Transformation of labeled cholesterol, 20 α-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (22R)-20 α, 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 14C and 3H) 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 −8M. 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.