Configurational analysis and relative binding affinities of 16-methyl-5alpha-androstane derivatives

The four possible isomers 16beta-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 1, 16alpha-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 2, 16beta-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 3 and 16alpha-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 4 with proven configuration were converted into the corresponding 16beta-methyl-5alpha-androstane-3beta,17beta-diol 5, 16alpha-methyl-5alpha-androstane-3beta,17beta-diol 6, 16beta-methyl-5alpha-androstane-3beta,17alpha-diol 7, 16alpha-methyl-5alpha-androstane-3beta,17alpha-diol 8, furthermore into the 16beta-methyl-17beta-hydroxy-5alpha-androstane-3-one 13, 16alpha-methyl-17beta-hydroxy-5alpha-androstan-3-one 14, 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3-one 15 and 16alpha-methyl-17alpha-hydroxy-5alpha-androstan-3-one 16. The steric structures of the resulting epimers were determined by means of 1H-, and 13C-NMR spectroscopy. In this way, comparison was possible with the C-16 epimers 5, 6 and 13, 14 prepared earlier by a different route, and the series of isomers could be completed with the steric structures of 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3beta-ol 7 and 16alpha-methyl-17alpha-hydroxy-5alpha 8 and with their 3-keto derivatives 15 and 16. The relative binding affinities of the 16-methyl-5alpha-androstane-3beta,17-diols 5, 6, 7, 8 and 17-hydroxy-16-methyl-5alpha-androstan-3-ones 13, 14, 15, 16 were studied. The introduction of a 16-methyl substituent into 5alpha-androstane molecules substantially decreases the binding affinity to the androgen receptor and 16alpha-methyl derivatives were always bound more weakly than the 16beta-methyl isomers.     

Metabolism of 17 alpha-methyltestosterone in the rabbit: C-6 and C-16 hydroxylated metabolites

17 alpha-Methyltestosterone and the reduced metabolites, 17 alpha-methyl-5 alpha-androstane-3 alpha, 17 beta-diol, 17 alpha-methyl-5 alpha-androstane-3 beta, 17 beta-diol and 17 alpha-methyl-5 beta-androstane-3 alpha, 17 beta-diol, together with three hydroxylated metabolites, 17 alpha-methyl-5 beta-androstane-3 alpha, 16 alpha, 17 beta-triol, 17 alpha-methyl-5 beta-androstane-3 alpha, 16 beta, 17 beta-triol and a new metabolite, 17 alpha-methyl-5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol, were isolated and identified in the urine of rabbits dosed with 17 alpha-methyltestosterone. No hydroxylated 5 alpha-metabolite of 17 alpha-methyltestosterone has been identified previously. No of 17 alpha-methyltestosterone has been identified previously. No evidence for epimerization at the C-17 position was observed.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by radioimmunoassay

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by radioimmunoassay using celite microcolumn purification. The antisera used for the assay were obtained by immunization of rabbits with 3 alpha,17 beta-dihydroxy-5 alpha-androstane-6-(O-carboxymethyl) oxime: BSA for 3 alpha-diol and 3 beta,17 beta-dihydroxy-5 alpha-androstane-15 alpha-carboxymethyl: BSA for 3 beta-diol. The concentrations (pg/ml +/- SD) of the two diols in normal male and female plasma are respectively: 216 +/- 51 and 49 +/- 32 for 3 alpha-diol, 239 +/- 76 and 82 +/- 45 for 3 beta-diol. Comparison of these results with published ones shows that 3 beta diol concentrations were significantly lower. The high specificity of the assay is due to chromatography on celite microcolumns, allowing elimination of 5-androstene-3 beta,17 beta-diol from the plasma sample.     

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.     

Species differences in 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat, monkey, and human prostate microsomes.

The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6 alpha-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol varied independently of both the 7 alpha- and 7 beta-hydroxylation, which varied in unison. The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with beta-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7 alpha- and the 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol without increasing the 6 alpha-hydroxylation. The 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human and monkey prostate microsomes, but not the 6 alpha-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human prostate microsomes, but not the 6 alpha-hydroxylation, was markedly inhibited (greater than 85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6 alpha-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: effects of antibodies and chemical inhibitors of cytochrome P450 enzymes.

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5 alpha-androstane-3 beta,17 beta-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5 alpha-[3H]androstane-3 beta,17 beta-diol with rat prostate microsomes were apparently 5 alpha-androstane-3 beta,6 alpha,17 beta-triol, 5 alpha-androstane-3 beta,7 alpha,17 beta-triol, and 5 alpha-androstane-3 beta,7 beta,17 beta-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol shifted from the 6 alpha- to the 7 alpha-position with increasing pH (6.8-8.0). The V(max) values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6 alpha-, 7 alpha-, and 7 beta-hydroxylation, respectively. Similar Km values (0.5-0.7 microM) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5 alpha-dihydrotestosterone, and 5 alpha-androstane-3 alpha,17 beta-diol did not appreciably inhibit the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

Effects of testosterone metabolites on serum gonadotropin concentrations in immature male rats.

The ability of testosterone, androsterone, 5alpha-androstane-3alpha,17beta-diol, and 5alpha-androstane-3beta,17beta-diol to prevent the castration-induced rise in serum gonadotropin levels was investigated in immature male rats. Rats castrated at 30 days of age were treated once per day by subcutaneous injection of 12.5-100 mug of the steroid per 100 g body weight per day for 3 days, beginning on the day of castration. The animals were sacrificed 24 h after the last injection. Testosterone propionate, androsterone propionate, and 5alpha-androstane-3alpha,17beta-diol dipropionate were also tested at the approximate molar equivalent of 100 mug of the free alcohol form per 100 g body weight per day. Testosterone propionate and 5alpha-androstane-3alpha,17beta-diol were the only compounds tested that prevented the castration induced rise in luteinizing hormone (LH) concentrations. Testosterone propionate also inhibited the rise in follicle stimulating hormone (FSH) concentrations whereas 5alpha-androstane-3alpha,17beta-diol inhibited the rise in FSH in one but not in another experiment. These were the only compounds tested that affected serum FSH concentrations. The lower doses of testosterone tested significantly increased serum LH, but not FSH concentrations compared to castrate control animals. The highest dose tested partially inhibited the rise in serum LH concentrations. Both androsterone and androsterone propionate maintained ventral prostate weights. Although neither compound prevented the castration induced rise in serum LH, two groups receiving androsterone had serum LH concentrations significantly lower than the castrate control group. 5alpha-Androstane-3beta,17beta-diol and 5alpha-androstane-3alpha,17beta-diol dipropionate failed to maintain ventral prostate weights or prevent the rise in serum gonadotropin levels. These results indicate that 5alpha-androstane-3alpha,17beta-diol is capable of preventing the castration induced rise in serum LH concentrations in the immature male rat and thus may participate in the regulation of LH secretion in these animals.     

Radioimmunoassays for androsterone, 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol

Androsterone (3alpha-hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol were conjugated at C-16 through sulfur to bovine and human serum albumin. Rabbits injected with these conjugates produced antibodies suitable for radioimmunoassays of these hormone metabolites. Samples were purified on Sephadex LH-20 columns. Levels of these steroids were measured in a rat blood serum pool and in ovarian tissue extract pools.     

Uptake and metabolism of androgens by bovine spermatozoa

Spermatozoa from bovine ejaculates and cauda epiditymidis were incubated with either tritiated 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol). Examination of the medium incubations demonstrated metabolic conversion of both DHT and 3 alpha-diol when these steriods were incubated with ejaculated sperm. In addition to this interconversion, the following metabolities were identified: 5 alpha-androstane-3 beta, 17 beta-diol, (3 beta-diol), androsterone and 5 alpha-androstane-3, 17-dione (5 alpha-A-dione). Incubations with cauda spermatozoa showed similar metabolic patterns. Androgen binding was exhibited by both sperm types. Examination of the washed cauda sperm pellet, following incubations with 3 alpha-diol showed that the incubated steroid was the most abundantly bound. DHT and 5 alpha-androst-16-en-3 alpha-ol (delta 16-3 alpha-ol1 were also detected. The major part of the radioactivity bound in the sperm pellet was identified as DHT when this steroid was used as the substrate; the remaining radioactivity consisted of 3 alpha-diol and delta 16-3 alpha-ol. Investigations of ejaculated sperm pellets gave similar results apart from the additional identification of 5 alpha-androst-16-en-3 one (delta 16-3-one) and 5 alpha-androst-16-en-3 beta-ol (delta 16-3 beta-ol (delta 16-3 beta-ol).     

The identification and characterization of a new C19O3 steroid metabolite in the rat ventral prostate: 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol.

This study represents the first report of the formation of 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol (6 alpha-triol) by prostatic tissue. The 6 alpha-triol has been identified by rigorous methods and a chemical synthesis of this triol has been accomplished. This 6 alpha-triol is the major metabolite of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the rat ventral prostate. A minor metabolite of 3 beta-diol has been identified as 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol (7 alpha-triol). Using a variety of C19 androstane substrates, the 6 alpha- and 7 alpha-triols were always found as the major components of the total 3 beta-hydroxy-5 alpha-androstane metabolites produced by the ventral prostate. Following intraperitoneal injection of 3H-3 beta-diol, both 6 alpha- and 7 alpha-triol were formed in vivo by the ventral prostate and found in the blood. The 6 alpha- and 7 alpha-triols were found to possess no androgenic activity when tested by the ventral prostatic growth bioassay in the castrate rat.     

Concentrations of unconjugated 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol and their precursor in human testicular tissue. Comparison with testosterone, 5 alpha-dihydrotestosterone, estradiol-17 beta, and with steroid concentrations in human epididymis

The concentrations of testosterone and its tissular metabolites were determined in testicular and epididymal tissue obtained from eleven male subjects (aged 65-85 years) after orchiectomy for prostatic cancer. The steroids were measured in different tissular compartments, i.e. testis, caput, corpus and cauda epididymis. The values (mean +/- SD; ng/g wet weight) were: Testosterone 724.0 +/- 286.0, 32.08 +/- 2.56, 41.45 +/- 1.77 and 32.24 +/- 2.14; 5 alpha-dihydrotestosterone 6.95 +/- 1.99, 9.76 +/- 2.33, 16.87 +/- 0.21 and 15.79 +/- 2.67; 5 alpha-androstane-3 alpha, 17 beta-diol 6.07 +/- 2.33, 2.17 +/- 0.24, 1.93 +/- 0.02 and 1.17 +/- 0.20; 5 alpha-androstane-3 beta, 17 beta-diol 56.66 +/- 20.97, 3.55 +/- 0.19, 2.21 +/- 0.27 and 3.34 +/- 0.32; estradiol-17 beta 5.36 +/- 3.0, 1.08 +/- 0.014, 1.44 +/- 0.038 and 1.47 +/- 0.03, respectively. Incubation of human testicular tissue with [3H]androst-5-ene-3 beta, 17 beta-diol or [3H]dihydrotestosterone showed that both androstane-diols were exclusively formed from dihydrotestosterone. Since high concentrations of 5 alpha-androstane-3 beta, 17 beta-diol are found in testicular tissue it is suggested that this steroid may be an index of seminiferous tubular function.     

The in vivo metabolism of 7 beta, 17-dimethyltestosterone-6,7-3H.

7 beta, 17-Dimethyltestosterone (17 beta-hydroxy-7 beta, 17-dimethyl-4-androsten-3-one) (I) was given to three subjects in oral doses of 400 mg per day for ten days. The initial dose contained the steroid tritiated in the 6 and 7 positions. Plasma levels and urinary excretion patterns were followed in all three subjects. Isolations were done on the urine, plasma, and stools of one patient. From the urine 7 beta, 17-dimethyl- 5 alpha-androstane-3 beta,17 beta-diol (VI) was isolated from the nonhydrolyzed fractions. Unchanged (I), 7 beta,17-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (III) and 7 beta, 17-dimethyl-5 beta-androstane-3 beta,17 beta-diol (IV) were isolated from the nonhydrolyzed and enzyme-hydrolyzed fractions. 7 beta,17-dimethyl-5 alpha-androstane-3 alpha,17 beta-diol (V) was isolated from the enzymatic fractions. From the stools were isolated unchanged (I), (III), (IV), (V), and (VI). Unchanged (I) and its 5 alpha-dihydro derivative (17 beta-hydroxy-7 beta,17-dimethyl-5 alpha-androstan-3-one) (II) were identified in the plasma. The total recovery of radioactivity in the one patient on whom the isolations were done was 57%; 40% from the urine and 17% from the stools.     

Characterization of two new enzymatic activities of the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase.

This study has characterized two new enzymatic hydroxylase activities specific for 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase (6 alpha-hydroxylase) and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase (7 alpha-hydroxylase). Both of these irreversible hydroxylase activities require NADPH and are localized in the microsomal fraction of the prostate. The apparent Km for 3 beta-diol is 2.5 microM for both the 6 alpha- and 7 alpha-hydroxylase activities. The apparent Km for NADPH is 7.6 microM for the 6 alpha-hydroxylase and 7.0 microM for the 7 alpha-hydroxylase. The pH optimum for both activities is 7.4. Several steroid inhibitors of these hydroxylase activities in vitro were identified including cholesterol, progesterone, and estradiol. Estradiol was found in vitro to be a noncompetitive inhibitor (Ki = 5 microM). Injection of estradiol into intact male rats, simultaneously receiving exogenous testosterone, also produced a significant lowering of the 6 alpha-plus 7 alpha-hydroxylase activities. Both the 6 alpha- and 7 alpha-hydroxylase were found to be androgen sensitive. Following castration there is a rapid decrease in both activities.     

Guinea pig liver 3-hydroxyhexobarbital dehydrogenase. Purification and properties.

3-Hydroxyhexobarbital dehydrogenase, which catalyzes the reversible oxidation of 3-hydroxyhexobarbital to 3-oxohexobarbital, has been purified 470-fold from the soluble fraction of guinea pig liver with a yield of 47%. The specific activity of the purified enzyme is 9.4 units/mg of protein. Results of polyacrylamide gel disc electrophoresis and isoelectric focusing indicated that the purified enzyme preparation is a single and homogeneous protein. NADP+ served as preferred co-factor, but NAD+ is also utilized in the presence of phosphate ion. The guinea pig liver enzyme possessed a relatively narrow substrate specificity in comparison with the rabbit liver enzyme. It is very distinctive that guinea pig liver 3-hydroxyhexobarbital dehydrogenase catalyzes the dehydrogenation of 17beta-hydroxysteroids such as testosterone, 4-androstene-3beta,17beta-diol, 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol, 5alpha-androstan-17beta-ol-3-one, and 5beta-androstane-3alpha,17beta-diol.     

The effect of short term treatment with cyproterone acetate or flutamide on the metabolism of 5 alpha-dihydrotestosterone in human testicular tissue

Testicular tissue obtained from ten patients orchiectomized for prostatic cancer was incubated with [3H]5 alpha-dihydrotestosterone (DHT) in order to study the metabolic transformation into 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Throughout 5 days before surgery four subjects were treated with cyproterone acetate (CA). To three patients flutamide (F) was administered for the same period of time. Three subjects remained untreated. Compared to the control group the administration of CA decreased the formation of 3 beta-diol whereas that of 3 alpha-diol increased. Treatment with F lead to an elevated formation of both diols. However, the 3 alpha/3 beta ratio did not change. As 3 beta-diol is considered to be an index of tubular function in the human testis it is concluded that CA has a direct inhibitory effect upon this testicular compartment whereas F has none.     

Age-related changes in conversion of 5 alpha-androstan-17 beta-ol-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol by rat testicular cells in vitro

Conversion of labelled 5 alpha-androstane-17 beta-ol-3-one (DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5--10 micrograms DHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro. 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol). Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3 alpha- and 3 beta-diols formed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3 alpha-diol, but thereafter 3 beta-diol was the predominant testicular metabolite of DHT.     

Androgen metabolism by rat epididymis. Metabolic conversion of 3H 5alpha-androstane-3alpha,17beta-diol, in vitro

The epididymis of adult rats metabolizes 3H 5alpha-androstane-3alpah,17beta-diol (3alpha-diol) by experiments in vitro. After incubation of tissue slices at 37 degrees C for 2 hours, 2% of the radioactivity was found in the water-soluble fraction whereas 98% was found to be ether soluble (free steroids). Further investigation of the free steroids showed the following to be present: 3alpha-diol 39.9%, DHT (17beta-hydroxy-5alpha-androstan-3-one) 33.7%, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) 9.2%, 3beta-diol (5alpha-androstane-3beta,17beta-diol) 2.6%, 5alpha-A-dione (5alpha-androstan-3,17-dione) 1.1%, delta 16-3alpha-ol (5alpha-androst-16-en-3alpha-ol) 1.0%, delta16-3beta-ol (5alpha-androst-16-en-3beta-ol) 2.6%, delta 16-3-one (5alpha-androst-16-en-3-one) 2.9%, and polar compounds 3.3%. When segments of the epididymis (caput and cauda) were incubated in the same way, qualitatively similar metabolites were formed but a greater amount of 3alpha-diol was metabolized by the cauda epididymis. This increase was mainly accounted for by an increased formation of delta 16 compounds (14.3% in cauda, 4.3% in caput). This is most probably due to the presence of larger numbers of mature spermatozoa, which, as we have previously shown, form delta16 steroids from 3alpha-diol and DHT (5).