Androstenedione Research Articles

Changes in the Activities of Steroidogenic Enzymes during the Development of Ovarian Follicles in Rana nigromaculata

Earlier studies showed that the predominant steroid (estradiol [E 2], testosterone [T], progesterone [P 4]) secreted by in vitro cultured amphibian ( Rana nigromaculata) ovarian follicles varied with their size. E 2 was mainly produced by medium-sized follicles, T by intermediate-sized follicles, and P 4 by the largest follicles. Experiments were carried out to ascertain whether the activities of steroidogenic enzymes changed during follicle development and were responsive to gonadotropic stimulation. Enzyme activities were measured indirectly by monitoring conversion of exogenously added substrates to products during in vitro culture of isolated follicles. Different stage follicles were cultured in the presence or absence of frog pituitary homogenate (FPH, 0.1 pituitary/2 ml) and/or various steroid precursors (25-200 ng/2 ml). Amounts of E 2, T, androstenedione (AD), 17α-hydroxyprogesterone (17α-OHP 4), or P 4 secreted into the medium were measured by RIA. Exogenous pregnenolone (P 5) was converted to P 4 by all types of follicles in a dose-dependent manner in the absence of FPH. Addition of FPH markedly enhanced medium levels of P 4 in all sized follicles. Highest levels of P 4 were presented in cultures containing the largest follicles. Such follicles were much less efficient than intermediate follicles in metabolizing P 4 to AD or T. EPH suppressed conversion of exogenous 17α-OHP 4 but not androstenedione to testosterone by the largest follicles. Exogenous T was converted to E 2 only by medium-sized follicles and FPH had little or no stimulating or inhibiting effect on this process in either medium- or intermediate-sized follicles. Taken together, the data suggest that (1) the transition from E 2 to T secretion (Class III to IV) is associated with a loss of aromatase activity, (2) the transition from T to P 4 production (Class IV to V) is correlated with a decrease in the activity of enzymes which metabolize P 4, and (3) activities of steroidogenic enzymes varied in their responsiveness to EPH stimulation.

Serum androgens and gonadotrophins in bronchial carcinoma

The concentrations of testosterone, free testosterone index derived by two independent techniques (FTI1, FTI2), dehydroepiandrosterone sulphate (DHAS), androstenedione (AND), luteinizing hormone (LH), follicle stimulating hormone (FSH), and sex hormone binding globulin (SHBG) were measured in 38 healthy male controls aged between 40 and 60 years (Group 1) and in 52 male patients with bronchial carcinoma of whom 28 were aged between 40 and 60 years (Group 2) and 24 were aged over 60 years (Group 3). Compared with Group 1, testosterone, FTI1, FTI2, and DHAS were significantly lower ( P <0·001), AND and LH were significantly higher ( P <0·001) and there was no significant difference in FSH or SHBG, in Group 2. No significant difference between Groups 2 and 3 was found for any variable. Analysis of covariance for Groups 2 and 3 combined, showed testosterone, FTI1 and FTI2 were significantly lower in the presence of metastases. These results suggest a more widespread abnormality of androgen metabolism in male patients with bronchial carcinoma than a simple reduction in serum testosterone; DHAS is also significantly reduced while AND is significantly increased. The primary abnormality appears to be at testicular and/or adrenal level as pituitary function appears normal as witnessed by compensatory increases in LH.

The production of monoclonal antibody to androstenedione and the effect of passive immunization on Xinjiang fine-wool sheep with it.

Five hybridomas stably secreting monoclonal antibodies (McAbs) to androstenedione were prepared by using artificially synthesized androstenedion-11 alpha-succinyl conjugate with bovine serum albumin (BSA) as antigen. These McAbs showed slightly high cross-reactivity with testosterone (8.1-12.3%) and estrone (0.8-2.5%) and high affinities ranging from 2.0 x 10(7) to 2.8 x 10(8) L/M. They were all of the IgG1 subclass. Xinjiang finewool ewes which were passively immunized with McAbs had higher circulating levels of progesterone (P) and luteinizing hormone (LH) than those of control ewes in two oestrous cycles and these changes led to increased ovulation rate and twin lambs born in young ewes.

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Stage-related production of 21-hydroxylated progestins by the dogfish (Squalus acanthias) testis.

Using shark (Squalus acanthias) testicular microsomes and [3H]progesterone ([3H]P) and [3H]17 alpha-hydroxyprogesterone ([3H]17 alpha-P) as substrates, two major products of 21-hydroxylase action were identified; respectively, 21-hydroxy-4-pregnen-3-one (11-deoxycorticosterone, DOC) and 17 alpha,21-dihydroxy-4-pregnen-3-one (11-deoxycortisol,S). Additional products of 17 alpha-hydroxylase/C-17,20-lyase action were detected: 17 alpha-hydroxyprogesterone (17 alpha P), testosterone (T), and androstenedione (AE). When microsomes derived from tissues in premeiotic (PrM), meiotic (M), and postmeiotic (PoM) stages of spermatogenesis were compared, maturation-related increases were observed with both 21-hydroxylase (3- to 20-fold) and 17 alpha-hydroxylase (2- to 6-fold). With [3H]P as tracer, the half-maximal substrate concentrations (Km = 0.2-1.0 microM) and maximal reaction velocities (Vmax = 9-25 pmol/mg protein/min) were similar for both enzymes when assayed in the same preparation, suggesting they compete for available substrate. Also, the presence of 1- or 10-fold molar excess radioinert DOC reduced conversion of [3H]P to 17 alpha-hydroxylated products. [3H]DOC itself was a substrate of 17 alpha-hydroxylation but not C-17,20-lyase action. Expression of 21-hydroxylase and 17 alpha-hydroxylase activities in cultured spermatocysts (intact germ cell/Sertoli cell units) was confirmed by detection of immunoreactive 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S),S,17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P), DOC, P and T in spent media. 20 beta-S and S secretion increased and 17 alpha,20 beta-P decreased progressively with stage of maturation, but DOC was similar in all stages. P secretion was maximal and T lowest in M-stage spermatocysts, but when DOC (0.1 microM) was added to PrM or PoM spermatocysts, T output decreased. Taken together, the data suggest that 21-hydroxylating pathways, via direct and indirect mechanisms, affect accumulation of bioactive steroids (P and T) differentially by stage of spermatogenic development. Whether 21-hydroxylated progestins produced in shark testis have paracrine or endocrine actions in their own right remains to be investigated.

Stage-related changes in steroid-converting enzyme activities in Squalus testis: synthesis of biologically active metabolites via 3β-hydroxysteroid dehydrogenase/ isomerase and 5α-reductase

To investigate the relationship between steroidogenesis and spermatogenesis, two key enzymes of androgen biosynthesis, 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and 5α-reductase, were compared at premeiotic (PrM), meiotic (M) and postmeiotic (PoM) stages. Staged tissues were obtained by dissection from the testis of the spiny dogfish Squalus acanthias, in which spermatogenesis is a simple diametric progression. Microsomal 3β-HSD activity was measured by conversion of [ 3H] dehydroepiandrosterone (DHEA) to androstenedione (AE). Reaction constants were: K m = 3.2 μM and V max = 243 pmol/min/mg protein. 3β-HSD increased progressively with maturation, resulting in three- to four-fold higher levels in PoM than in PrM stages. Absolute values and stage-related differences were the same, whether microsomes were derived from whole testis or from isolated spermatocysts (germ cell/Sertoli cell units), thus supporting microscopic studies showing that Sertoli cells are the primary steroidogenic elements of dogfish testis. In vitro conversion of [ 3H]testosterone to [ 3H]dihydrotesterone (DHT) was used to estimated 5α-reductase activity. Apparent substrate affinity was similar to that of 3β-HSD ( K m = 2.9 μM), but maximal product yields were two to three orders of magnitude lower ( V max = 208 fmol/min/mg protein). Also, the stage-related pattern of 5α-reductase activity ( PrM > PoM >> M) differed from that of 3β-HSD ( PoM >> M > PrM). Together with previously measured steroidogenic enzyme activities using the shark testis model, these results predict that de novo androgen biosynthesis is highest in testicular regions with mature spermatids and fully differentiated Sertoli cells, and also suggest that availability of T versus DHT for androgen receptor binding may be developmentally regulated during spermatogenesis.

Endocrinological profile of oligomenorrheic strenuously exercising adolescents

The endocrinological profile of 20 strenuously exercising oligomenorrheic adolescents divided into 2 groups (groups A and B), was correlated with that of 10 athletes (group C) with normal menstrual cycles and without strenuous exercise. Group A LH serum baseline values were found to be statistically significantly lower than those of group C (P < 0.001). FSH/LH basic values were 1.9- and 2.9-times higher in group A athletes than those of group B or C (P < 0.05 and P < 0.001, respectively). 17β-estradiol (E 2) and prolactin serum levels were found lower in group A and B athletes than those of group C (P < 0.01–0.05). Dehydroepiandrosterone sulfate and Δ4 androstenedione serum levels were found lower in group A athletes than those of group C (P < 0.001). The low LH and E 2 values indicate the anovulatory status of group A and B cases which were also confirmed by ultrasound. It is concluded that no severe endocrinological changes exist in strenuously exercising oligomenorrheic athletes in relation to menarche.

Contribution of aromatase to the deoxyribonucleic acid synthesis of MCF-7 human breast cancer cells and its suppression by aromatase inhibitors

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [ 3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14α-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.

Androstenedione therapy reinstates normal, not supernormal, song structure in castrated adult male zebra finches

In previous studies, androstenedione (AE) replacement therapy restored the highest levels and intensities of courtship song displays in castrated male zebra finches of any hormone tested. Furthermore, female zebra finches responded strongly to AE-treated males and preferred intact males given small AE implants to unsupplemented males. In this study, we asked whether AE treatment might alter song structure, since male song is an important cue in mate choice by female zebra finches. Songs of adult males were recorded. The males were then castrated and given AE therapy and recorded again. No differences were found between the courtship or undirected songs males sang before castration and after AE treatment. As in previous studies, the structure of a male's courtship song differed significantly from his undirected song, and the structural differences between these two song types were not altered by AE treatment.

The effect of DHAS on steroidogenesis of the human corpus luteum.

To examine whether or not dehydroepiandrosterone sulfate (DHAS) is a substrate for steroidogenesis in the corpus luteum, we studied 17 women in the luteal phase, the follicular phase, and after castration. Following suppression of adrenal function with dexamethasone, DHAS was administered intravenously and the serum levels of DHAS, dehydroepiandrosterone (DHA), androstenedione (ADS), testosterone (T), 17 beta-estradiol (E2) and progesterone (P) were measured serially for 24 h. An obvious increase in the serum levels of all steroids except for E2 and P was observed in each subject for at least 8 h after DHAS administration. To evaluate the effect of DHAS on the serum levels of the steroid hormones, the integrated response area (IRA) was calculated for each hormone in all the subjects. The IRA values for ADS, T and E2 (at 2 and 4 h) in the luteal phase group were significantly higher than in the other DHAS treated groups, and the IRA values for DHA and P tended to be higher than in the other groups. These results suggest that the corpus luteum utilizes serum DHAS as a substrate for steroidogenesis.

Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself. The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10 −9 M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10 −10 M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative. Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, n = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, n = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17α-, 21- and 11β-hydroxylases. The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response ( P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%. Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17α, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.

Rationale for using aromatase inhibitors to manage benign prostatic hyperplasia. Experimental studies.

Today, human benign prostatic hyperplasia (BPH) is considered primarily to be a disease of the stroma, in which estrogens are thought to play a considerable causative or permissive role. The growing incidence of BPH with increasing age coincides with a shift in the androgen:estrogen ratio in favor of estrogens, not only in terms of serum hormone values, but also in the prostate itself. Furthermore, evidence has been provided for a preferential accumulation of estrogens in the stroma of human hyperplastic tissue, and the presence of an estrogen receptor satisfying the classical criteria of high affinity and low capacity has been demonstrated. Also, animal studies have emphasized the potential role of estrogens in the pathogenesis of BPH. Experimentally, stimulation of the stroma, particularly of smooth muscle, can be induced by aromatizable substrates, such as androstenedione, in the prostates of beagles and cynomolgus monkeys. These effects can be antagonized by aromatase inhibitors, such as atamestane. In addition, the increase in intraprostatic estrogen concentrations and immunohistochemically detectable estrogen receptor content induced by androstenedione in intact dogs is completely reversed by simultaneous treatment with atamestane. In conclusion, clinical data, as well as that from animal models, emphasize an important role for estrogens in the development of BPH. Estrogen deprivation might, therefore, represent a useful treatment for human BPH.

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA (II)——HORMONE SECRETING ACTIVITY OF CYTO-TROPHOBLAST CELLS

The capability of cytotrophoblast cells to produce hCG, progesterone, estrogen, cGnRH and β-endorphin in vitro has been demonstrated in serum-free culture medium. Before experiment, a 24-h preculture was carried out in order to remove the endogenous hormones of the tissue. During a period of 8 days' culture, the cytotrophoblast cells could constantly produce a small amount of hCG. The production of progesterone rose rapidly and became doubled within six days. The estrogen secretion showed a similar pattern in the presence of androstenedione, a precursor of estrogen, indicating the elevation of aromatase activity in the cells. The elevation of the enzyme activity has been further demonstrated not to be induced by androstenedione. In both cytotrophoblast and syncytiotrophoblast cell cultures, cGnRH was only detected in the culture of cytotrophoblast cells, with a value up to 4 pg/105 cells/24 h. However, β-endorphin was identified both in cytotrophoblast and syncytiotrophoblast cells. Its content increased significantly in the medium of cytotrophoblast cell culture from the 4th to 6th days, but declined in the medium of syncytiotrophoblast cell culture. The results demonstrate clearly that the cytotrophoblast cells are the sole origin of GnRH in human placenta and are also able to synthesize β-endorphin and steroid hormones. The findings indicate that there is no such a sharp functional demarcation existing between these two kinds of trophoblast cells as suggested before. The data are of significance for a better understanding of the mechanism of hormonal regulation in placenta.

Altered testicular hormone production in infertile patients with idiopathic oligoasthenospermia.

We studied the kinetics of testicular response to human chorionic gonadotropin (hCG) in oligoasthenospermic and asthenospermic patients (OAZ-AZ). The responses of testosterone (T), androstenedione (A), 17 OH-progesterone (17OHP), and estradiol (E2) were evaluated in 60 OAZ-AZ patients and compared to those of 10 normal men. The responses of T, A, and 17OHP to hCG in the control group displayed a biphasic pattern with an initial peak at 4 hours and a second peak after 24 hours. The E2 response showed a single peak between 24 and 48 hours after hCG administration. OAZ-AZ patients had two types of T responses: group 1 (n = 40) had no first peak and group 2 (n = 20) had a normal response pattern. The response of A was similar to that of T, and the E2 response was normal in both groups. There were three types of 17OHP responses in group 1 (low, high, or normal); however, the 17OHP response was normal in group 2. Treatment of group 1 with aromatase inhibitors (aminoglutethimide or testolactone) induced an improvement of the acute T response only in patients with high or normal 17OHP response to hCG, whereas no effects were observed in patients with low 17OHP response. In group 2, the aromatase inhibitors induced no changes in the T response. These results demonstrate that in some OAZ-AZ patients (group 1, blunted T response) testicular hormone production is altered. They also suggest the presence of two enzyme blocks: one at the 17,20 desmolase level, mediated by E2, and another at early biosynthetic steps, not mediated by E2.

Microbial transformations in a cyclodextrin medium. Part 2. Reduction of androstenedione to testosterone by Saccharomyces cerevisiae

An intensive and systematic investigation of the reductive transformation of androstenedione (AD) to testosterone by Saccharomyces cerevisiae was undertaken in the presence of natural and chemically modified cyclodextrins (CD). The bioconversion was significantly larger in the presence of β- and ψ-CD and hydroxypropyl-β-CD b but only slight in the presence of α-CD and dimethyl- and trimethyl-β-CD. The performance of the various cyclodextrin media was interpreted in the light of the measured phase solubility diagrams of AD. Further investigation focused on biotransformation of the β-CD-androstenedione complex, the formation of which was studied by differential scanning calorimetry and X-ray powder diffractometry and stoichiometry determined by 1H-nuclear magnetic resonance. A mechanism whereby CDs reduce the effective inhibitory concentrations of substrate and product as well as facilitate transport of the complexed substrate through the yeast cell wall has been suggested for the CD-promoted biotransformation.

Wood duck hatch date: Relationship to pairing chronology, plasma luteinizing hormone, and steroid hormones during autumn and winter

We examined variation in courtship activity and hormone levels of male and female wood ducks ( Aix sponsa) in relation to hatch date. Young wood ducks were assigned in groups of 8 (4 males and 4 females) to 4 experimental pens; 2 pens contained early-hatched ducks (3–12 April) and 2 pens contained late-hatched ducks (7–16 June). Courtship behaviors occurred less frequently in October and November than in December and January-February for both early- and late-hatched groups. Early-hatched wood ducks participated in courtship more frequently and formed pair bonds sooner than late-hatched individuals. Testosterone (T) and androstenedione (AD) levels of males did not differ between treatment groups; however, average levels of luteinizing hormone (LH) were greater for early-hatched males. Differences in LH occurred in 2 of 12 samples (6 October and 3 November); otherwise, LH did not vary by treatment. Levels of T and LH in males varied temporally, but there was no significant temporal variation in AE levels. Temporal variation in hormone levels was similar for early- and late-hatched males. Estradiol (E), progesterone (P), and LH levels of females did not differ between treatment groups. There was temporal variation in levels of E and LH, but not of P; this variation was similar for early- and late-hatched females. These data indicate that behavioral differences occurring temporally and between early- and late-hatched wood ducks were not related to corresponding differences in hormone levels.

Assessment of the hypothalamic-pituitary-testicular function in male patients with Wilson's disease.

Sixteen male patients with Wilson's disease were studied to detect potential endocrine dysfunctions. There was no clinical evidence of feminization in any of the patients, and the patient group spanned most pubertal stages. Gonadotropin, testosterone, sex hormone binding globulin (SHBG), dehydroepiandrosterone sulphate, androstenedione, estradiol, prolactin, cortisol, thyrotropin, and free thyroxine levels were determined. Low or borderline luteinizing hormone (LH) levels were present in most of the patients. In six of the adult patients, a standard gonadotropin-releasing hormone (GnRH) test was performed. Five of the six patients had blunted LH and follicle-stimulating hormone (FSH) responses to GnRH. Increased androgen levels were found in eight of the patients. Sex hormone-binding globulin was normal in eight of nine tested patients. Three single-dose human chorionic gonadotropin (hCG) stimulation tests of six adult patients showed normal responses. Three other patients who had elevated baseline levels responded with modest increases. Since liver disease is usually associated with decreased androgen levels, it is difficult to account for the elevated androgen levels. Both increased androgen levels and copper accumulation in the hypophysis could be responsible for the blunted GnRH response.

Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6β-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver. 6β-Hydroxylation was the most important pathway of biotransfromation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2β-, 15β- and 15α-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6β-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6β-alcohol was the only metabolite formed from AD at a rate > 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti- P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2β-, 6β-, 15α- and 15β-positions (by 31–56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6β-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6β-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6β-hydroxylation.

Multiple steroid hormone levels in depressed patients and normal controls before and after exogenous ACTH

Forty depressed patients and 36 age- and sex-matched controls were given 250 μg ACTH 1–24 by iv bolus. Plasma steroid hormone levels were measured prior to and 60 min after ACTH administration. The depressed patients had significantly greater cortisol (F), 11-deoxycortisol (S), androstenedione (AD), and 17α-hydroxyprogesterone (17α-OHP) responses (delta; p<0.05) and a marginally greater 11β-hydroxyandrostenedione (11β-OHAD) response (delta; p=0.091) than the controls. There was no significant difference in the corticosterone (B) response between the two groups. With the exception of 11β-OHAD, all the steroid hormones were significantly negatively correlated with age in the controls, but only S and AD marginally demonstrated this relationship in the depressed patients. F, S, AD, 17α-OHP, and B, but not 11β-OHAD, were significantly positively correlated with each other in the controls, but only F was significantly correlated with AD in the depressed patients. These data suggest that the hypercortisolemia found in some depressed patients involves increased precursor and metabolite levels both at baseline and in response to exogenous ACTH, compared to controls. Furthermore, variability in these precursors is greater in depressed patients, and their relationship to age is lost. These findings are consistent with the hypothesis that adrenal products other than cortisol also could be related to affective symptoms.

Dynamics of androstenedione metabolite formation in rat liver microsomes during temporal phenobarbital induction

1. Formation of androstenedione (AD) 7α-, 16α-, 16β- and 6β-hydroxymetabolites produced in rat liver microsomes and differing by the duration of phenobarbital (PB) induction (temporal induction) has been studied. 2. Formation of 7α-, 16α- and 6β-metabolite is sexually differentiated during PB-induction. 3. The most dramatical changes were observed in the 16β-hydroxylase activity specific for cytochrome P-450b which increased in all rat groups investigated. 4. The immunochemical method using antibodies against P-450b/e was applied to measure its content in microsomes. 5. It was shown that the inicrosomal level of P-450b/e correlated ( r = 0.63) with a 16β-hydroxylase activity in a narrow range of enzyme concentrations (from 0.16 to 0.32 nmol mg ). 6. In microsomal preparations with a higher level of P-450b/e the correlation is lower ( r = 0.4). 7. The dependence of the P-450b catalytic activity on the P-450b to NADPH-cytochrome P-450 reductase relation is discussed.