Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: endogenous steroids urinary profile after multiple oral doses

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography–mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration.The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

In vitro conversion of testosterone-4-14C to androgens of the 5alpha-androstane series by a normal human ovary

In a previous communication (1) the identification of Δ⁴ -3-oxo-steroids and estrogens as metabolites of testosterone-4-¹⁴C incubated with normal post-ovulatory human ovaries was reported. Thin-layer chromatography of the extracts of those ovaries which contained no corpus luteum yielded zones of radioactivity which were not associated with any of these products. Detailed investigation of these zones from the extract of one of these glands resulted in identification of the following radiometabolites of the 5α-androstane series: 5α-androstane-3,17-dione, androsterone, 3β-hydroxy-5α-androstan-17-one, 17β-hydroxy-5α-androstan-3-one, 5α-androstane-3ga, 17β-diol and 5α-androstane-3β, 17β-diol. The capacity of a normal human ovary to produce these 5α-reduced androgens, especially the potent 17β-hydroxy-steroids, suggests a regulatory role of these compounds in ovarian function.     

Proton magnetic resonance spectra of 17ξ-Hydroxy-17ξ-methyl-5ξ-androstane C-3 ketone and c-3ξ alcohol isomers in chloroform-d and pyridine-d5

17α-Hydroxy-17β-methyl-5β-androstan-3-one, 17μ-methyl-5α-androstane-3α, 17α-diol, 17β-methyl-5α-androstane-3β, 17α-diol, 17α-methyl-5β-androstane-3β, 17β-diol, 17β-methyl-5β-androstane-3α, 17α-diol and 17β-methy1–5β-androstane-3β, 17α-diol were synthesized for the first time. ¹H NMR spectra of all four 17ξ-hydroxy/17ξ-methyl C-3 ketones and all eight C-3 alcohols were recorded in chloroform-d and pyridine-d₅. Pyridine-induced chemical shifts are discussed. Thin-layer Chromatographic data are given.     

Steroid metabolism by mouse submaxillary glands. I. in vitro metabolism of testosterone and 4-androstene-3,17-dione

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α, 17β-diol. Testosterone was converted primarily to 5α-androstane-3α, 17β-diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.     

Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS)

Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5α-androstan-3β-ol acetate (5α-A-AC), 5α-androstan-3α-ol-17-one acetate (androsterone acetate, A-AC), 5β-androstan-3α-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5α-cholestane (Cne). CU/USADA 34-1 contains 5β-androstan-3α-ol-17-one (etiocholanolone, E), 5α-androstan-3α-ol-17-one (androsterone, A), and 5β-pregnane-3α, 20α-diol (5βP). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute δ¹³C_VPDB and Δδ¹³C_VPDB values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2‰. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

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.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Synthesis and stereochemistry of C-3- and C-7-linked (O-carboxymethyl)-oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

The 3-(O-carboxymethyl)oximino derivative of 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) was prepared. Thin-layer chromatography of the corresponding methyl ester showed the presence of two syn (60%) and anti (40%) geometrical isomers of the oxime chain to the C-4 position, which were characterized by ¹³C nmr. The 3β-hemisuccinami-do-5α-androstan-17β-ol was obtained after selective saponification with potassium carbonate of the 17β-hemisuccinate group of the 3,17-dihemi-succinoylated derivative of the previously described 3β-amino-5α-androstan-17β-ol. This 3β-hemisuccinamide was purified as the corresponding methyl ester-17β-acetate and was regenerated after saponification. The 3,3'-ethylenedioxy-7-oxo-5α-androstan-17β-yl acetate was obtained in quantitative yield by catalytic hydrogenation over 10% palladium-oncharcoal of the Δ⁵-7-oxo precursor in a dioxane-ethanol mixture containing traces of pyridine. The exclusive 5α-configuration of this hydrogenated product was established from nmr data and was confirmed by the synthesis of methyl 3,3'-ethylenedioxy-7-oxo-5β-cholan-24-oate as 5β-H-reference compound. The preceding 5α-H-7-ketone was converted into the 7-(O-carboxymethyl)oximino derivative (syn isomer to the C-6 position, exclusively) which was esterified into the corresponding methyl ester. The selective hydrolysis of the 3-ethyleneketal group was achieved by a short treatment with a formic acid-ether 1:1 (v/v) mixture at 20°C. Saponification of the latter reaction product with ethanolic potassium hydroxide gave the 7-(O-carboxymethyl)oximino-17β-hydroxy-5α-androstan-3-one derivative, which was characterized as the corresponding methyl ester. The reduction of the oxime of the 5α-H-7-ketone with sodium in ethanol or with lithium-aluminium hydride gave respectively the 7β-amine or the 7α-amine as the major product. The 7β- and 7α-configurations were established from nmr spectra of the corresponding 7-acetamido derivatives. The 7β- and 7α-hemisuccinamido derivatives were prepared from the mixture of 7β- and 7α-amines, as described above for 3-derivatives and were isolated after thin-layer chromatography of the methyl esters, followed by saponification of the corresponding 17β-acetates.     

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.     

The synthesis of 17-substituted 2α-chloro-5α-androstan-3-ols

This paper describes the synthesis of 2α-chloro-3α-hydroxy-5α-androstan-17-one and 2α-chloro-5α-androstane-3α,17β-diol and their 3-epimers. The epimers were characterized by nmr spectroscopy.     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

C19-steroid 5β-reductase and 3- and 17-oxidoreductases of adult male hamster kidney

Male hamster kidney cytosol exhibited strong 5β-reductase activity. Incubation of cytosol with [4-¹⁴C]-testosterone at pH 6.7 yielded 5β-DHT with minor quantities of 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. Incubation with [4-¹⁴C]-androstendione yielded 5β-androstanedione and smaller quantities of testosterone, 5β-DHT, 3α-hydroxy-5β-androstan-17-one, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α,17β-diol. The two major metabolites were progressively increased with increase in the concentration of the respective substrates but the other metabolites showed very little change. The metabolism of the respective substrates was progressively decreased with changes in pH of the incubation mixture from 6.0–7.5 accompanied by a parallel decrease in the formation of the respective major metabolites. NADPH was much more effective than NADH as coenzyme. The microsomes exhibited a trace of 5β-reductase activity only with NADPH and androstenedione.The kidney homogenate at pH 10.1 effectively converted [4-¹⁴C]-testosterone to [4-¹⁴C]-androstenedione. The dehydrogenase activity was present in the cytosol and microsomes. NAD⁺ was more effective than NADP⁺ in the cytosol and the reverse was indicated for the microsomes. Spectrophotometric assay revealed not only NADP⁺-linked Hβ-dehydrogenase activity but also a lower 3α-dehydrogenase activity but no detectable 3β- or 17α-dehydrogenase activity. NAD⁺-linked activity was not explored because of the interference by the very high endogenous NAD⁺-reduetase activity.     

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.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

The metabolism of androgens in rat preputial glands

The metabolism of testosterone, androstenedione and dehydroepiandrosterone in the rat preputial gland has been studied. A high activity of 5α-reductase is present as shown by the formation of 17β hydroxy-5α-androstan-3-one and 5α-androstan-3, 17-dione as the major products from testosterone and androstenedione respectively. Other enzyme activities are present including 17β-hydroxy steroid dehydrogenase, but the amounts of testosterone and 17β-hydroxy-5α-androstan-3-one formed from androstenedione and dehydroepiandrosterone are low. The main product of dehydroepiandrosterone metabolism was androstenedione indicating a high level of 3β-hydroxy steroid dehydrogenase 4-5 isomerase activity. The metabolism was compared with that in rat skin where it was found that the extent of metabolism was much less. The possible significance of the various products formed and of differences between skin and preputial gland metabolism is discussed. Some differences were noted between the metabolism of androgens by rat skin and preputial gland and the metabolism of androgens by human skin.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Biotransformation studies of prednisone using human intestinal bacteria Part II: Anaerobic incubation and docking studies

Anaerobic incubation of prednisone 1 with human intestinal bacteria (HIB) afforded nine metabolites: 5β-androst-1-ene-3,11,17-trione 3, 3α-hydroxy-5α-androstane-11,17-dione 4, 3β,17α,20-trihydroxy-5α-pregnan-11-one 5, 3α,17α-dihydroxy-5α-pregnane-11,20-dione 6, 3α,17α-dihydroxy-5β-pregnane-11,20-dione 7, 3β,17β-dihydroxy-5α-androstan-11-one 8β, 3β,17α-dihydroxy-5α-androstan-11-one 8α, 3α,17β-dihydroxy-5α-androstan-11-one 9β, and 3α,17α-dihydroxy-5α-androstan-11-one 9α. The structures of these metabolites (3–9) were elucidated using several spectroscopic techniques. Computer-aided prediction of potential biological activities of the isolated prednisone metabolites (3–9) revealed potential inhibition of prostaglandin E2 9-ketoreductase (PGE2 9-KR). Docking studies applied to PGE2 9-KR allowed recommendation of the metabolites 4, 8β, and 8α for further pharmacological study as PGE2 9-KR inhibitors.