The formation of cholest-5-ene-3β,26-diol as an intermediate in the conversion of cholesterol into bile acids by liver mitochondria

1. When [(14)C]cholesterol was incubated with rat liver mitochondria, radioactive 26-hydroxycholesterol, 3beta-hydroxychol-5-enoic acid and other bile acids were isolated from the incubation mixture. 2. In the absence of added 26-hydroxycholesterol, the specific radioactivity of the 26-hydroxycholesterol formed from [(14)C]cholesterol during the incubation was higher than that of the 3beta-hydroxychol-5-enoic acid. Addition of increasing amounts of 26-hydroxycholesterol led to a progressive fall in the specific radioactivity, and to a progressive increase in the mass, of the 3beta-hydroxychol-5-enoic acid recovered at the end of the incubation. 3. It is concluded that 26-hydroxycholesterol is an intermediate in the formation of 3beta-hydroxychol-5-enoic acid from cholesterol. 4. Comparison of the specific radioactivity of the 26-hydroxycholesterol formed in the incubation mixture with that of the added [(14)C]cholesterol indicates that endogenous cholesterol in mitochondria is accessible to cholesterol 26-hydroxylase.     

The conversion of (24S)-24-ethylcholesta-5,22,25-trien-3β-ol into poriferasterol,both in vivo and with a cell-free homogenate of the alga Trebouxia sp.

[6-³H₁] (24S)-24-Ethylcholesta-5,22,25-trien-3β-ol added to the growth medium of a culture of Trebouxia sp. 213/3 was efficiently taken-up by the cells and converted into (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) which is one of the major sterols of this alga. A cell-free homogenate was obtained from Trebouxia which catalysed the NADPH-dependent reduction of [6-³H₁] (24S)-24-ethylcholesta-5,22,25-trien-3β-ol to yield poriferasterol. The δ²⁵-sterol reductase was found to be mainly localized in the microsomal fraction of the homogenate.     

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.     

The role of (25S)-5α-cholestan-3β, 26-diol and (25S)-5α-furostan-3β,26-diol in the biosynthesis of tomatidine and neotigogenin

(25S)-5α-Cholestan-3β,26-diol was incorporated into neotigogenin and tomatidine, and (25S)-5α-furostan-3β,26-diol only in neotigogenin,     

Conversion of a 24β-ethyl-25-methylene intermediate into poriferasterol by Trebouxia species

Clerosterol-[26-¹⁴C], a 24β-ethyl-25-methylene sterol [(24S)-24-ethylcholesta-5,25-dien-3β-ol], was incorporated into clionasterol and poriferasterol by cultures of the green algae Trebouxia sp. 213/3 and Trebouxia sp. 219/2. Degradation of the labelled poriferasterol showed that the ¹⁴C retained its identity and was not incorporated as a result of metabolism of the clerosterol-[26-¹⁴C] and randomisation of label. These results are consistent with the proposed production, and subsequent reduction, of a 24β-ethyl-25-methylene intermediate in 24β-ethyl sterol biosynthesis in algae of the order Chlorococcales.     

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.     

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.     

Simultaneous radioimmunoassay of 5α-amdrostane-3α, 17β-diol and 5α-androstane-3β, 17β-diol unconjugated and conjugated in human serum

The simultaneous determinations of both 3α and 3β epimers of 5α-androstane-3, l7β-diol as their glucuronides, sulfates and in their unconjugated forms are described. The diol estimation is carried out by radioimmunoassay with two specific immune sera after purification of the serum by use of chromatography on Sephadex LH-20. The values obtained (mean ± S.D.) in pg/ml for the unconjugated 3α and 3β epimers were, respectively, 267 ± 67 and 816 ± 76 for men; 114 ± 33 and 515 ± 177 for women; 142 ± 77 and 779 ± 200 for hirsute women. Among the conjugates, the most important were the sulfoconjugates, their rates being, respectively, (men ± S.D. in ng/ml) 41.3 ± 9.5 and 103 ± 40 for men; 12.4 ± 3.1 and 51.2 ± 14.9 for women and 36 ± 22 and 72 ± 36 for hirsute women. Differences in the conjugation of both epimers were also noticed.     

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.     

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.     

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.     

(+)-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.     

The isolation of cholest-5-ene-3β,26-diol from human brain (Short Communication)

Cholest-5-ene-3beta,26-diol, isolated from human brain, was further characterized by oxidation to 3-oxocholest-4-en-26-ol and to 3-oxocholest-4-en-26-oic acid. Identification was achieved by comparison (by t.l.c., g.l.c. and g.l.c.-mass spectrometry) with corresponding reference compounds derived from kryptogenin.     

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.     

The molecular structure of (20R)-20-phenyl-5-pregnene-3β,20-diol,an inhibitor of cholesterol conversion to pregnenolone

The crystal and molecular structure of (20R)-20-phenyl-5-pregnene-3β, 20-diol hemihydrate has been determined by X-ray analysis in order to establish the configuration and conformation at C(20). Interest in this compound was stimulated by its high affinity inhibitory binding to cytochrome P-450_SCC, the enzyme which catalyzes the biosynthesis of pregnenolone(3β-hydroxy-5-pregnen-20-one) from cholesterol. The results of the analysis suggest a possible conformation for the cholesterol side chain in the enzyme complex.     

Effect of taurocholate on the conversion of 3α, 7α, 12α-Trihydroxy-5β-Cholestan-26-OIC acid into cholic acid

To determine if the conversion of the intermediate, 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid (THCA), into cholic acid is influenced by taurocholate, two rats were infused intravenously with [³H] THCA until they reached a steady state. Taurocholate was then added and infused at a rate of 1 μmole/min/rat for 48 hours. The percentage of [³H] THCA recovered in the bile did not increase indicating that taurocholate does not suppress the conversion of THCA into cholic acid.     

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).