48-hour dexamethasone suppression test.

The 48-hour DST is the most widely used confirmatory procedure. Dexamethasone (Decadron) is a synthetic steroid with cortisone-like actions but is approximately 30 times more potent than cortisone, so that amounts too small for laboratory measurement may be given to suppress pituitary ACTH production. The test is preceded by two consecutive 24-hour urine collections as a baseline. If low doses (2 mg/day) are used, patients with normal adrenal function usually have at least a 50% decrease (suppression) in their 24-hour urine 17-OHCS values compared to baseline, whereas those with Cushing’s syndrome from any etiology have little if any change. This test result is usually normal in those patients whose low-dose overnight DST is abnormal (nonsuppressed) only because of obesity. If larger doses (8 mg/day) are used, about 85% (range, 42%-98%) of those with adrenal cortex hyperplasia due to pituitary oversecretion of ACTH have at least a 50% decrease (suppression) of their 24-hour urine 17-OHCS values. Adrenal cortisol-producing adenomas or carcinoma rarely decrease their urine 17-OHCS levels. Patients with the ectopic ACTH syndrome due to bronchial or thymus carcinoids have been reported to produce false positive test results (decrease in urine 17-OHCS levels) in up to 40% of patients. Patients with the ectopic ACTH syndrome from lung small cell carcinoma or other tumors rarely change urine 17-OHCS levels. Since the test takes a total of 4 days (48 hours at baseline and 48 hours of test duration) and requires 24-hour urine collections, and since there are a significant number of exceptions to the general rules, plasma ACTH assay is supplementing or replacing the high-dose DST for differentiation of the various etiologies of Cushing’s syndrome. Some investigators report that the metyrapone test (discussed later) is better than the 48-hour high-dose DST in differentiating pituitary oversecretion of ACTH from adrenal tumor.

A single-dose overnight version of the high-dose DST has been reported, similar to the low-dose overnight test. A baseline serum cortisol specimen is drawn fasting at 8 A.M.; 8 mg of dexamethasone is given at 11 P.M.; and a second serum cortisol specimen is drawn fasting at 8 A.M. the next day. Normal persons and patients with pituitary ACTH syndrome have 50% or more cortisol decrease from baseline. Cortisol-producing adrenal tumors and ectopic ACTH patients have little change. Limited evaluation of this test reported similar results to the standard high-dose dexamethasone procedure.

Metyrapone test. Metyrapone (Metopirone) blocks conversion of compound S to cortisol. This normally induces the pituitary to secrete more ACTH to increase cortisol production. Although production of cortisol is decreased, the compound S level is increased as it accumulates proximal to the metyrapone block, and 17-OHCS or radioassay CPB methods for cortisol in either serum or urine demonstrate sharply increased apparent cortisol values (due to compound S) in normal persons and those with pituitary-induced adrenal cortex hyperplasia. Fluorescent assay or RIA for cortisol do not include compound S and therefore yield decreased cortisol values. Adrenal tumors are not significantly affected by metyrapone. Some authorities recommend measuring both cortisol and compound S. An increase in compound S verifies that lowering of the plasma cortisol level was accompanied by an increase in ACTH secretion. This maneuver also improves the ability of the test to indicate the status of pituitary reserve capacity, and the test is sometimes used for that purpose rather than investigation of Cushing’s disease. To obtain both measurements, one must select a test method for cortisol that does not also measure compound S. Compound S can be measured by a specific RIA method. Phenytoin or estrogen administration interferes with the metyrapone test.

Adrenocorticotropic hormone stimulation test. Injection of ACTH directly stimulates the adrenal cortex. Patients with cortex hyperplasia and some adenomas display increased plasma cortisol and 17-OHCS levels. If urine collection is used, a 24-hour specimen taken the day of ACTH administration should demonstrate a considerable increase from preinfusion baseline values, which persists in a 24-hour specimen collected the day after ACTH injection. Normal persons should have increased hormone excretion the day ACTH is given but should return to normal in the next 24 hours. Carcinoma is not affected. The ACTH stimulation test at present does not seem to be used very frequently.

Serum adrenocorticotropic hormone. Serum ACTH measurement by immunoassay is available in many reference laboratories. At present, the assay techniques are too difficult for the average laboratory to perform in a reliable fashion, and even reference laboratories still have problems with accuracy.

There is a diurnal variation in serum ACTH levels corresponding to cortisol secretion, with highest values at 8-10 A.M. and lowest values near midnight. Stress and other factors that affect cortisol diurnal variation may blunt or eliminate the ACTH diurnal variation. Serum ACTH data in adrenal disease are summarized in Table 30-1.

Plasma adrenocorticotropic hormone in adrenal diseases

Table 30-1 Plasma adrenocorticotropic hormone in adrenal diseases

In Cushing’s syndrome due to adrenal tumor or micronodular hyperplasia, pituitary activity is suppressed by adrenal-produced cortisol, so the serum ACTH level is very low. In ectopic ACTH syndrome, the serum ACTH level is typically very high (4-5 times the upper preference limit) due to production of cross-reacting ACTH-like material by the tumor. However, some patients with the ectopic ACTH syndrome have serum levels that are not this high. In bilateral adrenal hyperplasia due to pituitary overactivity, serum ACTH levels can either be normal or mildly to moderately elevated (typically less than the degree of elevation associated with the ectopic ACTH syndrome). However, there is a substantial degree of overlap between pituitary tumor ACTH values and ectopic ACTH syndrome values. It has been suggested that ACTH specimens obtained at 10-12 P.M. provide better separation of normal from pituitary hypersecretion than do specimens drawn in the morning. Another study found that specimens drawn between 9:00 and 9:30 A.M. provided much better separation of normal from pituitary hypersecretion than specimens drawn at any other time in the morning. In summary, adrenal tumor (low ACTH levels) can usually be separated from pituitary-induced adrenal cortex hyperplasia (normal or increased ACTH levels) and from ectopic ACTH (increased ACTH levels). Pituitary-induced adrenal cortex hyperplasia has ACTH values that overlap with the upper range of normal persons and with the lower range of the ectopic ACTH syndrome. The time of day that the specimen is drawn may improve separation of normal persons from those with Cushing’s disease.

Corticotropin-releasing hormone test. About 85% of Cushing’s disease is due to pituitary hyperplasia or tumor, and about 15% is due to ectopic ACTH from a nonpituitary tumor. Corticotropin-releasing hormone (CRH) from the hypothalamus stimulates the pituitary to release ACTH (corticotropin). Ovine CRH is now available, and investigators have administered this hormone in attempts to differentiate adrenal tumor and the ectopic ACTH syndrome from pituitary overproduction of ACTH. Initial studies reported that after CRH administration, pituitary ACTH-producing tumors increased plasma cortisol levels at least 20% over baseline and increased their ACTH level at least 50% over baseline. Normal persons also increase their ACTH and plasma cortisol levels in response to CRH, and there is substantial overlap between normal response and pituitary tumor response. Primary adrenal tumors and the ectopic ACTH syndrome either did not increase cortisol levels or increased ACTH less than 50% and plasma cortisol levels less than 20%. However, several studies found that about 10% of pituitary ACTH-producing tumors failed to increase plasma ACTH or cortisol to expected levels. This is similar to the rate that pituitary tumors fail to suppress cortisol production as much as expected in the high-dose 48-hour DST. About 15% of patients with the ectopic ACTH syndrome overlap with pituitary ACTH tumors using the ACTH criteria already mentioned, and about 10% overlap using the plasma cortisol criteria. Therefore, differentiation of the etiologies of Cushing’s syndrome by the CRH test alone is not as clear-cut as theoretically would be expected. To prevent false results, the patients should not be under therapy for Cushing’s syndrome when the test is administered.

The CRH test has also been advocated to evaluate the status of pituitary function in patients on long-term, relatively high-dose corticosteroid therapy.

To summarize, the expected results from the CRH test after injection of CRH are (1) for a diagnosis of Cushing’s disease, an exaggerated response from adenoma of pituitary; (2) for Cushing’s syndrome of adrenal origin or ectopic ACTH syndrome, no significant increase in ACTH; (3) for the differential diagnosis of increased ACTH from pituitary microadenoma versus ectopic ACTH syndrome, inconsistent results. The CRH test is not completely reliable in differentiating primary pituitary disease from hypothalamic deficiency disease.

Cushing’s disease versus ectopic ACTH syndrome. The intracerebral inferior venous petrosal sinuses receive the venous blood from the pituitary containing pituitary-produced hormones; the right inferior petrosal sinus mostly from the right half of the pituitary and the left sinus from the left half. Several studies have suggested that catheterization of both inferior petrosal sinuses can differentiate ectopic ACTH production from the pituitary ACTH overproduction of Cushing’s disease in patients who do not show a pituitary tumor on computerized tomography (CT) scan or when the diagnosis is in question for other reasons. The most commonly used method is comparison of the ACTH level in the inferior petrosal sinuses with peripheral venous blood (IPS/P ratio) 3 minutes after pituitary stimulation by ovine CRH. Although several criteria have been proposed, it appears that an IPS/P ratio greater than 2.0 without CRH stimulation or a ratio of 3.3 or more in one of the inferior petrosal sinuses 3 minutes after CRH stimulation is over 95% sensitive and specific for Cushing’s disease versus ectopic ACTH syndrome (if technical problems are avoided). However, apparently this procedure is not as good in differentiating Cushing’s disease from pseudo-Cushing’s disease, since there is about 20% overlap with results from patients with some clinical or laboratory findings suggestive of Cushing’s disease (such as some patients with psychiatric depression) but without proof of pituitary hyperplasia or adenoma. In one study the same overlap was seen with clinically normal persons.

Computerized tomography

CT can frequently differentiate between unilateral adrenal enlargement (adrenal adenoma or carcinoma) and bilateral enlargement (pituitary hyperactivity or ectopic ACTH syndrome). However, it has been reported that nonfunctioning adrenal cortex nodules may occur in 1%-8% of normal persons, and one of these nodules could be present coincidentally with pituitary Cushing’s syndrome or ectopic ACTH. CT is very useful, better than pituitary sella x-ray films, in verifying the presence of a pituitary adenoma. Even so, third- and fourth-generation CT detects only about 45% (range, 30%-60%) of pituitary adenomas. In addition, it has been reported that 10%-25% of normal persons have a pituitary microadenoma, and some of these nonfunctioning nodules may be seen on CT and lead to a misdiagnosis of Cushing’s disease.

Summary of tests in Cushing’s syndrome

Currently, the most frequently utilized tests to screen for Cushing’s syndrome are the overnight low-dose DST and the test to detect abolishment of serum cortisol diurnal variation. Urine free-cortisol determination would provide more accurate information than the diurnal variation test. Confirmatory tests (if necessary) and tests to differentiate adrenal from nonadrenal etiology that are most often used are the 48-hour DST or the metyrapone test, serum ACTH assay, and CT visualization of the adrenals.

Conditions that affect the screening and confirmatory tests should be kept in mind. In particular, alcoholism (especially with recent drinking) and psychiatric depression can closely mimic the test results that suggest Cushing’s syndrome. Finally, there are some patients in each category of Cushing’s syndrome etiology who do not produce the theoretically expected response to screening or confirmatory tests.