Howard L. Kimball

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.

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.

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.

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.

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.

Synthesis of 7α-hydroxycholesterol-1, 2-3H

Abstract The known cholesta-1, 4, 6-trien-3-one was transformed to cholesta-1, 5-diene-3β, 7α-diol (3 steps) which was catalytically reduced with tritium to yield the title compound.