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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 (). 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 (-), 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 (). 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 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-β-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.

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.

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 () or ultrafiltration (, ) 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 (). 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.

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 (). 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% (, , ). 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 (). 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 (). It would be thus incorrect to make a diagnosis of adrenal insufficiency relying solely on 24-hour urine collections.

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 (, , -) Interestingly, mass spectrometry assays demonstrate high sensitivity, but low specificity (75%) for the diagnosis of CS (). 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 (). 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 (). 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.

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 (, ). 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.

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.

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.

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 (). This test is offered by major reference laboratories and is based on the radioimmunoassay format.

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.

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 (), 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 (, ). 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.

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 (). Patients with albumin <2.5 g/dL may also have a low cortisol level (, ). 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 (). However, there is some suggestion that new generation cortisol assays may have different cutoff values, but these have not been verified (). 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 ().

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 o C for a maximum of 4 months (). 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 (). Furthermore, this low dose test was helpful in identifying mild adrenal suppression in asthmatic children being treated with inhaled steroids (). 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 ().

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 (, ).

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-β 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 (). Serum 11-deoxycortisol and cortisol are measured at 8 AM the next morning; it is also recommended to measure plasma ACTH levels ().

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) (). 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 (). 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 (, ).

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 (). 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.