Urinary excretion of 5β-pregnane-3α,6α,20α-triol in human gestation

The 5β-pregnane-3α,6α,20α-iriol and 5β-pregnane-3α,6α,20β-triol obtained by reduction of 3α,6α-dihydroxy-5β-pregnan-20-one were converted to trimethylsilyl ether derivatives and analysed by gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS).After extraction, solvolysis and enzymatic hydrolysis of urinary steroid conjugates (from 19 normal pregnant women), the liberated steroids were separated by liquid-gel chromatography and were analysed by GLC and GLC-MS. The 5β-pregnane-3α,6α,20α-triol isolated and identified in the Sephadex LH-20 fractions 8 and 9, was present in urine from 15 pregnant women after the 16th week of gestation. After this time, this metabolite was found in a quantity between 0.20 to 2.90 mg/24 h, with a significant increase between the 26th to 30th week of the gestation.With the present in vivo data, it is not possible to establish the exact enzymatic pathway involved in the biosynthesis of the 5β-pregnane-3α,6α,20α-triol. However, it is probable that the immediate precursor of this compound was the 3α,6α-dihydroxy-5β-pregnan-20-one, and that urinary excretion of 5β-pregnane-3α,6α,20α-triol reflected one part of hepatomaternal metabolism of 6-hydroxyprogesterone formed in the foeto-placental unit.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

C-19-steroid 7α-hydroxylation by rat testes. isolation and identification of: 7α, 17β-dihydroxy-5α-androstan-3-one, 5α-androstan-3α,7α,17β-triol and 5α-androstane-3β,7α, 17β-triol

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7α-hydroxylated metabolites could be isolated and identified: 7α,17β-dihydroxy-4-androsten-3-one (7α-hydroxy-testosterone), 7α-17β-dihydroxy-5α-androstan-3-one (7α-hydroxy-Dht), 5α-androstan-3α,7α,17β-triol (7α-hydroxy-3α-A'DIOL) and 5α-androstane-3β,7α,l7β-triol (7α-hydroxy-3β-A'DIOL). To our knowledge this is the first demonstration of the formation of 5α-reduced-7α-hydroxylated metabolites of testosterone in the male gonad. These 5α-reduced-7α-hydroxylated metabolites could also be isolated after incubations of 5α-androstane-3α,17β-diol (3α-A'D10L) with testicular homogenates in the presence of a NADPH-generating system.Measured as the sum of 7α-hydroxy-testosterone, 7α-hydroxy-Dht. 7α-hydroxy-3α-A'DIOL and 7α-hydroxy-3β-A'DIOL formed using testosterone as substrate, total 7α-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3α-A'DIOL as substrate total 7α-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.     

Metabolism of 3α, 7α-dihydrexy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyi-5β-cholanoic acid in hamsters

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Synthesis of 3α,7α-dihydroxy-5β-androstan-17-one

A short and efficient method for the stereospecific synthesis of 3α,7α-dihydroxy-5β-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.     

Isolated Sertoli cells from immature rats produce 20α-hydroxy-pregn-4-en-3-one from progesterone and 3β,20α-dihydroxy-5α-pregnane from pregnenolone

Sertoli cells from 17 day old rats convert progesterone to 20α-hydroxy-pregn-4-en-3-one and pregnenolone to 3β,20α-dihydroxy-5α-pregnane after 72 hours $\text{in vitro}$. The metabolites were identified by several systems of thin layer and gas chromatography, derivative formation and crystallization with authentic steroids. The production of 20α-hydroxy-pregn-4-en-3-one and 3β,20α-dihydroxy-5α-pregnane amounted to 1380 and 740 pmoles/h/mg protein which can account for the total amounts of these steroids reported in the testis. It is the first direct evidence that Sertoli cells can metabolize progesterone and pregnenolone and suggests that Sertoli cells contain the major, if not the only, amounts of 20α-hydroxysteroid dehydrogenase in the immature rat testis.     

5α-Estrane-3β,17β-diol and 5β-estrane-3α,17β-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.     

Synthesis of 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their cytotoxic activities

A series of novel 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives were synthesized and characterized by NMR, HRMS. The pregnenolone (1) was first biotransformed by Mucor circinelloides var lusitanicus to 3β, 7α, 11α-trihydroxy-pregn-5-en-20-one (3), then 3 was treated with various benzaldehydes to produce 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives. These derivatives showed remarkable activity against EC109 cells. The absolute configuration of 3 was also confirmed by signal-crystal X-ray analysis.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).     

A simple synthesis of 3β-acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene

3β-Acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R, 23R)-sitost-5-ene-3β,22,23-triol in 66% overall yields.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

Synthesis of new steroid haptens for radioimmunoassay. Part III. 15β-carboxyethylmercaptosteroid-bovine serum albumin conjugates. Specific antisera for radioimmunoassay of 5α-dihydrotestosterone, 5α-androstane-3β, 17β-diol and 5α-androstane-3α, 17β-diol

The syntheses of 15β-carboxyethylmercapto-5α-dihydrotestosterone, 15β-carboxy-ethylmercapto-5α-androstane-3β, 17β-diol and 15β-carboxyethylmercapto-5α-androstane-3α, 17β-diol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of 5α-dihydrotestosterone (5α-DHT), 5α-androstane-3β, 17β-diol (3β3-diol) and 5α-androstane-3α, 17β-diol (3α-diol).     

3β,20α-羟基甾体脱氢酶的动力学性质

3β,20α-羟基甾体脱氢酶(3β,20α-Hydroxysteroid dehydrogenase,3β,20α-HSD)是从胎羊血中分离得到的。分子量为35kD。该酶以NADPH为辅酶,有两种底物。以孕酮为底物时,Km=30.8μmol/L,Vmax=0.7nmol min~(-1)(nmol enzyme)~(-1);以5α-二氢睾酮(5α-Dihydrotestosterone,5α-DHT)为底物时,Km=74μmol/L,Vmax=1.3nmol min~(-1)(nmol enzyme)~(-1)。5α-DHT竞争性抑制20α-还原活性,Ki=102μmol/L。16α-溴代乙酰氧基(16α-Bromo acetoxyprogesterone,16α-BAP)是3β,20α-HSD不可逆竞争性抑制剂,t_(1/2)=75min。对3β和20α还原活性的抑制常数Ki分别为23μmol/L和58μmol/L。