Biosynthesis of cholesterol and lanosterol in isolated dog hepatocytes: incorporation of [1,2-13C2]- and [2-13C2H3]-acetate, and [1-13C]acetate and [1-2H2]ethanol

Abstract

Since isolated dog hepatocytes have been shown to exhibit potent activity for the synthesis of cholesterol from acetic acid, an analysis of cholesterol and lanosterol biosynthesized from [1,2-¹³C₂]- or [2-¹³C²H₃] acetate in fresh isolated hepatocytes has been attempted in order to clarify the incorporation and distribution of the acetate carbon and hydrogen into these sterols. Lanosterol was obtained by adding SSF-109, a sterol biosynthesis inhibitor, to the hepatocytes suspension. ¹³C NMR spectroscopic analysis of the [¹³C]- and [¹³C²H]-labelling patterns of these compounds confirmed the following: (i) the involvement of two 1,2-hydride shifts, 20-H from C-17 and 17-H from C-13; (ii) two 1,2-methyl migrations, 13-methyl group (C-18) from C-14 and 14-methyl group (C-32) from C-8 in the cyclization of 2,3-oxidosqualene to form lanosterol; and (iii) stereospecific hydrogenation from the Si-face at C-25 in the conversion of lanosterol into cholesterol. The proton-decoupled ¹³C–¹H COSY spectrum confirmed the stereospecific hydrogen attack at C-7, C-15 and C-24. The deuterium atoms of [1-²H₂]ethanol were incorporated into lanosterol at C-2, C-6, C-11, C-12, C-16 and C-23, which arise from C-5 of mevalonic acid.