Oxidative demethylation of lanosterol in cholesterol biosynthesis: accumulation of sterol intermediates.

With [3H-24,25]-dihydrolanosterol as substrate, large-scale metabolic formation of intermediates of lanosterol demethylation was carried out to identify all compounds in the metabolic process. Utilizing knowledge of electron transport of lanosterol demethylation, we interrupted the demethylation reaction allowing accumulation and confirmation of the structure of the oxygenated intermediates lanost-8-en-3 beta,32-diol and 3 beta-hydroxylanost-8-en-32-al, as well as the demethylation product 4,4-dimethyl-cholesta-8,14-dien-3 beta-ol. Further metabolism of the delta 8.14-diene intermediate to a single product 4,4-dimethyl-cholest-8-en-3 beta-ol occurs under interruption conditions in the presence of 0.5 mM CN-1. With authentic compounds, each intermediate has been rigorously characterized by high performance liquid chromatography and gas-liquid chromatography plus mass spectral analysis of isolated and derivatized sterols. Intermediates that accumulated in greater abundance were further characterized by ultraviolet, 1H-NMR, and infrared spectroscopy of the isolated sterols.