ABSTRACT

Adult growth hormone deficiency (GHD) is a clinical syndrome that can manifest either as isolated or associated with additional pituitary hormone deficiencies. Its clinical features are subtle and nonspecific, requiring GH stimulation testing 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 perform a GH stimulation test 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 remains the gold standard test, while the glucagon stimulation test and macimorelin test are reasonable alternatives to the insulin tolerance test, whereas the arginine test is no longer recommended because arginine is a poor GH secretagogue that requires a very low peak GH cut-point of 0.4 μg/L. In this chapter, we discuss 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 based on the clinical suspicion of having adult GHD, and summarize current knowledge and change of status of availability of the oral macimorelin test in the United States.

INTRODUCTION

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) that 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.

DIAGNOSIS OF ADULT GH DEFICIENCY: CURRENT PERSPECTIVE

Adult GHD is a rare 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 many, but not all, 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 safety risks, particularly the development of diabetes mellitus, cancer and tumor recurrence, it is imperative that an accurate biochemical diagnosis is made so that appropriate GH replacement is offered to adults who are GH-deficient, and not for non-approved conditions (e.g., aging and sporting enhancement) (13, 14).

For the clinician, establishing the diagnosis of adult GHD is challenging because of the lack of a single biological end-point (e.g., growth failure 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 as each test has its pros and cons, 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 of when GH stimulation testing can be 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. Conversely, GH stimulation testing should not be performed in patients with commonly encountered, generalized, nonspecific symptoms of weakness, frailty, fatigue, or weight gain, without a history of organic hypothalamic/pituitary disease, as such patients are unlikely to benefit from GH therapy (8, 16, 18). These considerations are important for the clinician when deciding which patients to consider testing for possible adult GHD. 

All GH stimulation tests are based on the concept that a GH secretagogue agent acutely stimulates pituitary GH secretion, and peak serum GH levels are detected by sequential blood sampling of serum GH levels after administration of the 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, safety with minimal side-effects, affordability, and short test duration. It should also not be unpleasant to the patient and it should be 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 who are at risk of and/or with a history of cardio-/cerebrovascular disease and seizures.

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),  GHRH-arginine test became 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 document, we will discuss published evidence of the GH stimulation tests used in the United States and the inherent caveats and limitations of each individual test. The lower GH cut-point of 1 mg/L for the GST should be utilized to improve its diagnostic accuracy in some overweight and all obese patients. We will also summarize current knowledge of the oral macimorelin test as the only approved diagnostic test for adult GHD by the United States Food and Drug Administration (FDA) and the European Medicines Agency, and its change in status of availability in the United States.

GENERAL LIMITATIONS AND IMPORTANT CAVEATS WHEN INTERPRETING GH STIMULATION TESTS

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 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 (35-40), 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 up-regulate GH receptor and sensitivity. Furthermore, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis are now recognized as being highly prevalent in overweight and obese adults with GHD (44), with consequent lower serum IGF-I levels being associated with increased severity of the disease (7). Thus, these data suggest that BMI-specific cut-points should be considered when testing patients for adult GHD. Table 1summarizes the accepted GH cut-points for the GH stimulation tests used in the United States, as recommended by different consensus guidelines.

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.

GROWTH HORMONE STIMULATION TESTS USED IN DIAGNOSING ADULT GH DEFICIENCY

Insulin Tolerance Test

The ITT remains 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).

ACTH: adrenocorticotropic hormone, HPA: hypothalamic-pituitary-adrenal, IV: intravenous.

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). Glucagon is also a more potent GH secretagogue when administered intramuscularly or subcutaneously compared to the intravenous route (49). However, the mechanism/s of glucagon-induced GH stimulation remains unclear, and one hypothesis is 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.

IM: intramuscular, IV: intravenous.

¹Serum GH: peak GH levels tend to occur between 120-180 mins; ²blood 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. It is important to note that this test is not affected by age, BMI, or sex indicating its robustness for diagnosing adult GHD (64).

Main advantages of macimorelin are 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). Thus, 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.

However, in August 2022, a press announcement stated that Novo Nordisk Healthcare AG provided a 270-day notice period to terminate the amended development and commercialization license agreement for macimorelin (MacrilenÒ) in the United States (65). This means that as of May 23, 2023, Aerterna Zentaris regained its full rights in the United States and Canada to macimorelin but because it has yet to find a partner in the United States to market macimorelin, it was further announced that sales of macimorelin will be temporarily discontinued and use of the agent beyond May 2023 will continue until its supplies in the United States runs out (66).

Summary of Tests

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

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.

STANDARDIZATION OF GH ASSAYS

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) (67).

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 (68). 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.

CONCLUSIONS

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 GH stimulation testing is recommended. 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 remains the gold standard GH stimulation test, and the GST and macimorelin test (where available) are reasonable alternatives to the ITT. As the reliability of the GST GH cut-point of 3 mg/L in overweight/obese subjects and in those with glucose intolerance can 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 is an attractive test because it is easy to conduct with high reproducibility, safe, and has comparable diagnostic accuracy to the ITT and GHRH plus arginine test. The factors that limit its wider is its high cost (one 60 mg macimorelin packet costs approximately $4,500) (69) and the potential of drug-to-drug interactions that may cause QT prolongation. Following the announcement in August 2022 that macimorelin will be temporarily discontinued in the commercial market effective May 2023, after supplies of macimorelin runs out in the United States, the ITT and GST will only be the two GH stimulation tests available to clinicians, limiting the choices of tests that can be used.

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ABSTRACT

Abnormalities in the hypothalamic pituitary adrenal (HPA) axis are identified by a careful analysis of both direct and non-stimulated measurements of the hormones as well as provocative tests.  Dynamic testing is useful to determine if elevated levels are suppressible and whether there is sufficient hormone reserve when low levels are measured under stimulation. A combination of all these analyses can distinguish between normal physiology and the consequences of clinical disease in the HPA axis.  While clinical suspicion drives the testing performed, arrival at the correct diagnosis by laboratory testing is crucial for cure of the patient.  Knowledge of the methodologies used in measuring cortisol and ACTH and associated hormones and binding proteins is essential for correct interpretation of the tests.  In this review we compare methodologies available, sensitivity and specificity of the various assays and volumes of sample needed. There are at least 7 different types of dexamethasone suppression testing and they are compared and described in detail. Confirmation of the anatomic source of the hormone is necessary. Petrosal sinus sampling and adrenal vein sampling are reviewed and the clinical indications for each discussed. Finally, once the endocrine diagnosis is reached based on endocrine testing, imaging studies are then reviewed which can confirm the endocrine diagnosis. An abnormality in the HPA axis is a laboratory diagnosis and radiologic imaging is reserved for the last step in the diagnosis of endocrine disease.

NONSTIMULATED HORMONE MEASUREMENTS

Overview

In evaluation of the hypothalamic pituitary adrenal (HPA) axis, static measurement of hormones is seldom useful due to the variable nature of cortisol and ACTH secretion in normal physiological states. In general, if one is suspicious of hypofunction of the HPA axis, then measurement of morning cortisol at 8 am when it is expected to be at its peaks is a good screening strategy. Depending on the result, this might need to be followed by dynamic testing to stimulate either adrenocorticotrophic hormone (ACTH) or cortisol for confirmatory purposes. On the other hand, if one is concerned about Cushing’s syndrome (CS), an overproduction of cortisol or ACTH, then measurement of cortisol should be performed late at night, when it is expected to be at its nadir. Alternatively, one could test cortisol’s response to suppression with dexamethasone.

The American Endocrine Society Clinical Guidelines recommend one of the following tests for the initial CS testing: at least two measurements of urinary-free cortisol (UFC), two measurements of late night salivary cortisol (LNSC), 1 mg overnight dexamethasone suppression test (DST) or a longer low-dose DST (1). Cortisol measurement (serum, UFC or salivary) is the end point for each recommended test.

Despite recent literature reports describing utility of direct salivary and urine cortisol measurements in CS diagnosis (2-4), most clinicians prefer provocative testing due to the variable nature of cortisol and ACTH secretion in normal physiological states. Cortisol is secreted under the direction of ACTH and follows a diurnal variation, with peak values at 08:00 h and a nadir at 22:00 h. In CS, diurnal variation is lost and PM cortisol level is inappropriately elevated. Superimposed on this diurnal pattern are 8-10 pulsatile peaks released during the course of a 24-hour period. Therefore, depending on the instance when blood is sampled, there can be significant variation in the absolute values of ACTH and serum cortisol. Due to this variability of cortisol and ACTH levels, it may be challenging to distinguish pituitary-dependent Cushing’s disease from pseudo-Cushing’s states. Cunningham et al conducted a study where blood was sampled and cortisol measured every 20 to 30 minutes for 24 hours. The group demonstrated that both circadian and pulse amplitudes of cortisol secretion were decreased in Cushing’s disease (5).

This section provides and overview of methodologies commonly used in clinical laboratories for direct determinations of cortisol and ACTH, regardless of whether they are a part of a provocative testing series or direct, non-stimulated hormone assessment.

Cortisol

Methods currently available for measuring serum cortisol levels include automated immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

CORTISOL IMMUNOASSAYS (TOTAL CORTISOL)

Cortisol immunoassays are widely available, have been in use for a long time, and automated methods provide high throughput with minimal manual sample manipulations. Virtually all immunoassay methods are based on the competitive binding principle, where cortisol from the patient sample and exogenous, labeled cortisol compete for the binding sites available on the anti-cortisol antibody. The major difference between the assays is in the label design and chemistry enabling antibody-antigen binding. All currently available cortisol methods have limit of detection below 1 µg/dL, providing sufficient sensitivity to support interpretation of CS dynamic testing results.

A widely recognized disadvantage of immunoassays is a potential of interferences from auto-, anti-animal or heterophilic antibodies. In addition, the older generations of cortisol assays had significant cross-reactivity with other steroids, such as 6-b-hydroxycortisol or prednisolone, due to the use of less specific polyclonal antibody in the assay formulation. However, the majority of current immunoassay methods have transitioned to a more specific monoclonal antibody format, minimizing or eliminating cross-reactivity with other steroids. It should also be noted that some immunoassay vendors use biotinylated antibodies in their assay design. In these instances, biotin may interfere with the assay, causing spuriously elevated cortisol measurement. The presence and magnitude of interference is vendor-specific and the potential of biotin interference should be checked with the laboratory that performs the testing. In general, none of the assays manufactured by Abbott use biotin in reagent formulation, while all assays manufactured by Roche do. The Roche cortisol assay should not be used to measure serum cortisol in a patient taking daily doses of biotin exceeding 5 mg, unless blood is obtained at least 8 hours following the last biotin ingestion.

LC-MS/MS CORTISOL ASSAYS

The LC-MS/MS assays utilize liquid chromatography to separate cortisol from other serum/plasma components and tandem mass spectrometry to detect and quantify compounds of interest. LC-MS/MS based methods offer superior analytical sensitivity and specificity over immunoassays.

Serum Free Cortisol

In conditions where CBG concentrations are affected, such as pregnancy or critical illness for example, total serum cortisol may not always reflect the true pituitary-adrenal status. In these cases, assessment of serum free cortisol is preferred. Free serum cortisol concentration are directly measured by separating free serum cortisol fraction using equilibrium dialysis (6) or ultrafiltration (7, 8) followed by cortisol determination, usually performed using LC-MS/MS method. Alternatively, free serum cortisol can be estimated by calculating the ratio of serum cortisol and CBG to obtain serum cortisol index (6).  Although not affected by CBG levels, free cortisol is also secreted in episodic fashion and thus not much more useful than random total serum cortisol levels in assessment of HPA axis functionality.

Urinary Free Cortisol

Cortisol is excreted in urine in an unbound (free) form and, like free serum cortisol is unaffected by fluctuations in CBG levels. Properly collected 24-hour urine specimens can be used to eliminate fluctuations that would affect serum cortisol levels, due to the pulsatile nature of its release. Therefore, measurement of UFC from 24 hour urine collections has become a valuable diagnostic tool for evaluation of adrenal cortical function and it is one of the first line tests recommended for CS diagnostic testing (1). In the unstressed patient, with normal renal function, elevation of UFC in 24-hour urine specimen is usually sufficient to diagnose CS. A normal result is strong evidence against that diagnosis. Although this test has long been used, its utility in CS diagnosis still remains somewhat controversial. Studies show wide variability in clinical utility of UFC for diagnosis of CS with clinical sensitivity ranging from 53% to over 90% and specificity ranging from 79% to 90% (2, 3, 9). These differences are due to differences in study design, cut-off, and methodology used. Furthermore, in a careful study of normal subjects de Boss Kuil et al found that urinary excretion of free cortisol can differ by as much as 50% between the two consecutive urine collections, while the creatinine values can differ by as much as five fold (10). Since the ratio of free cortisol/creatinine also varies considerably (range 1.0-3.7; median 1.3), intra-variation in urinary cortisol excretion could not be attributed to variation in creatinine excretion. In addition to biological variation, other factors include difficulty in over or under collection of urine. Given such wide discrepancies in reported clinical sensitivity and specificity of UFC measurements and significant intra-individual UFC variability, this test may not be an ideal choice for initial screening of CS.

Methodology used for UFC quantitation is the same as for serum cortisol. In terms of specimen collection, an 8:00 AM to the following day’s 8:00 AM collection is desirable. Samples should be refrigerated during collection and, while preservatives are not required, boric acid is usually acceptable. Quantitation of urine cortisol with a more sensitive and specific LC-MS/MS method is generally preferred over immunoassays. Typically, all the LC-MS/MS UFC assays involve a sample pretreatment with an organic solvent which removes the interfering substances. However, some UFC assays immunoassays either do not include this pretreatment step or offer it as an optional step to the user. As a result, UFC reference ranges vary widely between the assay manufacturers, methodologies, and different laboratories. To increase sensitivity, it is recommended that the upper limit of normal for any UFC assay be used as a positive test (5). It would be thus incorrect to make a diagnosis of adrenal insufficiency relying solely on 24-hour urine collections.

Salivary Cortisol

Late-night (23:00-24:00 h) salivary cortisol (LNSC) is one of the first line tests used to screen for CS. Most studies report high diagnostic sensitivity of this test (80-90%), but there are discrepancies in reported specificities (70-90%), resulting mostly from difference in methodologies and populations studied (2, 3, 11-13) Interestingly, mass spectrometry assays demonstrate high sensitivity, but low specificity (75%) for the diagnosis of CS (11). One potential explanation, as postulated by Raff, is that higher analytical specificity of mass spectrometry actually leads to lower diagnostic specificity, suggesting that cortisol metabolites and precursors picked up by immunoassays may be diagnostically relevant (14). Kannankeril et al recently reported that LNSC has excellent negative predictive value (99.8%) but poor positive predictive value (16.8%) for diagnosis of ACTH-dependent CS (12). Thus, a negative LNSC can be used to rule out ACTH-dependent CS, but complementary tests of adrenal function are needed to establish the diagnosis.

Salivary cortisol concentration is not dependent on CBG and could therefore be useful during an ACTH stimulation testing in patients with increased CBG concentrations due to increased estrogen or decreased plasma binding globulins due to critical illness.

Similar to UFC, the assay methodology remains the same as serum cortisol with the differences in specimen collection.

ACTH

ACTH measurements, while subject to the same circadian variability as cortisol (actually it is the variability of the ACTH that is directly responsible for the variability of the cortisol), are not subject to the effects of CBG. Values of ACTH > 100 pg/ml in the setting of possible adrenal insufficiency are usually suggestive of primary adrenal insufficiency, while values >500 pg/ml are diagnostic. Low concentrations of plasma ACTH are not diagnostic, except for the undetectable levels observed in patients with cortisol producing adrenal adenomas. Plasma ACTH concentration is also low in patients taking exogenous steroids.

Unlike widely available cortisol assays, the availability of clinical ACTH assays is limited. All currently available methods are immunoassays based on the “sandwich” principle, where two antibodies that recognize different ACTH epitopes are utilized. The first antibody, designated as capture antibody, detects one specific site on ACTH molecule and is used to pull the antigen from the patient’s plasma. The second antibody that detects a different ACTH epitope is then used to “sandwich” the antigen and generate a signal.

As is the case with any immunoassay, ACTH assays are susceptible to heterophilic antibody interferences. Several cases have been described in literature where aberrant, falsely elevated ACTH results were inconsistent with clinical picture and lead to unnecessary testing, misdiagnosis, and in some cases surgical interventions. These cases emphasize the importance of interaction between clinicians and the laboratory to identify any interference present and ensure that each patient is appropriately managed (15, 16). In addition, just as is the case with cortisol immunoassays, some vendors use biotinylated antibodies in the capture antibody design. Unlike cortisol, however, biotin interference may result in falsely decreased ACTH levels. The two most commonly used ACTH assays are manufactured by Siemens and Roche. Siemens ACTH assay is not affected by biotin, while the recommendation for Roche ACTH assay is not to use the test in patients ingesting >5 mg biotin daily, unless at least 8 hours had elapsed following the last biotin dose (cf. Roche Elecsys ACTH Package Insert V 12.0, 2020-11).

The preferred specimen for ACTH is EDTA plasma. ACTH is heat labile, and if not collected and preserved on ice, will lead to proteolysis, which can reduce the plasma concentration leading to falsely lower values.

Miscellaneous Non-Stimulated Measurements

CORTISOL BINDING GLOBULIN (CBG)

As mentioned earlier, the majority of cortisol (~92%) is bound to CBG, a serum protein. CBG levels increase in pregnancy and patients on oral contraceptives or supplemental estrogen. CBG is decreased in hyperinsulinemic states, nephrotic syndrome, starvation, severe illness, and chronic liver disease. This test is useful for the assessment of unexpected serum cortisol values. It is offered by large reference laboratories and uses a radioimmunoassay method.

11-DEOXYCORTISOL (COMPOUND S)

This is the immediate precursor of cortisol and is typically increased when ACTH is elevated or in 11 beta-hydroxylase deficiency. The method for 11-deoxycortisol measurement is now available by LC-MS/MS technology and is offered by most reference laboratories.

ANTI-ADRENAL ANTIBODIES

The measurement of anti-adrenal antibodies has been suggested to be useful in detecting early evidence of adrenal insufficiency, before cortisol values are decreased even in response to stimuli. The only test currently clinically available is a test that detects 21-hydroxylase autoantibodies, which are present in the common autoimmune form of Addison’s disease (17). This test is offered by major reference laboratories and is based on the radioimmunoassay format.

CORTICOTROPHIN RELEASING HORMONE (CRH)

Serum concentration of CRH is markedly elevated in pregnancy, presumably due to the production of CRH by the placenta. High levels are associated with high levels of CRH binding protein. Although mentioned as useful in the diagnosis of ectopic CRH syndromes, little data is available in this regard. CRH testing is not commonly done and we have not been able to find a commercial laboratory that is currently performing this test.

DYNAMIC TESTING

Glucocorticoid Deficiency

Adrenal insufficiency is a life-threatening disorder and prompt diagnosis is important because adequate hormonal replacement therapy can be lifesaving.

Despite that more than 35 years have elapsed since the initial description of the use of the insulin tolerance test (ITT) to diagnose adrenocortical deficiency (18), and more than 200 scientific publications in this area, clinicians today still argue as to which is the most sensitive and specific test to diagnose adrenocortical deficiency. The ITT is still regarded as the gold standard upon which to compare all other tests of HPA axis function. Unfortunately, this test has a considerable spectrum of intra-individual and inter-individual variation (19, 20). Therefore, when comparing other tests to the "gold standard", if the standard is not reliable, how can one determine the effectiveness of the other forms of testing? The problem lies in the ability of a single laboratory to know what the values are for their tests. Therefore, ranges from an ITT test response in normal subjects performed in one laboratory may not be normal for another laboratory. Taking this into account there are some general guidelines that are available for evaluating patients with suspected adrenal insufficiency.

PRIMARY ADRENAL INSUFFICIENCY

High Dose ACTH Stimulation Test

WHEN TO USE THIS TEST: Patients acutely ill in the hospital or clinic who present with signs and symptoms suggestive of primary adrenal insufficiency. Patients who are thermodynamically unstable should be resuscitated with crystalloids and given dexamethasone prior to testing if the diagnosis of primary adrenal insufficiency is being considered.

PROCEDURE: An intravenous (IV) line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. The IV line is to be kept open with 0.9% sodium chloride (NaCl) at a rate of 50 ml/hr. Blood is drawn at 0 min for ACTH (2 ml in a lavender top tube on ice) and cortisol (2 ml in a red top tube). Cosyntropin, 0.25 mg is administered as an IV bolus over 2 minutes. The cosyntropin comes as a lyophilized powder which should be reconstituted with 1 ml of 0.9% NaCl. Thirty min after the injection, blood is obtained from the IV line (2 ml) for cortisol. The same is repeated at 60 min (2 ml) for cortisol.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day. If the patient is receiving hydrocortisone or cortisone acetate, the medication should be held for at least 12 hours prior to testing (if possible). Although the test can be performed while the patient is receiving dexamethasone, there is some cross-reactivity in some assays and cortisol levels may not be accurate. Each laboratory should determine for itself, the effect of dexamethasone on their assay.

Patients with known sensitivity to cosyntropin or its preservatives should not have it administered. Oral estrogen use may result in elevation of the total serum cortisol level due to increased corticosteroid binding globulin (21). Patients with albumin <2.5 g/dL may also have a low cortisol level (21, 22).

CONTRAINDICATIONS: Hypersensitivity to cosyntropin or any component of the formulation.

WARNINGS / PRECAUTIONS: Use with caution in patients with pre-existing allergic disease or a history of allergic reactions to corticotropin. Class C in pregnancy.

ADVERSE REACTIONS 1% to 10%: Cardiovascular: Flushing. Central nervous system: Mild fever. Dermatologic: Pruritus. Gastrointestinal: Chronic pancreatitis. <1%: Hypersensitivity reactions

DRUG INTERACTIONS: Decreased effect: May decrease the effect of anticholinesterases in patients with myasthenia gravis; nondepolarizing neuromuscular blockers, phenytoin and barbiturates may decrease effect of cosyntropin

INTERPRETATION OF RESULTS: Baseline cortisol values <5 µg/dl and ACTH concentrations >100 pg/ml are usually diagnostic of primary adrenal insufficiency. The normal peak cortisol value post stimulation should be an increment no less than 7µg/dl. A peak stimulated cortisol value of >18 µg/dl at 30 min is considered normal. Since 37% of subjects had a peak response to cosyntropin at 30 min and 63% had a peak response at 60 min, both time points are analyzed in all patients and if either the 30 min or 60 min sample reaches the criteria as noted above, the test is considered normal (23).  However, there is some suggestion that new generation cortisol assays may have different cutoff values, but these have not been verified (24). 

Free cortisol, instead of total cortisol can be measured using a value of >1.2 µg/dl at 30 or 60 min as a normal result. This can be indicated in patient with albumin levels <2.5 g/dL or those with low cortisol binding globulin.

Serum aldosterone can be measured in 0 min, 30 min and 60 min blood samples as ACTH stimulation of the adrenal cortex will also stimulate aldosterone. It has been suggested that a normal aldosterone response to ACTH in the presence of a suboptimal cortisol response is diagnostic of secondary adrenal insufficiency (25).

Low dose ACTH stimulation Test

WHEN TO USE THIS TEST: Patients with subtle signs of adrenal insufficiency or patients who have been treated with glucocorticoids in whom determination of adrenal reserve is necessary. Patients who have autoimmune disease and may have early adrenocortical insufficiency may be best assessed with this test.

PROCEDURE: An intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. The IV line is to be kept open with 0.9% NaCl at a rate of 50 ml/hr. Blood is drawn at 0 min for ACTH (2 ml in a lavender top tube on ice) and cortisol (2 ml in a red top tube).

Cosyntropin, 1 µg is administered as an IV bolus over 2 minutes. The injection material was prepared according to the method of Dickstein as follows: The cosyntropin was diluted with 50 ml of sterile saline to a stock concentration of 5 µg/ml. Aliquots of 0.2 ml were added into sterile plastic tubes and kept at 4^oC for a maximum of 4 months (26). Immediately prior to testing 0.8 ml of saline is added to the tube (final dilution 1 µg/ml) and 1 ml is injected into the patient. Thirty min after the injection blood is obtained from the IV line (2 ml) for cortisol. The same is repeated at 60 min (2 ml) for cortisol.

SPECIAL CONSIDERATIONS: Same as for high dose ACTH stimulation test, see above.

INTERPRETATION OF RESULTS: This test was originally developed to be more sensitive for diagnosing secondary adrenal insufficiency because it was more of a "physiologic" dose. It is much better at diagnosing secondary adrenal insufficiency than the high dose, although it is not at all recommended in acute or recent hypopituitarism when the intact adrenal glands can still respond normally to any dose of ACTH. Although probably not useful for the initial purpose of secondary adrenal insufficiency, it may be more sensitive at distinguishing milder forms of primary adrenal insufficiency (27). Furthermore, this low dose test was helpful in identifying mild adrenal suppression in asthmatic children being treated with inhaled steroids (28). As noted above, each laboratory should establish their normal values, however in general, a stimulated value at 30 or 60 min greater than 20 µg/dl would be considered normal.

A meta-analysis of 30 studies enrolling 1209 adults and 228 children with secondary adrenal insufficiency, evaluating the diagnostic accuracy of high and low dose ACTH stimulation concluded that they have similar diagnostic accuracy. They are both adequate to rule in, but not rule out, secondary adrenal insufficiency

SECONDARY ADRENAL INSUFFICIENCY (PITUITARY OR HYPOTHALAMIC)

Insulin Tolerance Testing (ITT)

WHEN TO USE THIS TEST: Patients in whom pituitary or hypothalamic disease may result in impaired corticotroph (or somatotroph) activity. Patients following pituitary surgery or pituitary radiation can be tested at any time, unlike the ACTH stimulation tests described above which are not useful in the acute setting. A random serum cortisol should be drawn prior to scheduling the test if the value is > 20 µg/dl, the test may not be necessary This test, can be performed in the outpatient clinic, however while relatively safe it requires a trained endocrine registered nurse to be present with the patient during the course of the test.

PROCEDURE: A 50 ml vial of 50% Dextrose should be at the patient's bedside in a syringe ready for injection before beginning the procedure.

An intravenous line is placed 30 minutes before the test for rapid phlebotomy, to eliminate a temporary rise in cortisol associated with a needle stick, and in order to have IV access for 50% Dextrose in the event of severe hypoglycemia. The IV line is to be kept open with 0.9% NaCl at a rate of 50 ml/hr. Blood is drawn at 0' for cortisol (2 ml in a red top tube) and glucose (1 ml in a gray top tube). Blood glucose is also checked at the bedside with a glucose monitor.

Regular (short acting) insulin is administered as an IV bolus at a dose of 0.1 units/kg. Blood is sampled for cortisol and glucose as noted above at 10min, 15min, 30min, 45min, 60min, 90min and 120min. A bedside nurse should monitor the blood glucose more frequently if glucose drops below 60 mg/dl on the glucometer or if the patient complains of neuroglycopenic symptoms, such as fatigue, diaphoresis, hunger, lightheadedness, or nausea. The test should continue until the blood glucose concentration drops below 40 mg/dl.

In patients with diabetes on insulin, consideration should be given that they may be insulin resistant. In which case, larger doses of insulin may be given. We usually begin with a single bolus of 0.1 U/kg and then re-bolus with insulin depending on the response to the initial dose (either give the same dose again if there was some response but insufficient, or double the dose if there was only minimal response to blood glucose, or give half the dose if the hypoglycemic response was close to the desired goal). This can be repeated several times until adequate hypoglycemia is reached.

Once the response goal of a glucose < 40 mg/dl is reached, patients can be fed a meal such as crackers and orange juice. Blood glucose should be checked at 5min, 10min and 15min post feeding. If there is no increase in glucose or a clinical response within 5min, intravenous glucose should be administered. If no response, then a repeat bolus of glucose is suggested. If no response or IV access is lost, glucagon 1 mg intramuscular can be given.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although due to the need for patients to be fasting it is most conveniently done in the morning. If the patient is receiving hydrocortisone or cortisone acetate, the medication should be held for at least 12 hours prior to testing (if possible). Unlike the ACTH stimulation tests, the ITT cannot be performed while the patient is receiving dexamethasone, due to suppression of the hypothalamic pathways necessary to respond to hypoglycemia.

In general ITT is not recommended in patients with uncontrolled seizure disorder or significant coronary artery disease.

In order to determine if the level of dysfunction is at the hypothalamus or at the pituitary this test is sometimes used in addition to the CRH stimulation test. When the ITT fails to stimulate cortisol, but the CRH test does stimulate it is likely that the patient is having hypothalamic dysfunction.

INTERPRETATION OF RESULTS: Serum cortisol should increase within 30 min of the hypoglycemic response to > 20 µg/dl. If the serum cortisol at baseline is 18 ug/dl the test may not be diagnostic. If the baseline serum cortisol is higher than 19 µg, adrenal insufficiency is unlikely. Although the response of cortisol is more reproducible than that of growth hormone in the ITT, intra-subject differences have been reported (20, 30).

Metyrapone Testing

WHEN TO USE THIS TEST: This test is perhaps the most sensitive to determine whether the HPA axis is intact. Although metyrapone is not generally available from your neighborhood pharmacy, it can be obtained by calling Novartis Pharmaceutical Corp. at 1-800-988-7768 on weekdays. Metyrapone blocks 11-b hydroxylase and results in the inhibition of conversion of 11-deoxycortisol to cortisol. Serum levels of cortisol decrease and concentration of 11-deoxycortisol increases, however 11-deoxycortisol does not down regulate ACTH. Therefore, in a normally functioning HPA axis there is an increase in 11-deoxycortisol. This metabolite can be directly measured in the serum or measured in the urine as 17-OH corticosteroids. This test can help differentiate primary adrenal deficiency from ACTH deficiency. It has a similar diagnostic performance to the ITT and it’s a potential alternative when there is a contraindication to ITT.

PROCEDURE: For assessment of adrenal or pituitary insufficiency the test can be performed as an overnight test. Metyrapone is given orally (30 mg/kg body weight, or 2 grams for <70 kg, 2.5 grams for 70 to 90 kg, and 3 grams for >90 kg body weight) at midnight with a glass of milk or a small snack (24). Serum 11-deoxycortisol and cortisol are measured at 8 AM the next morning; it is also recommended to measure plasma ACTH levels (31).

SPECIAL CONSIDERATIONS: The concurrent use of glucocorticoids will interfere with the test. Any medications that the patient is taking which increase the P450 enzymes will increase the metabolism and clearance of metyrapone (such as rifampin, phenobarbital, and phenytoin) (32). Similarly, hypothyroidism or hyperthyroidism will affect clearance of metyrapone and the adrenal responsiveness. Therefore, thyroid function tests should be measured prior to performing this test. Measurement of 11-deoxycortisol, like cortisol itself is dependent on CBG and drugs such as estrogens and oral contraceptives will falsely increase the concentrations of 11-deoxycortisol (33).

PREGNANCY IMPLICATIONS - Use during pregnancy only if clearly needed. Subnormal response may occur in pregnant women and the fetal pituitary may be affected.

LACTATION - Excretion in breast milk unknown/use caution

ADVERSE REACTIONS - Frequency not defined. Central nervous system: Headache, dizziness, sedation. Dermatologic: Allergic rash. Gastrointestinal: Nausea, vomiting, abdominal discomfort or pain. Hematologic: Rarely, decreased white blood cell count or bone marrow suppression.

INTERPRETATION OF RESULTS: 8 AM serum 11-deoxycortisol concentrations should be >7 µg/dL with serum cortisol less than 5 µg/dL (138 nmol/L), confirming adequate metyrapone blockade. The plasma ACTH concentration at 8 AM should exceed 75 pg/mL (17 pmol/L), confirming that any increases in serum 11-deoxycortisol concentrations are ACTH-dependent, thereby separating primary from secondary adrenal insufficiency (34, 35).

Glucocorticoid Excess

DEXAMETHASONE SUPPRESSION TEST

Measurement of endogenous cortisol production in response to exogenous dexamethasone suppression was the first provocative test and still remains among the most useful tests used for the evaluation of excess cortisol. Dexamethasone, due to its high affinity to the glucocorticoid receptor is a potent inhibitor of ACTH synthesis and release. In addition, most of modern immunoassays for cortisol (both urine and serum) utilize an antibody that does not cross-react with dexamethasone. Therefore, the combination of being able to use relatively low doses and at the same time not interfere with the measurement of cortisol make dexamethasone suppression useful for establishing the presence of a perturbation in the pituitary - adrenal axis and for diagnosing the etiology of hypercortisolism.

At least five different tests have been described using dexamethasone, which differ in the dose and timing of dexamethasone treatment and differ in whether there is measurement of urine or serum cortisol or 17-OH-corticostseroids (Table 1). Although the endocrine basis for the tests are in general the same, none are perfect. Confirming the diagnosis of patients with suspected hypercortisolism requires several tests for accurate diagnosis.

Note: To assure patient compliance and determine whether there is abnormal metabolism of the dexamethasone, serum levels of dexamethasone can be measured. However, this is not a common diagnostic test. Testing can be done by specialized laboratories, such as Esoterix inc. CA. The principle of the assay is RIA after chromatographic sample separation and requires 1 ml of serum sample.

All these tests require significant patient participation as the patients are required to self-administer the dexamethasone at inconvenient hours of the day (11PM) or up to 4 times a day. Sampling requires either collection of urine for 24 hours or coming to the physician's office at 8 AM for multiple blood sampling. Drugs that induce hepatic cytochrome P-450 enzymes, such as barbiturates, phenytoin, rifampin, and aminoglutethimide, increase the metabolism of dexamethasone and other steroids. Measurement of serum dexamethasone a few hours after the last dose will help determine if there is abnormal metabolism. All these caveats are in addition to the other problems associated with measurement of cortisol as noted above, including the variable diurnal variation as well as interference with concurrent administration of glucocorticoids, estrogen, or other medications that increase cortisol binding globulin.

A popular screening test for confirming hypercortisolism is the overnight 1 mg dexamethasone. A single dose of 1 mg is administered (or 0.3 mg/Kg for children (34) at 11PM and blood is obtained by 8 AM the following morning. The dexamethasone dose is given prior to the diurnal rise in endogenous ACTH release and therefore suppresses the early AM cortisol. A normal response would be a serum cortisol concentration of <1.8 mcg/dl, alternatively a cut point of < 5 µg/dl can be used which will yield more specificity with less sensitivity. If cortisol is >10 µg/dl the likelihood of hypercortisolism is high. The other dexamethasone suppression tests are reviewed in Table VIII. Patients with corticotroph macroadenomas or very active tumors, may have urine free cortisol in excess of 1000 µg/dl which will require higher doses of dexamethasone to confirm suppressibility and/or rule out ectopic ACTH production (36).

The two- day low dose dexamethasone suppression test can be used to differentiate Cushing’s syndrome from pseudo-Cushing’s which can present with many of the signs and symptoms associated with hypercortisolism in the setting of other clinical conditions such as depression, alcoholism, PCOS, obesity, and uncontrolled diabetes (37, 38). Dexamethasone 0.5 mg is delivered orally Q6 hours for 48 hours. Serum cortisol is measured 2 hours after the last dose and a cutoff level of <1.4 µg/dl is consistent with pseudo-Cushing’s. Measurement of 24 hour urine excretion of 17-hydroxycorticosteroid and creatinine during the administration of dexamethasone starting at 1200h, has also been suggested with a cut point of 11 umol/day or higher considered positive for Cushing’s syndrome (39). This test however, can misclassify as many as 15% of patients with Cushing’s syndrome and up to 15% of patients with pseudo Cushing’s.

The overnight high dose dexamethasone suppression test can help differentiate Cushing’s disease from ectopic ACTH syndrome in patients with ACTH-dependent Cushing’s syndrome. The basis for this differentiation is the fact that ACTH secretion in Cushing’s disease is only relatively resistant to glucocorticoid negative feedback inhibition. Cortisol levels will not suppress normally with overnight 1 mg but will suppress with a higher dose of 8 mg of dexamethasone. Serum cortisol concentration at 8 AM is <5 µg/dL in most patients with Cushing’s disease and is usually undetectable in normal individuals. A more than 50% decrease in cortisol on the day after taking 8 mg dexamethasone supports a diagnosis of Cushing’s disease over ectopic ACTH production. In patients with non-ACTH dependent hypercortisolism, a lack of suppression of cortisol by more than 50% with a low normal ACTH level (5-20 pg/ml) suggests an adrenal etiology.

CRH STIMULATION TEST

WHEN TO USE THIS TEST: This test is one of the most sensitive to determine if there is an abnormality in the HPA axis and for diagnosing the etiology of hypercortisolism in ACTH dependent Cushing’s.  Although CRH is expensive ($300), when one considers the cost of multiple urine collections and analyses of cortisol as well as the cost of a single MRI of the pituitary (which generally exceeds $1500), CRH is at least cost effective when one considers the overall expense in the evaluation of these patients.

PROCEDURE: An intravenous line is placed 30 min before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. Blood is drawn at -15' and 0' for cortisol and ACTH (2 ml in a lavender top tube on ice). CRH is then injected IV at a dose of 1 µg/Kg up to a maximum of 200 µg. Blood is obtained at 15, 30, 60, 90, 120, 180 and 210 min for cortisol and ACTH (2 ml in a lavender top tube on ice).

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although the initial studies describing the test have been done in the morning.

Side effects: The patient may experience slight nausea, metallic taste, urgency to urinate, a change in blood pressure (either increase or decrease), a change in heart rate, headaches, abdominal discomfort, facial flushing, and lightheadedness. These side effects are mild and last for only few minutes. Category C in pregnancy.

INTERPRETATION OF RESULTS: The mean ACTH concentrations at 15 and 30 min after CRH should increase by at least 35% above the mean basal value at -15 and 0 min in patients with Cushing's disease, but not in patients with ectopic ACTH secretion. This measure gave the best sensitivity (93%) and specificity (100%) (40, 41).  The best cortisol criterion was a mean increase at 30 and 45 min of 20% or more above mean basal values, which gave a sensitivity of 91% and a specificity of 88%. It should be noted that the criterion for Cushing's disease is based on the presence of hypercortisolism. The CRH test will not adequately differentiate subjects with pseudo-Cushing’s and those with true pituitary dependent Cushing's disease.

CRH TEST WITH DEXAMETHSONE

WHEN TO USE THIS TEST: Several investigators have found that modifications of the CRH stimulation test can increase further the sensitivity and specificity in the diagnosis of the etiology of Cushing's disease. While the simultaneous use of vasopressin can augment the response to CRH, dexamethasone can be used to suppress all but pathologic responses to CRH stimulation [33]. Without dexamethasone the sensitivity and specificity of the CRH test is 65 and 100%, respectively, while with dexamethasone the CRH test is 100% sensitive and specific. This test is also particularly useful to differentiate true Cushing’s from pseudo-Cushing’s state.

PROCEDURE: Dexamethasone, 0.5 mg is self-administered orally by the patient every 6 hours for 2 days, at 6 AM, 12 Noon, 6 PM and midnight. On the morning of the 3rd day an additional dose of dexamethasone is given at 6 AM. The patient arrives at the testing center by 8 AM and an intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. Blood is drawn at -15' and 0' for cortisol and ACTH (2 ml in a lavender top tube on ice). CRH is then injected IV at a dose of 1 µg/Kg up to a maximum of 200 µg. Blood is obtained at 15, 30 60, 90 120, 180 and 210 min for cortisol and ACTH (2 ml in a lavender top tube on ice).

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although it is usually done in the morning.

Side effects that the patient may experience are: slight nausea, metallic taste, urgency to urinate, a change in blood pressure (either increase or decrease), a change in heart rate, headaches, abdominal discomfort, facial flushing, and lightheadedness. These side effects are mild and last for only few minutes.

Similar to the dexamethasone suppression test, the results should be interpreted with caution in patients taking estrogen therapy as they can present with falsely elevated cortisol levels due to an increase of cortisol-binding globulin. Drugs such as phenytoin, phenobarbitone, carbamazepine, rifampicin and alcohol induce hepatic enzymatic clearance of dexamethasone, mediated through CYP 3A4, thereby reducing the plasma concentration and may be associated with false positive results (42).

INTERPRETATION OF RESULTS: A normal response would be a plasma cortisol concentration less than 1.3 µg/dl measured 15 minutes after the administration of CRH.  Values of cortisol greater than 1.3 µg/dl correctly identified all cases of Cushing's syndrome and all cases of pseudo-Cushing's states (100% specificity, sensitivity, and diagnostic accuracy). While this is a general recommendation, each laboratory should confirm based on the sensitivity of the respective cortisol assay. Furthermore, it is important to confirm the serum level of dexamethasone at the time of the blood draw to assure patient compliance with the dexamethasone regimen.  Patients with ectopic ACTH production will have nonsuppressed cortisol and ACTH levels that are not stimulated by CRH.

DDAVP STIMULATION TEST

WHEN TO USE THIS TEST: This test can be used as part of the workup of ACTH dependent hypercortisolism. It can be used in addition to the CRH stimulation test as studies have shown that the combination of these two tests performs better than either of the tests separately. It can also be performed in lieu to the CRH test in situations in which CRH is not available. The aberrant expression of vasopressin V2 receptor in pituitary ACTH-secreting adenomas is the rationale for the use of the desmopressin test to differentiate corticotroph adenomas (which should respond to desmopressin injection) from ectopic ACTH secreting tumors or pseudo Cushing’s (which should not respond)(43-45).

PROCEDURE: An intravenous line is placed 30 minutes before the test for rapid phlebotomy and to eliminate a temporary rise in cortisol associated with a needle stick. Blood is drawn at -15' and 0' for cortisol and ACTH (2 ml in a lavender top tube on ice). DDAVP is then injected IV at a dose of 5 to 10 ug. Blood is obtained at 15, 30, 60, 90, 120, 180 and 210 minutes for cortisol and ACTH (2 ml in a lavender top tube on ice) (45).

INTERPRETATION OF RESULTS: No definitive cutoff values have been standardized for the interpretation of this test. The published established criteria for this test have generally been based on studies with small series of subjects. Malerbi et al. proposed a cortisol increase over baseline of 12% to be consistent with diagnosis of Cushing’s disease (46). An absolute ACTH increase over baseline equal or greater than 6 pmol/L yielded higher sensitivity and specificity to differentiate Cushing’s disease from pseudo Cushing’s in a different study. Alternatively the criteria used for the CRH stimulation test can be used in  the interpretation of the results (47).

INFERIOR PETROSAL SINUS SAMPLING (IPSS) WITH CRH STIMULATION

WHEN TO USE THIS TEST: Once the diagnosis of ACTH dependent Cushing's syndrome has been made based on endocrinologic testing, the next step in the evaluation of such patients should be an MRI of the pituitary to confirm the presence of a pituitary mass. Unfortunately, MRI imaging of the pituitary as a primary diagnostic tool is distinctly unhelpful due to the fact that 10% of all normal individuals may have slight abnormalities of their pituitary and that in many subjects with Cushing's disease, the tumor may be too small to be imaged with MRI scans. However, subjecting a patient to surgical pituitary exploration in the absence of a demonstrable mass is likely to result in an unsuccessful surgery. Furthermore, if previous dexamethasone and/or CRH testing is equivocal, then IPSS should be performed to further confirm the pituitary as the source of the ACTH (34). Although this test is less reliable in lateralizing the ACTH source (i.e., left versus right), than it is in confirming that the ACTH is central in origin, it can rule out ectopic ACTH production by a tumor (although ectopic CRH secreting tumors would be difficult to distinguish from true Cushings' disease based on IPSS). Simultaneous measurement of prolactin in the central samples can normalize the data if there is any difference in the location of the catheters (48).

It is recommended that active hypercortisolism is confirmed by measuring a 24-hour UFC or overnight UFC the day preceding IPSS. Misleading results have been reported when this test is performed “out of cycle” in patients with cyclical Cushing’s.

PROCEDURE: This test is done in conjunction with a skilled interventional neuroradiologist. It is important that the endocrinologist is personally present in the room during the procedure so that assurance can be made that the proper blood tests were drawn at the specified times. The patient is brought to the angiogram suite without sedation. A large bore IV line is placed in an antecubital fossa (to be certain there is access to peripheral blood sampling and CRH injection). Catheters (5 French) are placed in the femoral veins and threaded under fluoroscopic guidance to the inferior petrosal sinus. Injection of IV contrast confirms proper placement of the catheters.

Patients are on constant, pulse, blood pressure and oxygenation monitors during the course of the procedure. Test tubes are prechilled in ice and labeled so that during the rapid sampling period, blood can be placed in the tubes without delay.

It is recommended to routinely obtain 4 baseline measurements at -15, -10, -5 and at 0 minutes. This allows for practice allowing proper coordination between the radiologists drawing blood from the IPSS and the individual drawing blood from the brachial vein. Appropriate amounts of blood should be removed to discard the dead space of the catheter (this varies depending on the size of the catheter used). 2 ml of blood is obtained in lavender top vacutainer tubes on ice for measurement of cortisol (on peripheral samples); ACTH and prolactin (on central samples).

At 0' CRH is then injected as described above for the peripheral CRH test. Alternatively, a combination of CRH and 10ug of desmopressin can be used, especially if the patient has had a negative response to a prior CRH test. If CRH is not available, IPSS can also be performed with desmopressin alone per the protocol described above. Blood is then sampled from both central and peripheral lines at 2', 5' 10' and 15'. After the 15' time point and right before the IPSS catheters are removed, repeat fluoroscopic localization of the catheters should be performed to confirm that there was no displacement during the sampling. However, sampling on peripheral blood may continue as described in the CRH test discussed above.

SPECIAL CONSIDERATIONS: The test can be performed at any time of the day, although it is usually done in the morning.

Side effects that the patient may experience are: slight nausea, metallic taste, urgency to urinate, a change in blood pressure (either increase or decrease), a change in heart rate, headaches, abdominal discomfort, facial flushing, and lightheadedness. These side effects are mild and last for only few minutes.

Patients greater than 300 pounds in weight may not be able to be supported by the standard fluoroscopic table. Furthermore, such large patients may have an abdominal pannus that precludes reasonable access to the femoral veins. In such instances the IPSS can be performed via catheters placed in the antecubital vein with the patient immobilized in the sitting position.

Strokes have been reported in the literature as a potential complication (36). To minimize this possibility, it is recommended that the catheters remain in the petrosal sinus for no more than 30 min.

Freeze/thawing can decrease the ACTH concentration (see above); therefore, we recommend that the samples be brought to the endocrine lab and analyzed within 24 hours with the plasma separated and kept on ice during this time. If the analysis is not possible within 24 hours, the samples should be aliquoted and frozen to minimize the amount of freeze/thawing.

INTERPRETATION OF RESULTS: Plasma ACTH values are normalized to the prolactin value in order to correct for possible different localization of the catheters, or movement of the catheters during the study. The post CRH ACTH/Prolactin value of the central catheters should be >2.1 fold the ACTH/Prolactin value of the peripheral sample. In most cases of pituitary dependent Cushing’s, the increase is > 5.0-fold. A central to peripheral ACTH gradient higher or equal to 2 before CRH administration or higher or equal to 3, 10 min after CRH infusion is considered diagnostic of a pituitary source of ACTH (Cushing's disease). Lateralization would mean that the ratio of the left to right side is >2.0. Frequently the ratio criteria can be met without the need for CRH stimulation, however, the diagnostic accuracy increases from 86% to 90% with CRH (37).

The workup of ACTH dependent Cushing’s to differentiate Cushing’s disease from ectopic ACTH source can be quite challenging and often times requires combination of different dynamic testing in addition to imaging that often ultimately led to costly and invasive diagnostic procedures such as IPSS to be able to establish an accurate diagnosis. A retrospective study involving 167 patients with Cushing’s disease and 27 patients with ectopic Cushing’s found that using thresholds of a cortisol increase >17% with an ACTH increase >37% during CRH test and a cortisol increase >18% with an ACTH increase >33% during desmopressin test, the combination of both tests gave 73% sensitivity and 98% PPV of Cushing’s disease. The PPV was 100% in patients with positive response to both tests, with a negative pituitary MRI and whole-body CT scan. The NPV was 100% in patients with negative response to both tests, with negative pituitary MRI and positive whole body CT scan. This combination of dynamic tests with imaging studies is proposed as an accurate, cost-effective diagnostic strategy for the workup of ACTH depended Cushing’s that might minimize the need for IPSS which can be invasive, costly and unavailable in all institutions (43)

ADRENAL VEIN SAMPLING

WHEN TO USE THIS TEST: Patients diagnosed with ACTH independent Cushing’s and found to have bilateral adrenal tumors on imaging pose a particular challenge to the clinician. The differential diagnosis in these cases includes unilateral cortisol secreting adenoma (or carcinoma) with contralateral non-functioning cortical adenoma, bilateral cortisol secreting adenomas, macronodular adrenal hyperplasia, and primary pigmented nodular adrenocortical disease. Adrenal vein sampling measuring cortisol can be very helpful in this scenario and give valuable information to elucidate the proper diagnosis and guide therapy.

PROCEDURE: This test is done in conjunction with a skilled interventional radiologist under sedation. The procedure is usually performed early morning after an overnight fast on the second day of either a low dose (0.5 mg orally every 6 hours) or high dose (2 mg orally every 6 hours) of dexamethasone administration. This eliminates the probability of endogenous ACTH secretion causing interference with the interpretation of autonomous adrenal gland cortisol secretion. The adrenal veins can be catheterized by the percutaneous femoral vein approach, the position of the catheter tip should be verified by venogram. Concentrations of cortisol and aldosterone should be measured in blood obtained from both adrenal veins and the external iliac vein (for the detection of peripheral venous concentrations) (49).

SPECIAL CONSIDERATIONS: Potential complications include thrombosis with subsequent infarction or hemorrhage adrenal insufficiency and hypertensive crisis, however these are rare (48).

The aldosterone concentrations are usually much higher on the right adrenal vein compared to the left, this is presumably due to the anatomy differences and the catheter proximity to the right adrenal medulla. For this reason, although plasma epinephrine is measured to confirm success of adrenal vein catheterization, it cannot be used to correct for blood sample dilution between the 2 adrenal veins. There have been few case reports in which aldosterone has been used for side-to-side dilution differences, however whether it can be used for this purpose remains unclear (49, 50).

INTERPRETATION: Catheterization of each adrenal vein can be considered successful if plasma aldosterone concentration in the adrenal vein exceeds peripheral venous concentration by more than 100 pg/ml. An adrenal-to-peripheral venous cortisol gradient greater than 6.5 can be considered consistent with a cortisol secreting adenoma. Lateralization can be determined by measuring the side-to-side cortisol gradient (high-side to low-side). A ration of 2.3 or greater is consistent with autonomous cortical secretion from predominately 1 adrenal gland (49).

IMAGING STUDIES IN THE HPA AXIS EVALUATION

The evaluation of the HPA axis function should always be approached through biochemical measurements. With few exceptions, imaging studies provide no information about hormonal function but can be very useful for the localization of tumors or lesions. Once a biochemical diagnosis of either deficiency or excess of glucocorticoid production has been established, imaging studies can complement and assists the hormonal evaluation, providing valuable information about etiology, prognosis, and management.

Pituitary Imaging

In the vast majority of cases of ACTH dependent Cushing’s syndrome (CS), the source of ACTH is in the pituitary (Cushing’s Disease), so performing imaging studies of the pituitary gland in this scenario to try to localize a tumor is appropriate. However given the high incidence of non-functioning pituitary adenomas in the general population (up to 10%) (51) and the increasing sensitivity of the high resolution imaging modalities available that can lead to false positive results, it is important to perform a thorough dynamic testing evaluation of each case and consider inferior petrosal sinus sampling if appropriate (see section above), before committing a patient to pituitary surgery. Adrenocorticotropic pituitary tumors represent about 10% of all pituitary tumors (52) and  ACTH-secreting adenomas are most commonly microadenomas (<1cm). In cases of macroadenoma, assessment of extrasellar extension including chiasmatic compression and cavernous sinus involvement is imperative (53).

The other scenario in which pituitary imaging is indicated and can be useful in the evaluation of the HPA axis function, is in patients diagnosed with secondary adrenal insufficiency who have no history of recent exogenous glucocorticoid exposure or any other clear explanation for the clinical presentation. In these cases, a mass lesion disrupting the HPA function should be suspected, especially if the patient presents with deficiencies of other pituitary hormones and/or elevated prolactin, as isolated adrenal insufficiency from a non-functioning tumor affecting the pituitary is very rare.

PITUITARY MRI

Magnetic resonance imaging (MRI) is the mainstay of pituitary assessment. MRI is more sensitive than computed tomography (CT) in detecting corticotroph adenomas, but still detects only about 50% of these tumors (54) and has a false positive rate of 12-19% (55, 56)]. Standard pituitary imaging protocols typically include thin-section (2 or 3 mm) of T1-weighted (w) spin echo sequences (SE) performed both in coronal and sagittal planes through the pituitary fossa, which are repeated after administration of intravenous gadolinium contrast medium, associated with a T2-weighted sequence in the coronal plane (57, 58). High spatial detail can be achieved by using thin slices, a fine matrix size and a small field of view focused on the pituitary (58). The classic MR features of a corticotroph adenoma include a less than 1 cm focal area of lesser enhancement on T1-w images following contrast administration, hyperintense or hypointense on T2-w images as compared with the normal pituitary gland, remodeling of the pituitary sella floor and deformity of the gland contour (59). Acquiring dynamic sequences in the first 1-2 minutes after contrast injection can increase the sensitivity (60), but this technique has not been unequivocally demonstrated to improve the usefulness of MR in Cushing’s (61). The use of three-dimensional (3D) spoiled gradient recalled acquisition in the steady state (SPGR) sequence allows for superior soft tissue contrast compared to conventional spin echo sequences, this technique can be further optimized with thin-slice imaging (<1mm) (58).  Compared to T1-w SE sequence, SPGR has been reported to increase sensitivity but also has a higher false positive rate (62, 63).

PITUITARY CT SCAN

Pituitary computed tomography (CT) scanning is less sensitive than MRI for the detection of pituitary adenomas (64)and it is usually reserved for those patients who cannot safely undergo brain MRI. Acquisition of 1 mm (or less) axial sections through the pituitary fossa with coronal reconstructions can be helpful in the assessment of macroadenomas (57). It is also very helpful preoperatively in patients planned for transsphenoidal pituitary surgery to delineate the bony anatomy (65)

Adrenal Gland Imaging

There are a couple of scenarios in which adrenal gland imaging plays a role in the evaluation of the HPA axis. It is indicated and particularly important in the evaluation of patients diagnosed with ACTH independent CS, which is most commonly caused by adrenocortical adenomas or carcinomas and less frequently bilateral micronodular and macronodular hyperplasia. It can also be considered in cases of primary adrenal insufficiency. Tumors in the adrenal are fairly common in humans, they have been found to be present in 3% of autopsies performed in persons older than 50 years of age (66) and have been reported to be incidentally discovered in up to 5% of cross-sectional abdominal imaging carried out for unrelated problems (67). Most of these incidentally found adrenal tumors are nonfunctioning, 10 to 15% secrete excess amounts of hormones (68) of these, adrenocortical tumors are the most common. On the basis of imaging characteristics alone, no distinction can be made between a benign hyperfunctioning and a non-functioning adenoma, and this can only be differentiated based on clinical and biochemical diagnosis. Adrenal carcinoma represents <10% of adrenal tumors, 30 to 40% of these are hyperfunctioning in adults (69).  There are multiple important imaging characteristics that can help differentiate benign adrenal adenomas from pheochromocytomas, adrenocortical carcinomas and metastasis, like percentage of lipid content, tumor size, homogeneity, border regularity, presence of calcifications, invasion of surrounding tissue, and lymph node enlargements (Table 2).

ADRENAL CT SCAN

Unenhanced thin- section CT scan followed by contrast-enhanced examination is the cornerstone of imaging of adrenal tumors. Unenhanced CT is important to provide density measurements of lesions (70). The rich intracytoplasmic fat in adenomas results in a low attenuation on nonenhanced CT. The Hounsfield (HU) scale is a semiquantitative method to measure radiograph attenuation. If an adrenal mass measures <10 HU on unenhanced CT, the likelihood that it is a benign adenoma is nearly 100% (71).  However up to 30% of benign adenomas might not contain large amounts of lipid and present with higher HU on nonenhanced CT scan. This is when measuring the contrast washout on delayed images is very useful. Ten minutes after the administration of contrast, an absolute medium washout of more than 50% has been reported to be close to a 100% sensitive and specific for benign adenoma (72). Non-adenomas include metastases, pheochromocytomas and carcinomas.

Adrenal carcinomas usually appear as a unilateral mass, >4 cm in size with an inhomogeneous appearance due to necrosis, hemorrhage, fibrosis, and calcification. Careful assessment of the draining venous structures is essential on imaging, together with identification of direct infiltration of adjacent viscera (57).

ADRENAL MRI

When lesions cannot be characterized adequately with CT, MRI evaluation (with T1 and T2-weighted sequences, chemical shift and fat-suppression refinements) can be sought. Adrenal adenomas usually show low homogeneous signal on T1-weighted images and a signal intensity equivalent or higher than the liver on T2-weighted images. Chemical shift imaging will readily identify the lipid rich adenomas with signal loss on the out-of-phase sequences (73). This loss of signal can be measured using the adreno-splenic-ratio (ASR) and the signal intensity index (SII). An ASR ratio of <70% has been shown to be highly specific for adenomas and has a 78% sensitivity. Using the SII, a minimum of 5% signal loss characterizes an adrenal adenoma with accuracy of 100% [61]. MRI can also be particularly useful to evaluate for local and distant invasion of adrenocortical carcinomas.

Primary pigmented nodular adrenocortical disease is a rare cause of Cushing’s syndrome that has a female predilection and may be familial or associated with Carney complex. On imaging the adrenal glands may appear normal or minimally hyperplastic with multiple, usually <5 mm, unilateral or bilateral benign cortical nodules. The adrenal nodules are macroscopically pigmented; they demonstrate a lower T1 and T2 signal intensity on MRI compared to surrounding atrophic cortical tissue. When nodules are 1-2 cm in size, there might be atrophy of the intervening cortex, which helps distinguish this condition from ACTH- dependent hyperplasia (57).

Another rare cause of Cushing’s syndrome is ACTH-independent macronodular adrenal hyperplasia, which has a male predilection. The imaging appearance of the adrenal glands is striking with massive bilateral adrenal enlargement, nodularity. and distortion of adrenal contour. Nodules can measure 1 to 5.5 cm. On MRI they are hypointense relative to liver on T1-w images and hyperintense or isointense in T2-w images. On chemical shift imaging, nodules lose signal intensity on out-of-phase due to their high lipid content (57).

OTHER ADRENAL IMAGING MODALITIES

Patients that harbor adrenal masses, which are not adequately characterized by CT or MRI, can be further evaluated with functional nuclear medicine modalities that include single photon emission computed tomography (SPECT) scintigraphy with various radionuclide tracers, and positron emission tomography (PET) scintigraphy with various radionuclides.  PET images provide a higher spatial resolution compared to SPECT (70).

PET scan with either Fluorodeoxyglucose (FDG) or ¹¹C-metomidate (MTO) can be useful in selected cases to differentiate benign adrenal adenomas from adrenocortical carcinomas. An elevated uptake on the FDG scan correlates with high metabolic activity and raises the suspicion for malignancy (74) with high sensitivity and specificity (75). Limitations of this technique include physiological excretion of FDG into renal inflammatory system and high metabolic uptake in inflammatory and infectious processes as well as in benign pheochromocytomas, leading to false positive results (64). Metomidate is an inhibitor of 11 beta-hydroxylase (CYP11B1) and aldosterone synthetase (CYP11B2), and based on this property its use can help differentiate tumors of adrenocortical origin from non-cortical lesions. Originally developed as a PET imaging agent radiolabeled with ¹¹C, more recently it has been labeled with ¹⁸F and ¹²³I, allowing SPECT and SPECT/CT imaging (76).

Integrated or “fused” PET-CT imaging allows to combine CT attenuation measurements with the intensity of FDG uptake, as described by the standardized uptake value (SUV), improving the performance of either imaging technique alone (77).

Scintigraphy with Iodine-131-Iodomethyl-19-norcholesterol (NP 59) is a functional nuclear medicine imaging modality that can be used to differentiate adrenal cortical adenomas from carcinomas. This is a labeled cholesterol analogue that specifically binds to low-density lipoproteins and after receptor-mediated uptake it is stored in the adrenocortical cells (70).  NP 59 uptake is regulated by ACTH and suppressed by dexamethasone, concentrating in hyperfunctioning cortisol and aldosterone secreting adenomas and showing low uptake in adrenocortical carcinomas because of the inefficient concentration of radiotracer by malignant tissue (78).

Other Imaging Modalities for Ectopic Cushing’s

Patients diagnosed with ACTH dependent Cushing’s whose biochemical dynamic tests suggests an ectopic source, pose a special challenge to the clinician. In 12 to 20% of these patients, the source remains undiscovered despite repeated biochemical and radiological investigations (55).

In the setting of ectopic ACTH production, imaging studies play a crucial role in trying to identify the source of the tumor causing the disease and guide management and prognosis. The optimal imaging study to detect these tumors has not been defined. CT, MRI, PET scan, ¹¹¹In-pentetreotide (OCT) scintigraphy at conventional or higher radionuclide doses, as well as newer molecular imaging techniques like ¹³¹I/¹²³-metaiodobenzylguanidine (MIBG), ¹⁸F-fluoro-2-2-deoxyglucose-positron emission tomography (FDG-PET), ¹⁸F-fluorodopa-PET (F-DOPA-PET), ⁶⁸Ga-DOTATATE-PET/CT or ⁶⁸Ga-DOTATOC-PET/CT scan (⁶⁸Gallium-SSTR-PET/CT) are complementary and have been shown to be useful in different scenarios with variable sensitivity and specificity (79-83). For the most part at least two different imaging modalities are needed to establish a diagnosis and sometimes, repeated imaging over several months is required to identify the source. The choice of imaging modalities is guided by the sensitivity of the procedure balanced with the risk of false-positive findings (72).

A good approach is to start by obtaining images of the chest, since most ACTH-secreting tumors are located in this area. The most common causes are bronchial carcinoid tumors and small cell lung cancer. Other sources of excess ACTH production include neuroendocrine tumors of the thymus, bowel and pancreas, medullary carcinoma of the thyroid, pheochromocytomas, and mesotheliomas.

CT of the chest, abdomen and pelvis with intravenous contrast medium injection is the most commonly used initial imaging test performed and is very useful in many cases. In patients with equivocal CT imaging findings, MRI can be useful, particularly for tumors within the abdomen. It is recommended to follow CT and MRI imaging with a functional imaging modality, being OCT scintigraphy the most widely used. Functional imaging reduces false-positive results because it relies on the specific properties of tumor cells, not just their anatomic characteristics. However, tumors lacking the relevant receptor can have false negative results (83). Site-specific differences occur and different imaging modalities might have higher sensitivity and specificity depending on this. A recent systematic review showed that FDG-PET can be very sensitive in the detection of neuroendocrine tumors with high proliferation index, particularly in the pancreas. This review also showed that ⁶⁸Gallium-SSTR-PET/CT had a 100% sensitivity but this is an imaging technique that has limited availability and was only performed in a minority of the patients in their series (80).

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