Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Biotransformation of progesterone to 14 alpha-hydroxypregna-1,4-diene-3,20-dione, a novel fungal metabolite, by Colletotrichum antirrhini

The fermentation of progesterone by Colletotrichum antirrhini SC 2144 was examined. Instead of 15 alpha-hydroxyprogesterone, the reported product, this fungus converted progesterone to androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 14 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 11 alpha-hydroxypregn-4-ene-3,20-dione, 14 alpha-hydroxypregn-4-ene-3,20-dione, and a hitherto undescribed compound, 14 alpha-hydroxypregna-1,4-diene-3,20-dione.     

Studies on an extract of rat testicular microsomal fraction that catalyses the transformation of progesterone into 17-hydroxyprogesterone and androgens

The supernatant obtained by centrifugation of Triton N-101-treated freeze-dried rat testicular microsomal fraction at 105000g(av.) for 2h transformed progesterone into testosterone via 17-hydroxypregn-4-ene-3,20-dione and androst-4-ene-3,17-dione. Hydroxylation at C-17 of 3beta-hydroxypregn-5-en-20-one and deoxycorticosterone was not observed. Non-haem iron protein, cytochrome P-450 and material with NADPH dehydrogenase activity were precipitated by 40% saturation of the supernatant with ammonium sulphate; however, it was not possible to establish the participation of these substances in the 17alpha-hydroxylase and side-chain-cleavage activities also present in the precipitate. The results of gel-filtration chromatography indicated that the Triton N-101 extract consisted primarily of a suspension of small particles of microsomes and that the progesterone 17-hydroxylase and the 17-hydroxypregn-4-ene-3,20-dione side-chain-cleavage enzyme were not in true solution.     

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.     

Large-Scale Transformation of Steroids by Fungal Spores

Spores of Aspergillus ochraceus and Septomyxa affinis were produced on a large scale by surface sporulation on moist wheat bran and barley. 11alpha-Hydroxylation of progesterone and Reichstein's compound S by spores of A. ochraceus and 1-dehydrogenation of compound S by spores of S. affinis were carried out in 5-liter fermentors. It was shown that, above a certain minimum, increase in aeration and agitation did not significantly affect steroid conversion. The industrial feasibility of the spore process was further demonstrated by 11alpha-hydroxylation of 6alpha-fluoro-16alpha,17alpha-dihydroxypregn-4-ene-3,20-dione in a modified 200-gal stainless-steel vessel with spores of A. ochraceus. Strict aseptic conditions are not necessary, either during harvesting of spores or during steroid transformation.     

Progesterone fate in rabbit cornea

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.     

Isolation,identification and quantitation of serum 5α-pregnane-3,20-dione and its relationship to progesterone in the pregnant mare

5α-pregnane-3,20-dione was isolated from pooled pregnant mare serum using Sephadex LH-20 column chromatography and identified by the use of radioimmunoassay, gas-liquid chromatography and gas-liquid chromatography-mass spectrometry analyses. 5β-pregnane-3,20-dione was not cross-reactive with the radioimmunoassay system and was not detected by gas-liquid chromatography. Peripheral blood levels of progesterone and 5α-pregnane-3,20-dione were determined by radioimmunoassay in four Quarter Horse mares for the first 150 days of gestation. Progesterone and 5α-pregnane-3,20-dione declined from a range of 6 to 15 ng/ml at day 10 to a range of 2 to 8 ng/ml at day 40. After day 40 there was an increase in progesterone and 5α-pregnane-3,20-dione concentration. Toward the end of 150 days of gestation progesterone tended to decline whereas, 5α-pregnane-3,20-dione levels were maintained or increased to levels as high as 35 ng/ml. Neither source nor function of 5α-pregnane-3,20-dione is known.     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors

The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5α-reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor.The 3,20-dioxopregna-4-ene-17α-yl acetate 4 containing an acetoxy group in C-17 and steroid 17α-hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes. On the other hand, 17α-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited only the type 1 enzyme. Steroid 4-chloro-17α-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at C-4 respectively, inhibited both types of 5α-reductase. These results indicate that an increase in the electronegativity of ring A produces a major inhibitory activity for 5α-reductase type 1; however this increase was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds 3,20-dioxo-4-chloropregn-4-ene-17α yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17α yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5α-reductase type 2 enzyme.Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however the IC₅₀ values of these compounds were higher as compared to that of finasteride.These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas compounds 6, 8a and 8b failed to do so.The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.     

Induction of C-reactive protein by cytokines in human hepatoma cell lines is potentiated by caffeine.

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum.     

Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

ORG-2058 as a ligand in the assay of progesterone receptor in breast cancer

Tritiated [(16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione)-6,7-3H] (ORG-2058) and 17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020) were compared as ligands in the assay of progesterone receptor in human and rat breast tumors. We found that ORG-2058 is a better ligand because of its low nonspecific binding. Most of the nonspecific binding of the other ligand R5020, is to proteins which bind corticosteroids. In cancerous tissue ORG-2058 binds to progesterone receptor linearly in a range of protein concentrations which are normally used in the receptor assay. On the other hand, R5020 exhibits binding linearity over a narrower protein concentration in many tumor biopsies, which may cause severe limitation in the assay procedure or frequent underestimation of receptor content.     

Steroidogenic correlates of pregnancy in the rock hyrax (Procavia capensis)

In pregnant rock hyraxes isolated leucocytes metabolise both [3H]pregnenolone and [3H]progesterone while whole blood, erythrocytes and an erythrocyte/leucocyte mixture only metabolised [3H]progesterone. Plasma displayed no tendency to metabolically convert any one of these two steroids. In whole blood [3H]progesterone appears to be converted to 5alpha-pregnane-3,20-dione and a compound with chromatographic properties similar to that of 5alpha-pregnan-3alpha-ol-20-one. 5Alpha-pregnane-3,20-dione exhibited a high relative binding affinity for the uterine progesterone eceptor (94%), but 5alpha-pregnan-3alpha-ol-20-one displayed very little affinity for the same receptor (0.4%). 5Alpha-pregnane-3,20-dione may therefore aid in the maintenance of pregnancy. Corpora lutea metabolised progesterone to 17alpha-hydroxyprogesterone, a compound exhibiting no progestational function because of its low relative binding affinity for the uterine progesterone receptor (2%). Progesterone appears to be the main product of the corpus luteum. However, 5alpha-pregnane-3,20-dione circulated at concentrations approximately 8.5 times higher than progesterone, probably due to the metabolic conversion of progesterone to 5alpha-pregnane-3,20-dione by the blood. We conclude that in the hyrax progesterone, produced by the corpora lutea, enters the circulation, where it is reduced to 5alpha-pregnanes. 5Alpha-pregane-3,20-dione may then be transported to the uterus where it binds to the progesterone receptor to assist in the maintenance of pregnancy. This mechanism appears to be analogous to that of the African elephant which is phylogenetically related to the hyrax, except that in the elephant the 5alpha-reduced metabolites are produced by luteal tissue and not the blood.     

A rapid approach to 11 alpha-hemisuccinylprogesterone synthesis

The synthetic hapten 11 alpha-hemisuccinylprogesterone (11 alpha-hemisuccinyl-pregn-4-ene-3,20-dione), when linked to the appropriate macromolecular carrier, has been used successfully as a solid-phase antigen for progesterone detection in immunoassay. In this study the synthesis of 11 alpha-hemisuccinylprogesterone from 11 alpha-hydroxyprogesterone has been improved by using 4-dimethylaminopyridine (DMAP in refluxing dioxane, a highly nucleophilic polar solvent.     

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6?nM, respectively.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

中国南海侧扁软柳珊瑚中孕甾烷类化学成分的研究(英文)

从南海侧扁软柳珊瑚Subergorgia suberosa的二氯甲烷-甲醇提取物中首次分离鉴定了8个孕甾烷类化合物,经波谱鉴定为3β-O-palmitoyl-pregn-5-ene-20-one-3-ol (1),3β-O-palmitoyl-5α-pregn-20-one-3-ol (2),5α-pregn-1-ene-3,20-dione (3),3β,5α-pregn-20-one-3-ol (4),3β-pregn-5-ene-20-one-3-ol (5),3β,5β-pregn-20-one-3-ol (6),5β-pregn-3,20-dione (7),pregn-4-ene-3,20-dione (8).其中化合物1,2为新化合物.     

Properties of 5-ene-3β-hydroxysteroid dehydrogenase in granulosa tissue from the domestic fowl (Gallus domesticus)

Microsomal 5-ene-3β-hydroxysteroid dehydrogenase activity from fowl preovulatory granulosa tissue was solubilised by treatment with 2.0 M KCl. The efficiency of the solubilisation procedure was assessed by measuring the amount of activity remaining in the supernatant after centrifugation at 105,000 g for 8 h. The microsomal activity was resolved into two components: a fraction solubilised by the salt treatment and a fraction remaining membrane bound. The results indicate that the enzyme exists in two different environments within the membrane. The time course for the thermal inactivation of the microsomal enzyme supported this conclusion. The properties of the solubilised enzyme were compared with the untreated microsomal preparation, with respect to steroidal inhibition. Progesterone. 4-androstene-3,17-dione and oestradiol-17β all inhibited the enzyme and no difference was observed between the two preparations. The results are discussed in relation to the involvement of 5-ene-3β-hydroxysteroid dehydrogenase in the control of progesterone production in fowl granulosa tissue.