17-Deoxygenation: a new pathway of cortisol metabolism. Isolation of 17-deoxycortolonic acids.

The existence of a 17-deoxylation pathway in the metabolism of cortisol has been established by isolation of two 17-deoxy metabolites of cortisol. They have been identified as the 20alpha and 20beta-hydroxy isomers of 3alpha,20-dihydroxy, 11-oxo-5beta-pregnan-21-oic acids by comparison of the nuclear magnetic resonance spectra (nmr) and high resolution mass spectra of their methyl esters with authentic samples and confirmed by reverse isotope dilution.

Placental production of 5 beta-pregnane-3 alpha,20 alpha-diol in goats.

A major product of progesterone metabolism by the goat placenta in vitro was found to be 5 beta-pregnane-3 alpha,20 alpha-diol. The concentration of this steroid has been measured by radio-immunoassay in the peripheral circulation during pregnancy. Peripheral plasma concentrations of 5 beta-pregnane-3 alpha,20 alpha-diol were low (less than 6 nmol/l) in anoestrous and nonpregnant ovariectomized goats, and during the first month of pregnancy but increased progressively after day 45 of pregnancy, reaching 78-94 nmol/1 between days 112 and 142. Thereafter levels declined before term. Changes in the plasma concentration of 5 beta-pregnane-3 alpha,20 alpha-diol during pregnancy in the goat therefore resembled those of progesterone in the sheep. Plasma concentrations of 5 beta-pregnane-3 alpha,20 alpha-diol between day 70 and term were not influenced by repeated administration of medroxyprogesterone acetate from days 51 to 82 or by lutectomy in goats treated with medroxyprogesterone acetate. Secretion of 5 beta-pregnane-3 alpha,20 alpha-diol by the uterus and its contents was indicated by a positive venous-arterial difference across the uterus between days 128 and 141 in three ovariectomized pregnant goats receiving medroxyprogesterone acetate. Comparison of the rates of metabolism of progesterone by homogenates of placenta in vitro showed that the placental tissue from goats was three time more active in this respect than was tissue from sheep. The ratio of the plasma concentrations of 5 beta-pregnane-3 alpha,20 alpha-diol and progesterone in late pregnancy in ovariectomized or lutectomized goats exceeded by a factor of 10 that in sheep at a comparable stage of gestation. It is suggested that reductive metabolism of progesterone before it is secreted may account for the inability of the placenta to maintain pregnancy after ovariectomy in goats.

Progesterone and 5 beta-pregnanediol production by isolated fetal placental binucleate cells from sheep and goats.

Enzymic dispersion and density gradient separation were used for the isolation of enriched populations (60-90%) of cells from the corpus luteum, placenta and peripheral blood of pregnant sheep and goats. Analysis of the steroids produced from radioactive pregnenolone demonstrated that placental binucleate cells can produce progesterone and 5 beta-pregnanediol whereas white blood cells were relatively inactive. Thus, sheep binucleate cells converted pregnenolone predominantly to progesterone as did sheep luteal cells. However, goat binucleate cells produced 5 beta-pregnanediol as the major metabolite, which is consistent with its production in vivo during pregnancy. Production of progesterone (sheep) or 5 beta-pregnanediol (goat) by binucleate cells was shown to be proportional to the number and viability of the cells. In contrast with the binucleate cells there was no evidence that trophectodermal uninucleate cells play a significant role in placental progesterone or 5 beta-pregnanediol synthesis in either species.

Sex differences in fetal sheep adrenal steroidogenesis.

The formation of several steroids was determined in vitro in adrenals removed from 18 female and six male fetuses of 113-115 days' gestation and in two female and two male fetuses at near term (137-143 days). Samples were incubated with 14C-acetate and the formation of labeled steroids was determined by two-dimensional paper chromatography. Protein and corticosterone concentrations were determined by chromophore absorption and acid hydrolysis fluorescence methods, respectively. Tissue corticosterone concentrations were significantly higher in female (0.145 +/- 0.010 microgram/mg protein) than in male (0.083 +/- 0.010 microgram/mg protein) adrenal tissue at both stages, whereas corticosterone formation was similar in both sexes. Cholesterol formation was significantly higher in female (0.103 +/- 0.079 muM/mg protein) than in male (0.044 +/- 0.011 muM/mg protein) adrenals at both stages. Both testosterone and estradiol were synthesized at higher rates in female than in male adrenals (52% and 33%, respectively), whereas pregnanediol formation was 21% higher in the male. These results indicate that significant sex differences exist in the formation of various adrenocortical hormones by fetal tissues. The relevance of these findings to better survival of female premature newborns from respiratory distress syndrome in contrast with male, is discussed.

Fetal adrenal steroidogenesis: drug effects in sheep.

The influence of ACTH (100 pg/ml), ethanol (10(-3)/M) and morphine (10(-6)/M) on the rate of formation of cholesterol and various steroids in female fetal adrenal tissue was studied in vitro. ACTH and ethanol had no effect on corticosterone formation, while morphine increased it significantly (0.586 +/- 0.049 to 0.799 +/- 0.027 microM/mg protein) (P less than 0.02). Cholesterol formation was increased significantly by ethanol (from 0.103 +/- 0.079 to 0.248 +/- 0.035 microM/mg protein) (P less than 0.01) but not by morphine. Both ethanol and morphine significantly decreased testosterone synthesis (from 0.079 +/- 0.043 to 0.019 +/- 0.002 and 0.006 +/- 0.003 microM/mg protein, respectively) (P less than .01, less than .001). Adrenocortical formation of 17-beta estradiol was similarly attentuated by both ethanol (from 0.372 +/- 0.056 to 0.948 +/- 0.024 microM/mg protein) and morphine (to 0.600 +/- 0.020 microM/mg protein). Adrenocortical pregnanediol formation was significantly decreased by both ethanol (50%; P less than .01) and morphine (36%; P less than .02). Thus, the effect of ethanol and morphine on testosterone, estradiol and pregnanediol was similar and consisted of suppression. The effects of these agents upon corticosterone and cholesterol formation were different. Ethanol, like ACTH, did not stimulate corticosterone formation, but such stimulation did occur with morphine. Ethanol, but not morphine, stimulated cholesterol formation. The data suggest that agents capable of placental transport affect the formation of adrenocortical hormones in the fetal adrenal.

Microbiological transformation of steroids. 1-Dehydrogenation of 17,21-dihydroxypregn-4-ene-3,20-dione with Glomerella cingulata.

The micro-organism Glomerella cingulata dehydrogenates 17,21-dihydroxypregn-4-ene-3,20-dione to 17,21-dihydroxypregna-1,4-diene-3,20-dione in high yield practically without by-products.

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.