Shlomo Burstein

Biosynthesis of pregnenolone.

Publisher Summary Pregnenolone is the C21 steroid closest in structure to cholesterol, which is the most abundant sterol in the Eutheria. C21 steroid is by far the major precursor of all the steroid hormones in the adrenal gland, ovary, and testis. Cholesterol sulfate converts to various steroidal sulfates in humans in vivo and to pregnenolone sulfate by bovine adrenocortical mitochondria in vitro. The chapter presents the most advanced scheme for the enzymatic transformation of cholesterol to pregnenolone. Cholesterol derivatives with only a hydroxyl at C-20 in the side chain appear not to have been isolated from natural sources. The premise that the hydroxylated derivatives of cholesterol served as better substrates than cholesterol itself was based on the more rapid transformation of the labeled precursors, which were not always studied at specified substrate concentrations. A better understanding of the mechanism of the conversion of cholesterol to pregnenolone awaits further investigation.

A preliminary report on the intermediates in the conversion in vitro of cholesterol to pregnenolone in adrenal preparations.

Abstract The reaction sequences cholesterol → 20α-hydroxycholesterol → 20α, 22R-di-hydroxycholesterol → pregnenolone, and cholesterol → 22R-hydroxycholesterol → 20α, 22Rrdihydroxycholesterol → pregnenolone have been studied quantitatively under conditions of first-order kinetics with adrenocortical preparations. The sequence of 22R-hydroxycholesterol was much more important than that of 20α-hydroxycholesterol, but only a small fraction of the pregnenolone formation from cholesterol via the monohydroxy derivatives could be accounted for. A much more substantial fraction could be accounted for by assuming an enzymatic, concerted attack of oxygen on cholesterol to form 20α, 22R-dihydroxycholesterol which then is oxidatively cleaved to pregnenolone. This study establishes several pathways leading from cholesterol to pregnenolone.

Side-chain cleavage of cholesterol to C6 and C8compounds by adrenal and testis tissue preparations

Abstract 1. 1. Using gas-liquid chromatography the conversion of [25,26-3H2]cholesterol to isocaproaldehyde, isohexanol, isocaproic acid, 2-methylheptan-6-oneand 2-methylheptan-6-ol has been studied with adrenal and testis preparations without the addition of “trapping” agents. 2. 2. With bovine adrenocortical mitochondrial soluble preparations, isocaproaldehyde, isohexanol and isocaproic acid were formed. In some of the bovine, preparations either isocaproaldehyde or isohexanol predominated, but in none was the isocaproic acid a major product. 3. 3. With human and chicken adrenal acetone-dried powders isohexanol was observed as the major product along with smaller amounts of isocaproaldehyde and isocaproic acid. 4. 4. [4,5-3H2] Isocaproaldehyde, of the same specific activity as the [25,26-3H2]-cholesterol used, was rapidly converted to isohexanol and isocaproic acid with a bovine acetone-dried powder preparation and to isohexanol with a human adrenal preparation, strongly indicating that isocaproaldehyde was the primary product of the cholesterol side-chain cleavage. These studies appear to be the first demonstration of the existence of an isocaproaldehyde reductase system in adrenal preparations. 5. 5. With a soluble bovine acetone-dried preparation in which cholesterol was converted predominantly to isocaproaldehyde and in which pregnenolone was not significantly further converted to other products, a 1:1 stoichiometric relationship was observed between this C21 steroid and the sum of the C6 compounds formed. A similar 1:1 stoichiometry was also observed with two human adrenal acetone-dried powders, in which the further metabolism of pregnenolone was insignificant. 6. 6. No 2-methylheptan-6-one or 2-methylheptan-6-ol was formed with a human adrenal mitochondrial acetone-dried powder preparation obtained from a patient with an androgen-secreting adrenal carcinoma. 7. 7. Incubation of rat testis and adrenal homogenates with [25,26-3H2]cholesterol after the procedure of Jungmann (Biochim. Biophys. Acta, 164 (1968) 110) yielded isocaproic acid as the only positively identifiable volatile product. The carrier C8 compounds 2-methylheptan-6-ol and 2-methylheptan-6-one added at the end of the incubation were isolated either devoid of radioactivity or containing insignificant counts as compared to the isocaproic acid. The mean maximal possible combined radioactivity in these two C8 compounds was 1 500 and 1 4000 of that found in the isocaproic acid obtained with testis and adrenal homogenates, respectively. This contrasted with the studies by Jungmann 7 who reported ratios of 1 2.4 and 1 15.4 for 2-methylheptan-6-one, with these two tissues, respectively.

Quantitative paper chromatography of C21O5 and C21O6 corticosteroids from guinea pig urine extracts

Abstract The development of a quantitative paper chromatographic method for the routine determination of reducing C21O5 and C21O6 corticosteroids from guinea pig urine extracts by means of a single chromatographic step and direct densitometry following reaction with blue tetrazolium has been described. Among a number of paper chromatographic systems tried, the system 1,2-dichloroethane-methanol-water was found to give the most suitable results. The phase diagram of this ternary system was constructed and a number of compositions and other variables affecting the resolution have been studied. An empirical system with a mobile phase consisting of 1–2% methanol in ethylene chloride saturated with H2O was found to give a good resolution and reproducibility. Treating the papers with 1,2-dichlorobenzene prior to reaction with blue tetrazolium caused the formation of a much bluer formazan with an absorbancy of roughly three times that obtained without treatment. The all-over precision achieved following cortisol (20–120 μg) deposition on paper, chromatography, and densitometry was on the average 10% (coefficient of variation).

Urinary corticosteroid excretion patterns in guinea pigs: 2 main phenotypes

Abstract Reducing corticosteroid excretion patterns were studied individually in short-haired English type guinea pigs of both sexes. According to the patterns two .main types were discerned: type A with relatively high urinary 2α-hydroxycortisol and type B with relatively low quantities of this steroid. Type A animals also excreted a larger amount of 6β-hydroxycortisol and other as yet unidentified corticosteroids of similar Chromatographic mobility. The differences in corticosteroid excretion were increased during maximal ACTH stimulation. Among type A animals, the strain 13 guinea pigs exhibited the highest amounts of urinary 2α-hydroxycortisol. Hartley animals were of both types. These results demonstrate the importance of studying animals individually.

Studies on the solubilization and purification of rat liver microsomal steroid sulfatase

Abstract Digestion of rat liver microsomal preparations with heat-treated snake venom led to a mean 27% solubilization of the steroid sulfatase activity. Further purification by (NH 4 ) 2 SO 4 precipitation followed by chromatography on DEAE-Sephadex A-25 afforded a 1.5-fold increase in the specific activity over that of untreated microsomes. The highest purification achieved with some preparations following chromatography on DEAE-Sephadex A-50 was 3-fold. The soluble preparation was associated with high molecular weight material (about 600 000, as judged by filtration on Sephadex G-200) and exhibited a tendency to form insoluble aggregates.

Urinary cortisol and 6β-hydroxycortisol in the monkey, Cebus albifrons: Normal variation and the effects of ACTH and phenobarbital☆

Abstract The urinary excretion of unconjugated cortisol and 6β-hydroxycortisol has been studied in the New World monkey Cebus albifrons. The mean values of cortisol in male 3-year-old (2–3 kg) untreated monkeys as determined by the Porter-Silber reaction after chromatographic purifications (using tracer tritiated cortisol to correct for procedural loss) was 170 μg/24 hours. The respective mean daily excretion of 6β-hydroxycortisol (using the mean cortisol recovery to correct for procedural loss) was 100 μg/24 hours. There was a considerable day-to-day variability in the urinary excretion of both steroids exhibiting a mean coefficient of variation of 60%. ACTH administration caused a seven- and a five-fold increase in the excretion of cortisol and 6β-hydroxycortisol, respectively, over control levels. Stress of restraint in a chair elicited a five- and three-fold increase, respectively. The average cortisol: 6β-hydroxycortisol ratios obtained for the control, ACTH and stress periods were 1:0.6, 1:0.4, and 1:0.3. Phenobarbital in two consecutive experiments caused a significant increase in the urinary excretion of 6β-hydroxycortisol and led to an inversion of the ratio of cortisol: 6β-hydroxycortisol in favor of the latter. Cortisol and 6β-hydroxycortisol were eluted and identified from the chromatograms used in the determinations. The response to phenobarbital in Cebus albifrons was comparable to that observed in man and indicates the value of this species for further investigations concerning 6β-hydroxylation of cortisol.

Synthesis of 2α- and 2β-hydrqxycortisol

Abstract The synthesis (in 1 gm. amounts) of 2α-hydroxy- (I) and 2β-hydroxycortisol (II), their 2,21-diacetates (I-diac. II-diac.) and 2,11β,21-triacetates (I-triac. II-triac.) is described. Cortisol 21-acetate was acetoxylated with lead tetraacetate in glacial acetic acid. Following saponification in methanolic potassium hydroxide a mixture of I and II was obtained by partition chromatography in approximately 13% yield. Reacetylation of the purified mixture of I and II with acetic anhydride and pyridine at room temperature and subsequent partition chromatography yielded pure I- and II-diac. and 5–10% of I- and II-triac. I and II were obtained by saponification of I- and II-diac. in methanolic potassium hydroxide without inversion. The structure of the isolated compounds was assigned from their elemental analyses, molecular rotation, NMR, ultraviolet and infrared spectra.

Biosynthesis of Pregnane Derivatives

Publisher Summary Pregnane derivatives are not only important in their own right but also occupy a central position in steroid biochemistry because they serve as precursors of steroids possessing other types of biological activities—such as the androgenic and estrogenic hormones in vertebrates and the cardenolides. Pregnane compounds appear in nature both as free, as conjugates with sulfuric and glucuronic acid, with N-acetylglucosamine, as acetate esters, and as glycosides. Pregnane derivatives are widely found in nature. This chapter focuses on the type of C 21 steroids isolated, their distribution, their precursors and steroids closely related to them, and the distribution of the enzymes concerned with their formation. The major contributions responsible for the advances made in this field include the preparation of appropriate steroid substrate intermediates, both unlabeled and labeled, and the development of analytical techniques for their chromatographic separation and measurement. The chapter reviews the present knowledge of how pregnenolone is formed from cholesterol in steroid-producing tissues.

Preparation and properties of labelled 2α-, 2β- and 6β-hydroxycortisol

Abstract 1,2-Tritiated 2α- and 2β-hydroxycortisol were prepared by acetoxylation of 1,2-tritiated cortisol with lead tetraacetate. 1,2-Tritiated 6β-hydroxycortisol was synthesized by catalytic tritiation of 6β, 11β, 17, 21-tetrahydroxypregna-1,4-diene-3,20-dione 6,21-diacetate. The radiochemical purity of the prepared radioactive steroids was established by determining specific activities following chromatographic separations of the free and the acetylated steroids and following reduction to cortisol with zinc and oxidation to 11β-hydroxyandrost-4-ene-3,17-dione with sodium bismuthate. The tritium in the steroids was stable to partition chromatography in the usual systems, to chromatography on thin layer silica gel, to zinc dust deacetoxylation of the C-2α, C-2β and C-6β acetoxyl groups, to alkaline saponification of the acetates in methanol and to side-chain cleavage with sodium bismuthate. Loss of tritium occurred following reflux in aqueous methanolic alkali under conditions which led to destruction of the 17,21-dihydroxy-20-oxo side chain. The fraction of alkali exchangeable tritium in 2α-, 2β- and 6β-hydroxycortisol (determined by alkali treatment of the derived 11β-hydroxyandrost-4-ene-3,17-diones) was 13.9 ± 0.9, 16.0 ± 0.4 and 37 ± 1% (mean ± S. E.), respectively. No exchange of tritium occurred following the injection of tritiated cortisol and 2α-hydroxycortisol to guinea pigs. During acetoxylation of cortisol with lead tetraacetate, no significant interconversion between the diacetates of 2α- and 2β-hydroxycortisol took place.