Marcel Gut

Transformation of labeled cholesterol, 20α-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (22R)-20α,22-dihydroxycholesterol by adrenal acetone-dried preparations from guinea pigs, cattle and man: I. Establishment of radiochemical purity of products

Abstract The preparation of radiochemically pure tritiated 20α-hydroxy-cholesterol, (22R)-22-hydroxycholesterol, (22S)-22-hydroxycholesterol and (22R)-20α,22-dihydroxycholesterol has been described. The enzymatic transformation of cholesterol to 20α-hydroxycholesterol, (22R)-22-hydroxycholesterol, (22S)-22-hydroxycholesterol, (22R)-20α,22-dihydroxy-cholesterol and pregnenolone has been studied using adrenal acetone-dried powder preparations from guineapigs, cattle and man. No significant conversion of cholesterol to (22S)-22-hydroxycholesterol took place. The amount of radioactivity appearing in 20α-hydroxy-cholesterol was exceedingly small and the radiochemical purity of this product was not firmly established. Using reverse isotope dilution techniques, involving chromatographic separations, derivative formation, specific reactions and crystallization, the conversion of cholesterol to (22R)-22-hydroxycholesterol and (22R)-20α,22-dihydroxycholesterol has been established for the first time without the use of “trapping” procedures. The further conversion of 20α-hydroxycholesterol and (22R)-22-hydroxycholesterol to (22R)-20α,22-dihydroxycholesterol and pregnenolone and that of (22R)-20α,22-dihydroxycholesterol to pregnenolone also has been investigated. The transformation of (22S)-22-hydroxycholesterol to (22S)-20α,22-dihydroxycholesterol and pregnenolone was insignificant in comparison to the transformation of the (22R) isomer.

Transformation of labeled cholesterol, 20α-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (22R)-20α, 22-dihydroxycholesterol by adrenal acetone-dried preparations from Guinea pigs, cattle and man: II. Kinetic studies

Abstract A general theoretical approach utilizing tracer methodology has been presented for the quantitative study of homogeneous irreversible consecutive reaction sequences under conditions of first-order kinetics. From the empirical overall disappearance rate constants of substrates and products, and the net formation of products from each preceding substrate (obtainable with the aid of isotopic tracers), the first order rate constants of all the individual steps of a sequence could be obtained. From the rate constants of formation and disappearance, the theoretical formation of products then could be calculated and compared with the experimental data thus allowing the assessment of the importance of a suggested pathway. For experimental designs with two different tracers (such as 14 C and 3 H) the theoretical approach enabled the calculation of the tracer ratios in the intermediates and the final product. Certain general relationships invariant with respect to reaction time were implicated. First-order reaction kinetics appear to have been obtained with bovine adrenal preparations at substrate concentrations of less than 2×10 −8 M. Under these conditions were determined the rate constants of the individual steps of the two reaction sequences: cholesterol20 α -hydroxycholesterol(22R)-20 α , 22-dihydroxycholesterolpregnenolone, and cholesterol(22R)-22-hydroxycholesterol(22R)-20 α , 22-dihydroxycholesterolpregnenolene, with bovine, guinea pig and human adrenal mitochondrial acetone dried preparations. In all species studied the reaction sequence involving (22R)-22-hydroxycholesterol was considerably more important than that involving 20 α -hydroxycholesterol. However, only a relatively small fraction of the pregnenolone formation from cholesterol could be accounted by the enzymatic reaction sequences given above.

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.

The synthesis and stereochemistry of (22R)-20α,22- and (22S)-20α,22-dihydroxycholesterol

Abstract An improved synthesis of (22 R )-20α,22- and (22 S )-20α,22-dihydroxycholesterol from pregnenolone acetate is described and the stereochemistry at C-20 and C-22 established. The title compounds have been labeled at C-1 and C-2, using 20α-hydroxycholesta-1,4-diene-3,22-dione as starting material.

Enzymatic formation of (20R,22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: Random incorporation of oxygen atoms☆

Abstract [4-14C]Cholesterol was incubated with an adrenocortical preparation in the presence of 16O2 and 18O2 devoid of significant 16O18O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of m e 289, 291, and 293 (comprising the C8 C-20 to C-27 side-chain and containing, respectively, 16O2, 16O18O, and 18O2) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of m e 291 would have been absent.

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.

Steric considerations regarding the biodegradation of cholesterol to pregnenolone.-exclusion of (22S)-22-hydroxycholesterol and 22-ketocholesterol as intermediates

Abstract (22 S )-[22-3H1]-Cholesterol was incubated with an adrenocortical preparation and the isolated (22 R )-[22-3H1]-22-hydroxycholesterol had a small loss of radioactivity, proving that direct replacement of the hydrogen from the now hydroxylated position occurred. In addition [1-3H1]-4-methylpentanol was isolated, which also had incurred a relatively small loss of its specific activity, thereby excluding (20 R )-3β,20-dihydroxycholest-5-en-22-one as an important metabolite in the degradation of cholesterol to pregnenolone by adrenal tissue.

Formation of 4-methyl-3-pentenoic acid from cholesta-5, 24-dien-3β-ol by adrenal enzyme

Abstract Cholesta-5, 24-dien-3β-ol-26- 14 C was incubated with an enzyme preparation obtained from calf adrenal and 4-methyl-3-pentenoic acid- 14 C was identified. The result suggests that cholesta-5, 24-dien-3β-ol can be a direct precursor of 3β-hydroxypregn-5-en-20-one.

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.