted MRI with contrast in a patient with an intrasellar GH secreting adenoma. Arrows indicate the common finding of “cutis gyrata”. B. Sagittal T1 weighted MRI in the same patient with arrows indicating the frontal bossing and the enlarged frontal sinus, and * the tumor.

Cushing's disease causes changes in body habitus with characteristic increased weight gain, truncal obesity, "buffalo hump", and moon facies. Skin changes are also common and include purple striae, easy bruisability, ruddy complexion, and increased body and facial hair. Patients suffer from fatigue, proximal muscle weakness, osteoporosis, psychological/psychiatric disorders, high blood pressure, and impaired glucose metabolism. They often have headache, menstrual disorders, and cognitive dysfunction.


Women with prolactinomas classically present with amenorrhea or oligomenorrhea and galactorrhea. Most are in their childbearing years, and are more likely to pursue medical attention for infertility and menstrual irregularity. Men, and women beyond their reproductive years, more often have headache, visual symptoms, sexual dysfunction, and signs of decreased pituitary function. Amenorrhea and galactorrhea are not specific to prolactinomas, however. Prolactin secretion is under constant inhibitory control from the hypothalamus. Any lesion that imposes pressure upon the portal venous connection of the pituitary stalk (infundibulum) connecting the hypothalamus and pituitary gland can interrupt these inhibitory dopaminergic signals.  This, in turn, causes an increase in serum prolactin levels, and mimics a prolactinoma, i.e. a 'pseudo-prolactinoma'. In such cases serum prolactin levels are usually only moderately elevated. As a general rule, serum prolactin levels over 200 ng/ml (3600mU/L) are indicative of prolactinomas [32].




Tumor growth impairs the normal secretory function of the anterior pituitary and causes hypopituitarism. Common complaints include diminished sex drive, fatigue, weakness, and hypothyroidism. Pituitary insufficiency generally develops slowly over time.  However, acute pituitary insufficiency may occur in the setting of pituitary apoplexy, a condition in which the tumor infarcts or has internal bleeding (Fig 3). Apoplexy can be particularly devastating because it combines acute hypopituitarism and adrenal insufficiency with a rapidly expanding intracranial mass, and often causes visual loss or even sudden blindness.

Figure 3. Pituitary apoplexy. Sagittal T1 weighted MRI without contrast in a patient presenting with pituitary apoplexy. Note the fluid-fluid level within the tumor indicative of the apoplectic tumor.

Neurological Dysfunction


Neurologic signs and symptoms develop as adenomas grow beyond the confines of the sella turcica and exert pressure upon adjacent brain structures. As tumors enlarge, they compress the optic nerves and optic chiasm, and patients experience visual deficits and diminished visual acuity. Classically this causes a bitemporal hemianopia, i.e. visual loss in the temporal fields of each eye. Tumor growth may also affect other nerves (such as the 3rd, 4th, 5th, or 6th cranial nerves) and cause facial pain and/or double vision or drooping of the eyelid. Headache, although a non-specific complaint, can occur when a tumor stretches the dural sac that surrounds the pituitary gland. Headache from pituitary lesions is usually frontal or retro-orbital – it may be bitemporal or radiate to the occipito-cervical region.  Many patients will have been previously diagnosed with “migraine”, or “tension-headache” [33].




A panel of endocrinological tests can often confirm the clinical diagnosis of pituitary adenoma. Serum GH and IGF-1 levels screen for acromegaly. Failure to suppress GH levels after an oral glucose load (oral glucose tolerance test [OGTT]) can further confirm the diagnosis. Although any macroadenoma may cause moderate increases in serum PRL, levels greater than 200 ng/ml (3600 mU/L) are highly suggestive of a prolactin secreting adenoma. Dilution of the samples for assay may be necessary to avoid the “hook effect” related to macroprolactinemia.

Endocrinologic studies that suggest Cushing's disease includes an elevated ACTH and late night salivary or elevated 24-hour urine free cortisol (UFC), loss of the normal diurnal variation in cortisol levels, and suppression of serum cortisol levels after high dose dexamethasone but failure to suppress after low dose dexamethasone. Inferior petrosal vein sampling after corticotropin-releasing hormone (CRH) stimulation (i.e., Inferior Petrosal Sinus Sampling; IPSS) may be required to confirm and localize the pituitary source. At times, prior to diagnosing Cushing's disease, other ectopic sources of excess ACTH, such as bronchogenic or pancreatic carcinoma and pulmonary carcinoid tumors, must be excluded. This can often be accomplished with a CT scan or MRI of the chest and abdomen and with novel nuclear imaging tests [34, 35]. Obesity, alcoholism, and depression also elevate serum cortisol levels, and the diagnosis of Cushing's disease should be made with caution in these “pseudo-Cushing’s” settings [36].




Although some incidentally-discovered microadenomas that do not cause symptoms may be followed clinically and with repeated MRI, patients with macroadenomas generally need medical or surgical intervention. Therapeutic goals are improved quality of life and survival; elimination of mass effect and reversal of related signs and symptoms, normalization of hormonal hypersecretion; preservation or recovery of normal pituitary function, and prevention of recurrence of the pituitary tumor.




Medical therapy is available for most hypersecretory tumors [37-40]. Most prolactin-secreting adenomas are effectively treated with dopamine agonists (bromocriptine and cabergoline). Cabergoline is generally preferred due to a better side-effect profile, and between 80-90% of patients can achieve hormonal control [37]. Surgical intervention is ordinarily reserved for those who are intolerant of medical therapy due to side effects (e.g. nausea, headache, impulsive or compulsive behavior), whose prolactin levels remain elevated, or whose tumors continue to grow despite maximal medical treatment.


Medical treatment using somatostatin analogues (octreotide, lanreotide, and pasireotide) and dopamine agonists (cabergoline) have varying degrees of efficacy for treating GH adenomas.  The growth hormone receptor antagonist, pegvisamont, can be used in combination with other agents [41-43], and hormonal control can generally be achieved in about 60-90% of patients [37].  Although medical therapy is most often reserved for those patients awaiting surgery or those with persistent disease postoperatively, some advocate primary medical therapy, particularly for invasive tumors [44, 45]. There is some conflicting evidence that pre-surgical medical therapy may improve surgical outcome [46].


Ketoconazole and/or metyrapone therapy can normalize serum cortisol levels in patients with Cushing's disease preoperatively 50-75% of the time. Metyrapone and ketoconzaole inhibit enzymes in the adrenal gland required for steroid synthesis. Like in acromegaly, surgery remains the first-line therapy. Clinical trials have also demonstrated some role for medical therapy with cabergoline or pasireotide, and with mifepristone (cortisol receptor blocker) in selected cases [47, 48].


The disadvantage of medical treatment of hypersecretory syndromes is that it is suppressive in nature and not cytotoxic. Tumors often recur when medications are discontinued, or they become resistant to therapy. Potential new targets are being explored but have not yet reached clinical practice [49-51].




Radiotherapy is most often employed in conjunction with medical or surgical therapy. Fractionated external beam radiation therapy can reduce excessive hormone production and can reduce the incidence of tumor recurrence [52]; however, it can be replaced by  stereotactic radiotherapy with focal conformal fractionated delivery. Gamma knife, Cyberknife, proton beam or linear accelerator stereotactic radiosurgery is increasingly applied to pituitary tumors, and is also effective in normalizing hormonal hypersecretion and preventing recurrence  [53-55]. Whether by fractionated external beam or radiosurgery, the effects of radiotherapy are delayed. Patients require continued suppressive medical therapy during the period between treatment and effect. There is also a significant incidence of radiation-induced delayed hypopituitarism [52]. There is no evidence to date that one of these various modalities is superior to another in efficacy, risks of complications, recurrence rates, or incidence of hypopituitarism. For more information on radiotherapy for pituitary tumors, see the corresponding chapter in Endotext.




Indications for Surgery


For most pituitary tumors, surgery remains the first-line treatment of symptomatic pituitary adenomas. Large or invasive asymptomatic tumors may also warrant surgical consideration. It is sometime possible to estimate a tumor’s invasiveness on an MRI using the Knosp grading system [56]. Asymptomatic tumors with evidence of radiographic invasion or displacement of the optic apparatus may benefit from surgery to prevent neurological deficits and progressive pituitary dysfunction. Surgery is also chosen secondarily when medical treatment fails for the treatment of prolactinoma. Regardless of the tumor type, surgery provides prompt relief from excess hormone secretion and mass effect. There is evidence to suggest that debulking of medically refractory prolactinomas and GH adenomas can return these tumors to a responsive state [57, 58]. Rarely is surgery recommended as first line therapy for prolactinomas [59].  Surgery may be indicated in pituitary apoplexy with acute vision loss £ 72 hours due to mass effect on the optic chiasm from hematoma formation. Studies have shown that some patients with pituitary apoplexy can be successfully treated without operative intervention, but they are often confounded by selection bias, and the ideal patient has not been conclusively established for operative versus non-operative treatment [60-62].


Peri-Operative Management


A major component of the surgical management of patients with pituitary tumors actually occurs in the peri-operative period. Detailed information on peri-operative management of pituitary tumors can be found elsewhere [63]. Briefly, pre-operative planning is very important in order to avoid complications and achieve optimal outcomes. It is obligatory to note any prior nasal surgery, review prior imaging, and obtain adequate pre-operative imaging for integration with neuronavigational systems. Typically, a high resolution T1 post contrast MRI is adequate for neuronavigational registration. The authors advocate additional imaging that includes (1) coronal and sagittal T1-weighted pre and post contrast images with at least 3mm slice thickness through the parasellar region for identification of the tumor, pituitary gland/stalk, cavernous sinus, and vasculature, (2) axial T2-weighted images of the sella to measure intercarotid distance, and (3) a coronal and sagittal strong T2-weighted Constructive Interface in Steady State (i.e., CISS, also known as FIESTA) through the parasellar region to identify midline structures and the optic chiasm. For revision surgery, a CT scan of the sinuses can be helpful to identify abnormal osseous anatomy. The imaging should be reviewed to identify normal gland and pituitary stalk, look for cavernous sinus invasion, identify arachnoid diverticula, and verify anatomical landmarks. Finally, it is critical to assess pre-operative pituitary function and replete necessary hormones (especially cortisol and thyroid hormones) prior to surgery. Remember to replete cortisol before thyroid hormone to avoid precipitating an adrenal crisis. For more information on the evaluation and management of pituitary hormone deficiency, see corresponding chapters in Endotext.


Post-operative management varies from routine to very complicated depending on the lesion size and extent of the operation and post-operative pituitary function. Patients with complete removal of intrasellar non-functioning tumors and intraoperative preservation of the normal pituitary gland without a cerebrospinal fluid (CSF) leak can have a relatively benign post-operative course. It is important to monitor closely for diabetes insipidus (DI), check a fasting morning cortisol to rule out secondary adrenal insufficiency, and restrict fluids as appropriate to prevent the syndrome of inappropriate anti-diuretic hormone (SIADH) [64]. Patients with larger suprasellar or invasive tumors and/or those with CSF leaks requiring more extensive skull base reconstructions may require ICU care [65, 66]. For information on the management of endocrine dysfunction and post-operative care in Cushing’s disease and Acromegaly, please see the corresponding chapters in Endotext.


Surgical Technique


The minimally invasive transsphenoidal approach can be used effectively for 95% of pituitary tumors. Exceptions are those large tumors with significant temporal or anterior cranial fossa extension. In such circumstances, transcranial approaches are often more appropriate. Occasionally, combined transsphenoidal and transcranial approaches are used. Nevertheless, some surgeons extend the basic transsphenoidal exposure in order to remove some of these tumors and avoid a craniotomy (Fig. 4) [67-70].


The transsphenoidal approach is a versatile method for treating pituitary tumors (Table 4). Endoscopic approaches may be used in isolation or as an adjunct to the other transsphenoidal approaches (Fig. 4) [71-78]. Computer-guided neuronavigational techniques are nearly ubiquitous at major pituitary centers in lieu of traditional fluoroscopic guidance (Fig. 5) [79, 80]. The role of neuronavigation is most pertinent in recurrent adenomas in which the midline anatomy has been distorted by previous transsphenoidal surgery. Intraoperative MRI is increasingly available and appears to be most applicable for large tumors [81].  There are three basic variations of the transsphenoidal approach.

Figure 4. Endoscopic approach. Intra-operative photograph of one surgeon (left) driving the endoscope while the main surgeon (right) resects the tumor.

Figure 5. Modern operating room set up for endoscopic endonasal surgery. Stereotactic navigation system shown to the left (A) and endoscopy screens in the center (B) facing the surgeon who will stand to the patient’s right just next to the shoulder. The patient is positioned supine in a beach chair position (C) with arms tucked and head in a skull clamp. Preoperative images may be stored and used for image guidance during operative procedures after configuring the navigation reference frame and registering the patient’s head using surface landmarks.

Table 4. Transsphenoidal Surgery for Pituitary Adenomas: Personal Summary of 3744 Cases over a 36 year period

Type of Adenoma

Number of Patients (%)

Functioning adenomas


GH adenoma (Acromegaly)

662 (17.7)

PRL adenoma

975 (26.0)

ACTH adenoma (Cushing's disease)

680 (18.2)

TSH adenoma

45 (1.2)

Non-functioning adenomas

1382 (36.9)



The patient is placed in a lawn-chair position and a hemi-transfixion incision is made just inside the nostril so that the scar cannot be seen after surgery (Fig. 6). Most often the entire procedure can be accomplished endonasally. Conversion to a sublabial approach may be necessary for large macroadenomas and children in whom the exposure through one nostril is sometimes inadequate. A submucosal plane is developed along the nasal septum back to the level of the sphenoid sinus. Bone of the septum can be harvested for use later in the operation. The bone in front of the pituitary gland is also removed, the dura opened, and tumor is extracted in fragments (Fig. 7). Afterwards the saved bone, cartilage, or artificial material can be used to refashion the normal housing of the pituitary gland. Closure is rapid and consists of several interrupted absorbable sutures in the nasal mucosa and temporary nasal packing to promote healing of the mucosa.

Figure 6. Standard positioning for the endonasal approach (above). Below left, endonasal hemitransfixion incision; below right, direct sphenoidotomy technique.

Figure 7. Left, standard endonasal approach showing the trajectory to sella in sagittal view; Right, sequential steps used in tumor removal and repair of the sellar floor common to all techniques.


This approach uses incisions deeper within the nasal cavity (Fig 6, lower right. The incision for the septal pushover technique is made at the junction of the cartilaginous and bony septum. Submucosal tunnels are developed on either side of the bony septum until the sphenoid sinus is reached. Another option to reach the sphenoid sinus is by performing a direct sphenoidotomy. Using this method, no incision is made in the septum. Instead, the posterior part of septum just in front of the sphenoid sinus is deflected laterally and the sphenoid sinus is entered directly. There are several advantages to these techniques. Because there is no submucosal dissection of the cartilaginous septum, the risk of an anterior nasal septal perforation is eliminated. In addition, there is less need for nasal packing postoperatively, a frequent cause of postoperative pain and discomfort. The main drawback of these more direct approaches is that the exposure is not as wide as can be achieved by the standard endonasal transseptal approach in which the cartilaginous septum can be more extensively mobilized.



The pure endoscopic approach has much appeal and is becoming the procedure of choice at many pituitary centers  [82, 83]. Surgery begins at the sphenoid rostrum where a direct anterior sphenoidotomy is performed after identifying the natural sphenoid os within the sphenoidoethmoidal recess. Some surgeons prefer to perform the surgery using a single nostril. A binostril approach, however, provides more maneuverability and two-handed microdissection. To achieve an adequate exposure for the binostril approach, the middle and superior turbinates are lateralized and the bony septum just in front of the sphenoid sinus is removed. The sphenoidotomy is widened from the midline inferior vomer to the ethmoid air cells superiorly and then laterally until the carotid arteries are easily visualized (Fig 8-A). This allows instruments to be used in both nostrils simultaneously. Although a specialized endoscope holder may be used during tumor removal, the “3-hand” technique is advocated by many surgeons. The “3-hand” or “4-hand” technique requires two surgeons; one surgeon maneuvers the endoscope while another has both hands free to remove the tumor using microsurgical techniques. The surgical team is typically a neurosurgeon and otolaryngologist with experience in skull base surgery. Extended approaches are more commonly performed by teams rather than individuals [80, 84]. The endoscope provides panoramic magnified views of the sellar anatomy during both the approach to and resection of tumors (Fig 8 – A, B). The option of using angled endoscopes allows surgeons to inspect for residual tumor, particularly along the cavernous sinus walls and the suprasellar region [85] (Fig 8 – C, D). No nasal packing is required as the procedure is performed posterior to the septum. The main disadvantages are the procedure’s learning curve and that the depth of field may problematic for some surgeons. There are 3D endoscopes and continued development of High Defintion (HD) imaging that may help to alleviate this potential problem. A recent international survey showed that about 7% of surgeons report using the 3D endoscope for transphenoidal surgery. Advances in patient specific anatomical modeling is increasingly available for integration with the neuronavigation in the form of “augmented reality” which helps the surgeon visualize otherwise hidden anatomical structures [86]. Finally, given the importance of vision preservation during endonasal surgery, especially with extended approaches, new developments in visual evoked potential monitoring are being studied [87]. The clinical benefit of these new technologies is promising but still uncertain.

Figure 8. Endoscopic views. A. After the anterior wall of the sphenoid sinus is opened, the endoscope provides a panoramic view of the sella and surrounding anatomy. B. Endoscopic view of the tumor bed after resection. C. Endoscopic view of the right cavernous sinus wall using the 0 degree endoscope. D. Note the dramatically improved view of the right cavernous sinus wall in the same patient using the 45 degree endoscope. (arrowhead= carotid artery)



Surgical outcomes after surgery for pituitary adenomas can be divided into functional outcomes and oncologic outcomes. Functional outcomes goals include the relief of symptoms and improvement or preservation of pituitary and visual function, and improved quality of life [88-90]. Visual deficits in patients with non-functioning pituitary adenomas are improved in approximately 80-90%. Some visual deterioration may occur in 0-4%. Most patients with intact pituitary function preoperatively retain their normal function. Those with preoperative pituitary deficiency regain function in 27% of the cases. The remaining patients are managed with hormone replacement therapy. Oncologic outcomes relate to tumor resection, recurrence, and biochemical remission from hormonal excess. Ten-year recurrence rates are approximately 16%, although only 6% require additional treatment (Table 5). On long-term follow-up, 83% of patients are alive and well without evidence of disease.


Table 5. Results of Transsphenoidal Surgery, Personal Summary of 3093 Cases over a 28 year period. Proportions (%) represent cumulative incidence.



10-year Recurrence

Non-functioning adenoma

Not applicable*


GH adenoma








PRL adenoma








ACTH adenoma





12% (Adults)

42% (Pediatric)



 *Visual improvement occurs in 87% of those with preoperative visual loss.


Currently, using strict criteria for remission and in expert hands, transsphenoidal surgery obtains remission in 85-90% of patients with acromegaly with microadenomas and 65% of those harboring macroadenomas. For functional tumors, remission rates vary by tumor size and tumor type [91]. Microadenomas typically have higher biochemical remission rates and remission rates are highest for microprolactinomas (92.3%) and lowest for somatotroph macroadenomas (40%). In our hands, acromegalic symptoms are improved in 95% and recurrence is less than 2 percent at ten years. Ninety seven percent of patients have preserved normal pituitary function  [92]. Modern criteria for remission include normal IGF-1 levels and either GH suppression to less than 0.4 ng/ml with oral glucose tolerance test or GH random less than 1.0 ng/ml. Using these criteria, surgical biochemical remission is over 60% [93]. Both repeat surgery and medical therapy are options for those with residual disease and/or biochemical recurrence [37, 94].


Patients with prolactinomas who present for surgery are most often those who have failed medical management. Endonasal surgery for prolactinomas is associated with additional risks due to tumor fibrosis from dopamine agonist therapy but remission rates are still quite good. Prolactin levels are normalized in about 87% of microadenomas and 56% of macroadenomas (Table 5). The recurrence rate among those patients who are normalized after a transsphenoidal operation is 13% at ten years. Preserved pituitary function occurs in all but 3%.


Surgical management of Cushing's disease achieves a 91% remission rate for microadenomas, but falls to 65% for those with macroadenomas. Some 10-20% of adults experience recurrence after ten years. Postoperative stereotactic radiosurgery has achieved remission in approximately 60-70% of patients whose disease either did not remit following surgery or recurred [95].


As pituitary surgeons, like all health professionals, strive in the pursuit of excellence in the care of our patients, it is becoming clear that criteria must be developed in order optimize surgical outcomes. Recently, a consensus statement on Pituitary Tumor Centers of Excellence (PTCOE) was released [96]. In brief, PTCOE should be independent non-for-profit organizations, widely recognized by endocrinologist and pituitary surgeons, aimed at the advancement of pituitary science and the highest quality of patient care. They should also be recognized by external societies and act as resident training centers.


Complications of Transsphenoidal Surgery


Complication avoidance is central to transsphenoidal surgery given the close proximity of major neurologic and vascular structures [97, 98]. Recently, surgical checklists for endonasal transsphenoidal surgery have been developed in order to optimize surgical outcomes and avoid complications [99]. The overall mortality rate for transsphenoidal surgery is less than 0.5% (Table 6). Major morbidity (cerebrospinal fluid leak, meningitis, stroke, intracranial hemorrhage, and visual loss) occurs in between 1 and 3% of cases. Less serious complications (sinus disease, nasal septal perforations, and wound issues) occur in approximately 1-7%. Larger invasive tumors and giant adenomas are associated with a higher morbidity. In the modern era, more aggressive extended approaches to large invasive tumors has led to a higher incidence of CSF leak, but the use of the pedicled nasoseptal flap has been largely successful in preventing recurrent leaks with a success rate of up to 98.6% [100]. The nasoseptal flap can also be reused in certain revision cases with good results [101].


Table 6. Complications of Transsphenoidal Surgery (1972-2017). Personal historical series and a modern results covering a 45 year period and 4,246 cases.

 Outcome Measure

Cumulative Incidence (%)



1992-2017 [102]




Major complication: (CSF leak, meningitis, ischemic stroke, intracranial hemorrhage, vascular injury, visual loss)


CSF leak 2.6%

Other 3.2%

Minor complication: (sinus disease, septal perforations, epistaxis, wound infections and hematomas)






Pituitary adenomas are a complex set of benign tumors that present with characteristic hypersecretory syndromes and mass effect. Although medical and radiotherapy offer effective treatment for particular functional tumors in specific situations, transsphenoidal surgery continues to provide optimal outcomes for non-prolactin secreting adenomas with a low incidence of major morbidity.




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