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.     

Easy stereoselective synthesis of 5α-estrane-3β,17α-diol, the major metabolite of nandrolone in the horse

5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α-diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance.     

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.     

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.     

Quantification of urinary 3α,21-dihydroxy-5β-pregnan-20-one and 5-pregnene-3β, 20α-diol by mass fragmentography

A sensitive and accurate method is described for measuring urinary corticosteroids by gas chromatography-mass spectroscopy (GC-MS). Using single peak monitoring (mass fragmentography) and electron impact ionization, the acetates of 3α,21-dihydroxy-5β-pregnan-20-one (tetrahydrodeoxycorticoster-one) and 5-pregnene-3β,20α-diol were estimated with deuterio-acetate carriers as recovery markers. With this technique, the coefficient of variation did not exceed 3% for GC-MS analyses of the urinary corticosteroid samples by single peak monitoring. An evaluation of the trimethylsilyl ether derivatives of the two steroids by chemical ionization was also made. Secretion rates determined for deoxycorticos-terone derived from specific activities of urinary tetrahydrodeoxycorticosterone and excretion levels of 5-pregnene-3β,20α-diol were slightly lower than those obtained by other methods.     

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).     

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.     

Measurement of 5α-androstane-3α,17β-diol in human spermatic venous plasma by mass-fragmentography

Five alpha-androstane-3α,17β-diol (3α-diol) an active metabolite of testosterone (T) was measured in the spermatic and peripheral venous blood of 6 normal males using mass-fragmentography. Using this method 3α-diol was clearly separated from the following isomers: 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. The mean concentrations (±SE) of 3α-diol in spermatic and peripheral venous blood were respectively 100 ± 38 ng/100 ml and 7.7 ± 1.9 ng/100 ml. The existence of a significant (P < 0.01) gradient between spermatic and peripheral vein clearly demonstrates that the human testis secretes 3α-diol.     

5α-androstane-3β,17β-diol in feedback between gonads and hypothalamo-pituitary system of male rats

Serum level of LH and levels of LH-RH in the arcuate nucleus and in the median eminence of hypothalamus were measured by radioimmunoassay in castrated male rats 1, 3, 6, 12, 24 h after single administration of 25 μg/100 g b.w. testosterone, 5α-androstane-3α,17β-diol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol).The levels were then measured 6 h after single administration of 3α-diol and 3β-diol in different doses: 5 μg; 25 μg; 100 μg; 250 μg; 2500 μg/100 g b.w.The results suggest basic differences between the influence of testosterone and of its 5α-reduced metabolites upon the hypothalamo-pituitary system as to speed and strength of effect. Both 3α-diol and 3β-diol are active in the negative feedback between the gonads and the hypothalamo-pituitary system, but their effect depends on the dose. 3β-Diol is active in the low doses we have studied as opposed to its 3α-epimer.     

Incorporation of 5α-furostan-3β,26-diol into tigogenin by digitalis lanata

2,2′,4,4′-³H₄-dihydrotigogenin was converted by Digitalis lanata plants into tigogenin.     

Degradation of 5-pregnene-3β, 20β-diol by a Nocardia sp.

Summary With growing cells of a Nocardia sp., isolated from soil, the degradation of 5-pregnene-3, 20-diol into 3-[5-oxo-7a-methyl-1 (1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid was investigated. The results show that iron is essential for production of the perhydroindanpropionic acid, that this production is greatly enhanced by the presence of calcium and that it is maximal in the pH range 7.0–7.5.Abbreviations used in the text PD 5-pregnene-3, 20-diol (pregnendiol) - PDSA 3-[5-oxo-7a-methyl-1(1-hydroxo)-ethyl-3a-perhydroindane-4]-propionic acid (pregnendiol-secoacid) - PSA 3-[5-oxo-7a-methyl-1-acetyl-3a-perhydroindane-4]-propionic acid (progesterone-secoacid) - EDTA Ethylendiamintetracetic acid - DMSO Dimethylsulfoxide     

Biosynthesis of 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol in Datura

Datura meteloides; plants were fed with tiglic acid-[-¹⁴C] via the roots and after 2 days the percentage incorporation into the alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, meteloidine and 3α,6β-ditigloyloxytropan-7β-ol were 15·2, 1·82, 2·2 and 1·8 respectively. 3α,6β-Ditigloyloxytropane was partially hydrolysed to 6β-hydroxy-3α-tigloyloxytropane which contained 58·1% of the radioactivity of the original base, whereas 3α,6β-ditigloyloxytropan-7β-ol gave meteloidine containing only 9·2% of the original activity. The results suggest that the di- and tri-hydroxytropanes may be formed by different routes.     

Concentrations of unconjugated 5α-androstan-3β,17β-diol in human peripheral plasma as measured by radioimmunoassay

5α-androstane-3β,17β-diol was measured in human peripheral plasma using a specific antibody generated against a carboxymethyloxime BSA conjugate linked at position 7. Concentrations were significantly higher in normal men than women. Preliminary results suggest that plasma 5α-androstane-3β,17β-diol concentrations might be a useful clinical parameter in cases of hirsutism and male infertility.     

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.     

(+)-Eudesm-3-ene-6β,7α-diol from the liverwort Lepidozia reptans

The structure of a new sesquiterpene diol from the liverwort Lepidozia reptans has been established as eudesm-3-ene-6β,7α-diol on the basis of its ¹H and ¹³CNMR spectroscopic properties.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

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.     

Differential metabolism of androst-5-ene-3β,17β-diol between rats, canines, monkeys and humans

The potent anti-inflammatory activity of exogenous dehydroepiandrosterone (DHEA) in rodents has not translated to humans. This disparity in pharmacological effects has been attributed to factors such as differences in expression and function of molecular targets and differential metabolism. Hepatocytes from rats, dogs, monkeys, and humans were used to measure species-specific metabolism of a related compound, androst-5-ene-3β,17β-diol (5-AED) using reversed-phase radio-HPLC, to explore the metabolic contribution to this interspecies disparity. We found that rat hepatocytes transformed 5-AED predominantly into an array of highly oxidized metabolites. Canine metabolites overlapped with rat, but contained a greater abundance of less hydrophilic species. Monkey and human metabolites were strikingly less hydrophilic, dominated by 5-AED and DHEA conjugates. From the accumulating evidence indicating that the DHEA anti-inflammatory activity may actually reside in its more highly oxidized metabolites, we advance a hypothesis that the virtual absence of these metabolites in humans is central to the failure of exogenous DHEA to produce a potent pharmacological effect in clinical investigations. Accordingly, emulation of its anti-inflammatory activity in humans will require administration of an active native metabolite or a synthetic pharmaceutical derivative.     

The roles of pregn-5-ene-3β,20α-diol and 20α-hydroxy steroid dehydrogenase in the control of progesterone synthesis preceding parturition and lactogenesis in the rat

1. The activity of 20α-hydroxy steroid dehydrogenase in rat ovarian corpora lutea increased at least 50-fold between 2 days before and 2 days after parturition, and then fell gradually during lactation. The activity of 3β-hydroxy Δ⁵-steroid dehydrogenase decreased by 50% at parturition but remained constant at other times. 2. The 20α-hydroxypregn-4-en-3-one/progesterone concentration ratio in the ovary fell tenfold between 1 day before and 1 day after parturition, in contrast with the increase of the ratio for these steroids in plasma. 3. Pregnenolone was metabolized in intact cells or cell-free systems either to pregn-5-ene-3β,20α-diol and then to 20α-hydroxypregn-4-en-3-one by 20α-hydroxy steroid dehydrogenase and 3β-hydroxy Δ⁵-steroid dehydrogenase respectively, or directly to progesterone by the latter enzyme. The relative activities of these pathways appeared to reflect the relative amounts of the two enzymes and the concentrations of their respective coenzymes NADPH and NAD⁺. 4. From these and other observations it was concluded that the cessation of progesterone secretion, which precedes parturition and lactogenesis at the end of pregnancy, is partly due to the redirected metabolism of pregnenolone away from progesterone and towards 20α-hydroxypregn-4-en-3-one as the secreted end product. This is primarily the consequence of the sharp increase in the activity of 20α-hydroxy steroid dehydrogenase. This mechanism is super-imposed on the already declining rate of net Δ⁴-steroid release by the ovary. 5. A relationship of these pathways to subcellular compartments of luteal cells is proposed.     

Sterol synthesis. Synthesis of 15-oxygenated 5α, 14β-cholest-7-en-3β-ol derivatives

Treatment of 3β-benzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3β-benzoyloxy-5α, 14β-cholest-7-en-15-one with the unnatural C ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15α, 14β-cholest-7-en-3β, 15α-diol and 5α, 14β-cholest-7-en-3β, 15β-diol in 9% and 81% yields, respectively.