An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Synthesis and release of steroids in intestines from cynomolgus monkeys (Macaca fascicularis)

To examine the synthesis and release of steroids in intestinal tissues from cynomolgus monkeys (Macaca fascicularis), we performed the following experiments: 1) incubated prepared intestinal tissues with [(3)H]testosterone to study the conversion to other steroids; 2) used a radioimmunoassay to determine steroid levels in six segments of intestinal tissues and contents (duodenum, jejunum, ileum, cecum, colon, and rectum); 3) localized testosterone in the six intestinal segments by immunofluorescence histochemistry; and 4) determined steroid levels in feces from males and females of various ages by radioimmunoassay to examine a correlation between steroid levels and age or sex. In prepared intestinal tissues, testosterone was converted into androstenedione, 5 alpha-dihydrotestosterone, and an unidentified substance; all of these steroids were detected in all segments of the intestinal tissues and contents by radioimmunoassay. Immunofluorescence showed that testosterone was located in all segments of intestinal epithelia. Androstenedione, testosterone, 5 alpha-dihydrotestosterone, and the unidentified substance were also detected in feces, and their levels were not affected by the age or sex of the animal. The present findings in cynomolgus monkeys led us to conclude that 1) steroids were synthesized in the intestines; 2) intestinal steroids were released from the six intestinal tissues to the intestinal cavities and excreted outside the body with feces; and 3) intestinal steroids were released irrespective of age or sex of the animal. Intestinal steroids seem to be paracrine or exocrine agents and to have different characteristics from classical serum steroids.     

Sex hormones and corticosteroids in pollen of Pinus nigra

In continuation of earlier investigations on steroid hormones in the pollen of pine, we have isolated and quantitatively determined the following steroids by radioimmunoassay and fluorimetric methods: testosterone, testosterone together with epitestosterone, respectively, and androstenedione; progesterone was determined by radioimmunoassay alone. In addition, we have tried to isolate cortisol, cortisone, 11-deoxycortisol, corticosterone and 11 -deoxycorticosterone and to characterize these compounds by specific reactions. The intensity of these reactions were used to estimate the amounts of corticosteroids in pollen.     

Radioimmunoassay of androstenedione and testosterone in cow plasma at the time of luteolysis and oestrus

Androsteredione and testosterone were measured by radioimmunoassay after chromatography on micro-columns of Lipidex-5000 in jugular plasma samples taken every 2-3 h at the time of luteolysis and oestrus in 3 dairy cows. The concentrations of androstenedione and testosterone varied between 5 and 60 pg/ml and 5 and 80 pg/ml respectively and no consistent pattern in the fluctuations of either steroid was observed.     

Developmental patterns of serum luteinizing hormone, gonadal and adrenal steroids in the sooty mangabey (Cercocebus atys)

Serum levels of luteinizing hormone (LH), testosterone, dehydroepiandrosterone sulfate (DHAS), androstenedione and cortisol were determined in multiple samples from 86 sooty mangabeys of varying ages (0-17 years). Testosterone, androstenedione, DHAS and cortisol were measured by radioimmunoassay; LH was determined by in vitro bioassay. Serum LH concentrations were elevated in neonates (less than 6 months) and in animals older than 72 months of age. The higher LH levels were associated with increased circulating concentrations of testosterone in males but not females. The pubertal rise in serum testosterone at approximately 55-60 months of age in males was coincident with rapid body growth. No pubertal growth spurt was observed in females. Serum levels of androstenedione and DHAS were highest during early postnatal life (less than 6 months) with androstenedione exceeding 600 ng/dl in males and 250 micrograms/dl in females, but declined rapidly in both sexes to a baseline of 150 ng/dl by 19 months of age. Serum androstenedione did not fluctuate significantly in adult animals. The pattern of age-related changes in serum DHAS paralleled those of serum androstenedione, whereas serum cortisol values did not change significantly with age. Developmental changes in serum LH, testosterone and body weight suggest that the sooty mangabey matures substantially later than the rhesus monkey. The pattern of serum gonadal and adrenal steroids during sexual maturation is similar to that seen in the baboon with no evidence of an adrenarche.     

A radioimmunoassay of androstenedione

A dextran-coated charcoal radioimmunoassay for androstenedione (4-androsten-3, 17-dione) is reported which uses an anti-testosterone antiserum raised in sheep, against a testosterone-17-hemisuccinate-Bovine Serum Albumin conjugate. It is more sensitive and rapid than previously published double dilution, gas chromatographic and competitive protein binding assays. Androstenedione is separated from cross-reacting Steroids by Celite column chromatography. The intra-assay and interassay coefficients of variation were 10.7 and 11.6 per cent, respectively. Using this method serum androstenedione in men was 1.15 ± 0.35 ng/ml; in women, 1.41 ± 0.30 ng/ml; in post-menopausal women, 0.88 ± 0.34 ng/ml; in ovariectomized women, 0.67 ± 0.17 ng/ml; and in ovariectomized-adrenalectomized women, 0.14 ± 0.05 ng/ml. The blank of the method was usually 4 to 5 pg, but ranged between 0 and 12 pg. The sensitivity of the standard curve was 8 pg.     

Excretion of non-conjugated androstenedione and testosterone in human urine.

A method for the measurement of unconjugated testosterone and androstenedione in human urine is described. The method uses chromatographic separation followed by radioimmunoassay and has been examined for reliability. The mean 24-hour excretion of androstenedione by adult male subjects was 2.5 mug and of testosterone was 0.8 mug. For women, the mean excretion was 2.9 mug of androstenedione and 0.25 mug of testosterone. In pregnancy, androstenedione excretion was occasionally elevated above the normal range, but testosterone excretion was quite commonly increased. Some hirsute subjects exhibited an increase in androstenedione excretion, which was decreased by administration of dexamethasone. The results suggest that the amount of unconjugated testosterone in urine is not a direct reflection of the plasma free testosterone, but urinary androstenedione may be a useful reflection of plasma androstenedione levels.     

Dexamethasone in resting and exercising men. II. Effects on adrenocortical hormones.

This study presents the reactions of adrenocorticosteroids (cortisol and aldosterone) and sex steroids [testosterone, androstenedione, and dehydroepiandrosterone and its sulfate (DHAS)] 1) to a dexamethasone (Dex) treatment, which is expected to lower steroid levels via the ACTH blockade, and 2) to an exercise bout at maximal O(2) consumption, which is expected to increase steroid production via ACTH stimulation. Consistent with the decrease in ACTH, all steroids except testosterone reacted negatively to Dex, independently of the dose (0.5 and 1.5 mg administered twice daily for 4.5 days). After exercise, plasma ACTH rose to 600% of basal value, resulting in a significant increase in aldosterone and adrenal androgens, but cortisol and DHAS were unaffected. This apparently surprising result can be explained by differences in peripheral metabolism: a theoretical calculation predicted that after 15 min the increase in hormone concentration may only reach 12% for cortisol and 2% for DHAS. For cortisol and adrenal androgens, assays were carried out using plasma and saliva. The consistent results obtained from the two matrices allow us to consider salivary assays as a useful tool for steroid abuse detection.     

Simultaneous radioimmunoassay of testosterone and dihydrotestosterone

A radioimmunoassay, which simultaneously measures both testosterone (T) and dihydrotestosterone (DHT) in the same serum sample, is presented. Celite column chromatography is employed to separate T from DHT, and these two steroids from other potentially cross-reacting and interfering steroids. The normal values for men, women in the follicular phase, women in the luteal phase, ovariectomized and adrenal ectomized women, post-menopausal women and ovariectomized women for T are 5, 140 ± 1190 pg/ml, 307 ± 97 pg/ml, 285 ± 46 pg/ml, undetectable (<5 pg/ml), 262 ±47 pg/ml and 199 ±44 pg/ml; and for DHT 470 ± 165 pg/ml, 160 ±45 pg/ml, 147 ±44 pg/ml, undetectable (<5 pg/ml), 168 ± 27 pg/ml, 94 ± 15 pg/ml. The maximum sensitivity of the method was 10 pg/ml for T and 14.3 pg/ml for DHT when 1 ml was extracted. The blank in most assays was undetectable, but rarely exceeded 10 pg.     

A sensitive and specific enzymeimmunoassay for serum testosterone.

A very sensitive enzymeimmunoassay for testosterone was developed using testosterone-penicillinase conjugate and an antibody to testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin. The specificity of the assay was demonstrated by the fact that estradiol-17 beta, estrone, estriol, progesterone, 17 alpha-hydroxy-progesterone, dehydroepiandrosterone, androstenedione, cortisol and cortisone were ineffective in crossreacting with testosterone while dihydrotestosterone was 8 times less crossreactive as compared to testosterone. The minimum detectable amount of testosterone was 10-15 pg per assay tube. Intra-assay and inter-assay coefficients of variation for samples containing 0.3-6ng/ml of testosterone were 6-8% and 8-10%, respectively. A high degree of correlation (r = 0.97) was observed between serum testosterone values obtained by enzymeimmunoassay and radioimmunoassay. The levels of testosterone in the sera of normal men and women and those in hypogonadal males following stimulation with human chorionic gonadotropin determined by this enzymeimmunoassay appear similar to those reported by other investigators.     

Production of steroids by in vitro superfusion from adrenals of the Mongolian gerbil (Meriones unguiculatus): effect of acute stress

1. The output of steroids by in vitro superfusion from adrenals of normal gerbils was studied; the glands secreted the following amounts of steroids (ng/animal/hr): 272 (glucocorticosteroids), 60 (aldosterone), 5.0 (progesterone), 1.0 (androstenedione) and 1.0 (testosterone). 2. Glucocorticosteroid and progesterone output from superfused glands of animals stressed by exposure to a novel environment (a) on concentrated ether vapor (b) was significantly higher than that of control animals (glucocorticosteroids: a: 3-9 min, b: 3-30 min after start of superfusion; progesterone: a, b: 3 min after start of superfusion). 3. Aldosterone output was not affected by the stressors applied. 4. Glucocorticosteroid plasma levels of 204 ng/ml were found in control animals. Exposure to a novel environment or concentrated ether vapor resulted in significantly elevated glucocorticosteroid concentrations (511 ng/ml and 760 ng/ml, respectively). 5. Neither testosterone nor progesterone plasma levels were changed by these stressors.     

Enzyme immunoassay for testosterone and androstenedione in culture medium from rabbit oocytes matured in vitro

Two enzyme immunoassays (EIAs) were validated to determine testosterone and androstenedione levels in culture medium (Brackett's medium with or without the addition of IGF-I, hormone and serum-free), without previous extraction, from rabbit oocytes matured in vitro. Polyclonal testosterone (C917), and androstenedione (C9111) antibodies were raised in rabbits using testosterone 3-carboxymethyloxime:BSA, and androstenedione 3-carboxymethyloxime:BSA. Horseradish peroxidase was used as label, conjugated to testosterone 3-carboxymethyloxime, and to androstenedione 6-hemisuccinate. Standard dose response curves covered a range between 0 and 1 ng/well. The low detection limits of the technique were 11.43 pg/ml for testosterone, and 2.32 pg/ml for androstenedione. Intra- and inter-assay coefficient of variation percentages were < 6.4 and < 7.1 for testosterone, and < 5.1 and < 6.3 for androstenedione, respectively (n= 10). The recovery rate of known testosterone or androstenedione concentrations added to pools of culture maturation medium samples averaged 97.58 +/- 2.11%, and 95.73 +/- 1.59%, respectively. Compared with RIA, EIA values were in close agreement for testosterone (n= 15, r= 0.96, P< 0.001), and androstenedione (n= 15, r= 0.94, P< 0.001). Culture medium samples were obtained at the end of oocyte in vitro maturation (14-16 h). Mean +/- SE culture maturation medium concentrations (ng/ml) were 1.80 +/- 0.09 and 0.52 +/- 0.01 for testosterone, and 1.70 +/- 0.04 and 0.24 +/- 0.01 for androstenedione in both the oocytes with and without cumulus cells, respectively. We concluded that our EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive and nonradiometric alternative to RIA for determining testosterone and androstenedione concentrations in oocyte maturation culture medium.     

Testicular androgens and spermatogenetic cycles in the viviparous lizard

Androstenedione, testosterone and dihydrotestosterone levels were measured in the testis of 36 males of the viviparous lizard throughout a period (from end of May to end of July) characterized by the transition between two spermatogenetic cycles and by very low levels of plasma testosterone. The sudden rise of testicular testosterone and androstenedione in June is concomitant with a degeneration of the seminiferous epithelium. It coincides with a transient appearance of testicular dihydrotestosterone. During the next decline in the levels of testosterone and androstenedione, it occurs a restoration of the seminiferous tubules which resume spermatogenesis (proliferation of spermatogenia and prophase of first meiotic division). The part played by some testicular steroids in the control of spermatogenesis is discussed.     

Effect of injection of gonadotrophin-releasing hormone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Hypogonadal (hpg) mice were injected once daily with 10 ng, 50 ng or 1 microgram GnRH for 5, 10 or 20 days or 12 times daily with 4.2 ng GnRH for 5 days. Basal and hCG-stimulated production in vitro of androstenedione, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol (androstanediol) were measured by radioimmunoassay. All doses of GnRH increased testicular weight and in-vitro androgen production although seminal vesicle weights were unchanged and serum testosterone concentrations remained undetectable. After 5 days' treatment androstenedione and androstanediol were the dominant androgens produced, the latter indicating the presence of high levels of 5 alpha-reductase. By 20 days testosterone production was predominant after treatment with higher doses of GnRH. Total androgen production (androstenedione + testosterone + androstanediol) after 5 and 10 days was similar at all concentrations of GnRH used. After 20 days' treatment total androgen production was significantly greater with 50 ng GnRH/day than with 10 or 1000 ng/day. Multiple daily injections of 4.2 ng GnRH (total dose 50 ng/day) had no greater effect on androgen production in vitro compared to single daily injections of 50 ng. This suggests that under the conditions used in this study the testis does not require pulsatile release of the gonadotrophins. The pattern of [3H]pregnenolone metabolism was measured after 5 days injection of 50 ng GnRH/day. Compared to control hpg animals there was a significant increase in formation of C19 steroids, synthesis being solely through the 4-ene pathway. These results show that GnRH treatment of hpg mice will induce testicular steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intratesticular steroid levels and their hormonal control

Litter-mate adult male rats were treated with daily intramuscular injections of ACTH (10.5 micrograms), dexamethasone (2.0 mg), ethynyl estradiol (1.7 micrograms) and hCG (5 IU) for three consecutive days. The animals were sacrificed on the fourth day and the intratesticular and peripheral plasma steroid levels were analyzed. The steroids measured by radioimmunoassay included pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone. In addition, the sulphoconjugated forms of pregnenolone, dehydroepiandrosterone, testosterone and dihydrotestosterone were estimated in the peripheral blood. The administration of ACTH diminished the intratesticular levels of all steroids studied. Also dexamethasone and ethynyl estradiol treatment suppressed all intratesticular steroid levels, except that of pregnenolone (the former) and of 17-hydroxyprogesterone (the latter). The suppressive effect of ethynyl estradiol was strongest on the levels of the delta 5-steroids and that of dexamethasone on the delta 4-steroids; the latter was significantly stronger than the effect of ACTH. The stimulatory effect of hCG was limited to the metabolism of progesterone and was restricted to the sequence: 17-hydroxyprogesterone----androstenedione----testosterone---- dihydrotestosterone. Dexamethasone-suppression, and hCG-stimulation of the intratesticular levels of delta 4-steroids, was mirrored by corresponding changes in the peripheral plasma levels, with the exception of the plasma levels of androstenedione which were not influenced by any of the treatments studied. Also the suppression of intratesticular testosterone and dihydrotestosterone levels by ACTH, dexamethasone, or ethynyl estradiol was closely reflected by their plasma levels both in the unconjugated and sulphoconjugated forms. On the hand, the administration of ACTH diminished the intratesticular levels of pregnenolone and progesterone but significantly increased those in the plasma. Moreover, both ACTH and ethynyl estradiol reduced the levels of all delta 5-steroids in testicular tissue, but not in the peripheral plasma, although they decreased the circulating levels of pregnenolone sulphate and dehydroepiandrosterone sulphate. The data are interpreted as suggesting that the hormonal agents studied interfere with testicular steroidogenesis through different mechanisms.     

Progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone concentrations in specific regions of the human brain

The concentrations of progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone were determined in tissue samples from the human hypothalamus, anterior pituitary, pineal, amygdala and parietal cortex, taken at autopsy from male (n = 4) and female cadavers (n = 4) of various ages. The measurements were performed using radioimmunoassays for the individual steroids after the chromatographic purification of solvent extracts of tissue samples on Lipidex-5000TM. Preliminary qualitative analyses of the chromatographic profiles of various steroids by radioimmunoassay demonstrated the presence of these steroids in various regions of the brain, but an immunoreactive peak corresponding to 17-hydroxyprogesterone was not found. The concentrations (ng/g tissue wet wt.) of all steroids measured were either very low or below the limit of detection in brain tissues taken from male and female infants. In the adult brain, there was no difference in the distribution of steroids between the various regions studied. There was no sex difference in the brain tissue steroid concentrations, with the exception of testosterone which was clearly much higher in brain tissues from men as compared to women. Although testosterone was undetectable in most samples taken from adult women. 5 alpha-dihydrotestosterone could be measured in almost all samples, which suggests that this is the most important androgen in the human brain. When brain tissue steroid levels are compared with serum concentrations, it can be postulated that a state of equilibrium exists between the fraction of serum steroids which are not bound to high-affinity binding proteins and the amount of steroids in brain tissues.     

A specific radioimmunoassay for androstenedione with reduced bridge-binding

Antibody used in a steroid radioimmunoassay raised against a steroid hapten-carrier protein conjugate may recognize both the hapten and the chemical bridge to the protein. Use of the same bridge in the radio-isotopic label may lead to higher affinity binding to the label than to the native steroid. Inhibition curves under these conditions are shallow and generally not acceptable for radioimmunoassay procedures. We have developed a radioimmunoassay for androstenedione that employs different bridges at the 11 beta position of the steroid for the protein conjugate and label. The resulting assay has greatly reduced bridge-binding, has an acceptable slope for the standard curve and is very specific as evidenced by low crossreactivies to other steroids.     

Plasma androgens in idiopathic hirsutism

The plasma concentrations of testosterone, androstenedione,5-androstene-3β, 17β-diol, 5α-dihydrotestosterone and 17β-hydroxy-androgens have been measured in normal women and in patients with idiopathic hirsutism. The mean levels of all the compounds studied were higher in the group of patients with hirsutism than in the controls and apart from dihydrotestosterone, this difference was statistically significant. The correlation between the plasma levels of the various steroids was examined. In the normal subjects, testosterone and androstenedione levels were well correlated, but in the hirsute patients, the correlation is poor. It is suggested that this is related to altered binding of testosterone to plasma proteins in these patients. No one of the steroid levels measured was consistently abnormal in hirsutism, but in 40% of the patients, plasma androstenedione levels were above the normal range.     

Circulating androgen levels in the developing boar

Circulating levels of serum androgens were studied for 11 Duroc boars. Jugular blood samples were collected at 2-wk intervals, beginning at 5 wk of age and continuing until 27 wk of age. Testosterone and androstenedione values were determined by radioimmunoassay. Analysis of variance indicated a significant difference among ages in testosterone and androstenedione concentrations. Plasma levels of testosterone were 1.5 to 1.9 ng/ml at 5 to 7 wk, decreased to 0.3 to 0.6 ng/ml between 7 and 17 wk, and then increased to 3.7 ng/ml by the 27th wk of age. Plasma androstenedione tended to be elevated during the 5th through 7th wk (3.5 to 4.9 ng/ml), decreased to 0.9 to 1.6 ng/ml through the 19th wk and then gradually increased through the 27th wk (1.4 to 2.4 ng/ml). A highly significant correlation was observed between testosterone and androstenedione (r=0.39). Testicular volume was shown to be highly correlated with testosterone concentration (r=0.48). During the early life of the pig, the predominant androgen is androstenedione with testosterone becoming the predominant androgen as the boar reaches maturity.     

Endogenous C19-steroids and oestradiol levels in human primary breast tumour tissues and their correlation with androgen and oestrogen receptors

Endogenous levels of testosterone, 5 alpha-dihydrotestosterone (5 alpha-DHT), androstenedione and oestradiol as well as levels of androgen (AR) and oestrogen (ER) receptors were measured in human primary breast tumour samples. The purification procedure developed allowed simultaneous quantitation of the four steroids, by radioimmunoassay, in small samples with adequate precision, sensitivity and accuracy. The majority of the tumours analysed contained detectable levels of the four steroids in the homogenate or cytosol fractions. There was no significant correlation between steroid content of the tissue and the age of the patient for any of the four steroids. A positive correlation (r = 0.71) was found between the levels of 5 alpha-DHT and testosterone in tumours. In general, tissue steroid concentrations decreased with an increase in dedifferentiation. Fifty-two per cent of the tumours analysed for receptor content were found to be ER positive, and a similar proportion were AR positive. No relationship was observed between AR status and age although receptor concentration was significantly (P = 0.004) higher in post-menopausal women when only receptor positive tumours were evaluated. The mean values for AR and ER were higher in tumours containing both receptors than in tumours showing either receptor alone; there was, however, no significant relationship between concentrations of the two receptors. No correlation was observed between tumour AR or ER status and any of the four steroids measured in either fraction. In addition, the ratio between the combined levels of 5 alpha-DHT and testosterone compared to oestradiol in the same tumour, only showed a maximum value of 40. Thus, in vivo these two androgens are unlikely to influence oestrogen action in human primary breast tumours by interfering with the association of oestradiol with its receptor.