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Hirsutism is a common condition of elderly women, but its aethiopathogeny and its clinical implications remain unclear. We therefore studied circulating androgen concentrations in elderly women. In addition, this study aims to define a possible relationship between hirsutism and anthropometric determinations, bone mass and serum lipids. Androgen levels were determined at basal state, after adrenocorticotrophin (ACTH) stimulation and after dexamethasone administration in 10 hirsute elderly women and compared to 10 age-matched non-hirsute women. Anthropometric determinations included measurements of skinfold thickness and body mass index. Spinal bone mass density was assessed using dual photon absorptiometry. Hirsute women presented significantly higher levels of testosterone than controls (1.49 +/- 0.38 vs 0.59 +/- 0.05 nmol/l, mean +/- SEM; p < 0.05) and dihydrotestosterone (0.54 +/- 0.07 vs 0.32 +/- 0.03; p < 0.02). 17-hydroxyprogesterone levels after ACTH stimulation tended to be higher in hirsute women than in controls. No differences were observed between the two groups in serum oestrogen concentrations, plasma lipid pattern or bone mineral density. Hirsute women had a lower body mass index and lower calculated percentage body fat than the control group. We conclude that: (i) hirsutism of elderly women is associated with increased androgen levels, probably from adrenal origin; (ii) in some cases, enhanced response in 17-hydroxyprogesterone after ACTH stimulation suggests a partial adrenal 21-hydroxylase deficiency; (iii) hirsute women present anthropometric characteristics compatible with the known anabolic effect of androgens on fat-free mass.

The response of the pituitary-adrenal axis to corticotrophin-releasing hormone (CRH) and the adrenal response to adrenocorticotrophin hormone (ACTH) stimulation were studied during infection of Boran (Bos indicus) cattle with Trypanosoma congolense. For CRH, 15 animals were challenged during preinfection and infection phases, while for ACTH 10 animals were challenged during pre-infection, infection and post-treatment phases of the experiments. The axis showed a reduced responsiveness after CRH challenge during patent parasitaemia, manifested by low (p < 0.05) plasma ACTH and cortisol concentration as compared to the pre-infection CRH challenge concentrations. Cortisol concentration after ACTH challenge during pre-infection, infection and post-treatment phases did not differ. The reduced pituitary-adrenal response to CRH challenge and the normal adrenal response to ACTH challenge found during trypanosomiasis indicates pituitary dysfunction due to infection.

Electron microscopy was used to assess the cytological maturity of the zona fasciculata cells in the adrenal cortex of fetal sheep at 105 days of gestation, following several ACTH infusion regimes. The aim of this study was to correlate the morphological appearance of the fetal adrenal zona fasciculata cells with the expression of the steroid hydroxylase genes and the fetal plasma cortisol concentrations in a parallel study. Immediately following infusion of ACTH for 24 or 72 h, the zona fasciculata cells at the cortico-medullary junction were more mature than those in the saline-infused controls. When ACTH infusions were withdrawn for 24-72 h prior to the termination of the experiment, the deep cortical cells appeared less mature than those in fetuses which had received ACTH right up until the time of tissue collection. Following ACTH administration, mitochondrial changes preceded changes in the smooth endoplasmic reticulum, and when ACTH was withdrawn, the smooth endoplasmic reticulum responded before the mitochondria. The study demonstrated a correlation between the cytological maturity of the deep zona fasciculata cells and the expression of the genes for the steroidogenic enzymes P-450(17)alpha and P-450scc in the 105-day fetal sheep adrenal following ACTH infusion.

To investigate the direct effect of corticotropin (ACTH) on the renin-angiotensin-aldosterone system, isolated guinea-pig kidneys with adrenal glands were perfused with various doses of ACTH (0.1-1000 micrograms/l) and 0.3 mmol/l of dibutyryl cyclic AMP (cAMP) through each cannula inserted into the abdominal aorta and the inferior caval vein. Perfusate renin activity was increased in a dose-dependent manner by the addition of ACTH in a range of 0.1-1000 micrograms/l, and reached a plateau at 20 min with each dose. The perfusate cAMP level was dose-dependently increased with 10-1000 micrograms/l of ACTH. Perfusate renin activity was also markedly increased by the addition of dibutyryl cAMP. The same effects of ACTH on renin and cAMP secretions were observed in the kidney perfusion model from which the adrenal glands were excluded. Aldosterone secretion failed to respond to 0.1 micrograms/l of ACTH, and was increased by higher concentrations (1-1000 micrograms/l) in the same experiments. These results demonstrate that ACTH has a direct effect on renal renin release in a physiological concentration (0.1 micrograms/l), and that the action of ACTH is probably mediated by cAMP. The sensitivity of renin release to ACTH stimulation is no less than that of aldosterone secretion during ACTH infusion, so it is possible that ACTH is an important stimulator of the renin-angiotensin system.

Arginine vasopressin stimulates the secretion of adrenocorticotropin. A direct stimulatory effect of AVP on cortisol as well as aldosterone secretion has been postulated by several investigators. To study the possible role of a direct stimulatory action of AVP on the adrenal cortex, normal volunteers were treated with incremental injections of ACTH or with incremental infusions of AVP which raised plasma AVP levels to a maximum of 256 +/- 16 pmol/l. In both situations, a significant (p less than 0.001) linear correlation between plasma ACTH and plasma cortisol was observed. The regression coefficients were not different (p greater than 0.5). Plasma aldosterone was stimulated by both treatments, but the weakly positive correlation between plasma ACTH and plasma aldosterone was not significant for either stimulus. Thus, in man, a direct stimulatory effect of AVP on cortisol secretion cannot be demonstrated. A direct effect of AVP on aldosterone cannot be definitely excluded, but is certainly of minor importance.

Gossypol, an antifertility agent, has inhibitory actions on many membrane-associated enzymes, suggesting that this agent might have a generalized effect on cell membranes. This hypothesis was examined in the present study using membranes and dispersed cells prepared from human and rat adrenal glands. Four parameters were determined: microviscosity as measured by fluorescence polarization of human adrenal microsomal- and mitochondrial-enriched membranes, adrenal steroidogenic enzymes; and cAMP and cortisol responses to ACTH. It was found that gossypol increased the polarization constants of microsomes and mitochondria in a dose-dependent manner. Of the three adrenal enzymes tested, both 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4 isomerase and 11-hydroxylase were inhibited by gossypol, but not 21-hydroxylase. Using intact human adrenocortical cells, high doses of gossypol also inhibited the ACTH-stimulated cAMP and cortisol levels. The in vivo corticosterone response to ACTH in rats subjected to chronic gossypol treatment was also found to be reduced. These findings suggest that gossypol has multiple effects on adrenal function. Its effects on membrane microviscosity, adrenal steroidogenesis, cAMP and corticosterone responses to ACTH stimulation probably occur through a generalized membrane effect.

Effects of deficiency in ascorbic acid on in vivo production of corticosterone and testosterone were examined using a mutant strain of rats unable to synthesize ascorbic acid. The adrenal weight of scorbutic rats was larger, and corticosterone levels in plasma and adrenal tissues were higher than those of ascorbic acid-supplied (ascorbutic) rats. Acute and chronic stimulation with ACTH increased corticosterone levels in both ascorbutic and scorbutic rats. In contrast, weights of seminal vesicles and ventral prostates in unstimulated scorbutic rats were smaller, and testosterone levels in plasma and testicular tissues were lower than those in ascorbutic rats. Acute stimulation with hCG increased testosterone levels only slightly in plasma and not in testicular tissues of scorbutic rats, when testosterone levels in ascorbutic rats reached a maximum. Chronic stimulation with hCG increased testosterone levels remarkably in both ascorbutic and scorbutic rats. These findings seem to indicate that ascorbic acid is not essential for the synthesis of steroid hormones. The scurvy seems to increase plasma ACTH levels secondary to the stress, resulting in the stimulation of the adrenals. In contrast, a prolonged deficiency in ascorbic acid appears to decrease plasma gonadotropin levels, and may reduce the sensitivity of testes to gonadotropins.

Changes in plasma aldosterone, plasma renin activity, plasma cortisol, serum sodium and potassium concentrations were studied in 9 patients with thromboembolic diseases treated with heparin. Heparin was administered at doses of 700-1000 units/h for 7-10 days. Plasma aldosterone decreased from 239 +/- 33 to 114 +/- 25 pmol/l during heparin therapy and returned to basal levels after discontinuation of the therapy. In addition, responses to a low sodium intake (3 g/day) and ACTH were examined in 5 patients during and 2 weeks after heparin therapy. The increase in plasma aldosterone caused by low sodium intake was significantly attenuated during heparin therapy (124 +/- 5% increase from baseline) as compared with that 2 weeks after heparin therapy (148 +/- 7%, p less than 0.05). On the other hand, ACTH stimulated plasma aldosterone similarly during and at 2 weeks after heparin therapy (increase from baseline: 190 +/- 20% vs 193 +/- 9%). These results suggest that heparin decreased plasma aldosterone owing to attenuation of the angiotensin II-induced aldosterone production.

Between 90 and 120 days of gestation (term = 147 +/- 5), when plasma cortisol concentrations in the fetus are at a minimum, levels of mRNA encoding the steroidogenic enzymes 17 alpha-hydroxylase (P-450(17 alpha] and cholesterol side-chain cleavage (P-450scc) are also very low. Over the following 30 days, P-450(17 alpha) and P-450scc gene expression increases concurrent with increasing fetal cortisol concentration. The hypothesis tested in this study was that cortisol biosynthesis is minimal in the period 90-120 days because of insufficient ACTH. Fetuses were cannulated between 98-102 days of gestation. Following recovery, 7 fetuses received 24-h ACTH infusions (12 micrograms/24 h) and 5 fetuses received 24-h vehicle infusions; 4 ACTH-infused and 4-vehicle-infused fetuses were then sacrificed immediately after cessation of the infusion. The other fetuses were left in utero for 3 days prior to sacrifice. Fetal blood samples were analysed for ACTH and cortisol and the adrenals processed for hybridization histochemistry and Northern blot analysis. ACTH, but not vehicle, induced significant increases in the width of the adrenal cortex and in the levels of P-450(17 alpha) and P-450scc mRNA. Concurrently, fetal plasma ACTH and cortisol concentrations also increased significantly. In adrenals from fetuses left in utero for 3 days after cessation of the ACTH infusion, P-450(17 alpha) and P-450scc mRNA levels returned to control levels. Plasma ACTH and cortisol levels also approximated basal values. P-450c21 mRNA levels did not vary significantly at any time with the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Quartered rat adrenal glands transformed labelled 21-deoxyaldosterone into aldosterone in vitro. 21-deoxyaldosterone was released from the quartered rat adrenals in vitro in amounts 10 times lower than those of aldosterone and 18-hydroxycorticosterone. The production of all three steroids was qualitatively dependent on the same regulatory elements (electrolytes, ACTH, exogenous precursors, zonal specificity). However, quantitative differences could be observed. The results support a role for 21-deoxyaldosterone as a facultative precursor of aldosterone and indicate that the regulation of its production shows similarities to that of aldosterone.

In patients with anorexia nervosa 24-h mean plasma concentration of cortisol were 0.44 +/- 0.09 mumol/l (normal less than 0.28 mumol/l). Following stimulation by ACTH (1-24) urinary excretion rates of cortisol were stimulated from 0.22 +/- 0.08 to 4.85 +/- 2.78 mumol/24 h. Similarly, plasma concentrations of the glucocorticoid metabolite, tetrahydrocortisone, increased from 23.3 +/- 9.0 to 47.3 +/- 30.2 nmol/l; urinary excretion rates of tetrahydrocortisone increased from 3.61 +/- 0.90 to 8.40 +/- 1.72 mumol/24 h. The relative share of the sulphate, glucuronide and free fractions of tetrahydrocortisone in the patients' urine did not indicate any defect in metabolization of this steroid metabolite. Excretion rates of the four glucocorticoid tetrahydro-metabolites, tetrahydrocortisone, allotetrahydrocortisone, tetrahydrocortisol, and allo-tetrahydrocortisol, expressed as percent of total steroid excretion, were similar in patients with anorexia and in healthy women under basal conditions (24 +/- 6 vs 23 +/- 6%) and during stimulation by ACTH (1-24) (36 +/- 10 vs 45 +/- 6%). The share of the two androgen metabolites, androsterone and etiocholanolone, was 24 +/- 5% of total steroid excretion (basal; healthy women: 27 +/- 8%) and 13 +/- 2% (ACTH stimulation; healthy women: 12 +/- 4%) in patients with anorexia nervosa. Thus, analysis of urinary steroid excretion rates did not indicate a shift in adrenocortical function. The results confirmed enhanced secretion of cortisol in patients with anorexia nervosa under basal conditions and during/following stimulation by ACTH. The ACTH-induced increase in the concentrations of the tetrahydro-glucocorticoid metabolites in urine was less pronounced than that of cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II, ACTH and potassium chloride were administered to rats for 6 days and the effects on adrenal renin-like activity and adrenal angiotensin II/III immunoreactivity were investigated. Rats infused with angiotensin II (140 pmol/min) either ip or sc showed increases in adrenal angiotensin II/III immunoreactivity (p less than 0.05) and plasma aldosterone concentration (p less than 0.05), but no change in adrenal renin-like activity. Captopril treatment of angiotensin II-infused rats caused a slight decrease in angiotensin II/III immunoreactivity which did not reach statistical significance. In contrast, rats treated with ACTH (Cortrosyn-Z, 3 IU/day, sc) showed an increase in adrenal renin-like activity (p less than 0.01), but no significant change in adrenal angiotensin II/III immunoreactivity. Rats treated with KCl in drinking water showed increases (p less than 0.05) in adrenal renin-like activity, adrenal angiotensin II/III immunoreactivity, and plasma aldosterone. These results suggest that angiotensin II, ACTH and potassium, three major regulators of aldosterone secretion by the adrenal gland, have different effects on the adrenal renin-angiotensin system when administered in vivo.

The objective of this study was to determine whether anti-oestrogens (nafoxidine, MER-25) would block the suppressive effects of ACTH on gonadotrophin secretion in immature rats. Female rats were castrated at 25-26 days of age, and an Alzet osmotic minipump containing ACTH (1-24) or saline was implanted in each animal. ACTH was administered at a rate of 1 IU/day by constant infusion. Beginning on the day of surgery, animals were injected daily for 5 days with 0.25, 5 or 25 micrograms/100 g body weight of nafoxidine or 5 mg MER-25 and sacrificed on the sixth day following castration. ACTH lowered serum LH concentrations and increased pituitary LH levels. Serum androstenedione concentrations were more than two times greater in ACTH-infused than in control rats, but serum oestrone levels were not affected. Serum testosterone and oestradiol concentrations in ACTH-infused rats remained below levels of detection. Administration of 0.25 micrograms of nafoxidine prevented the suppressive effects of ACTH on serum LH. Serum levels of LH in these animals were comparable to saline-treated controls (418 +/- 94 vs 443 +/- 73 ng/ml). The two higher doses of nafoxidine and MER-25 were ineffective in suppressing the actions of ACTH on serum LH. MER-25 reduced serum LH values in both controls and ACTH-infused rats. Serum FSH concentrations were not altered by ACTH or nafoxidine treatment. MER-25 elevated pituitary FSH concentrations in both control and ACTH-infused rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of chronic ACTH administration on the growth and aldosterone secretion of the zona glomerulosa of sodium-loaded timolol maleate-treated rats was investigated by stereological and radioimmunological techniques. Combined sodium loading and timolol maleate administration induced a significant atrophy of the zona glomerulosa and its cells. The plasma concentration of aldosterone was significantly decreased, while that of corticosterone did not vary. ACTH administration completely reversed zona glomerulosa atrophy, and the plasma concentration of corticosterone was noticeably enhanced, whereas that of aldosterone underwent a further decrease. This data suggests that in the absence of the other adrenoglomerulotrophic factors, ACTH stimulates the growth of the zona glomerulosa, but transforms its parenchymal elements to a functional fasciculata cell type.

Effect of intramuscular administration of ACTH or dexamethasone on blood serum levels of testosterone, LH and FSH was examined in intact and castrated, adult, male rats. Six IU ACTH or 1 mg dexamethasone were given daily for 7 days. Corticotrophin treatment had no influence on circulating testosterone, LH and FSH in intact or castrated male rats. Dexamethasone administration resulted in a slight elevation of serum FSH in intact animals but not in castrates. LH and testosterone remained normal in both intact and castrated animals injected with dexamethasone. Under our conditions of study the secretions from the adrenal gland appear to be insignificant for the regulation of pituitary secretion of gonadotrophins in the male rat.

The in vitro effects of theophylline and aminogluthetimide upon basal and ACTH stimulated cAMP, cortisol and aldosterone responses of normal human adrenocortical tissue were evaluated. Theophylline increased basal cAMP levels and cortisol output, however, basal aldosterone output was depressed. Theophylline in concert with ACTH depressed cortisol and aldosterone output. Aminogluthetimide alone did not affect basal cAMP levels, however, the normal cAMP response to ACTH was delayed in aminogluthetimide pre-treated adrenals. Aminogluthetimide also depressed basal and ACTH stimulated cortisol and aldosterone output with the latter being more sensitive. The findings indicate that both theophylline and aminogluthetimide produce effects upon the adrenal in addition to inhibition of phosphodiesterase and cholesterol side-chain cleavage, respectively. Further, theophylline depression of ACTH stimulated steroid output may be helpful in understanding the interplay between a number of factors in the control of adrenal steroid biosynthesis and release.

Serum cortisol response to synthetic corticotrophin (B1-24-ACTH) was evaluated in 21 normal subjects. There was significant negative correlation (r = -0.633, P less than 0.001) between the basal serum cortisol and the increase in serum cortisol level in response to ACTH. Two subjects had an increase in serum cortisol of less than 194 nM/1 (7.0 microgram/100 ml) and two additional subjects had an increase of less than 278 nM/l (10.0 microgram/100 ml). All subjects had an absolute peak value greater than 583 nM/l (21.0 microgram/100 ml) at 30 or 60 min post-ACTH. Inverse correlation between basal serum cortisol and the increase in serum cortisol suggests that in patients who have high basal serum cortisol levels, endogenous ACTH may already have reduced the cortisol reserve of the adrenal gland.

In two children with isolated congenital hyperreninaemic hypoaldosteronism, as well as in their relatives, plasma levels of aldosterone (Aldo), corticosterone (B), deoxycorticosterone (DOC), 18-OH-B and 18-OH-DOC were measured before and after an iv bolus of 0.25 mg Synacthen (Ciba). A corticosterone methyl oxidase deficiency type II was demonstrated in one child. Her normoreninaemic parents (no consanguinity) had plasma values consistent with heterozygosity. The results in the other child and one asymptomatic sib were compatible with a partial corticosterone methyl oxidase deficiency type I. His parents were consanguine but had normal Aldo levels. Overnight dexamethasone administration did not suppress any of the steroids measured except cortisol, suggesting synthesis of these steroids by the zona glomerulosa.

Isolated adrenocortical cells from 6 patients with a 'normal' zona fasciculata, 4 patients with a 'normal' zona glomerulosa, and tumour cells from 1 adrenocortical adenoma and 1 carcinoma were incubated with and without increasing concentrations of ACTH 1-24 (10(-13) M to 10(-9) M) or Asp1-Ile5-angiotensin II (10(-11) M to 10(-7) M). In 4/5 'normal' cases, cortisol was clearly stimulated by 10(-13) M ACTH. The maximum of the dose-response curve (5-fold stimulation) was reached at 10(-10) M ACTH. Angiotensin II (AII) started to stimulate 'normal' cells at 10(-11) M with a maximum (2-fold stimulation) at 10(-9) M. Aldosterone production by 'normal' cells was less markedly stimulated by ACTH and AII, although the threshold doses for both peptides were similar to those of the cortisol response curves. The cells of the adrenocortical adenoma from a patient with Cushing's syndrome produced large amounts of cortisol and small amounts of aldosterone, both steroids being clearly stimulated by ACTH and AII. The adrenocortical carcinoma cells produced small amounts of cortisol and no aldosterone. Cortisol production responded to ACTH, but not to AII. The results suggest that an activated renin-angiotensin system may stimulate the zona fasciculata, since 10(-11) M AII (= 10 pg AII/ml) is a normal plasma AII concentration on an unrestricted diet. Clinical evidence supporting this thesis is reviewed. However, cortisol production itself will rarely be increased by AII in vivo, since a down-regulation of ACTH would occur.

The effects of the recognized diabetogenic hormones, growth hormone and ACTH, administered in vivo, on (pro-)insulin biosynthesis and secretion in isolated islets of normal rats were studied. Rats were treated with these hormones for 4 weeks. Afterwards, their collagenase-isolated islets were incubated with [3H]leucine for 3 h. (Pro-)Insulin biosynthesis was estimated for the incorporation data into islet protein fractions. Islets of treated rats released significantly more insulin and incorporated significantly less [3H]leucine into proinsulin and insulin compared with controls when tested at 100 mg glucose/100 ml. At 200 mg glucose/100 ml, no significant differences were found. The findings demonstrate an impact of these hormones on the B-cells derived from normal pancreas of intact rats. The alterations are, however, not very pronounced and can be easily compensated under a strong glucose stimulus. It appears that the mechanisms for (pro-)insulin biosynthesis and release in rats are more resistant against the diabetogenic actions of pituitary hormones than in other species.

Cells from adrenal glands of 140-160 days foetal, neonatal and infant Rhesus monkeys (Macaca mulatta) were prepared, by collagenase digestion and incubated with 10 pg-16 ng/ml ACTH. The production of cortisol, androstenedione, corticosterone, progesterone and prostaglandins was measured. The cortisol production in the 140 day and 160 day foetuses and in the neonatal adrenal cells was comparable. It was 2-fold higher in adrenal cells of 6 month infant monkeys. In all the groups there was an increasing production of cortisol with increasing ACTH concentration, and a response to low physiological concentrations of ACTH. The androstenedione production was significantly greater in the 160 day foetuses than in either those of 140 days or of the neonate which demonstrated a poor response to increasing ACTH concentrations. It responded well to increasing ACTH in adrenal cells from 6 month infant monkeys. Corticosterone output was 1/10th of cortisol with only the 140 day foetuses showing an increase in production with increasing ACTH concentrations. The results demonstrate that cells of the primate foetal adrenal gland are not inherently unresponsive to ACTH stimulation as regards cortisol production, which per/micrograms DNA does not appear to change over the last 25 days before term.

The roles of growth hormone (GH) and insulin-like growth factor I (IGF I) were studied in 9 German Shepherd dwarf dogs. GH deficiency was evidenced in all dogs by an absence of increase in GH levels in response to clonidine administration. While the mean IGF I concentration in normal adult German Shepherds was 280 +/- 23 ng/ml and 345 +/- 50 ng/ml in immature animals, the mean IGF I concentration in the dwarf dogs was 11 +/- 2 ng/ml (mean +/- SEM, P less than 0.001). In the affected animals, plasma thyroxine (T4) levels were only slightly subnormal and there was an increase in these levels in response to thyroid stimulating hormone (TSH) administration. The findings indicate 1) that dwarfism in German Shepherds is caused by primary GH-deficiency resulting in low circulating levels of IGF I and 2) that IGF I levels in the dog as in man are subject to control by GH.

To test the hypothesis that the trophic action of angiotensin II on the adrenal zona glomerulosa may allow a sustained stimulation of aldosterone by ACTH by preventing the morphological changes of the zona glomerulosa cells into zona fasciculata-like elements we investigated the effects in rats of a 6-day treatment with ACTH (100 micrograms/kg/day) alone or combined with angiotensin II (300 ng/kg/day) on corticosterone and aldosterone production and adrenal morphology. The responsiveness of both steroids to an acute ACTH dose was also studied on the last day of long-term treatment. Morphologic data showed that prolonged ACTH treatment stimulated the growth of zona glomerulosa cells, though it transformed the tubulo-lamellar cristae of mitochondria into a homogeneous population of vesicles. Angiotensin II furthered the trophic effects of ACTH but prevented the mitochondrial transformation. Despite its ability to conserve the well differentiated aspect of the zona glomerulosa cells, the administration of angiotensin II was unable to prevent the fall in the secretion of aldosterone caused by chronic ACTH treatment and its subsequent unresponsiveness to ACTH stimulation.

An accurate and relatively simple radioimmunoassay for the determination of aldosterone concentration in peripheral plasma has been developed, 0.5-2.0 ml plasma with added [1,2-3H]aldosterone is extracted with dichloromethane. Purification of the extract is achieved by thin later chromatography in the system benzene-acetone 1:1. Recovery of [1,2-3H] aldosterone is 58 +/- 6 (SD)%. Bound and free fractions are separated by dextran-coated charcoal. The intra-assay reproducibility is 8.8% and the inter-assay reproducibility varies from 11.4-16.1%. The sensitivity of the assay for a 5 ml plasma sample can be put at 0.2 ng/100 ml. Normal values determined in 52 healthy children of different age groups are presented. Furthermore the aldosterone stimulating effect of low sodium diet (17 children), severe and prolonged vomiting (19 children) and synthetic ACTH (10 children) has been studied by our modified method.

A 22 year old XX female patient with primary amenorrhoea and sexual infantilism was studied. Persistently elevated serum LH and FSH concentrations and exaggerated LRH pituitary responsiveness indicated deficient ovarian hormonal production. Serum levels of C21 and C19 steroids measured by specific radioimmunoassays before and after appropriate stimulations demonstrated an impairment of adrenal and ovarian steroid biosynthesis. Baseline levels of androstenedione (delta 4-A), testosterone (T), and oestradiol-17 beta (E2) were persistently below the normal range for healthy women at early follicular phase, whereas progesterone (P) and 17 alpha-OH-progesterone (17-OH-P) serum levels were significantly higher than those observed for normal women. Adrenal and gonadal stimulation with ATCH and hCG, respectively, resulted in a considerable rise in serum P and 17-OH-P without any significant change in circulating levels of delta 4-A, T, and E2. These findings were consistent with the diagnosis of 17,20 steroid desmolase deficiency at both adrenal and ovarian levels. This is the first report of a 17,20 desmolase deficiency in an XX individual, and is in line with previous suggestions that familial occurrence of the disorder would fit an autosomal recessive pattern of inheritance.

To examine the nature of adrenal abnormalities in diabetic patients with hyporeninemic and normoreninemic hypoaldosteronism, responses of plasma 18-hydroxycorticosterone and plasma aldosterone to angiotension II infusions and ACTH injection were investigated in 8 diabetic patients with hyporeninemic hypoaldosteronism and 9 diabetic patients with normoreninemic hypoaldosteronism compared to 11 control subjects. In both the patients with hyporeninemic and normoreninemic hypoaldosteronism, plasma 18-hydroxycorticosterone and plasma aldosterone were low, whereas plasma cortisol and plasma corticosterone were within normal ranges. Percent increments of plasma 18-hydroxycorticosterone and plasma aldosterone above their baseline levels after angiotensin II infusions were low or somewhat low in the patients with hyporeninemic hypoaldosteronism and low in the patients with normoreninemic hypoaldosteronism. Percent increments of plasma 18-hydroxycorticosterone and plasma aldosterone above their baseline levels after ACTH injection were similar in three groups. These results suggest that in diabetic patients with isolated hypoaldosteronism, the adrenal abnormality, regardless of whether it is primary or secondary, is mainly due to impaired adrenal responsiveness to angiotension II and atrophy and the zona glomerulosa.

A very unusual case of Cushing's syndrome is presented. Most of the preoperative biochemical and radiological examinations were compatible with Cushing's syndrome owing to a right adrenal adenoma. Exceptional findings include normal concentrations of adrenocorticotrophin (ACTH) in plasma as well as a disturbance of its circadian rhythmicity and a significant adrenocortical responsiveness to exogenous ACTH. Secretory patterns of ACTH did not change even after right adrenalectomy. Studies in vitro revealed that the adenoma itself, but not the surrounding normal adrenal, was the source of cortisol secreted in response to ACTH. Post mortem examinations disclosed unexpectedly a hormonally inactive left adrenal adenoma and a focal hyperplastic lesion of the anterior pituitary with an ACTH concentration 53 times higher than that of the remaining tissue of the gland. It is a possibility that this case may have represented a transition between pituitary-dependent adrenocortical hyperplasia and adrenal adenoma to this date reported in only one similar case.

A simple method for measuring the free, non-protein-bound steroid fraction in plasma by equilibrium dialysis is described. The alteration occurring in the volume of the inner phase (undiluted plasma) is corrected by the difference in weight before and after dialysis. Total cortisol was determined by radioimmunoassay. Although it is not possible to differentiate between values for total cortisol after ACTH stimulation (242.2+/-shing's syndrome (n: 15)), and women treated with oestrogens (211.8+/-42.0 ng/ml (n: 20)), there were significant differences for free cortisol (ACTH stimulation: 29.3+/-5.6 ng/ml; Cushing's syndrome: 31.5+/-8.6 ng/ml; women under increased oestrogenic activity: 10.0+/-2.1 ng/ml; pregnant women: 13.7+/-5.1 ng/ml plasma). Compared with normal values for healthy women and men (9.3+/-1.4 ng/ml), women with increased oestrogenic activity showed slight elevations of free cortisol. Compared to cortisol, the percentage of dialysable testosterone and oestradiol was lower in women than in men (1.57 vs. 2.08% for testosterone and 1.68 vs. 2.15% for oestradiol). In healthy men the concentration of free steroid was 13.1+/-1.0 ng free testosterone/100 ml plasma (0.44+/-0.045 pg free oestradiol/ml plasma), in healthy women 0.64+/-0.07 ng/100 ml (0.98+/-0.10 pg/ml) and in women receiving oestrogens 0.37+/-0.04 ng/100 ml (0.01+/-0.015 pg/ml). When the method described here for determing the free fractions of cortisol (n: 45), oestradiol (n: 18) and testosterone (n: 18) at 37 degrees C is compared with the method of centrifugal filtration, the correlation was r: 0.80 r: 0.86 and r: 0.91, respectively. In practice, equilibrium dialysis with undiluted plasma is simple, fast and can be applied to all steroid hormones. It allows direct measurements of non-protein-bound steroids under nearly physiological conditions.

The renin-angiotensin system was examined in Fischer rats at 7, 11 and 14 days after Leydig cell tumour transplantation, and in age matched controls. Mean arterial blood pressure (MAP), active plasma renin and serum calcium were higher (P less than 0.01) in the tumour transplant rats than in the controls at 11 days after transplantation. There was a positive correlation of both active renin and MAP with serum calcium at this time. Although inactive renin levels were elevated in the tumour transplanted rats, the ratio of inactive to active renin was decreased in comparison to controls. Plasma norepinephrine, active renin and plasma angiotensin II were higher in tumour rats at 14 days. Nevertheless, basal levels of aldosterone and MAP as well as aldosterone responses to graded infusion of angiotensin II, ACTH and KCl were decreased in the tumour rats at 14 days. Moderate hypercalcaemia (day 7 and 11), induced by Leydig cell transplantation in the Fischer rat, is associated, therefore with elevated blood pressure which appears to be related, in part, to activation of the renin-angiotensin system. However, severe hypercalcaemia (day 14) was associated with hypotensive hyperreninaemic hypoaldosteronism state.

The concentration of cortisol and ACTH in the umbilical vein plasma of the foetal guinea pig has been measured over the later half of gestation. Between 55--60 days there is a large increase in the concentration of both. Injection of ACTH1-24 into the foetal guinea pig elicits a relatively small increase in plasma cortisol at 46--55 days, a somewhat greater response at 56--60 days and a large response at 61--66 days. Between 47--55 days and 58--63 days cells prepared from the foetal adrenal show a large increase in their responsiveness to ACTH stimulation as measured by cortisol and androstenedione output. However, there was no clear difference in responsiveness between 58--60 days and 61--63 days. Thus between 56--60 days the foetal adrenal appears less responsive to ACTH stimulation in vivo than in vitro. ACTH in the circulation of the guinea pig is present in high-molecular-weight and low-molecular-weight forms and the ratio of concentration of ACTH1-39 to that of the high-molecular-weight forms rises after 60 days. It is proposed that this is an important factor in inducing an increased steroid response from the foetal adrenal.

The spontaneous glucocorticoid production in control adrenal cells (N = 10) and in the adenoma cells (N = 15) exhibited comparable geometric mean values: 1.896 nmol/ml/4-5 x 10(5) cells per 2 h (confidence limits: 0.428-8.391) and 1.852 nmol/ml (0.326-12.241), respectively. The same results were obtained for the three samples of nodular hyperplasia cells. When cortisol and corticosterone were measured separately, there was no significant difference between the outputs for control cells and those for pathological cells. Baseline aldosterone production in control cells showed a geometric mean of 2.525 pmol/ml (0.236-27.192). In the 15 adenomas, spontaneous production was extremely important: 57.297 pmol/ml (3.357-976.692). The difference was highly significant (P less than 0.0005). Aldosterone levels in the 3 samples of nodular hyperplasia cells were not different from the control values. In 9 out of the 15 adenomas, aldosterone responses to 10(-10) mol/l ACTH, expressed as stimulated/basal production, were above normal: 3.58 +/- 0.86 (SEM) against 1.48 +/- 0.08 (P less than 0.025). In the remaining 6 and in the 3 samples of nodular hyperplasia cells, there was a slight or no response. Angiotensin II (AII) stimulated both adenoma and nodular hyperplasia cells to varying degrees, without any obvious difference between these two categories. A combination of ACTH (10(-12) mol/l) and AII (10(-12) mol/l) had a synergistic action on aldosterone production in cells classed in the adenoma group. These findings demonstrate that despite the abnormal rate of aldosterone formation in adenoma cells, the production rate of corticosterone and cortisol remains normal. They unmask two functional categories with regard to ACTH in the adenoma group. Finally, they underline the relative insensitivity of nodular hyperplasia cells to ACTH.

Ten IU of ACTH (1-24) per day was infused for 34 h (starting at 7 a.m.) into 8 normal men on a constant diet containing 135 mM Na+ per day. All subjects retained between 152 and 181 mM of sodium. Potassium balance was negative. Plasma renin activity (PRA) and plasmaangiotensin II (P-A-II) started to rise in most subjects after 6 to 8 h of infusion, reached a maximum after 24 h and then tended to decline. As shown previously, the rise in PRA is not due to a rise in plasma renin substrate concentration. Systolic, but not diastolic blood pressure increased significantly on the second day of ACTH-infusion. Plasma cortisol (P-F) was continuously stimulated by ACTH. Plasma aldosterone (P-aldo) increased rapidly 1 h after ACTH administration, then tended to fall, and increased again in most subjects, roughly in parallel with PRA. No significant changes in electrolyte balance, PRA, P-A II, P-F, and P-aldo occurred in 3 subjects receiving 'sham' infusions. Additional experiments in subjects treated with propranolol or indomethacin allowed the conclusion that the effect of ACTH on PRA and P-A II is not mediated by renal beta-adrenergic receptors, but perhaps (partially?) by prostaglandins. Since the infusion rate of ACTH was not much higher than the secretion rate of ACTH in the early morning hours, it is possible that ACTH is physiologically involved in the regulation of renin secretion.

The work presented here is an investigation of the effect of elevated levels of corticosteroids on the biophysical properties of skin, aorta and muscle tendon. Rats were given corticotrophin injections for 10, 30 and 60 days to elevate the level of plasma endogenous corticosteroids. The corticotrophin treatments did not change the water or collagen content of specimens from dorsal skin, thoracic aorta and peroneal muscle tendons, tested mechanically. Changes became evident after longer treatment times. For both skin and aorta, the tensile strength, elastic stiffness and failure energy were increased after 60 days of treatment. The corticotrophin treatment did not influence the mechanical properties of muscle tendons. Complete reversibility of changes in the mechanical properties induced by 30 days of corticotrophin treatment was found after an additional period of 30 days of saline injections. This study indicates that an increased level of plasma corticosteroids elicited by corticotrophin treatment may increase the stiffness of the connective tissue of the organism. In the aorta this results in loss of capacitive function with increased haemodynamic strain on the aortic wall.

To study the effect of hypokalaemia in the regulation of aldosterone secretion, repeated injections of frusemide (3 mg/kg) plus saline with or without simultaneous infusion of potassium chloride (1 mEq/kg/h) were performed in 24 conscious female rabbits for 7 h. Without potassium supplementation, the plasma renin activity (PRA) remained elevated throughout the study, while an initial increase (1 h to 3 h) in plasma aldosterone (PA) gradually returned to normal with reduction of the serum potassium. In rabbits on potassium supplements to prevent the development of hypokalaemia, both PRA and PA remained elevated. The incremental aldosterone response to administration of potassium chloride, angiotensin II or ACTH, was considerably smaller in potassium-depleted rabbits than in potassium-repleted rabbits. These results suggest that serum potassium modulates the effects of angiotensin II or ACTH on aldosterone secretion, and that a certain level of potassium is necessary to maintain the aldosterone secretory capacity of the adrenal gland.

The regulatory function of glucocorticoids on thyroid hormone concentrations was studied in patients with adrenocortical insufficiency (ACI, n = 8) and in healthy subjects (n = 6). In patients with ACI withdrawal of glucocorticoid substitution for 84 h led to an increase in serum concentrations of total triiodothyronine (TT3) from 110 +/- 20 to 133 +/- 22 ng/dl and a decrease of serum reverse-T3 (rT3) from 23 +/- 6 to 18 +/- 6 ng/dl, whereas subsequent administration of dexamethasone (0.5 mg po q.i.d. for 3 days) induced a fall in TT3 (129 +/- 22 to 88 +/- 16 ng/dl) and a rise in rT3 (17 +/- 5 to 37 +/- 11 ng/dl) concentrations. Serum levels of total thyroxine (TT4) were unchanged by either withdrawal or re-administration of glucocorticoids. Basal plasma thyrotrophin (TSH) concentrations were unchanged by glucocorticoid withdrawal and fell from 2.2 +/- 1.5 to 1.1 +/- 0.8 mU/l during subsequent dexamethasone therapy. In healthy subjects a decrease of TT3 (89 +/- 8 to 69 +/- 8 ng/dl) and an increase in rT3 (19 +/- 5 to 32 +/- 8 ng/dl) concentrations were seen after stimulation of endogenous cortisol production by prolonged infusion of ACTH1-24 (0.5 mg, t = 8 h on 2 consecutive days), whereas concentrations of TT4 remained unchanged. During subsequent administration of dexamethasone serum level of both. TT3 and rT3 returned to basal levels. Thus, changes in thyroid hormone concentrations are induced by alterations of substitution therapy in patients with ACI. In healthy subjects the application of 2 mg dexamethasone/day following prolonged maximal stimulation of endogenous cortisol by iv ACTH may represent a state of relative glucocorticoid deficiency, thus explaining the observed hormonal changes, which are inverse to those generally induced in healthy man by endogenous or exogenous glucocorticoids.

In male Sprague-Dawley rats the influence of salt loading (1% NaCl), deoxycorticosterone acetate (2 X 15 mg/kg/day resp. 250 mg/kg sc), corticosterone (2 X 20 mg/kg/day sc) and adrenocorticosterone (0.5 mg/kg/day tetracosactid sc) on the activity of renal kallikrein and renal renin activity was investigated. Salt loading lowered renal kallikrein activity, deoxycorticosterone stimulated its activity and in combination they had no effect on renal kallikrein activity. The time course of kallikrein stimulation by deoxycorticosterone showed no relationship to the escape phenomenon of the kidney from the sodium retaining effect of the mineralocorticoid hormone. Reduction of endogenous mineralocorticoid hormones by adrenalectomy caused a marked reduction of urinary and renal kallikrein activity. Corticosterone suppressed the activity of the renal kallikrein-kinin system at the same time as the reduction in urinary aldosterone excretion. Adrenocorticotrophin caused the same decrease in the activity of renal kallikrein as corticosterone. Urinary aldosterone excretion, however, was significantly stimulated. Thus, the known positive correlation between kallikrein and aldosterone was missing. In all experiments the urinary excretion of kallikrein correlated highly with the kallikrein activity measured in renal cortical tissue. However, no correlation was found between kallikrein and urine volume or urinary excretion of sodium and potassium. In our experiments no relationship between the activity of the renin-angiotensin system and that of the renal kallikrein-kinin system was observed. Furthermore, no clear relationship was found between systemic blood pressure and the activity of the renal kallikrein-kinin system.

A virilizing adrenocortical adenoma was demonstrated in a yound female. Urinary 17-ketosteroid excretion and subfractions, plasma testosterone level and urinary 17-ketogenic steroid excretion were markedly increased. Dehydroepiandrosterone was the main constituent of the androgen excess. Otherwise the adrenocortical function was found to be normal as evaluated from measurements of cortisol, corticosterone and their metabolites under basal conditions and during dynamic tests. The androgen excess showed an unexpected response to trophic hormones. Human chorionic gonadotrophin stimulation resulted in a pronounced increase in androgen production, whereas no gonadotrophin-dependency could be demonstrated by means of the oestrogen suppression test. Similarly, no corticotrophin-dependency could be demonstrated by corticotrophin stimulation and suppression tests. Removal of the tumour resulted in normalization of the androgen production and no abnormal response upon human chorionic gonadotrophin administration could now be found. The inappropriate response of tumours to trophic hormones is discussed. It is concluded that the reliability of stimulation and suppression tests in determining the site of excessive androgen production should be accepted with reservation.

Studies were conducted to determine the effect on corpus luteum (CL) function of ACTH or hydrocortisone administered by ovarian perfusion on day 11 of the oestrous cycle or by carotid infusion on days 2-9 of the oestrous cycle. No significant local effects on CL function were observed during the ovarian perfusion study. Continuous carotid infusion of ACTH or hydrocortisone on days 2-9 resulted in a decreased slope of the progesterone increase during the infusion period. However, no difference in plasma progesterone level on days 9-11 between control and treated cycles was detected and there was no difference between treated and control CL at day 11 with regard to CL weight or progesterone content. The data suggest a possible direct effect of hydrocortisone on either the hypothalamus or pituitary which results in decreased progesterone secretion but not CL development. No residual effect was observed.

This study provides evidence that 22S-OH-cholesterol inhibits the conversion of 25-OH-cholesterol but has no effect on the conversion of 22R-OH-cholesterol. The latter sterol is an intermediate in the cholesterol side-chain cleavage, whereas for the conversion of 25-OH-cholesterol into pregnenolone the complete side-chain cleaving enzyme system is necessary. This complements a previous study in which it was shown, that 22S-OH-cholesterol has an inhibitory effect on the ACTH-induced conversion of cholesterol into corticosterone in isolated rat adrenal cells. The available evidence thus suggests an inhibition by 22S-OH-cholesterol of the first step in the cholesterol side-chain cleavage. The results, obtained from the experiments with rat adrenal cells and with bovine adrenal mitochondria, allow the hypothesis, that a causal relationship exists between conversion of 22S-OH-cholesterol and production of corticosterone, respectively pregnenolone. We conclude, that 22S-OH-cholesterol is a substrate for steroid production in the adrenal cell. This sterol inhibits the ACTH-stimulated corticosterone production. The site of this inhibition is located at one of the first steps in the cholesterol side-chain cleavage, probably the binding of cholesterol to the cytochrome P450-complex.

A 9 year old Mexican boy presented with severe hypertension, hypokalaemia and features suggesting acute glomerulonephritis. Nephrosclerosis was present on renal biopsy. Aldosterone levels were unresponsive to variations in dietary salt intake and plasma renin activity was suppressed. Following oral dexamethasone therapy (2 mg/day), plasma aldosterone decreased to undetectable levels, serum potassium normalized and plasma renin activity gradually increased. Dexamethasone also restored the normal responsiveness of the renin-aldosterone system to postural stimuli. The patient exhibited a marked response to a single dose of ACTH with a rise in plasma aldosterone. Long-term blood pressure control and normal potassium levels have been achieved with oral prednisone therapy (5 mg/day) for a period of one year. This case of dexamethasone suppressible hyperaldosteronism (DSH) illustrates that the degree of hypertension in this syndrome may produce severe renal microvascular lesions. DSH should be considered in all children who present with low renin hypertension.