Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.

The evolution of American Medical Association policies concerning health care of veterans.

Relationships between the American Medical Association (AMA) and the Department of Veterans Affairs (VA) have been reviewed from the perspective of evolving AMA policies regarding the care of veterans, including educational and research policies. During the first two decades of VA hospital development between 1925 and 1945, the AMA opposed government participation in the care of veterans. During the next three decades there was a reluctant acknowledgement by the AMA of the need for federally housed care of veterans with service-connected illnesses. At the same time, the AMA recognized the value of the mission of the VA in training specialists through postgraduate medical education. More recently the AMA has been especially supportive of VA-medical school affiliations and VA activities in education and research. The reluctance of the AMA to support veterans' health care was paralleled by reluctance of VA staff physicians to join the AMA. In recent years the AMA has recognized the need to diversify its membership as increasing numbers of physicians have been trained in part in VA medical centers. It appears to be a time of enhanced opportunity for the AMA to work more closely with the Department of Veterans Affairs.

Inhibition of dexamethasone binding to human glucocorticoid receptor by New World primate cell extracts.

To determine if New World primates express an inhibitor that influences glucocorticoid receptor (GR) binding characteristics, we examined [3H]dexamethasone binding in cytosol prepared from B95-8 lymphoid cells, derived from the cotton top tamarin (Saguinus oedipus), in combination with cytosol prepared from human or rat tissues. B95-8 cytosol inhibited specific binding of [3H]dexamethasone (P < 0.01) when mixed with cytosol prepared from either a human lymphoid cell line (HL) or rat thymus. The inhibitory activity was heat labile and trypsin sensitive. Peak inhibitory activity was found in the 150-200 kd fractions after Sephacryl G-200 ultrafiltration. Scatchard analysis of [3H]dexamethasone binding using mixed cytosol showed a diminished GR apparent binding affinity when compared to HL cytosol. Kinetic studies using mixed cytosol indicated that B95-8 cytosol did not affect the apparent dissociation rate of [3H]dexamethasone. These data demonstrate that B95-8 cells contain a competitive inhibitor that prevents binding of dexamethasone to its cognate receptor.

Influence of ethanol dependence on regional brain content of beta-endorphin in the mouse.

beta-Endorphin-like immunoreactivity (BE-LI) was measured in 7 brain regions of Swiss-Webster mice after 24, 48 and 72 h of exposure to ethanol vapor following a priming injection of ethanol and daily injections of pyrazole HCl to inhibit ethanol metabolism. Control mice in identical chambers received pyrazole injections but breathed air only. Ethanol dependence was confirmed by scoring additional groups of mice for handling-induced convulsions during withdrawal after each exposure duration. Measurement of anterior and neurointermediate (NIL) pituitary BE-LI, alpha-MSH and ACTH and plasma corticosterone confirmed earlier results showing NIL depletion of all 3 peptides at 24 h and increased plasma corticosterone concentrations at 72 h in ethanol-exposed mice. In brain extracts from ethanol-dependent mice, BE-LI was significantly reduced in the hypothalamus and midbrain with the greatest reduction occurring at 24 h. In forebrain, cerebral cortex, septum and hippocampus, pyrazole treatment significantly reduced BE-LI relative to an unhandled control group, and ethanol exposure tended to reverse this effect. HPLC of hypothalamic extracts revealed no differences in proportions of molecular forms of beta-endorphin-like peptides between 24 h control and ethanol-exposed groups. The predominant BE-LI peak in both groups co-eluted with opiate-active unmodified beta-endorphin. Ethanol dependence in mice is associated with regionally selective decreases in brain beta-endorphin concentration.

Ethanol-stimulated endorphin and corticotropin secretion in vitro.

Although acute administration of ethanol in vivo results in increased plasma glucocorticoid concentration, it is unclear whether this effect is mediated by corticotropin (ACTH) from the anterior pituitary. Secretion of beta-endorphin-like (BE-IR) and corticotropin-like (ACTH-IR) immunoreactivity from perifused, dispersed mouse adenohypophyseal cells was used to evaluate the effect of 17 mM ethanol on secretion of pituitary peptides. Cells were also exposed to 10 nM synthetic corticotropin-releasing factor (CRF), 1 microM vasopressin, 54 mM KCl, 100 nM corticosterone, and calcium-free medium, separately and in combination. Secretion of BE-IR and ACTH-IR were markedly sensitive to low concentrations of ethanol. Exposure to 17 mM ethanol produced 3-fold stimulation of the rate of hormone release. This represented one-third to two-thirds that of the rate of maximum stimulation by CRF. Unlike CRF-stimulated secretion, ethanol-stimulated secretion was transient. Further, a second ethanol exposure 1 h after the first did not stimulate peptide secretion. Similar to CRF-stimulation, ethanol-stimulated peptide secretion required extracellular calcium and was inhibited by the glucocorticoid corticosterone. We suggest that this system is a useful model for investigation of the actions of low concentrations of ethanol at the cellular level.

The relationship of endogenous ACTH levels to visual-attentional functioning in patients with congenital adrenal hyperplasia.

The within subject experimental approach of either doubling cortisone medication dose or withdrawing steroid treatment for 36 hr preceding behavioral testing was found to induce corresponding significantly elevated or suppressed plasma ACTH levels, as measured by radioimmunoassay, in six of eight adults diagnosed as having congenital adrenal hyperplasia (CAH). During the session characterized by elevated ACTH levels, the CAH patients exhibited significantly reduced median reaction times on the Sternberg Item Recognition Task. Their response pattern was suggestive of facilitated visual attentional functioning and/or overt motor response capacity rather than alteration of simple cognitive processing. Moreover, Sternberg performance was significantly correlated with endogenous ACTH levels but not with levels of plasma cortisol or cortisone replacement medication. This enhancement of performance paralleled a previous finding of improved performance on the Sternberg paradigm by normal adults following exogenous administration of ACTH 4-10 (Ward et al., 1979). Further analysis of the Sternberg performance suggests that other variables such as mineralocorticoid treatment, type of CAH impairment, and sex may act to moderate the degree of ACTH-related facilitation on this task. Performance on other visual and verbal attention and memory tasks, found earlier to be sensitive indices of exogenous administration of ACTH 4-10 and related fragments, was not significantly altered by manipulation of endogenous ACTH levels in these CAH patients.

Potassium modulation of corticosterone secretion from perifused mouse adrenal cells.

Potassium has long been recognized as a significant modulator of aldosterone synthesis and secretion. Its role as a regulator of glucocorticoids is less clear. Using a perifusion of dispersed mouse adrenal cells, we found a dose-related effect of extracellular potassium (K+) on secretion of the major rodent glucocorticoid, corticosterone. The maximal elicitable responses was 33% of the maximal response to ACTH. An increase in K+ concentration enhanced the effect of ACTH, while K+-free medium depressed the response to ACTH. The temporal characteristics of the corticosterone response to K+ were similar to that of ACTH and consistent with a primary effect on biosynthesis rather than on discharge of a preformed pool of hormone. The results indicate that a significant ion-mediated mechanism modulates ACTH-stimulated glucocorticoid secretion and suggest that further studies of endogenous substances operative by ionic mechanisms might be warranted.

Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development.

We have investigated the molecular weight forms of pro-opiomelanocortin (POMC)-derived peptides present in rat pituitaries during fetal and early postnatal development (embryonic Day 14 to 3 day neonate). At all early ages examined, the major immunoreactive form of corticotropin (ACTH) was POMC. Only during late fetal and early postnatal stages did progressively larger amounts of 4.5K ACTH, a major POMC processing end product, appear. This form was found almost exclusively in isolated anterior lobes. In contrast, 3.5K size endorphin(s), another POMC derivative, were present in whole glands even at early stages (Day 14), and were the major POMC derivative(s) found in isolated intermediate-posterior lobes of older fetuses. Despite the early appearance of 3.5K endorphin(s), alpha-MSH did not appear until Day 19 and was detected only in isolated intermediate-posterior lobes. We have also cultured dispersed fetal pituitary cells in the presence of radioactive amino acids. After immunoprecipitation using affinity-purified antisera, followed by fractionation of the radiolabeled products, we found that POMC biosynthesis does occur in cultures of Day 14 embryonic pituitary cells, and that the major POMC-derived end product produced is 3.5K size endorphin(s). These findings demonstrate that POMC is synthesized at least by Day 14 of rat pituitary development and that lobe-specific processing characteristic of the corresponding adult lobe is apparent at the earliest stages that the lobes can be separated. The presence of 3.5K-sized endorphins at early ages is consistent with the possibility that POMC synthesis first occurs in the intermediate lobe. The noncoordinate appearance of alpha-MSH, 1-39 ACTH, and endorphins implies that the activities of certain cleavage enzymes and acetylation enzymes responsible for lobe-specific post-translational POMC processing may be expressed at different times during development.

Endotoxin shock: prevented by naloxone in intact but not hypophysectomized rats.

Previous studies established that naloxone reverses hypotension in endotoxin, hemorrhagic, and spinal shock. We studied endotoxin shock in hypophysectomized (Hx) rats, which have little circulating beta-endorphin. Hx or intact rats received surgically implanted jugular catheters for drug injection and aortic catheters for arterial blood pressure (MAP) recording. On the second day after implantation, rats were pretreated with either naloxone or saline. Two minutes later each rat received endotoxin. Following endotoxin, all rats showed a brief biphasic hypertensive-hypotensive response followed by stabilization of MAP near baseline. Within 20 min, all Hx rats, regardless of pretreatment, and the saline-treated intact rats, showed progressive hypotension (P less than 0.005). Only the naloxone-pretreated intact rats maintained a stable MAP. Plasma endorphin measured at 20 min was undetectable in Hx rats in contrast to intact rats (P less than 0.001); plasma corticosterone levels were likewise suppressed in the Hx rats (P less than 0.01). Thus (1) naloxone protected only the rats with an intact pituitary-adrenal-sympathetic system, and (2) pituitary endorphin is not required to generate endotoxin shock in hypophysectomized rats.

Potassium-modulated secretion of immunoreactive melanocyte-stimulating hormone and endorphin from mouse neuro-intermediate lobes: evidence for stimulus-secretion uncoupling and rate sensitivity.

UNLABELLED: A multi-chamber perifusion system, capable of detecting transient secretory events, was used to define the roles of stepwise changes and gradients of K+ concentration in modulation of alpha-MSH and endorphin secretion. Fifteen dispersed mouse neuro-intermediate lobes per chamber were perifused with Dulbecco's Modified Eagle Medium at 0.5 ml/min. One-min fractions were collected. Ten min of 67 mM K+ elicited an immediate, very brief 4-fold increase in secretion of both hormones. Surprisingly, the return to normal K+ elicited a similar increase in secretion. Ten min K+-free medium produced an immediate decrease in secretion. Exposure to a 10-min 0-67 mM K+ gradient did not produce an increase in secretion; however, the stepwise return to normal K+, identical to that in the first experiment, elicited an immediate, brief increase in secretion. CONCLUSIONS: 1) The rapid decline in secretory activity during 67 mM K+ cannot be explained either by "down regulation" of receptors, since this secretagogue is not receptor-mediated, or by depletion of labile hormone, since a second secretory episode occurred immediately following termination of high K+. This suggests that some other cellular mechanism "uncouples" stimulus-secretory mechanisms. 2) Although depolarization with high K+ and hyperpolarization with K+-free medium were associated with increases and decreases, respectively, in secretion, it appears that it is the rate of ion flux rather than polarization which is responsible for stimulus-secretion coupling.

Dynamics of adrenocorticotropin (ACTH) secretion in cyclic Cushing's syndrome: evidence for more than one abnormal ACTH biorhythm.

We have studied a 57-yr-old woman with cyclic Cushing's syndrome of apparent pituitary origin who had a predominant cycle of 2-6 days. The patient also demonstrated an abnormal circadian rhythm, with afternoon peaks of plasma ACTH and plasma cortisol. In addition to these abnormal biorhythms, Fourier analysis showed what appeared to be a separate 35-day cycle. After 35 days of consecutive urinary free cortisol measurement, the patient was given cyproheptadine. During therapy with this agent, the urinary free cortisol levels fell dramatically, but cyclic secretion continued, albeit with a diminished amplitude. During general anesthesia for a bilateral adrenalectomy, there was a striking increase in the plasma ACTH level, and the ACTH concentration remained high in both the immediate and late postoperative periods. These observations indicated that stress could overcome cyclic ACTH secretion and that cortisol exerted feedback suppression on ACTH secretion. Although this is the predictable response for classic pituitary-dependent Cushing's syndrome, it is of interest in cyclic Cushing's syndrome, since previous studies of this entity have implied that cortisol secretion is independent of stimulation or feedback.

Hypothalamic control of endocrine thermogenesis.

Hypothalamic and ruminal cooling raised serum thyrotropin (TSH), adrenocorticotropin (ACTH), norepinephrine (NE), and glucose in conscious goats in 20 degree C ambient temperature. Cooling of the preoptic anterior hypothalamus (POAH) for 2 h initially evoked shivering and vasoconstriction, leading to 1.5 degree C rise in rectal temperature (Tr). Pituitary-thyroid activation by POAH cooling was shown by peak rises in TSH of 60% at 40 min, in triiodothyronine (T3) of 54% at 80 min, and in thyroxine (T4) of 40% at 140 min. At 60 min, ACTH and NE peaked at 57 and 65%, respectively. TSH, ACTH, and NE declined during the 2nd h of POAH cooling as Tr plateaued; when POAH cooling was stopped, these hormones fell below basal level as vasodilation and panting restored Tr to normal. In contrast to the core hyperthermia evoked by POAH cooling, ruminal intubation with O degree C water (1 liter/10 kg) led to general hypothermia, Tpoah and Tr falling 1.6 degree C at 40 min. Pituitary-thyroid responses were less but ACTH and NE more, compared with POAH cooling. TSH peaked at 37% at 20 min, T3 at 55% at 60 min, and T4 at 18% at 200 min. ACTH peaked at 250% at 30 min and NE at 120% at 20 min. Thermosensitive neurons in the POAH seem to mediate more sensitive and complete control over TSH than over ACTH, or NE release, whereas extrahypothalamic core thermosensitivity (e.g., brain stem, spinal cord, abdomen) may influence ACTH and NE more than TSH release.

CSF prolactin determination in patients following operation for pituitary tumor.

Serial cerebrospinal fluid (CSF) and plasma prolactin concentrations were determined from patients during prolactin stimulatory testing with thyrotropin-releasing hormone or during pneumoencephalographic stress. Six patients had been operated on for suprasellar extension of pituitary tumor and one had been irradiated for suprasellar extension of a pituitary tumor. Prior to testing, four patients had had no clinical evidence of tumor recurrence and 3 patients had had tumor recurrence. One of the recurrent tumors had again extended into a suprasellar location. Basal CSF prolactin was undetectable in all patients who had had no recurrence. In 3 of the 4 patients without recurrence, however, prolactin became detectable in CSF during stimulatory testing. CSF prolactin values also increased during stimulatory testing in the patient with suprasellar recurrence of the tumor. A basal CSF-to-plasma prolactin ratio was 0.1 or less in all patients without recurrence. In the 2 patients with recurrence but without suprasellar extension, the CSF-to-plasma prolactin ratio was 0.18 or less. The patient with suprasellar recurrence had a strikingly elevated CSF-to-plasma prolactin ratio of 1.1. Thus, an increase of CSF prolactin during stimulatory testing does not necessarily indicate suprasellar recurrence of a pituitary tumor. However, an elevated CSF-to-plasma prolactin ratio appears to remain a valid indicator of suprasellar extension despite prior pituitary surgery.

Corticotrophin, growth hormone and prolactin deficiencies with hypoaldosteronism and corticosteroid-reversible hypothyroidism in Sheehan's syndrome. Clinical and anatomical correlations.

Deficiencies of corticotrophin (ACTH), growth hormone, and prolactin were documented in a woman with diabetes mellitus and Sheehan's syndrome. The patient's ACTH deficit appeared to be secondary to a hypothalamic abnormality since on two occasions the patient had a marked plasma ACTH response to vasopressin but not to insulin induced hypoglycaemia. It is postulated that the deficits of these three adenohypophysial hormones were instrumental in causing a severely impaired aldosterone secretory capacity in response to sodium restriction and an angiotensin infusion. In addition, the patient had an unusual form of thyroid dysfunction that was in part reversed with hydrocortisone replacement. The patient's unfortunate death during a hypoglycaemic crisis allowed correlation between her extensive antemortum endocrine testing and her pathologic anatomy.

Beta-endorphin and adrenocorticotropin in extrapituitary sites: gastrointestinal tract.

Beta-Endorphin and ACTH immunoassays were employed to examine the concentrations, distributions, and character of those peptides in rat gastrointestinal tissues. Sections of the gastrointestinal tract were obtained from fasted and fed animals and were extracted in 5 N acetic acid containing proteolytic enzyme inhibitors. Aliquots immunoassayed for beta-enddorphin and ACTH revealed highest concentrations to be present in the small bowel, with stomach and colon containing little immunoreactivity. Tissues from fasted animals contained more immunoreactivity than did those from fed animals. Gel chromatography showed the presence of large molecular weight forms of beta-endorphin and ACTH in gut extracts. Concanavalin A affinity chromatography revealed that approximately 5% of gut immunoreactivity contained carbohydrate. Therefore, beta-enddorphin and ACTH immunoreactivities are present im the gut. The demonstration of large molecular weight and glycosylated forms of immunoreactivity suggests the presence of biosynthetic precursors of beta-endorphin and ACTH. The increase in immunoreactivity in response to fasting suggests that these peptides play a role in gut physiology.

The distribution of forms of adrenocorticotropin and beta-endorphin in normal, tumorous, and autopsy human anterior pituiary tissue: virtual absence of 13K adrenocorticotropin.

To begin to define the nature of the biosynthesis and processing of ACTH and beta-endorphin in the human, anterior pituitary tissue (fresh normal and adenomatous, and autopsy) was extracted in acetic acid in the presence of protease inhibitors and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel slice eluates were assayed for ACTH and beta-endorphin immunoactivity. Human anterior pituitary tissue contained four major size classes of ACTH and three major size classes of beta-endorphin. We found that in all tissue sources examined there was a virtual absence of 13-15K ACTH, which is a major form in the rat and mouse. When comparing extracts obtained from fresh normal or adenomatous anterior pituitary tissue, we also found a drastic decrease in beta-lipotropin and beta-endorphin in extracts of autopsy human anterior pituitaries. These results suggest that the biosynthesis and processing of pituitary ACTH and beta-endorphin in the human may be different than in the mouse, and because of apparent postmortem proteolysis of beta-endorphin, human pituitary obtained at autopsy is probably not a good source of material for biochemical studies of pituitary tissue.

Cushing syndrome: current concepts of diagnosis and therapy.

A variety of diagnostic advances including radioimmunoassay of adrenocorticotropic hormone (ACTH) have increased the number of methods for laboratory investigation of Cushing syndrome.* However, experience with these procedures has led to a recognition of their limitations. We have developed an algorithm which incorporates these newer techniques and minimizes the number of procedures required to diagnose the various causes of Cushing syndrome. At present, we recommend pituitary surgical operations for pituitary-dependent Cushing syndrome because we believe this disease is caused by the development of a pituitary ACTH-secreting tumor.

Concentration of human chorionic gonadotropin in the cerebrospinal fluid of patients with germinal cell hypothalamic tumors.

Plasma and cerebrospinal fluid (CSF) specimens were measured simultaneously for human chorionic gonadotropin (HCG) in two patients with HCG-secreting choriocarcinoma. In the patients with hypothalamic tumors, the CSF HCG levels were higher than the plasma HCG concentrations. In the patient with gestational choriocarcinoma with no known cerebral metastases, the plasma HCG level greatly exceeded the CSF HCG concentration. The finding of a CSF HCG concentration that approaches or exceeds the plasma value would be a useful screening procedure in localizing a pathologic source of HCG secretion in patients with a suspected hypothalamic tumor. An unexpected finding in the patient who also had a hypothalamic embryonal cell carcinoma and hypocortisolism was an extremely high concentration of a biologically inactive adrenocorticotropic like substance in the CSF.

Nelson's syndrome and spontaneous pituitary tumor infarction.

Large, adrenocorticotrophic hormone-secreting pituitary tumors (Nelson's syndrome) developed in four of 12 patients treated with a bilateral adrenalectomy for Cushing's disease. Two of the patients with Nelson's syndrome suffered spontaneous pituitary tumor infarctions. One patient improved under close observation and subsequent radiation therapy, although she ultimately died from her locally invasive tumor. The condition of the other patient-which had stabilized-appeared to be worsened by surgical intervention. The high incidence of these tumors after bilateral adrenalectomy, their large and agressive nature, and their apparent propensity to undergo spontaneous infarction supports the position that initial therapy for Cushing's disease should be directed to the pituitary gland.