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In order to determine the role of microfilaments in adrenal steroidogenesis, we have studied the effect of cytochalasin B, a microfilament-disrupting agent, on the kinetics of [3H] pregnenolone conversion to labelled metabolites by frog interrenal tissue in vitro. Cytochalasin B (5 x 10(-5)M) induced a 50 to 70% decrease in corticosterone, 18-hydroxycorticosterone and aldosterone biosynthesis while the formation of progesterone and 11-desoxycorticosterone was not affected. These results suggest that microfilaments interfere in the conversion of 11-desoxycorticosterone to corticosterone probably by controlling the movement of 11-desoxycorticosterone from the reticulum to the mitochondria.

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The obligate aerobe, Pseudomonas putida ATCC 31752, efficiently utilises bile acids as a source of carbon and energy for growth and maintenance. When aeration is considerably restricted, a consequence to the catabolism of the bile acids in a fermentor is an accumulation of certain steroidal catabolites. Evidence is presented to show that among these are hydroxy-9,10-seco-1,3,5 (10)-androstratriene-9, 17-diones and those from four of the common bile acids, cholic, chenodeoxycholic, hyodeoxycholic and deoxycholic acids have been isolated and their structures determined. The product of catabolism of hyodeoxycholic acid appears to exist in a hemi-acetal form which readily forms an acetal during isolation procedures. All but one of these are described for the first time.

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Trilostane is known as an inhibitor of 3 beta-hydroxysteroid dehydrogenase. Conflicting data published on this drug led us to look for the effects of 0.02 to 0.5 mM of trilostane on the in vitro steroid synthesis in sheep adrenals and human adrenals (Cushing's or Conn's syndrome) in the presence of an NADPH-generating system. The synthesis of 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from dehydroepiandrosterone or 4-androstenedione or 11 beta-hydroxyandrostenedione. The synthesis of 11-deoxycortisol, cortisol, cortisone, 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from 17-hydroxyprogesterone or 11-deoxycortisol or cortisol. This study showed that trilostane inhibited 3 beta-hydroxysteroid dehydrogenase whereas it had no effect on 21-, 11- and 17-hydroxylase. Trilostane was responsible for an increased 11 beta-hydroxysteroid dehydrogenase activity in vitro, resulting in low yields of cortisol and 11 beta-hydroxyandrostenedione, and high yields of cortisone and 11-ketoandrostenedione. This unexpected effect of trilostane allowed us to show that erroneous conclusions (in this case: pseudo inhibition of 11 beta-hydroxylase) can be drawn if all the metabolic pathways from a determined precursor are not exhaustively documented when studying the effects of drugs on steroid synthesis in vitro. The decrease of cortisol synthesis by trilostane may thus be related to the effects of the drug on both 3 beta-hydroxysteroid-dehydrogenase (inhibitory effect) and 11 beta-hydroxysteroid-dehydrogenase (stimulatory effect). This latter effect was found to be species-dependent.

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Preparations of dispersed human fetal adrenal cells from the inner third of the gland and from the subcapsular area were maintained in culture, and their ultrastructure and steroid production were studied. The former type of preparation contained only fetal zone cells, while the latter contained definitive zone cells together with varying numbers of fetal zone cells. Both types could be cultured with equal ease, but during short term culture, fetal and definitive zone cells became morphologically indistinguishable. The patterns of steroid production and, in particular, the relative production of delta 4,3-ketosteroids and delta 5,3 beta-hydroxysteroids were similar in both preparations, as were their dose-response relationships during incubation with alpha ACTH-(1-24). Although considerable variability in total steroid production was observed between cells from different adrenal glands, in no specimen was any evidence for functional zonation of the fetal adrenal cortex observed in vitro. The results suggest that the apparently unique histological appearance and function of the fetal adrenal cortex may only reflect intense stimulation by ACTH secondary to the combined influences of a rapid cortisol MCR and of some inhibitor of fetal adrenal 3 beta-hydroxysteroid dehydrogenase activity.

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[14-14C]16 alpha-Hydroxy-C-18- and C-19-steroid hormones were obtained in good yields by microbiological hydroxylation of correspondingly labelled steroids by Streptomyces roseochromogenes NRRL B-1233. Trace quantities of the labelled substrates were incubated on a rotary shaker (220 rpm) at 27 degrees C. The radioactive products were chromatographically separated, identified and the radiochemical purity was established by isotopic dilution analysis. The specific activities of 16 alpha-hydroxy-steroids obtained were assumed to be the same as those of the substrates, namely, 57.5 mCi/mmole for 16 alpha-hydroxy-4-androstene-3,17-dione, 57.5 mCi/mmole for 5-androstene-3 beta,16 alpha,17 beta-triol, 57.5 mCi/mmole for 16 alpha-hydroxy-dehydroepiandrosterone, 55.7 mCi/mmole for 16 alpha-hydroxy-estrone, and 57.5 mCi/mmole for 16 alpha-hydroxy-testosterone.

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The introduction of a 16 alpha-hydroxyl function into the steroid nucleus was studied in resting cells of Streptomyces roseochromogenes NRRL B-1233. The oxidation product of dehydroepiandrosterone (DHEA) was identified as 16 alpha-hydroxy DHEA by using thin-layer and gas-liquid chromatography. A linear relation between cell concentration and 16 alpha-OH-DHEA formation was observed. 16 alpha-Hydroxylase showed good activity at pH 8.0 for 16 alpha-OH-DHEA formation. The enzyme showed good activity at 3.1 x 10(-4) M DHEA. The oxidation products of pregnenolone, 4-androstene-3,17-dione, estrone, and 5-androstene-3 beta,17 beta-diol as well as of other substrates were identified as the 16 alpha-hydroxy steroid, respectively. The rates of microbial 16 alpha-hydroxylation were as follows: 76.9% for DHEA, 50.4% for pregnenolone, 43.9% for 4-androstene-3,17-dione, 34.3% for estrone, and 19.6% for 5-androstene-3 beta,17 beta-diol. The organism tested catalyzes 16 alpha-hydroxylation of a wide variety of steroids.

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The effects of oleate and hydroxycitrate on the rate of long-chain fatty acid and 3-beta-hydroxysterol synthesis were measured in perfused rat livers. Metabolite measurements show that in livers from fed animals inhibition of fatty acid synthesis by oleate or hydroxycitrate is associated with an increase in the tissue content of glucose 6-phosphate and fructose 6-phosphate, and a diminution in glycolytic intermediates from fructose diphosphate to phosphoenolpyruvate. Oleate also causes an increase in the tissue content of long-chain fatty acyl-CoA and citrate. The increase in long-chain fatty acyl-CoA is larger in livers from starved as compared to fed rats, while the increase in citrate is larger in livers from fed as compared to starved rats. However, the increase in the citrate content of livers from fed rats occurs in a range where it causes no further activation of acetyl-CoA carboxylase in vitro. Ketogenesis by livers from fed rats perfused without free fatty acids is strongly inhibited by hydroxycitrate. However, ketogenesis is not inhibited by hydroxycitrate when livers from starved rats are perfused with oleate, and ketogenesis is increased somewhat by hydroxycitrate when livers from fed rats are perfused with oleate. These results are interpreted in terms of an extramitochondrial pathway of ketogenesis which operates in carbohydrate-fed animals. The intramitochondrial pathway predominates in starved animals, or when the concentration of fatty acids is high, or both. Other interpretations, which cannot be ruled out at present, are also considered.

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A comparison of the activity of rabbit liver microsomes fortified with 0.1 mM NADPH to promote 12alpha-hydroxylation over 60 minutes with appropriate sterols provided the following relative order of activities: kalpha-cholestane-3alpha, 7alpha-diol, 100; 7alpha-hydroxy-5alpha-cholestane-3one, 76; 7alpha-hydroxycholest-4-en-3-one, 64; 5alpha-cholestane-3-3beta, 7alpha-diol, 26; allochendoexycholate, 22. If the more polar products formation. Investigation by gas-liquid chromatography-mass spectrometry of the nature of these more polar products derived form 5alpha-cholestane-3alpha, 7alpha-diol showed that a series of tetrols was dormed; i.e., 5alpha-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol as the major product and lesser amounts of 5alpha-cholestane-3alpha, 7alpha, 12alpha, 24- and 3alpha, 7alpha, 12alpha, 23tetrols. No significant amount of 26-hydroxylated product was formed.

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Ovarian homogenates from 10-150-day-old rats were incubated with [3H]progesterone and NADPH. Also, ovarian homogenates from 28-day-old rats were incubated for 5-180 min with either [14C]progesterone, [3H]5alpha-pregnane-3,20-dione or [14C]progesterone plus [3H]5alpha-pregnane-3,20-dione. Following incubation, radioactive metabolites were isolated, identified, and measured by column and paper chromatography, with derivative formation and recrystallizations to constant specific activity. Prepubertal ovaries (10, 20, and 28 days of age) converted 15-60% of progesterone to C21-17-hydroxysteroids and C19-steroids. At 40 and 150 days of age (postpubertal), the formation of these steroids decreased to less than 2%. At 10 and 150 days of age, the major C19-steroids formed from progesterone were androstenedione and testosterone. At 20 and 28 days of age, however, no accumulation of these C19-delta4-3ketosteroids was found (less than 0.1% of each), at which time the conversion of progesterone to 5alpha-reduced C19-steriods, such as androsterone and 5alpha-androstane-3alpha,17beta-diol, reached 30%. In ovaries of 28-day-old rats, the results from incubation studies for the detection of metabolic pathways indicated two biosynthetic pathways leading to 5alpha-reduced C19-steroids, one from progesterone via 5alpha-reduced C21 steroids, such as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one, and a second via 17-hydroxyprogesterone, androstenedione, and testosterone. It seems that the active 5alpha-reduction of C19-delta4-3-ketosteroids and the formation of 5alpha-reduced C19-steroids by the pathway through 5alpha-reduced C21-steroids, are present in the ovaries of older prepubertal rats and may be the biological significance.

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The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.

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The behavior of 20 beta-hydroxysteroid dehydrogenase in a two-phase system consisting of an emulsion of water with an immiscible organic solvent was investigated. The effect of several organic solvents on the stability, activity, and kinetic profile of the enzyme was considered. The most suitable system for carrying out the enzymatic reaction proved to be water-butyl acetate. The production of high quantitied of 20 beta-hydroxysteroids in 100% yield using catalytic amount of cofactor was achieved by coupling the 20 beta-hydroxysteroid dehydrogenase-and the alcohol dehydrogenase-catalyzed reactions.

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Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.

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