Investigation of the Component Reactions of Oxidative Sterol Demethylation

Abstract

The participation of 3-ketosteroids in the biosynthesis of cholesterol was investigated with preparations of rat liver microsomes. Without exogenous NADPH, microsomal enzymes catalyze the demethylation of one methyl group from 4,4-dimethyl-5α-cholest-7-en-3β-ol to yield carbon dioxide and 4-methyl-5α-cholest-7-en-3-one. Equal molar quantities of the two products are formed when either the amount of microsomal protein or the length of incubation is varied. The labeled methyl ketone was identified by chromatography and derivative formation with carrier 4α-methyl-5α-cholest-7-en-3-one. 5α-Cholest-7-en-3-one is formed similarly from 4α-methyl-5α-cholest-7-en-3β-ol. Measurement of the stoichiometry of formation of carbon dioxide and cholest-7-en-3-one is complicated somewhat by reduction of the product ketone to an alcohol. 4α-Methyl-, 4,4-Dimethyl-, and 4,4,14α-Trimethyl-Δ7-cholestenols and -cholestenones were synthesized. The efficiency of demethylation of the 3-alcohols by microsomes is inversely proportional to the number of methyl groups on the substrate. Hydroxylation is the first step of demethylation; the 4-methyl groups of 4,4-dimethyl-5α-cholest-7-en-3-one and 4α-methyl-5α-cholest-7-en-3-one are not hydroxylated. The 4-methyl groups of the corresponding alcohols are hydroxylated. Thus, the 4-methyl ketone that is produced by demethylation of one methyl group from the 4,4-dimethyl sterol must be reduced to an alcohol before further metabolism. Although both alcoholic and ketonic forms of the methyl steroids are demethylated by either crude homogenates or unwashed microsomes from rat liver, the ketones are not demethylated by washed microsomes. Addition to washed microsomes of either the supernatant fraction from centrifugation of crude homogenates for 1 hour at 105,000 × g or an NADPH-generating system simultaneously restores the reduction and demethylation of ketones by microsomes. Microsomes contain an NADPH-dependent 3-ketosteroid reductase. This evidence supports the conclusion that 4-monomethyl and 4-demethyl ketones are obligatory intermediates of cholesterol biosynthesis. Dimethyl and trimethyl ketones probably are in equilibrium with the corresponding 3-alcohols, but the C29- and C30-ketones probably are not obligatory intermediates of C27-sterol biosynthesis.