The Fate of the 6α-Hydrogen of 5α-Cholest-7-en-3β-ol in the Conversion to 7-Dehydrocholesterol by Rat Liver Microsomes

Abstract

Abstract 2-14C-Mevalonic acid has been incubated with a rat liver homogenate in the presence of the inhibitor trans-1,4-bis(2-chlorobenzylaminoethyl) - cyclohexanedihydrochloride yielding 1,7,15,22,26-14C5-cholesta-5,7-dien-3β-ol which was reduced with lithium and ammonia to 14C5-5α-cholest-7-en-3β-ol (Δ7-cholestanol). Separation from endogenous cholesterol was achieved by oxidation with chromic acid, and subsequent borohydride reduction of the isolated Δ7-3-ketone. 6β-3H-5α-Cholest-7-en-3β-ol has been converted in 3% yield to 6-3H-cholesta-5,7-dien-3β-ol by a rat liver microsomal preparation containing (trans-1,4-bis(2-chlorobenzylaminoethyl)-cyclohexane dihydrochloride. 6α-3H-14C5-5α-Cholest-7-en-3β-ol has been incubated with a rat liver microsomal preparation, being converted in 5.5% yield into 14C5-cholesta-5,7-dien-3β-ol with loss of tritium. Less than 1% of the liberated tritium was found in isolated NADH, the remainder being isolated as 3H2O. In a control experiment, synthetic NADH-4-3H2 was incubated with a microsomal preparation as before. The resulting NADH, isolated in 30% yield, contained 70% of the 3H (corrected to 100% isolation); the remainder of the radioactivity was isolated as 3H2O. Clearly then in the conversion of 5α-cholest-7-en-3β-ol into 7-dehydrocholesterol, the 6α-hydrogen is removed as a proton, and is not directly transferred to NAD+. Had NADH-3H been formed, not more than a 30% loss of 3H would have been expected during the isolation of the NADH.