Possible indices for the detection of the administration of dihydrotestosterone to athletes.

Dihydrotestosterone (DHT) can be used by an athlete as an anabolic steroid to evade the current International Olympic Committee approved drug tests. To investigate the possibility of a method for its detection, the heptanoate ester of DHT was administered to two male subjects (150 mg i.m.). Urine samples, collected before and after the injection, were subjected to enzymatic hydrolysis and the excretion rates of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and testosterone (T) were determined by radioimmunoassay. Relative changes in the excretion of DHT, 3 alpha-diol, 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol), T and epitestosterone (17 alpha hydroxyandrost-4-en-3-one; Epi-T) were determined by gas chromatography-mass spectrometry (GC-MS). Following administration of DHT, the urinary excretion rates of DHT, 3 alpha-diol and 3 beta-diol increased when compared to those of T, Epi-T, 5 beta-diol and luteinizing hormone (LH). Concentrations of DHT in the plasma increased whereas those of T, LH and follicle stimulating hormone decreased. The changes following such modest doses of DHT suggest that these ratios of urinary hormones may be used for the detection of doping with DHT.     

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.     

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.     

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.     

Reductive metabolism of 5 alpha-dihydrotestosterone by rat ventral and dorsolateral prostate: kinetic parameters of the enzymes

In male sex accessory organs the active androgen 5 alpha-dihydrotestosterone (DHT) is metabolized to 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) by the reductase activities of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR; EC 1.1.1.50) and 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR; EC 1.1.1.51). After separation of radiosubstrate and products by HPLC, these enzymes activities in subcellular preparations of rat ventral and dorsolateral prostate were determined from the conversion of [3H]DHT to the radiometabolites 3 alpha-diol and 3 beta-diol and 3 beta-triols (5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol plus 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol). Whereas both enzymes were found in the dorsolateral prostate, 3 beta-HSOR reductase activity was near the limit of detection in ventral prostate. Unlike the equal distribution of 3 alpha-HSOR reductase between the microsomal and cytosol fractions of the ventral prostate, both 3 alpha- and 3 beta-HSOR reductase activities of the dorsolateral prostate are mainly confined to its cytosol fraction. Km and Vmax of the 3 alpha- and 3 beta-HSOR reductases in dorsolateral prostate cytosol were 1.8 microM, 24.6 pmol.mg-1 min-1 and 25.4 microM, 45.7 pmol.mg-1 min-1, respectively. We surmise from these and earlier studies that 3 beta-HSOR reductase is the rate-limiting prostatic enzyme in the catabolic disposition of intracellular DHT.     

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).     

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).     

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.     

Prostatic cytosol binding of 5 alpha-androstane-3 beta, 17 beta-diol and estradiol-17 beta

The goal of the present research was characterization of the interaction of 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) with prostatic estradiol-17 beta(E2) binding sites to address the role of this 5 alpha-dihydrotestosterone(DHT)a metabolite in prostatic regulation. Using dextran-charcoal assay we demonstrated specific 3 beta-diol and E2 binding sites in rat ventral prostate cytosol (RVPC) and dog prostate cytosol (DPC). In both cytosols, E2 binding is of high affinity (Ka congruent to 10(9) M-1; RVPC:68 fmol/mg protein), DPC:170 fmol/mg protein), and 3 beta-diol binding is of moderate affinity (Ka congruent to 10(8) M-1; RVPC:62 fmol/mg protein, DPC:165 fmol/mg protein). Unlabeled 3 beta-diol competes effectively for cytosolic 3H-E2 binding sites, whereas unlabeled DHT, 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and testosterone (T) are poor competitors for 3H-E2 binding sites. Using DNA-cellulose column chromatography, we separated prostatic androgen and estrogen binding activities. The E2 binding activity which adhered to DNA-cellulose was displaced by 100-fold excess 3 beta-diol but not by DHT. Thus data from two assay procedures show competition of 3 beta-diol for 3H-E2 binding sites in rat and dog prostate.     

5 alpha-DHT metabolism in monolayer cultures of distinct pituitary cell populations

5 alpha-Dihydrotestosterone (DHT) metabolism into 5 alpha-androstane-3 alpha, 17 beta-diol (alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (beta-diol) was studied in monolayer cultures of distinct cell populations from prepubertal male rats pituitaries. Cells were characterized through immunocytochemistry with the various antihormone antisera. Centrifugal elutriation was used to prepare a gonadotrope-enriched population "G" and a gonadotrope-depleted population "L", containing most lactotropes and somatotropes. Using centrifugation on Percoll gradient, two sub-populations, P1 and P2, were prepared by further fractionation of the "L" population. Cells were incubated for 48 h with [3H]DHT (1 microM, sp. act. 0.9 Ci/mmol) and metabolites extracted from the whole cell and medium. DHT was metabolized to about the same extent (30-40%) in all cell fractions. Compared with unfractionated population, the conversion of DHT into alpha-diol increased significantly in the P1 fraction, consisting of lactotropes, somatotropes and highly depleted in gonadotropes. This increase was lower in the somatotrope-enriched P2 fraction in which the amount of lactotropes was similar to P1 but that of gonadotropes slightly higher. In contrast, the conversion of DHT into alpha-diol decreased significantly in the "G" population compared with total or "L" fractions, whereas androstanedione formation, low in every population, increased significantly. The increase in alpha-diol formation could be related either to the decrease of gonadotropes or to a role of non-immunoreactive cells. As the beta-diol formation was constant in all cell types, the beta-diol/alpha-diol amount increased significantly in gonadotropes. Then, beta-diol and DHT could be both active steroids in gonadotrope regulation inasmuch as specific binding sites were identified for these two steroids. It can be concluded that DHT action at the pituitary level is subject to complex control mechanisms involving a specific balance of its metabolites in each particular cell type.     

The effect of infusions of 5 alpha-dihydrotestosterone or estradiol-17 beta on the concentration of some steroids in the human testicular vein and artery

The concentrations of testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstan-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, estradiol-17 beta and testosterone-glucosiduronate were measured in the plasma of the testicular vein and artery simultaneously with the estimation in peripheral venous and arterial plasma 60 min after an infusion of 3000 micrograms dihydrotestosterone (DHT) or estradiol (E2), respectively, in patients undergoing orchiectomy for prostatic cancer. The results were as follows; following infusion of DHT or E2, both steroids were completely metabolized by the testes. After DHT the testicular secretion of E2 was significantly reduced. In peripheral plasma 3 alpha-diol concentration was increased. Following E2 a transient elevation of testosterone in the spermatic vein was observed, whereas a slight decrease of DHT and an increase especially of 3 beta-diol levels occurred. It is assumed that DHT as well as E2 plays a role as intratesticular regulator of steroid synthesis and metabolism.     

Urinary 5 alpha-androstane-3 alpha,17 beta-diol levels in normal and hirsute women: discriminating power and relation to other urinary steroids

The urinary levels of seven steroids, 5 alpha-androstane-3 alpha,17 beta-diol, 5 beta-androstane-3 alpha,17 beta-diol, androsterone, etiocholanolone, tetrahydrocortisone, tetrahydrocortisol and allotetrahydrocortisol were measured in both normal (n = 18) and hirsute (n = 24) women. The results confirmed 5 alpha-androstane-3 alpha,17 beta-diol as the most significant steroid with respect to discrimination between hirsute and normal subjects. Investigation of the inter-steroid relationships, using multivariate techniques established that the mode of steroid metabolism was different between the two groups. Whereas in normal women the strong correlation amongst all the androgen metabolites inferred a predominant hepatic route to 5 alpha-androstane-3 alpha,17 beta-diol formation, the same analogy was not applicable to the hirsute subjects. Excellent agreement was found for the predicted vs actual excretion of 5 alpha-androstane-3 alpha,17 beta-diol in normal women, based on a regression model involving the six other steroids as independent variables. When the same model was used for estimation of 5 alpha-androstane-3 alpha,17 beta-diol levels in thirteen hirsute subjects, misclassified as "normal", 50% gave values which were considerably less than actually measured. It is suggested that this discrepancy, with respect to these hirsute subjects is a reflection of extrahepatic production of 5 alpha-androstane-3 alpha,17 beta-diol due to increased 5 alpha-reductase activity.     

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.     

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.     

Androgen metabolism in rat L6 myoblast cells; high formation of 5 alpha-androstane-3 alpha,17 beta-diol from testosterone

We have studied androgen metabolism in L6 rat myoblasts. 4-androstene-3,17-dione (Adione), testosterone, 5 alpha-dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) were used for substrates and the amounts of metabolites formed from the respective substrates in the medium were determined. Conversion of Adione to testosterone was dominant over the reverse conversion. DHT formation from testosterone was low and did not change with the duration of incubation, whereas 3 alpha-diol formation increased in a time-dependent manner. Major metabolite of testosterone was not DHT but 3 alpha-diol. A large amount of 3 alpha-diol was formed from DHT, however, DHT formation from 3 alpha-diol was very low. These data indicate that L6 cells have high 5 alpha-reductase activity and suggest that DHT formed from testosterone is rapidly metabolized to 3 alpha-diol in these cells.     

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.     

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)     

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.     

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.