Oxidation of corticosteroids to steroidal 20-hydroxy-21-oic acids by mouse liver.

We have studied the enzyme catalyzed oxidation of 11-deoxycorticosterone to 20-hydroxy-3-oxo-4-pregnen-21-oic acid (pregnolic acid) in mouse liver. Enzyme activity, though variable, was higher than that of other species. Pregnolic acid was identified as the free acid, as the methyl ester by thin layer chromatography, and as the p-bromophenacyl ester by high performance liquid chromatography. With [4-14C, 21-3H]-DOC as substrate, exchange of tritium with water (interpreted as due to the reversible isomerization of the ketol to aldol form by the side chain) and the overall conversion of the ketol side chain to hydroxy acid was catalyzed by the post-microsomal supernatant fraction. Although we could not physically separate tritium exchange and acid production, pregnolic acid formation could be decreased or eliminated while tritium exchange was retained, consistent with our previous conclusion that isomerization to aldol was a precondition for acid formation. In preparations that made no acid, [4-14C]-DOC was recovered, depleted of tritium. The rate of exchange of [21S, 21-3H]-DOC with water was faster than [21R, 21-3H]-DOC. The stereochemistry of pregnolic acid at C-20 was 85-90% R (i.e.. 20 alpha-hydroxy-21-oic acid). The Km for isomerase with [21RS-21(3) H-DOC was 4.3 x 10(-5); Km for pregnolic acid formation was 8.0 x 10(-5) M. Corticosterone was oxidized to acid metabolites at 20% the rate of DOC.

Different biological action of corticosteroids, corticosterone and cortisol, as a base of zonal function of adrenal cortex.

The effects of corticosterone and cortisol in concentrations attainable in the adrenal gland were studied on ACTH-induced steroidogenesis in cultured cortical cells of foetal human and rat adrenals. Corticosterone at a concentration of 5.8 x 10(-5) mol/l clearly inhibited cortisol production (65.5%; P less than 0.005) and simultaneously increased androgen production in tissue culture of foetal human adrenals. Cortisol at a concentration of 2.8 x 10(-4) mol/l clearly inhibited 18-OH-DOC (74.0%, P less than 0.001) and aldosterone (83.7% P less than 0.005) production in tissue culture of foetal rat adrenals. In primary culture of foetal human adrenals cortisol did not decrease aldosterone production absolutely, but it significantly decreased the relative amount of aldosterone with respect to corticosterone. Cortisol did not inhibit corticosterone production in either culture. The results demonstrate that cortisol and corticosterone have qualitatively different effects on adrenal steroidogenesis and that these steroids may play a basic role in the functional zonation of the adrenal gland.

Progesterone biotransformation by plant cell suspension cultures.

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid.

A study of the effects of methyl mercury, cadmium, arsenic, selenium, and a PCB, (Aroclor 1254) on adrenal and testicular steroidogeneses in vitro, by the gray seal Halichoerus grypus.

An in vitro study on the effects of the contaminants polychlorinated biphenyl (Aroclor 1254) (PCB), methyl mercury (MeHg), arsenic (As), cadmium (Cd), and selenium (Se) on the biosynthesis of steroid hormones in the gray seal (Halichoerus grypus) indicated altered steroid biosynthesis. Biotransformed delta4-androstene-3, 17-dione (delta 4A), dehydroepiandrosterone, 11-ketotestosterone (11-KT), and testosterone (T) were detected in all seal testicular incubates. Yields of 11-KT were greatly increased in the presence of Aroclor 1254. All contaminants except As and Se stimulated the in vitro biosyntheses of T, with the greatest increase in production of T being in the Cd-treated tissue. Cortisol (F), corticosterone (B), aldosterone (ALDO) but no cortisone (E), were biosynthesized by the seal adrenal tissue. Corticosterone (B) was the principal transformation product in all incubations with less B produced by the treated adrenals than by the control. The lowest yeild of B was achieved by the Se-treated adrenal. The yeild of ALDO was also lower in all contaminant treated incubations, with Se and Cd giving the greatest inhibition. More F was biosynthesized by all the treated adrenals than by the control. The greatest increase of production of F(6-fold) from progesterone was by the As-treated adrenal.

Metabolism of progesterone in uterine tissue of non-pregnant rats in vitro.

The metabolism of labelled progesterone was studied in vitro in uterine tissue of non-pregnant rats with particular emphasis on the influence of substrate concentration. Neither a qualitative nor quantitative difference was found for a steroid tissue ratio between 15 X 10(-6) and 4.2 X 10(-9) to 1 g (substrate amounts between 57.73 and 0.02 nmol); with both concentrations 42 to 44 per cent of progesterone was metabolized to about 35 per cent monohydroxymonoketonic steroids and 4-6 per cent dihydroxylated C21O2-compounds. In both sets of incubations we have isolated and identified the following steroids: 3alpha-hydroxy-5alpha-pregnan-20-one, 3beta-hydroxy-4-pregnen-20-one, 3alpha-hydroxy-4-pregnen-20-one, 20alpha-hydroxy-4-pregnen-3-one, 5alpha-pregnane-3alpha,20alpha-diol and 4-pregnene-3alpha,20alpha-diol. The most abundant metabolite formed in these incubations was 3alpha-hydroxy-4-pregnen-20-one which corresponds to about 30 per cent of the total activity recovered. It is the first time that the presence of 20alpha-hydroxysteroid-oxidoreductase activity is definitely established in this type of tissue. The identification of three allylic alcohols as progesterone metabolites in the rat uterus confirms that delta4-3-hydroxysteroids are important intermediates in the in vitro uterine metabolism of steroids.

Metabolism of 20beta-hydroxy-4-pregnen-3-one in uterine tissue of non-pregnant rats in vitro.

In vitro experiments carried out with uterus preparations of ovariectomized adult rats indicate the presence in this tissue of a 20beta-hydroxy-steroid-oxidoreductase which catalyzes the conversion of 20beta-hydroxy-4-pregnen-3-one to progesterone. Since a hepatic 20beta-hydroxysteroid-oxidoreductase is absent in adult female rats, the myometrial enzyme can be responsible for the biological activity of 20beta-hydroxy-4-pregnen-3-one in these animals. Besides progesterone five metabolites were isolated and identified after incubation of [4-14C]20beta-hydroxy-4-pregnen-3-one with uterine tissue: 20beta-hydroxy-5alpha-pregnan-3-one, 20beta-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3alpha,20beta-diol, 4-pregnene-3alpha,20beta-diol and 4-pregnene-3beta,20beta-diol. The conversion of 20beta-hydroxy-4-pregnen-3-one to progesterone permits us to regard all five steroids isolated as progesterone metabolites in the rat uterus. 20beta-hydroxy-5beta-pregnan-3-one is the first C21-metabolite with a 5beta(H)-configuration isolated in the rat uterus, which indicates the presence of 5beta-reductase in this tissue.

Ovarian enzymatic divergence in patients with polycystic ovary syndrome excreting urinary pregnanetriolone.

When ovarian mitochondria from patients with polycystic ovary syndrome (POS) were incubated with [7-3H]17alpha-hydroxypregnenolone and [4-14C]-17alpha-hydroxyprogesterone, 11beta-hydroxylated metabolites were obtained. The mitochondria, prepared from pooled, frozen, polycystic ovarian tissue of 5 patients, converted [7-3H]17alpha-hydroxypregnenolone to 3beta, 11beta, 17alpha--trihydroxy-5-pregnen-20-one (yield 0.065%) and to 3beta, 17alpha-dihydroxy-5-pregnene-11,20-dione (0.22%), while [4-14C]17alpha-hydroxyprogesterone was converted to 21-deoxycortisol (0.1%). Incubation of mitochondria, prepared from 4 pooled samples of frozen, normal ovarian tissue, yielded no evidence of 11beta-hydroxylation of either of the substrates. Mitochondria obtained from fresh, polycystic ovarian tissue of a single patient with POS converted [7-3H]17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (2.1%) and [4-14C]17alpha-hydroxyprogesterone to 21-deoxycortisol (0.1%). When the same mitochondrial preparation was incubated simultaneously with [7-3H]17alpha-hydroxypregnenolone and [4-14C]11-deoxycortisol, it converted 17alpha-hydroxypregnenolone to 3beta,17alpha-dihydroxy-5-pregnene-11,20-dione (1.9%), but no 11beta-hydroxylated derivatives of 11-deoxycortisol were found. These results demonstrate that ovaries of patients with POS contain an 11beta-hydroxylase active towards C-21-deoxysteroids but inert to C-21-hydroxysteroids such as 11-deoxycortisol.

Considerations of delta 5-pregnenediol formation in ovarian tissue.

20Alpha-Hydroxypregn-4-ene-3-one (20alpha-DHP) in ovarian tissue could be formed directly from progesterone (Pathway 1) or from pregnenolone via a delta 5-intermediate, pregn-5-ene-3beta, 20alpha-diol (Pathway 2). The participation of Pathway 2 is demonstrated to the extent that pregnenediol can be formed from pregnenolone and that the delta 5-diol, in turn, can be a precursor to 20alpha-DH P in vitro, using ovarian tissue from cycling rats by both flask incubation and superfusion techniques. Pathway 2 would allow greater flexibility and local regulation with respect to potential steroid sulfate conjugates of delta 5-3beta-OH intermediates. The existence of two pathways leading to 20alpha-DH P could explain the occurrance of variable levels of this steroid without a necessary direct or inverse relationship to progesterone secretion levels at different stages of the estrous cycle.

Some biochemical aspects of the enzymic transformation of cortisol with Bacillus cereus.

The role of a variety of compounds including organic acids, vitamins, growth promoting substances, purines and pyrimidines in the bioconversion of cortisol with Bacillus cereus was investigated. The transformation of cortisol to prednisolone and pregn-4-en-11beta, 17alpha,20beta, 21-tetrol-3-one was affected by these compounds in different manners. The enzymatic delta1-dehydrogenation reaction was greatly induced with fumarate, menadione, and xanthine treatments. However, the enzymic reduction of the 20-carbonyl to the 20beta-ol was specifically stimulated with fumarate, nicotinic acid amide, and uracil treatments.