Oxidative Demethylation in Sterol Metabolism

Oxidative demethylation of 4-methyl sterols by rat liver microsomes has been shown to be inhibited by low levels of NADH. Using snake venom-treated microsomes to remove endogenous NADH-utilizing activities, we observed inhibition of overall demethylation at concentrations of NADH as low as 2.5 p ~ . At 20 p~ NADH, overall demethylation had been reduced to 60% of optimal activity. In the presence of NADH, oxidative metabolism of [30,31-’4C]4,4-dimethyl-5a-cholest-7-en-3/3-ol resulted in formation of an intermediate which was metabolized anaerobically to 14COa in the presence of NAD’. This intermediate and its acetylated derivative had coincident mobility on silicic acid thin layer chromatograms with 3~-hydroxy-[’4C]4/3-methyl-5a-cholest-7-en-[’4C] 4a-carboxylic acid sterol and its acetylated derivative. The inhibition by NADH is transitory. Normal rates return after -5 min if NADH/NAD+ = 0.2, and after -15 min if NADH/NAD+ = 2. At an NADH/NAD+ ratio of 10, the return to normal rates is even slower, exceeding 30 min. Further, the inhibition, though transitory, occurs again upon introduction of additional NADH to the assay mix.