Peter C. Avgerinos

Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa: pathophysiologic mechanisms in underweight and weight-corrected patients

To study the pathophysiology of hypercortisolism in patients with anorexia nervosa, we examined plasma ACTH and cortisol responses to ovine corticotropin-releasing hormone before and after correction of weight loss. We also studied patients with bulimia whose weight was normal, since this disorder has been suspected to be a variant of anorexia nervosa. Before their weight loss was corrected, the anorexic patients had marked hypercortisolism but normal basal plasma ACTH. The hypercortisolism was associated with a marked reduction in the plasma ACTH response to corticotropin-releasing hormone. When these patients were studied three to four weeks after their body weight had been restored to normal, the hypercortisolism had resolved but the abnormal response to corticotropin-releasing hormone remained unchanged. On the other hand, at least six months after correction of weight loss their responses were normal. The bulimic patients whose weight was normal also had a normal response to corticotropin-releasing hormone. We conclude that in underweight anorexics, the pituitary responds appropriately to corticotropin-releasing hormone, being restrained in its response by the elevated levels of cortisol. This suggests that hypercortisolism in anorexics reflects a defect at or above the hypothalamus. The return to eucortisolism soon after correction of the weight loss indicates resolution of this central defect despite persistence of abnormalities in adrenal function.

The Ovine Corticotropin-Releasing Hormone Stimulation Test and the Dexamethasone Suppression Test in the Differential Diagnosis of Cushing's Syndrome

We gave a standard dexamethasone suppression test and an ovine corticotropin-releasing hormone (CRH) stimulation test to 41 patients with adrenocorticotrophic hormone (ACTH)-dependent hypercortisolism to determine the efficacy of each test in the differential diagnosis of Cushings syndrome. Twenty-nine of thirty-three patients with Cushings disease and 0 of 8 patients with ectopic secretion of ACTH responded to the ovine CRH test with increased levels of cortisol. When a cortisol response was judged as positive for Cushings disease, the CRH test had a diagnostic sensitivity, specificity, and accuracy of 88%, 100%, and 90%, respectively. Twenty-nine patients with Cushings disease and 1 patient with ectopic secretion of ACTH responded to the dexamethasone suppression test. A combined-test strategy requiring negative results from both tests to exclude a diagnosis of Cushings disease yielded superior sensitivity (100%) and diagnostic accuracy (98%). Thus, the ovine CRH test works as well as the standard dexamethasone suppression test in discriminating between Cushings disease and ectopic ACTH secretion. The diagnostic power of each test is enhanced when the two tests are combined.

The metyrapone and dexamethasone suppression tests for the differential diagnosis of the adrenocorticotropin-dependent Cushing syndrome: a comparison.

The diagnosis of suspected Cushing syndrome involves several phases: the confirmation of hypercortisolism, the differentiation between pseudo-Cushing states and the true Cushing syndrome, the differentiation between adrenocorticotropin (ACTH)-independent and ACTH-dependent causes of the Cushing syndrome, and the differentiation between pituitary and ectopic sources of the ACTH-dependent Cushing syndrome [1, 2] (Figure 1). The distinction between an ACTH-producing pituitary microadenoma and an occult ectopic ACTH-secreting tumor is the most difficult problem in the differential diagnosis of the Cushing syndrome. Although inferior petrosal sinus sampling has been shown to be highly effective in making this differential diagnosis [3], availability of this test is limited. Moreover, the test is invasive and may have adverse effects [2, 4]. Figure 1. A diagnostic approach to confirm the existence of the Cushing syndrome and to determine its cause. Three decades ago, two noninvasive tests for the differential diagnosis of the Cushing syndrome were introduced by Liddle [5, 6]: the high-dose dexamethasone suppression test and the metyrapone test. New diagnostic criteria for the dexamethasone test have recently been introduced to improve its accuracy [7]. However, even with these new criteria, 17% of patients with the pituitary Cushing syndrome suppressed excretion of 17-hydroxycorticosteroid and urine free cortisol to a lesser extent than did patients with the ectopic ACTH syndrome [7]. The metyrapone test has been less widely used than the dexamethasone suppression test and has been done and interpreted less consistently [8-15]. Additionally, its utility for determining the cause of the ACTH-dependent Cushing syndrome has been questioned [9, 16, 17]. Because of the continuing need for improved noninvasive means of distinguishing pituitary from ectopic ACTH-producing tumors, we reexamined the usefulness of the metyrapone test in a large series of patients with the ACTH-dependent Cushing syndrome. Our objectives were to determine optimal criteria for the diagnosis of the Cushing syndrome by the standard metyrapone test, to determine the relative usefulness of plasma 11-deoxycortisol and 24-hour urine 17-hydroxysteroid (17-OHS) levels as end points for the test, to compare the diagnostic accuracy of the metyrapone test with that of the dexamethasone suppression test, and to evaluate whether combining the results of the two tests would further improve sensitivity and diagnostic accuracy. Methods Patients The participants of this pilot study were selected from among patients referred to the National Institutes of Health (NIH). Nearly all were found to have hypercortisolism before referral. In clinical practice, only a few patients screened for suspected Cushing syndrome have hypercortisolism. To qualify for the study, patients had to have had at the NIH the dexamethasone suppression test, the metyrapone test, and a surgical procedure to remove the source of excessive ACTH. Metyrapone and Dexamethasone Tests For the metyrapone test, 24-hour urine specimens (starting at 0600 h) and venous blood samples (5 mL starting at 0800 h) were obtained for 4 consecutive days. On days 1 and 2 of the test, patients received no metyrapone; starting at 0800 h of day 3 and ending at 0400 h of day 4, patients received 6 doses of metyrapone at 4-hour intervals. Seven hundred and fifty milligrams per dose was administered to all patients except for an 8-year old boy in whom the dose was adjusted for body surface area to 500 mg per dose [18]. For the dexamethasone suppression test, 24-hour urine specimens (starting at 0600 h) were obtained for 6 consecutive days. On days 1 and 2 of the test, patients received no dexamethasone; starting at 0600 h of day 3 and ending at 2400 h of day 4, all patients received eight 0.5-mg doses of dexamethasone orally every 6 hours (low-dose); starting at 0600 h of day 5 and ending at 2400 h of day 6, all patients received eight 2.0-mg doses of dexamethasone every 6 hours (high-dose), with the exception of an 8-year old boy in whom the dose was adjusted for weight to 0.25 mg for days 3 to 4 and 1.0 mg per dose for days 5 to 6 [18]. The 24-hour urine specimens were refrigerated and sent to the NIH clinical chemistry laboratory. After October 1988, an aliquot was also sent to Hazleton Laboratories (Vienna, Virginia). Urine volume and creatinine levels were measured to confirm the completeness of collection. The 0800 h blood samples obtained during the metyrapone test were refrigerated and separated, and the plasma was sent to Hazleton Laboratories for measurement of plasma 11-deoxycortisol levels. Assays and Calculations An aliquot of each urine collection was used for measurement of excretion of 17-OHS by a modification of the Porter-Silber method [19-21]. The intra-assay and interassay coefficients of variation were 5.9% and 7% to 14%, respectively. The normal range for urine excretion of 17-OHS is 2 to 6 mg/d. Another aliquot of urine was frozen and sent either to SmithKline Bioscience Laboratories (King of Prussia, Pennsylvania) or, after October 1988, to Hazleton Laboratories for measurement of cortisol by radioimmunoassay. The Premix kit (Diagnostic Products Corporation, Los Angeles, California) or the Quanticoat kit (Kallestad Laboratories Incorporated, Austin, Texas) were used by these laboratories, respectively. The intra-assay and interassay coefficients of variation were 5.4% and 9.3%, respectively, for the former and 5.8% and 7.5%, respectively, for the latter. The normal range for urine excretion of free cortisol is 20 to 90 g/d for both laboratories. After extraction and chromatography, the plasma 11-deoxycortisol level was measured by radioimmunoassay [22, 23]. The intra-assay and interassay coefficients of variation were 11.7% and 18.2%, respectively. In most patients, the 11-deoxycortisol levels before and after the administration of metyrapone were measured in the same assay. We calculated the percentage of stimulation of 17-OHS after metyrapone was administered to each patient using the following formula: ([stimulated level baseline level] 100)/baseline. The stimulated 17-OHS values were calculated in five different ways. In the first method, the higher 17-OHS value between days 3 and 4, which is the conventional outcome measure, was used to interpret the test result [6]. To optimize the sensitivity of the test, we also evaluated the day 3 value of 17-OHS, the day 4 value, the lower of the days 3 and 4 values, and the mean of the day 3 and day 4 17-OHS values as alternate measures of the stimulated level. To test the sensitivity of a shortened test, the day 2 values were also used as a baseline in addition to the average of the values of days 1 and 2. Thus, when the five measures of response were combined with the two baseline measures, 10 different measures for interpreting the 17-OHS response to metyrapone were evaluated. We calculated the percentage of suppression of urine free cortisol levels after the administration of metyrapone for each patient using the following formula: ([baseline level -suppressed level] 100)/baseline. Suppressed levels were the values of day 3 and baseline levels, the average of days 1 and 2. The 11-deoxycortisol response to metyrapone was expressed either as the stimulated value on day 4 [13, 17] or as the ratio of the stimulated to baseline values as previously described [12, 15]. For the latter measure, the day 4 stimulated level was divided by the average baseline 11-deoxycortisol levels on days 1, 2, and 3 (11-deoxycortisol on day 3 was drawn before metyrapone administration). To test the diagnostic efficiency of a shortened test, the average of the days 2 and 3 11-deoxycortisol levels or the day 3 11-deoxycortisol level alone, were substituted as alternate measures of the baseline 11-deoxycortisol level. Thus, we assessed four different measures of the plasma 11-deoxycortisol response, that is, the absolute level of the day 4 plasma 11-deoxycortisol and the ratio of the day 4 plasma 11-deoxycortisol level to each of three different baseline measures (day 3 alone, the mean of days 2 and 3, and the mean of days 1, 2, and 3). We calculated the percentage of suppression of 17-OHS or urine free cortisol after the administration of high-dose dexamethasone for each patient using the following formula: ([baseline level suppressed level] 100)/baseline. Suppressed levels were the values on day 6, and baseline levels were the average of days 1 and 2 as previously described [7]. Statistical Analysis Estimates of sensitivity (true-positive results/[true-positive results + false-negative results]), specificity (true-negative results/[true-negative results + false-positive results]), and diagnostic accuracy ([true-positive + true-negative results]/total number of patients) were determined for each steroid at various suppression levels based on the diagnosis obtained at surgery as the gold standard. For each analysis, the presence of pituitary disease was considered to be a positive response (diseased) and the presence of ectopic ACTH secretion, a negative response (non-diseased). For the purpose of determining the specificity and diagnostic accuracy relative to the gold standard of pituitary surgery, the 15 patients who lacked a confirmed diagnosis after unsuccessful pituitary surgery were considered nondiseased. We determined likelihood ratios at each suppression level by dividing the sensitivity by 1 minus the specificity [24]. Because one aim of our study was to develop criteria by which the need for trans-sphenoidal surgery could be established by a noninvasive test, the criteria set for the end points of the metyrapone and dexamethasone tests were chosen so as not to misclassify any of the patients with surgically proven ectopic ACTH syndrome as having the pituitary Cushing syndrome. Thus, the criteria were set so that a positive response would indicate the presence of p

Abnormal acth and cortisol responses to ovine corticotropin releasing factor in patients with primary affective disorder

To further explore hypothalamic pituitary adrenal regulation in patients with affective illness, we administered 1 microgram/kg of synthetic ovine corticotropin releasing factor at 2000h to 26 drug-free patients with this disorder and to 15 healthy controls. Compared to controls, depressed patients (N = 12) showed a significant elevation in baseline cortisol and significant reductions in the net ACTH and cortisol responses to corticotropin releasing factor. These findings were normal in manic (N = 6) and improved (N = 8) subjects. An additional finding was that baseline cortisol and net ACTH and cortisol responses to CRF were negatively correlated in the entire group of patients and controls as well as in the patients alone. These data indicate that the reduced ACTH and cortisol responses to CRF in depression reflect normal functioning of the pituitary corticotroph cell (i.e., that the negative feedback effect of cortisol on ACTH secretion in depression is physiologically intact, effectively serving as a brake on the ACTH response to exogenous CRF. Thus, the hypercortisolism of depression may be due to a hypothalamic defect, possibly involving hypersecretion of endogenous CRF. This possibility may be of particular interest in light of clinical observations that depression can often be precipitated by stress and by data in experimental animals that CRF may influence several processes known to be altered in the overall symptom complex of depression.

Corticotropin releasing factor: Basic studies and clinical applications

Corticotropin releasing factor (CRF) is a newly sequenced peptide first isolated from sheep hypothalami and thought to be an important modulator of both the pituitary-adrenal axis and the sympathetic nervous system. We administered intravenous, intramuscular, and intracerebroventricular CRH to non-human primates and measured plasma ACTH, beta endorphin, cortisol, GH and PRL responses to CRF. In addition, we determined the pharmacokinetic properties of I125 in these primates. We administered CRF as an intravenous bolus or as a continuous infusion to normal volunteers and as an intravenous bolus to patients with disorders of the hypothalamic-pituitary-adrenal axis, such as Cushings syndrome and adrenal insufficiency, and patients with endogenous depression and mild hypercortisolism, and assessed their plasma ACTH, cortisol, GH and PRL responses. In addition, we determined the pharmacokinetic properties of CRF in man by measuring CRF immunoreactivity in plasma. CRF given intravenously to primates or man is a slowly metabolized, long-acting, secretagogue of ACTH, beta-endorphin and cortisol. When given intracerebroventricularly to primates it stimulates the hypothalamic-pituitary-adrenal axis without escaping into the plasma and it is actively cleared in the CNS. It does not cross the blood brain barrier appreciably when given intravenously. CRF given to primates and men as an intravenous continuous infusion has only mild ACTH stimulating effects and this may be due to an intact cortisol negative feedback system. Finally, CRF causes characteristic plasma hormone responses in patients with Cushings disease, adrenal insufficiency and depression.

The CRH stimulation test in bereaved subjects with and without accompanying depression

We studied recently bereaved individuals with the corticotropin-releasing hormone (CRH) stimulation test. Subjects with a bereavement complicated by a depressive illness (n = 9) had significantly higher basal plasma cortisol levels and smaller plasma adrenocorticotropic hormone (ACTH) responses to CRH than either subjects with an uncomplicated bereavement (n = 19) or normal controls (n = 34). Subjects with depressed bereavement showed ACTH responses to CRH similar to those of depressed patients (n = 30). Bereaved subjects who had received psychotropic medications in the past (n = 13), compared with those who had not (n = 15), showed significantly smaller plasma ACTH responses to CRH. Significantly more subjects with bereavement complicated by depression, as compared to subjects whose bereavement was uncomplicated, had a past history of treatment for depression. These results suggest that predisposed individuals may respond to the stress of bereavement with a depressive illness accompanied by dysregulation of the hypothalamic-pituitary-adrenal axis.

A comparison of the overnight and the standard metyrapone test for the differential diagnosis of adrenocorticotrophin-dependent Cushing's syndrome.

OBJECTIVE We wished to develop optimal criteria for interpreting the single‐dose overnight metyrapone test and to compare the diagnostic efficiency of the overnight and the standard 6‐dose metyrapone tests for the differential diagnosis of ACTH‐dependent Cushing’s syndrome.

ADAPTATION OF THE HYPOTHALAMIC-P ITU ITARY-ADRLNAL AXIS IN LATE-ONSET CONGENITAL ADRENAL HYPERPLASIA

Patients with late-onset congenital adrenal hyperplasia(LOCAH)due to partial 21-hydroxylase deficiency have no clinical evidence of hypocortisolism, unlike patients with classical forms of CAR, who have elevated plasma ACTH and low serum cortisol levels. In order to study the interactions of the various components of the hypothalamic-pituitary-adrenal axis in LOCAH. we administered ovine corticotropin releasing hormone(oCRH), 1 ug/kg, at 20:00h to 10 untreated patients with LOCAH (8 females, 2 males, ages 7-35y). Baseline(basal, B) and oCRH-stimulated(peak, P) ACTH and cortisol responses did not differ from normal and the integrated ACTH:cortisol response ratio was also normal. Stimulated serum 17-hydroxyprogesterone responses were significantly greater than normal (p<0.005). Stimulated serum Δ 4-androstenedione responses in 5 postpubertal female patients moderately exceeded those of 5 age-matched control females(p=NS). [Data shown as mean + SBM]We conclude that patients with LOCAH compensate for the cortisol biosynthetic defect at the expense of a moderate increase in adrenal androgen secretion. The normal ACTH and cortisol responses to oCRH suggest that a new equilibium has been established between the pituitary and the adrenal gland.