Assay of cholesterol 7α-hydroxylase utilizing a silica cartridge column and 5α-cholestane-3β,7β-diol as an internal standard

A simplified and accurate assay of cholesterol 7α-hydroxylase activity using 5α-cholestane-3β,7β-diol as an internal standard is described. Endogenous microsomal cholesterol was used as the substrate. Following incubation and addition of the internal standard, lipids extracted from the incubation mixture were applied to Bond-Elut silica cartridge columns. 7α-Hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol and the internal standard were quantitatively recovered by eluting the column with 6 ml of benzene—ethyl acetate (2:3, v/v) after removal of cholesterol with 6 ml of benzene—ethyl acetate (9:1, v/v). After trimethylsilylation, the mass of 7α-hydroxycholesterol was determined by capillary gas chromatography with selected-ion monitoring. The method permits a faster, easier and more sensitive determination of the activity of cholesterol 7α-hydroxylase in small samples.