Steroids in newborns and infants identification by combined gas chromatography-mass spectrometry of the major steroids excreted by a newborn chimpanzee (Pan troglodytes)

The following steroids have been identified by combined gas chromatography-mass spectrometry in a urine specimen collected from a newborn chimpanzee; 5-androstene-3β, 17α-diol, 3β,16α (and 16β)-dihydroxy-5-androsten-17-one, 5-androstene-3β, 16α, 17β-triol, 5-androstene-3β, 16β, 17α-triol, 5-pregnene-3β, 20α-diol, 5-pregnene-3β, 20α, 21-triol, 3β,21-dihydroxy-5-pregnen-20-one, 3β, 16α-dihydroxy-5-pregnen-20-one, 5-Piegnene-3β, 16α,20α, 21-tetrol, 5-pregnene-3β,17α, 20ξ, 21-tetrol androstenetriolones and androstenetetrols.     

Metabolism of progesterone by chorionic cells of the early sheep conceptus invitro

Progesterone-4-¹⁴C was extensively metabolized during incubation with dispersed trophoblast prepared from chorionic membranes of the 21-day sheep conceptus. Of the metabolites formed, 17,20α-dihydroxypregn-4-en-3-one, 20α-hydroxypregn-4-en-3-one, 20(β-hydroxypregn-4-en-3-one, 5α-pregnane-3α,17,20α-triol, 5β-pregnane-3ga, 17,20α-triol, 5β-pregnane-3g,20α-diol, 3β-hydroxy-5α-pregnan-20-one, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-5β-pregnan-3-one, 5α-pregnane-3,20-dione and 5β-pregnane-3,20-dione were identified. These findings indicate that the sheep conceptus acquires extensive steroid metabolizing capability very early in pregnancy.     

A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas-liquid chromatography

A method is described for simultaneous determination of seven common urinary 17-ketosteroids, pregnanediol (5β-pregnane- 3α, 20α-diol) and pregnanetriol (5β-pregnane-3α,17α,20α-triol) by gas-liquid chromatography. The essential steps include hydrolysis of conjugates by an extract from $\text{Helix}$$\text{pomatia}$, use of 5β-cholestan-3α-ol as an internal standard, silylation and gas chromatography using 3% XE-60 as a stationary phase. The chromatograms were clear and the peaks easily identifiable. Laborious purification procedures were not necessary. 5β-pregnane-3α,20α-diol and 5β-pregnane-3α, 17α,20α-triol were measured in 22 adult males and 17 adult females. The subjects were trained hospital personnel, healthy and without medications. The results, in general, were in agreement with those reported by others.     

Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

Antiserum to 5alpha-dihydrotestosterone: production, characterization and use in radioimmunoassay

5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (K_a= 1.4 × 10⁹ 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women.     

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.     

Four new diterpenes from Sideritis serrata

Four new diterpenes have been isolated from Sideritis serata: lagascol (4, ent-8,5-friedopimar-5-ene-15S,16-diol), tobarrol (8, ent-15-beyerene-12α,17-diol), benuol (12, ent-15-beyerene-7α,17-diol) and serradiol (18, ent-16R-atis-13-ene-16,17-diol). The previously known diterpenes lagascatriol (1, ent-8,5-friedopimar-5-ene-11β,15S,16-triol), jativatriol (2, ent-15-beyerene-1β,12α,17-triol), conchitriol (3, ent-15-beyerene-7α,12α,17-triol) and sideritol (17, ent-16R-atis-13-ene-1β,16,17-triol) have also been obtained from the same source.     

Identification and quantification of unconjugated neutral steroids in adrenal and liver tissue of early and mid-term human fetuses

In order to study further the metabolism of neutral steroids in human fetal adrenal and liver tissue the fractions of unconjugated neutral steroids isolated from these tissues were analyzed by gas-liquid chromatography and gas chromatography — mass spectrometry. In the adrenals, pregnenolone and 17-hydroxypregnenolone, but no corticoids, were detected. In the liver, pregnenolone, 3α-hydroxy-5β-pregnan-20-one, 5β-pregnane-3α, 20α-diol and 3β, 16α-dihydroxy-5β-pregnan-20-one were found. Thus, all the free steroids detected were C₂₁ compounds. From these results and those obtained earlier by the analysis of the sulfate-conjugated steroids present in these tissues it is concluded that in the fetal adrenals $\text{in situ}$ both sulfated and unconjugated steroids are actively metabolized. Regarding the liver it is obvious that the conjugated metabolites of progesterone are rapidly eliminated from this tissue. Here, pregnenolone is present both in the free and sulfate conjugated form, whereas its metabolites are found only as sulfate conjugates.     

Metabolism of 5β-pregnane-3,20-dione and 3β-hydroxy-5β-pregnan-20-one by Digitalis suspension cultures

Digitalis purpurea normal callus suspension culture is capable of metabolizing 5β-pregnane-3,20-dione (1) to 3β-hydroxy-5β-pregnan-20-one (2), 3α-hydroxy-5β-pregnan-20-one (3), 3β-hydroxy-5β-pregnan-20-one glucoside (7) and 3α-hydroxy-5β-pregnan-20-one glucoside (8). Digitalis purpurea habituated callus suspension culture is also capable of metabolizing 1 to 2, 3, 5β-pregnane-3β,20β-diol (5), (7), (8), 5β-pregnane-3β,20α-diol monoglucoside (9) and 5β-pregnane-3α,20α-diol monoglucoside (11). Furthermore, it was observed that 3β-hydroxy-5β-pregnan-20-one (2) is converted to 7, 9 and 11 by both suspension cultures. At the same time, 1, 3, 5 and 8 were detected in normal callus, while 5β-pregnane-3β,20α-diol (4) and 5β-pregnane-3β,20β-diol monoglucoside (10) were present in the habituated callus culture.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four Δ⁵-3β-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17α-hydroxypregnenolone, dehydroepiandroste rone and 5-androsterone-3β,17β-diol), and their four Δ⁴-3keto products (progesterone, 17α-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid In extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17,20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

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.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

In vitro conversion of 5alpha-reduced metabolites of testosterone by the seminal vesicles, ventral prostate glands and testes of adult rats

In order to ascertain the ability of rat seminal vesicles, testes and ventral prostate glands to interconvert 5α-reduced androgens, these three organs were incubated with either tritiated 17β-hydroxy-5αandrostan-3-one (5α-dihydrotestosterone,DHT), 5α-androstane-3α, 17βdiol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol). The incubation environment utilized (Krebs-Ringer bicarbonate glucose buffer) was selected because the histologic appearance of the tissue at the conclusion of the incubation was indistinguishable from tissue fixed immediately after sacrifice of the animal, thereby approximating the physiologic conditions as closely as possible. In incubations of rat seminal vesicles, ³H.-3β-diol was not metabolized while 26.7 ± 3.8% of ³H-3α-diol appeared as DHT and 17.2 ± 1.5% of ³H-DHT was metabolized to 3α-diol. A small amount (7.5 ± 0.8%) of ³H-DHT was, however, converted to 3β-diol. In incubations of rat testes, the major metabolite, regardless of substrate, was 3α-diol. The conversion of 75.7 ± 2.1% of ³H-3β-diol to 3α-diol has demonstrated, for the first time, that this steroid can be metabolized by the rat testis. Rat ventral prostate glands metabolized $\text{18.5 ± 2.5\%}\text{of}{}^3\text{H-3β-}\text{diol}$ to DHT and 61± 2.9% of ³H-3α-diol to DHT. When ³H-DHT served as the substrate, 83.2 ± 1.5% remained unmetabolized. The prostate glands are, therefore, capable of metabolizing 3β-diol to DHT.     

Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

Solanolide,a steroid lactone sapogenin from Solanum hispidum

Solanolide, a new C₂₂ steroid lactone sapogenin isolated from the leaves of Solanum hispidum Pers., has been characterized as 3β, 6α, 16β-trihydroxy-5α-pregnane-20S-carboxylic acid (22, 16)-lactone from ¹H and ¹³C NMR analyses and correlation with neochlorogenin.     

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.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

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.     

Diterpenes from Dysoxylum lukii Merr

Six new diterpenes and two known compounds were isolated from the stems of Dysoxylum lukii Merr and characterized on the basis of spectral data. Dysokusone F(1), 2-Oxoneoclerod-3, 13Z-dien-15-ol (2), 3α-(4-Hydroxy-3,5-dimethoxy-benzoyloxyl)-clerod-14-ene-4β,13-diol (3), 3α-(4-Hydroxybenzoyloxyl)-clerod-14-ene-4β,13-diol (4), and 3α-(4-Hydroxy-3-methoxybenzoyloxyl)-clerod-14-ene-4β,13-diol (5) exhibited inhibitory effects against PTP-1B with IC₅₀ values of 51.62±6.55, 56.74±7.96, 17.04±3.43, 28.96±4.59, and 19.70±2.57 μM, respectively.     

Metabolism of 17α-methyl-5β-dihydrotestosterone in the rabbit

17α-Methy1-5β-androstane-3α, 17β-diol together with the hydroxylated metabolites 17α-methyl-5β-androstane-1β, 3α, 17β-triol, 17α-methyl-5β-androstane-3α, 12β, 17β-triol, 17α-methyl-5β-androstane-3α, 16α, 17β-triol and 17α-methyl-5β-androstane-3α, 16β, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5β-dihydrotestosterone. Biotransformations differ from the 5α-series where hydroxylation occurred at C-6 and C-15. In both series, the C-3 equatorial epimer was the major urinary excretion product among the non-hydroxylated metabolites. The 5β-compound was more resistant to metabolic hydroxylation than the 5α-compound. No evidence for epimerization at the C-17 position was observed.