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The activities of the hepatic microsomal 2α-, 2β-, 18- and 7α-hydroxylase systems active on 5α-[4-14C] androstane-3α,17β-diol were studied in male and female rats which had been castrated and spayed at 14, 24, 35 and 45 days of age, treated for 5 days with 500 μg of estradiol benzoate per kg body weight and killed 6 days after gonadectomy. The hepatic microsomal 15β-hydroxylase enzyme system active on 5α-[1,2-3H] androstane-3α, 17β-diol 3,17-disulphate was also measured in these animals as well as in an additional group of animals that were gonadectomized at 56 days of age, treated with estradiol benzoate and killed at 62 days of age. The hydroxylase systems active on the free steroid substrate were all relatively unresponsive towards enstradiol in the developing rat, in contrast to the strong effects on hepatic hydroxylase activities previously noted following treatment of adult male rats with estradiol. On the other hand, the 15β-hydroxylase system active on disulphurylated 5α-androstane-3α, 17β-diol was inducible in both male and female rats of 41 and 51 days of age and in male rats of 61 days of age.

Analogs of 4-androstene-3,17-dione and testosterone were tested as substrates for the aromatizing enzyme complex of human placenta. Compounds modified in rings B, C and D were found to be aromatized via a pathway similar to that postulated for 4-androstene-3,17-dione and testosterone, in which oxidation to the 19-hydroxy and 19-oxo (or corresponding gem-diol) intermediates occurs. No evidence of additional intermediates was obtained.

[4-14C]Androstenedione and [4-14C]progesterone were perfused through isolated livers from male and female rats. The metabolites in bile, perfusate and liver tissue were identified by gas chromatography-mass spectrometry. Livers from male rats metabolized androstenedione and progesterone predominantly to 2-hydroxylated and 16-hydroxylated compounds, i.e. isomers of 3,16-dihydroxyandrostan-17-one, androstane-3,16,17-triol, androstane-2,3,16,17-tetrol, 2,3-dihydroxypregnan-20-one, 3,16-dihydroxypregnan-20-one, pregnane-2,3,20-triol and 2,3,16-trihydroxypregnan-20-one. Most of these metabolites were found in the glucuronide fraction. Livers from female rats metabolized androstenedione mainly to 7-hydroxylated and 15-hydroxylated compounds, i.e. isomers of 3,7-dihydroxyandrostan-17-one, androstane-3,7,17-triol and androstane-3,15,17-triol. These compounds were mainly present in the glucuronide fraction. Progesterone was predominantly metabolized into sulphates and “glucuronides” of 3α,15α-, 3α,15β-, and 3α,16α-dihydroxy-5α-pregnan-20-one. When increasing amounts of progesterone were perfused through livers from female rats less sulphates and more “glucuronides” were formed. The results are discussed in relation to previous results regarding the sexual differences in the excretion of steroids in urine, faeces and bile from rats.

This study has investigated the decomposition of pregnene 17α-hydroperoxides by adrenocortical microsomal fractions. The chemical identities of the metabolites were established by means of combined gas chromatography-mass spectrometry. Incubation of progesterone 17α-hydroperoxide with adrenocortical microsomes under aerobic as well as anaerobic conditions gave 17α-hydroxyprogesterone as the major conversion product together with a small amount of androstenedione. Similarly, pregnenolone 17α-hydroperoxide was converted to the corresponding 17α-hydroxy analog and to its expected cleavage product, dehydroepiandrosterone. With boiled enzyme preparations, both hydroperoxides remained virtually unchanged revealing the enzymic nature of the reaction. Incubation of progesterone 17α-hydroperoxide under nitrogen with adrenocortical microsomal fractions fortified with NADPH or NADH also resulted in the formation of 17α-hydroxyprogesterone as the major metabolite. In the presence of air, however, 17α,21-dihydroxyprogesterone became the major conversion product suggesting that the oxygen atom at the C-21 position was supplied by molecular oxygen by way of an active C-21 hydroxylase. In contrast, no 17α,21-dihydroxy analog of pregnenolone 17α-hydroperoxide was detected under similar incubation conditions and in this case, 17α-hydroxypregnenolone was the main metabolite formed. These results are discussed in light of previous suggestions that steroid hydroperoxides may be intermediates in biological hydroxylation reactions.

The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5α-[4-14C]androstane-3α,17β-diol, 4,16-[7α-3H]androstadien-3-one, [4-14C]cholesterol, and 7α-hydroxy-4-[6β-3H]cholesten-3-one was studied in the microsomal fraction of livers from adult male and female rats which had been castrated neonatally or postpubertally. The effect of testosterone propionate treatment on postpubertally castrated male and female rats was also studied with respect to the hepatic microsomal metabolism of the mentioned steroid substrates. It was found that the different microsomal enzyme activities could be grouped into three categories according to their specific characteristics. One category of enzymes (represented by the 2β-hydroxylase active on 5α-androstane-3α,17β-diol, the 6β-hydroxylase active on 4-androstene-3,17-dione and 4-pregnene-3,20-dione, and the 18-hydroxylase active on 5α-androstane-3α, 17β-diol) was more active in male than in female rats. This sexual difference was completely abolished both by postpubertal and neonatal testectomy. The activities of the enzymes in this group were stimulated by treatment with testosterone propionate. A second category of enzymes (the 2α-hydroxylase active on 4-pregnene-3,20-dione and 5α-androstane-3α,17β-diol, the 16α-hydroxylase active on 4-androstene-3,17-dione, the Δ16-C19 steroid epoxidase active on 4,16-androstadien-3-one, and the 17β-hydroxysteroid reductase active on 4-androstene-3,17-dione) was generally much more active in male than in female rats. Only neonatal testectomy completely abolished this sexual difference, whereas postpubertal testectomy reduced but did not abolish the differences in enzyme activities between male and female rats. Testosterone propionate treatment led to increased activities of these enzymes. The third category of enzymes (the 7α-hydroxylase active on 4-androstene-3,17-dione and cholesterol, the 12α-hydroxylase active on 7α-hydroxy-4-cholesten-3-one, and the 16α-hydroxylase active on 4-pregnene-3,20-dione) was generally slightly more active in female than in male rats. The activities of the enzymes in this group were not significantly affected by neonatal or postpubertal gonadectomy or by treatment with testosterone propionate. Based on these findings the microsomal steroid-metabolizing enzyme activities may be grouped into three classes with regard to the mechanisms regulating their activity: (a) enzymes with a basal activity level regulated by nongonadal factors but reversibly inducible by androgens; (b) enzymes irreversibly "imprinted" or "programmed" by androgens during the prepubertal period and reversibly stimulated by androgens postpubertally; and (c) enzymes primarily regulated by nongonadal factors and only slightly affected by androgens. The 5α-reductase active on 4-androstene-3,17-dione, 4-pregnene-3,20-dione and 7α-hydroxy-4-cholesten-3-one is basally regulated by nongonadal factors in both male and female rats. In addition, the activity of this enzyme in male rats, which is about 8 to 10 times lower than that in female rats, is suppressed by testicular androgens by two mechanisms: (a) irreversibly by prepubertal imprinting or programming; and (b) reversibly by postpubertal suppression.

SummaryThe liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione and 5α-[4-14C] androstane-3α, 17β-diol was studied in germfree, conventional and exgermfree rats with a normal indigenous microflora. Germfree rats were significantly more active than conventional rats in hydroxylating 4-androstene-3,17-dione in position 6β (P < 0.02) and 5α-androstane-3α,17β-diol in positions 2β (P < 0.05), 7α (P < 0.02) and 18 (P < 0.02). In addition 7α-, 16α- and 16β-hydroxylation of 4-androstene-3,17-dione and 2α- and 7β-hydroxylation of 5α-androstane-3α,17β-diol tended to be greater in germfree rats than in conventional rats. In most cases the exgermfree rats, which had been reared outside the germfree isolators for 4 wk, showed similar activities of the microsomal hydroxylase systems as their conventional counterparts. Thus, exgermfree rats showed significantly less efficient 16β-hydroxylation of 4-androstene-3,17-dione (P < 0.02) and 2β- (P < 0.02) and 7α- (P < 0.05) hydroxylation of 5α-androstane-3α,17β-diol ...

We report the isolation and characterization of a steroid, 1,4-androstadiene-3,17-dione from the urine of a 15-yr-old boy with a history of epileptic convulsions and premature puberty. To our knowledge, this is the first report of detection of this steroid in human urine. A substance from the urine of an epileptic boy, under prolonged treatment with diphenylhydantoin and phenobarbital, was characterized and shown to be 1,4-androstadien-3,17-dione. This characterization depended preliminarily on examining the behaviour of the substance and its derivatives on thin-layer, paper and gas Chromatographie systems, and conclusively by physico-chemical analysis of its spectra in U.V., I.R., circular dichroism, nuclear magnetic resonance, and gas chromatogram mass spectrometer. The question of whether the appearance of this substance in urine was caused by the therapeutic handling or whether this steroid is a true intermediate during aromatization of testosterone and androstenedione is discussed.

The metabolism of testosterone in human foetal adrenal glands at mid-gestation was investigated in vitro. After incubations with microsome preparations in a medium supplemented with NADPH-regenerating system, the metabolites were isolated by thin-layer and gas-liquid chromatography, and identified by gas chromatography-mass spectrometry. 4-Androstenedione, 3β-hydroxy-4-androsten-17-one and 4-androstene-3α (and 3β),17β-diols were identified in both incubation series. The presence of 1β-, 2α-, 2β-, 6α- and 6β-hydroxylase activities in foetal adrenal glands was demonstrated by the isolation of the corresponding testosterone derivatives. The biosynthesis of these steroids in human adrenal gland has not been demonstrated before. Because 3β-hydroxy-4-androsten-17-one and epimeric 4-androstene-3α (and 3β), 17β-diols could be isolated in these incubation studies the presence of 3α, 3β- and 17β-hydroxysteroid oxidoreductase in the adrenal gland preparation is very likely. Neither 5α(H)- nor 5β(H)-ring reductase activities have been detected.

The steroid composition of and steroid conjugation in human cadaver and boar testes were investigated by analyzing the endogenous steroids. Gas-liquid chromatography and gas chromatography-mass spectrometry were used to identify the steroids and to determine the position of the sulfate group in sulfate conjugates. For the latter purpose, the steroids were first acetylated and subsequently solvolyzed and converted to trimethylsilyl ethers. In addition to the compounds previously identified as endogenous components, human testis was also found to contain progesterone, 17α-hydroxyprogesterone and 20α-hydroxy-4-pregnen-3-one in the free steroid fraction and 3β-hydroxy-17α-5-pregnen-20-one in the monosulfate fraction.

Incubations of testes of adult rats with testosterone yield rather important amounts of a very polar metabolite which is identified as 7α-hydroxytestosterone. The identification of the metabolite is based on chromatography, spectrophotometry, fluorimetry, counter current distribution and NMR spectrometry.

An alkali-washed diethyl ether extract of 1300 ml of pooled adult human plasma was fractionated by passage through a single column of Sephadex LH-20. Without further purification the various fractions were subjected to mass spectrometrie analysis. The presence of 17β-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 3β-hydroxy-5-androsten-17-one, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3 (α or β), 17β-diol was noted. Since testosterone and DHEA occurred together with cholestadiene as did androstenedione with cholesterol, further separation was carried out by t.l.c. to confirm the identifications individually.

A general method for rendering Δ3-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7α-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17α-hydroxyprogesterone through the respective 4, 6-dienes. Immunization of rabbits with testosterone-7α-carboxymethyl-thioether-BSA and the homologous testosterone-7α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×109 1/mol) that showed little cross-reaction with androstenedione ( epit estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5α-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5α-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%). A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.

[4- 14 C + 7-D 0.44 ]Androstenedione and [4- 14 C + 7β-D 0.42 ]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M + ) and (M + + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting 14 C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.

The metabolism of [ 3 H]-testosterone in perfused dog adrenals and in adrenal minces of a normal dog has been studied. Three principal metabolites were studied and identified by isotope dilution and gas-liquid chromatography-mass speetrometry: androstenedione, 11β-hydroxyandrostenedione and 11 β-hydroxytestosterone. The last of these metabolites is obtained in high yield which is further increased after ACTH administration.

17β-hydroxysteroid dehydrogenase (EC 1.1.1.64) activity was studied in immature mice ovarian tissue. The substrates employed in this investigation were 4-androstene-3.17-dione (androstenedione) and oestradiol-17β. The products of the enzymic reaction. 17β-hydroxy-4-and rosten-3-one (testosterone) and oestrone respectively were identified by thin-layer chromatography, gas-liquid chromatography, mass spectrometry and derivative formation. The radiochemical purity was established by recrystallization to constant specific activity. For the reduction of androstenedione to testosterone NADPH was the electron donor whereas the oxidation of oestradiol-17β to oestrone utilized NADP+ as well as NAD+ as electron acceptor. Triphosphopyridine nucleotide was the preferred cofaetor for the latter reaction. The results of the present experiments demonstrate that the ovaries of immature mice devoid of corpora lutea are capable of converting androstenedione to testosterone and oestradiol-17β to oestrone indicating the presence of 17β-hydroxy steroid dehydrogenase in such tissue.