Measurement Of ACTH Research Articles

Effect of continuous intravenous injection of interleukin-6 and pretreatment with cyclooxygenase inhibitor on brain c-fos expression in the rat.

The aim of the present study was to assess potential brain sites of stimulation by peripheral interleukin (IL)-6 of the hypothalamo-pituitary-adrenal (HPA) axis in the rat, using c-fos protein as a marker of cellular activation. Involvement of prostaglandins in IL-6-induced ACTH secretion and c-fos expression was also investigated. IL-6 was infused continuously (40 ng/min) for 90 min to conscious male rats. Blood samples were taken before the infusion and at 30 and 90 min for measurement of plasma ACTH. Expression of c-fos in the brain was examined by immunohistochemistry. Administration of IL-6 significantly elevated plasma ACTH levels at 30 min (495 +/- 105 vs. 117 +/- 17 pg/ml in controls, p < 0.05). Elevated levels were still present at 90 min (596 +/- 139 vs. 113 +/- 20 pg/ml in controls, p < 0.05). Infusion of IL-6 (3.6 micrograms/rat) markedly triggered c-fos expression in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), as well as in the central amygdaloid nucleus (CeA), the nucleus tractus solitarius and the locus coeruleus. Pretreatment with the cyclooxygenase inhibitor indomethacin (10 mg/kg, i.v.) suppressed the ACTH response induced by IL-6. The number of IL-6-induced immunoreactive cells in the PVN was significantly reduced by indomethacin pretreatment (p < 0.01), but the number of IL-6-induced c-fos-positive cells in the SON and CeA remained unchanged. These findings suggest that circulating IL-6 may exert central actions by acting directly or indirectly on brain neurons. In addition, the ability of IL-6 to activate the HPA axis may depend upon the release of prostaglandins, probably in the brain.

The Kappa‐Opioid Receptor Agonist MR‐2034 Stimulates the Rat Hypothalamic‐Pituitary‐Adrenal Axis: Studies in vivo and in vitro

There is increasing evidence that opiates not only have analgesic properties, but also regulate mechanisms activated during the stress response, such as the hypothalamic-pituitary-adrenal (HPA) axis. Indeed, opioid-containing neurons innervate the paraventricular nucleus and the median eminence, thus modulating inputs to ACTH-controlling neurons. In addition, dynorphin (the endogenous ligand of the kappa-opioid receptor)-like peptides have been found co-localized with corticotrophin-releasing hormone (CRH) and are believed to be co-secreted with it in the hypophyseal portal circulation to modulate ACTH release. In this study, we evaluated the effects of the selective kappa-opioid receptor agonist MR-2034 [(-)-N-(2-tetrahydrofurfuryl)-normetazocine] on the HPA axis in vivo and in vitro. MR-2034 was given intravenously to catheterized, freely moving, male Sprague-Dawley rats and serial blood samples were collected for ACTH and corticosterone (B) measurements. We evaluated also the site of MR-2034 action on the HPA axis in vivo, after the administration of alpha-helical CRH9-41, a CRH receptor antagonist, on hypothalamic CRH, pituitary ACTH, and B release in vitro. MR-2034 increased plasma ACTH and B levels in a dose-related fashion and this effect was antagonized by the selective kappa-opioid receptor antagonist MR-1452. In the presence of alpha-helical CRH9-41, the responses of plasma ACTH and B to MR-2034 were blunted significantly, suggesting that this compound activates the HPA axis through a CRH-dependent mechanism. Accordingly, MR-2034 stimulated hypothalamic CRH release in vitro in a concentration-dependent fashion and this effect was antagonized dose-dependently by MR-1452. However, the stimulatory effect of MR-2034 on plasma ACTH and B in vivo was not completely abolished by alpha-helical CRH9-41, suggesting that an additional, CRH-independent, mechanism was involved. Indeed, MR-2034 was able to stimulate basal ACTH output in a dose-dependent manner and this effect was antagonized by MR-1452 in vitro. On the other hand, MR-2034 did not have any effect on B release from adrenocortical cells or adrenal quarters in vitro. These results show that the benzomorphan MR-2034 stimulates the HPA axis in the rat by acting at the hypothalamic and the pituitary level. We hypothesize that endogenous kappa-opioid peptides not only act at the pituitary level to increase ACTH output, but may also act at the hypothalamic level to increase CRH release through an autocrine and/or ultrashort positive feedback mechanism.

131I-6 beta-iodomethylnorcholesterol scintigraphy: an assessment of its role in the investigation of adrenocortical incidentalomas.

Most incidentally discovered adrenal tumours ('incidentaloma') are benign adrenocortical adenomas. It has been suggested that 131I-6 beta-iodomethylnorcholesterol (IMC) scan could specify the degree of functional autonomy of such adenomas depending on whether they prevent contralateral adrenal tracer uptake. Our purpose was to examine this hypothesis in a correlated scintigraphic and endocrine study. Prospective study evaluating the prevalence of unilateral IMC uptake (tumour uptake with no visualization of the contralateral adrenal gland) and bilateral uptake (uptake in both the tumoral and the contralateral adrenal glands) in patients with unilateral incidentaloma. Comparison of adrenocortical function and of IMC scan after dexamethasone (DXM) in the two scintigraphic groups thus defined. Thirty-five patients with a unilateral mass highly suggestive of benign adrenocortical adenoma on CT scan. The IMC scan was performed in basal conditions (baseline scan) and after DXM (suppression scan). Adrenocortical function assessment included basal measurements of 11-deoxycortisol, 17 alpha-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulphate (DHEAS), plasma cortisol and ACTH, urinary free cortisol (UFC), overnight and low-dose DXM suppression test, and CRH test. The baseline scan showed 16 patients (46%) with unilateral uptake (group A) and 19 (54%) with bilateral uptake (group B). Patients in group A exhibited lower ACTH values at 0800h (P = 0.05) and higher cortisol values after an overnight DXM suppression test (P = 0.02), than did patients in group B. In addition, 3 patients in group A failed the overnight and the low-dose DXM suppression tests. Adrenal masses were larger in group A than group B (P = 0.04) and an inverse correlation was found in the whole population between tumour size and ACTH value at 0800h (P = 0.05). On the suppression scan performed in 14 patients (7 in each group), patients in group A continued to exhibit unilateral tumour uptake and bilateral uptake was suppressed in 72% of patients in group B. An adrenal mass was removed in 3 patients of group A with confirmed benign adrenocortical adenomas. In the post-surgical period, the contralateral gland was again visualized in a baseline scan and the hormonal evaluation returned to the normal range. Unilateral 131I-6 beta-iodomethylnorcholesterol tumour uptake is a frequent feature in benign adrenocortical adenomas. Hormonal data and scintigraphic profiles obtained after dexamethasone, as well as hormonoscintigraphic changes observed after surgery, provide evidence that unilateral uptake is related to functioning adenomas with various degrees of autononomy and suggest that the 131I-6 beta-iodomethylnorcholesterol scan could be a valuable tool for screening 'subclinical' Cushing's adenomas.

Effect of androstenedione administration on the maternal hypothalamo-pituitary-adreno-placental axis in the pregnant rhesus monkey.

To assess the interaction among androgens, placenta, and the hypothalamo-pituitary-adrenal axis we studied effects of androstenedione administered intravascularly to the pregnant monkey on maternal plasma CRH, ACTH, dehydroepiandrosterone sulfate (DHEAS), cortisol, and estradiol concentrations. Ten monkeys (128 +/- 3 days gestation; mean +/- SEM) were instrumented under general halothane anesthesia with maternal femoral artery and venous catheters and uterine electromyogram electrodes. At 137-144 days gestation, baseline maternal femoral artery samples for CRH, ACTH, DHEAS, cortisol, and estradiol measurements were taken at 1.5-h intervals for 7 h starting 2 h before darkness. On the following day, a continuous iv androstenedione infusion (0.3 mg/kg.min at 0.25 ml/h) in 10% intralipid was started at 0930 h in four monkeys; the other six animals received vehicle alone at the same rate starting at the same time. Maternal blood sampling was repeated 1 and 3 days after androstenedione or vehicle administration. Maternal plasma CRH, ACTH, DHEAS, cortisol, and estradiol levels were unaffected by intralipid. In contrast, androstenedione infusion produced a sustained increase in maternal plasma estradiol and a sustained fall in maternal plasma ACTH, but did not affect maternal plasma CRH, DHEAS, or cortisol concentrations. These results provide evidence for negative feedback regulation by androgens at the hypothalamo-pituitary-adrenal axis in the pregnant monkey. Lack of inhibition of maternal plasma CRH after androstenedione administration supports differential regulation of hypothalamic and placental CRH by androgens.

Effect of androstenedione administration on the maternal hypothalamo- pituitary-adreno-placental axis in the pregnant rhesus monkey

To assess the interaction among androgens, placenta, and the hypothalamo-pituitary-adrenal axis we studied effects of androstenedione administered intravascularly to the pregnant monkey on maternal plasma CRH, ACTH, dehydroepiandrosterone sulfate (DHEAS), cortisol, and estradiol concentrations. Ten monkeys (128 +/- 3 days gestation; mean +/- SEM) were instrumented under general halothane anesthesia with maternal femoral artery and venous catheters and uterine electromyogram electrodes. At 137-144 days gestation, baseline maternal femoral artery samples for CRH, ACTH, DHEAS, cortisol, and estradiol measurements were taken at 1.5-h intervals for 7 h starting 2 h before darkness. On the following day, a continuous iv androstenedione infusion (0.3 mg/kg.min at 0.25 ml/h) in 10% intralipid was started at 0930 h in four monkeys; the other six animals received vehicle alone at the same rate starting at the same time. Maternal blood sampling was repeated 1 and 3 days after androstenedione or vehicle administration. Maternal plasma CRH, ACTH, DHEAS, cortisol, and estradiol levels were unaffected by intralipid. In contrast, androstenedione infusion produced a sustained increase in maternal plasma estradiol and a sustained fall in maternal plasma ACTH, but did not affect maternal plasma CRH, DHEAS, or cortisol concentrations. These results provide evidence for negative feedback regulation by androgens at the hypothalamo-pituitary-adrenal axis in the pregnant monkey. Lack of inhibition of maternal plasma CRH after androstenedione administration supports differential regulation of hypothalamic and placental CRH by androgens.

Clinical similarity and biological diversity in the response to alprazolam in patients with panic disorder and generalized anxiety disorder.

Thirty-six patients with panic disorder (PD) and 35 patients with generalized anxiety disorder (GAD) participated in an open alprazolam treatment phase that preceded controlled withdrawal from alprazolam. Clinical ratings, blood pressure and heart rate were obtained along with plasma measurements of cortisol, ACTH, growth hormone and catecholamines. A similar clinical response profile was evident in both groups with rapid onset of improvement within the first week. The two diagnostic groups differed in their biological response to alprazolam. PD patients had a significant reduction in blood pressure, plasma cortisol and a trend toward significant reduction in plasma epinephrine, which were not seen in the GAD patients. GAD patients showed a significant reduction in plasma norepinephrine. These findings provide further evidence that PD and GAD are biologically distinct syndromes.

Factitious Cushing’s Syndrome: Discovery with Use of a Sensitive Immunoradiometric Assay for Corticotropin

Factitious Cushing's syndrome is an unusual problem that may be clinically and biochemically indistinguishable from endogenous hypercortisolism. Results of biochemical studies may be misleading because of cross-reactivity of synthetic corticosteroids or their metabolites with plasma or urine cortisol. Because of its lack of specificity, radioimmunoassay (RIA) for corticotropin (adrenocorticotropic hormone or ACTH) may not show completely suppressed results in patients with surreptitious use of glucocorticoids. A new sensitive and specific immunoradiometric assay (IRMA) for ACTH demonstrates reliably suppressed levels after administration of glucocorticoids. In this report, we describe a 37-year-old woman with typical clinical and biochemical features of ACTH-dependent hypercortisolism. Metabolic evaluation had shown urinary free cortisol excretion of 7,499 nmol/day and plasma ACTH-RIA levels of 13.2 and 33.0 pmol/L on two separate occasions. The use of IRMA for the measurement of ACTH during inferior petrosal sinus sampling revealed a very low peripheral ACTH concentration (=0.4 pmol/L) and a considerably blunted response to ovine corticotropin-releasing hormone, findings that suggested ACTH-independent Cushing's syndrome. Plasma samples obtained during inferior petrosal sinus sampling were assayed for prednisolone and showed a concentration of 360 nmol/L; thus, the presence of factitious Cushing's syndrome was confirmed. Some commercial ACTH-RIA measurements may be unreliable in distinguishing ACTH-dependent from ACTH-independent hypercortisolism. ACTH-IRMA levels are low in patients with ACTH-independent Cushing's syndrome and will be helpful in identifying factitious Cushing's syndrome.

Adrenarche does not occur in treated patients with congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency.

There have been few studies of adrenarche in patients with congenital adrenal hyperplasia (CAH). We have therefore sought to detect the onset of adrenarche in CAH patients and to investigate whether its evolution was influenced by the severity of the disease, the age at the onset of substitution therapy, or both. Sixteen female CAH patients were studied longitudinally for 4-11 years. They were all given substitution therapy and treatments were well controlled as judged by repeated hormonal evaluations. The patients were divided into two groups: group A consisted of 10 girls with a severe classic (congenital) form, while group B included 6 girls presenting with a non-classic form. Circulating levels of dehydroepiandrosterone sulphate (DHEAS), were determined as an indicator of adrenarche. Hormonal assessments included measurements of 17-hydroxyprogesterone (17-OHP), testosterone, ACTH and plasma renin activity. All were estimated by conventional specific assays. Mean levels were analysed in consecutive two-year age periods. In group A, DHEAS levels were significantly lower at any age than in control subjects, and lower than in patients with non-classic CAH. DHEAS levels showed no increment with age. In group B, plasma DHEAS levels were surprisingly high for the age at the time of diagnosis, declining gradually on substitution therapy, although they remained somewhat higher than in group A. The high DHEAS levels observed in untreated girls of group B are probably the result of chronic hypersecretion of ACTH. Under well controlled, non-suppressive substitution therapy, patients with congenital adrenal hyperplasia showed no rise in DHEAS levels at the physiological age of adrenarche whatever the degree of the enzyme defect and whatever the age at onset of therapy.

Corticotropin-releasing factor alone, but not arginine vasopressin alone, stimulates the release of adrenocorticotropin in the conscious intact sheep

These studies compared the relative potencies of CRF and arginine vasopressin (AVP) as ACTH secretagogues in the sheep. Dose-response curves to CRF (10, 25, 50, and 100 micrograms/h) and AVP (0.3, 1.0, 3.0, and 10.0 micrograms/h) were obtained in five adult sheep, with arterial blood samples taken for CRF, AVP, ACTH, and cortisol measurements to determine the pituitary-adrenal response. It was found that the first dose of CRF increased plasma CRF levels to 444 +/- 79 pg/ml. ACTH levels rose significantly from 28 +/- 5 to 186 +/- 46 pg/ml, with a concurrent rise in cortisol levels from 7 +/- 3 to 44 +/- 9 ng/ml. Although plasma ACTH and cortisol levels remained elevated, higher doses of CRF failed to produce further increases. AVP, at all of the doses studied, did not produce an increase in ACTH levels, although cortisol levels rose significantly by the second dose. In a second series of experiments, animals received a continuous infusion of CRF (10 micrograms/h) alone or in combination with graded doses of AVP (0.3, 1.0, 3.0, and 10.0 micrograms/h for 60 min each). It was found that AVP at the highest dose was able to potentiate the ACTH response to CRF. Lower doses of AVP, which did not stimulate a further increase in ACTH levels were, however, associated with a significant rise in cortisol levels. In conclusion, it was found that although CRF alone was a potent stimulator of ACTH secretion, AVP at the doses studied was not able to elicit an ACTH response in the conscious intact sheep. However, a dose of AVP that was not stimulatory in itself was able to augment the ACTH response to CRF.

Dissociation of plasma adrenocorticotropin and cortisol levels in critically ill patients: possible role of endothelin and atrial natriuretic hormone.

The regulatory mechanisms of the hypothalamo-pituitary-adrenal system were studied in critically ill, intensive care unit patients. Serial measurements of immunoreactive ACTH-(1-39) (ACTHi), cortisol, endothelin-1 (ETi), and atrial natriuretic hormone (ANHi) were performed in blood samples of 18 patients with clinically defined sepsis, 12 critically ill patients after multiple trauma, and 15 hospitalized matched control subjects without acute illness for 8 consecutive days after admission. On admission, plasma levels of cortisol and ACTHi were significantly elevated in patients with sepsis (1.32 +/- 0.21 mumol/L and 130.0 +/- 38.2 pmol/L, mean +/- SD) and with multiple trauma (1.23 +/- 0.28 mumol/L and 123.7 +/- 41.3 pmol/L) compared to those in the control subjects (0.37 +/- 0.08 mumol/L and 15.6 +/- 5.8 pmol/L, respectively). The plasma cortisol levels of critically ill patients remained high (> 0.8 mumol/L) during the whole observation period. In contrast, plasma ACTHi levels decreased between days 3-5, reaching significantly lower levels on day 5 compared to those in the control group and remained below 5.0 pmol/L during the rest of the observation period. Plasma levels of ETi and ANHi were significantly elevated during the whole period in both patient groups (ETi, > 10 ng/L; ANHi, > 250 ng/L) compared to those in control subjects (< 5 and < 50 ng/L, respectively). The high plasma concentration of ETi observed in our patients may stimulate the steroid secretion of the adrenal cortex directly or potentiate the adrenal effect of ACTH. On the other hand, the increased concentration of ANHi found in critically ill patients together with the increased plasma cortisol level may explain the inhibition of ACTH secretion. Accordingly, we speculate that the high ET level exerts a positive drive on the adrenocortical level, that the high ANH level has an inhibitory effect on the hypothalamo-pituitary level, and that both mechanisms play a role in regulation of the hypothalamo-pituitary-adrenal axis during critical illness.

Intraoperative measurement of adrenocorticotropin (ACTH) during removal of ACTH-secreting bronchial carcinoid tumors.

The optimal treatment for ectopic ACTH syndrome is the complete removal of the tumor secreting ACTH. These tumors are often occult, with their location suggested but not proven with imaging techniques. The intraoperative measurement of ACTH by immunoradiometric assay in five patients with the occult ectopic ACTH syndrome during removal of suspicious intrapulmonary lesions is reported. A significant ACTH gradient was detected in the pulmonary veins of the affected lobes in two patients. ACTH had decreased significantly in all five patients by 10 and 15 min after tumor removal. All five patients had histologically proven ACTH-secreting bronchial carcinoid tumors, suppressed plasma ACTH by 24 h after tumor removal, and subsequent secondary adrenal insufficiency indicating successful surgical therapy (five of five true-positive). In one patient, previous surgery was not curative and did not result in a decrease in intraoperative measurement of ACTH (one of one true-negative). It was demonstrated that a rapid ACTH immunochemiluminescence assay with a 15-min incubation time has sufficient sensitivity and precision to detect decreases in ACTH described above. These results demonstrate that complete removal of ACTH-secreting bronchial carcinoid tumors can be detected intraoperatively by a decrease in arterial ACTH by 15 min. The modification of the ACTH immunochemiluminescence assay to 15 min incubation allows the documentation of a successful tumor removal in the operating room. It may also be used to locate the tumor intraoperatively by selective pulmonary vein sampling. This protocol may be applicable to the intraoperative measurement of ACTH during pituitary microadenomectomy for Cushing's disease.

Problems in the diagnosis of pituitary adenoma (Cushing's syndrome) in horses

Thyrotropin releasing hormone (TRH) stimulation tests were carried out on a number of horses, including one clinically affected horse whose age and clinical signs suggested that a pituitary adenoma was most unlikely. The results of these tests indicated that, according to criteria published overseas, the majority of these horses had pituitary adenomas. The fact that clinically normal or affected horses may have marked increases in cortisol concentrations indicates that the TRH stimulation test is not suitable for the diagnosis of pituitary adenoma. Other tests, including alterations in glucose metabolism and, if available, ACTH measurements, are probably more useful for diagnosis. However, most diagnoses will probably rely upon the characteristic clinical signs.

Evidence that activation of the hypothalamo-pituitary-adrenal axis by electrical stimulation of the noradrenergic A1 group is not mediated by noradrenaline.

The paraventricular nucleus (PVN) of the hypothalamus, where the CRF-containing neurosecretory cells controlling the hypothalamo-pituitary-adrenal (HPA) axis are located, receives a dense noradrenergic innervation from the A1 group of the caudal ventrolateral medulla. In the present study we studied the relationship between release of noradrenaline (NA) in the PVN and activation of the HPA axis in response to electrical stimulation of the A1 region. In the urethane-anesthetized male rat, extracellular NA in the PVN was monitored on line by electrochemical recording while the activity of the HPA axis was estimated by measurement of ACTH in blood samples. A 1 min, 10 Hz stimulation evoked a significant increase of extracellular NA in the PVN as well as an ACTH surge in blood. The NA and ACTH response evoked by stimulation in the 3- to 14-Hz range were found to be frequency dependent. However, whilst the NA response increased in an exponential manner with respect to frequency, the ACTH response appeared to plateau between 10 and 14 Hz. Specific lesions of the noradrenergic terminals in the PVN, by bilateral local administration of 6-hydroxydopamine, markedly reduced the ACTH response to stimulation. Intracerebroventricular injection of desmethylimipramine, a NA uptake inhibitor, enhanced the increase in extracellular NA evoked by submaximal stimulation about 2.5-fold but did not modify the corresponding ACTH response. Combined intracerebroventricular injection of alpha- and beta-adrenergic antagonists, phentolamine and propanolol respectively, did not prevent the ACTH response evoked by stimulation. Following stimulation of the caudal ventrolateral medulla, the ACTH response thus appears to result from the stimulation of the A1 noradrenergic group projecting to the PVN. However, the inability of pharmacological manipulations which enhance or block central noradrenergic transmission to influence the ACTH response suggests that the noradrenergic endings in the PVN originating from the A1 group use a transmitter other than NA to activate the HPA axis at the PVN level.

Time-Course of Hypothalamic CRH and Pituitary ACTH Contents, and Pituitary Responsiveness to CRH Stimulation After Bilateral Adrenalectomy

The pituitary ACTH and hypothalamic CRH alterations at different periods after adrenalectomy (ADX) or Sham ADX were studied by measurement of ACTH and CRH contents by radioimmunoassay (RIA) in rats. We also studied the corticotroph alterations by immunohistochemistry and the in vitro pituitary responsiveness to CRH. Plasma ACTH presented a triphasic response after ADX. Anterior pituitary (AP) ACTH content decreased 3 h and 1 day after surgery, then rose over this period. Immunohistochemistry demonstrated an initial degranulation of the corticotrophs with a progressive increase in cells immunostained for ACTH and a positive correlation (r = 0.88) with AP ACTH content measured by RIA. Hypothalamic CRH content decreased after ADX, but returned to sham value 3 weeks later. Basal ACTH secretion in incubation medium was correlated with the AP ACTH content observed in vivo. The pituitary responsiveness to CRH was not the same at all times after ADX. It was absent 1 day after, presented an increase of 51%, 117% and 26% when compared with the basal ACTH output 3 h, 3 and 14 days after ADX, respectively. Our data suggest that after ADX the corticotroph undergoes a transitory decrease in ability to secrete ACTH after store depletion. During a later phase, however, there is a relative hyporesponsiveness of the corticotroph to CRH stimulation. These data indicate that CRH may be acting on a down-regulated pituitary or more additional factors play a role in the sensitization of the pituitary after ADX.

Adrenocorticotropin hyperresponse to the corticotropin-releasing hormone-mediated stimulus of naloxone in patients with myotonic dystrophy.

We previously showed that CRH-mediated stimuli, including exogenous CRH, cause ACTH hypersecretion in many myotonic dystrophy (DM) patients. We confirmed this by giving naloxone, a stimulator of endogenous CRH release, to a large number of DM patients and controls. DM patients, first degree relatives, and normal controls received i.v. naloxone at 1400 h, and blood was taken for ACTH (RIA) and cortisol (high pressure liquid chromatography) measurements from 15 min before to 120 min after naloxone treatment. DM patients had basal ACTH levels approximately twice those of controls, and their ACTH responses were 4 times those of controls. In contrast, DM basal cortisol levels were not significantly different from those of relatives and were slightly higher than those of normal subjects. Cortisol responses were similar in the three groups, probably due to attenuation at high levels of adrenocortical stimulation, although some patients with inappropriately low cortisol responses for their level of ACTH stimulation warrant further investigation. Nineteen of the 36 patients whose ACTH responses were greater than 3 SD above the normal mean were classed as hyperresponders. Seven patients, who were tested more than once, had reproducible responses relative to those of the normal subjects. We conclude that ACTH hypersecretion after CRH-mediated stimuli, including naloxone, is an inherent, but variable, feature of DM, caused by expression of the genetic mutation at the anterior pituitary. The mechanism is probably a defect in the intracellular pathway initiated by CRH-receptor interaction as a result of abnormal levels of a cAMP-dependent kinase, DMPK, the product of the gene undergoing mutation in DM.

Endothelin stimulates ACTH secretion in the ovine fetus.

The endothelins are a series of peptides with potent vasoconstrictor effects in vascular tissue; however, they may also have a role as neuroendocrine secretagogues because endothelin and endothelin receptors have been found in hypothalamic and pituitary tissues. Since activation of the fetal hypothalamo-pituitary-adrenal axis is crucial to the process of parturition, the aim of this study was to determine whether endothelin could activate the hypothalamo-pituitary-adrenal axis in the ovine fetus. Catheters were inserted into the carotid artery, jugular vein and lateral cerebral ventricle of six fetal lambs at 118-122 days' gestation. After a 5-day recovery period, endothelin-3 or placebo (saline) was infused intravenously (i.v.) or intracerebroventricularly (i.c.v.) over 30-60 min. The dose of endothelin-3 employed was 0.01 and 0.1 microgram min-1 i.c.v. and 0.1 and 1.0 microgram min-1 i.v. Arterial blood was taken from the fetus before, during and for 1 h after the infusion for measurement of ACTH and cortisol. Blood gas analysis was also performed. Intravenous endothelin-3 produced a dose-dependent increase in ACTH and cortisol concentrations in fetal plasma and was associated with transient fetal hypoxia and acidosis. pH correlated inversely with plasma ACTH (r = -0.701, P < 0.001) and cortisol (r = -0.308, P < 0.001) concentration. An increase in ACTH and cortisol concentrations in fetal plasma was also induced by endothelin-3 administered i.c.v. at 0.1 microgram min-1, and this was not associated with fetal acidosis These data suggest that endothelin administered i.v. will stimulate ACTH and cortisol release indirectly through vasoconstriction and acidosis; however, the response to i.c.v. endothelin administration suggests that it may also act as a central secretagogue for ACTH in the fetus and could therefore play a role in the process of parturition.

A comparison of the standard high dose dexamethasone suppression test and the overnight 8-mg dexamethasone suppression test for the differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome.

To improve the overnight 8-mg dexamethasone (DEX) suppression test (DST) for differential diagnosis of Cushing's syndrome and to develop optimal criteria for its interpretation, we increased the number of blood samples and measured the suppression of both plasma ACTH and cortisol. Forty-one patients who were subsequently proven at surgery to have Cushing's syndrome were studied (34 Cushing's disease and 7 ectopic ACTH secretion). DEX (8 mg, orally) was administered at 2300 h. Blood samples for ACTH and cortisol measurements were obtained at 0800, 0830, and 0900 h on the day before and at 0700, 0800, 0900, and 1000 h on the morning after DEX treatment. The conventional 6-day DST was also performed, with measurement of both urinary free cortisol and urinary 17-hydroxysteroids as indices of suppression. Optimal criteria for the diagnosis of Cushing's disease were developed for both the overnight 8-mg and the 6-day tests using receiver operating characteristic curves. The results were compared with those using the previously published criteria for diagnosis of Cushing's disease by the overnight 8-mg test (> 50% suppression of plasma cortisol at 0700-0800 h). In our patients, the previously published criterion for the overnight 8-mg test yielded high sensitivity (88%), but low specificity (57%), in making the diagnosis of Cushing's disease. When the time of cortisol measurement and the diagnostic criteria for Cushing's disease were revised to achieve 100% specificity, the sensitivity of the overnight 8-mg test was 71%, which was not significantly different from that of the 6-day test (79%; P = NS). Addition of plasma ACTH levels to the test did not improve diagnostic accuracy compared to that with measurement of plasma cortisol levels alone. When the revised 8-mg overnight dexamethasone suppression test was combined with the 6-day dexamethasone suppression test, sensitivity increased to 91%, with a specificity of 100%, which was significantly better than that of the overnight 8-mg test alone (P < 0.002). We conclude that the overnight 8-mg DST has low specificity for the diagnosis of Cushing's disease when performed as originally described. However, with revised sampling times and diagnostic criteria, the overnight test has sensitivity and specificity similar to those of the conventional 6-day DST. The diagnostic performance of a criterion that combines the results of both tests is better than the diagnostic performance of either test alone.

A comparison of the standard high dose dexamethasone suppression test and the overnight 8-mg dexamethasone suppression test for the differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome

To improve the overnight 8-mg dexamethasone (DEX) suppression test (DST) for differential diagnosis of Cushing's syndrome and to develop optimal criteria for its interpretation, we increased the number of blood samples and measured the suppression of both plasma ACTH and cortisol. Forty-one patients who were subsequently proven at surgery to have Cushing's syndrome were studied (34 Cushing's disease and 7 ectopic ACTH secretion). DEX (8 mg, orally) was administered at 2300 h. Blood samples for ACTH and cortisol measurements were obtained at 0800, 0830, and 0900 h on the day before and at 0700, 0800, 0900, and 1000 h on the morning after DEX treatment. The conventional 6-day DST was also performed, with measurement of both urinary free cortisol and urinary 17-hydroxysteroids as indices of suppression. Optimal criteria for the diagnosis of Cushing's disease were developed for both the overnight 8-mg and the 6-day tests using receiver operating characteristic curves. The results were compared with those using the previously published criteria for diagnosis of Cushing's disease by the overnight 8-mg test (> 50% suppression of plasma cortisol at 0700-0800 h). In our patients, the previously published criterion for the overnight 8-mg test yielded high sensitivity (88%), but low specificity (57%), in making the diagnosis of Cushing's disease. When the time of cortisol measurement and the diagnostic criteria for Cushing's disease were revised to achieve 100% specificity, the sensitivity of the overnight 8-mg test was 71%, which was not significantly different from that of the 6-day test (79%; P = NS). Addition of plasma ACTH levels to the test did not improve diagnostic accuracy compared to that with measurement of plasma cortisol levels alone. When the revised 8-mg overnight dexamethasone suppression test was combined with the 6-day dexamethasone suppression test, sensitivity increased to 91%, with a specificity of 100%, which was significantly better than that of the overnight 8-mg test alone (P < 0.002). We conclude that the overnight 8-mg DST has low specificity for the diagnosis of Cushing's disease when performed as originally described. However, with revised sampling times and diagnostic criteria, the overnight test has sensitivity and specificity similar to those of the conventional 6-day DST. The diagnostic performance of a criterion that combines the results of both tests is better than the diagnostic performance of either test alone.

The Biochemical Diagnosis of Hypercortisolism

The diagnostic evaluation of Cushing's syndrome proceeds in two stages: first the definitive diagnosis of pathological hypercortisolism (Cushing's syndrome), and second the differential diagnosis of pituitary, adrenal, and ectopic causes. Proper initial diagnosis of Cushing's syndrome depends on correlation of clinical data with results of 24-hour urine free cortisol and responses to low-dose dexamethasone suppression. Although responses to corticotropin-releasing hormone (CRH) administration differ between patients with Cushing's syndrome and those with pseudo-Cushing's states such as stress, depression, and alcoholism, this test is not, at present, useful in the initial diagnosis of Cushing's syndrome. When the diagnosis of Cushing's syndrome has been secured, measurement of ACTH or β-LPH provides a reliable distinction between ACTH-dependent and adrenal-based etiologies. New criteria for the standard 48-hour high-dose dexamethasone test identify approximately 80% of patients with Cushing's disease. The CRH stimulation test may provide additional diagnostic accuracy. Inferior petrosal sinus sampling is a highly accurate test for ACTH-dependent Cushing's syndrome when other test results are inconclusive.

A Radioimmunoassay for Adrenocorticotropic Hormone (ACTH) in Unextracted Plasma of Various Animals

A radioimmunoassay of plasma ACTH, useful for various mammalian species was devel-oped. The RIA for ACTH involves use of commercially available antiserum and 125I labelled hormone, and permits measurement of immunoreactive ACTH in unextracted plasma of various mammals. Physiologi-cal validation of the method was obtained from the following studies. Intact male rats had elevations of immunoreactive plasma concentrations of ACTH after the onset of immobilization stress. A dose related increase in plasma concentrations of ACTH was obtained in adult male rats by the injection of ACTH releasing hormone (CRH).The specific steps in the procedure of assay were following. All steps were performed under cool condi-tion (4C). Basal buffer was 0.063 M Na2HPO4 containing 0.013M ethylenediaminetetraacetic acid 2Na salt (EDTA) and 0.02% NaN3 adjusted to pH 7.4. RIA buffer (basal buffer containing 0.1% Triton X-100 and 250 KIU/ml aprotinin) was made into BSA•RIA buffer (for dilution of standards or samples: containing 1% protease free bovine serum albumin), NRS•RIA buffer (for dilution of antiserum to ACTH: contain-ing 0.4% normal rabbit serum) and PEG•RIA buffer (for dilution of antiserum to rabbit gamma globulin: containing 4% polyethylene glycol). BSA•EDTA buffer (separation buffer) was also prepared by adding 1% BSA to basal buffer.Plasma or serum (1-40 μl) were diluted to 100μl with BSA•RIA buffer. Standards or samples (100μl) were put in the polystylene tubes and 100 μl of antiserum were added to each tubes. After incubation at 4C for 24 h, 100μl of labelled ligand were added and incubated for another 24 h. Then, antiserum to rabbit gamma globulin was added to each tube. After further incubation for 24 h, 1 ml of separation buffer was added to each tube and antibody bound hormone was separated from the free hormone by centrifugation at 4C, 1700 g for 30 min. The supernatant was carefully decanted and the precipitate was counted in auto-matic gamma counter. Plasma or serum concentrations of ACTH were calculated using a standard curve.