Simultaneous assay of the activities of two key enzymes in cholesterol metabolism by gas chromatography—mass spectrometry

Abstract

A very sensitive and specific method for the simultaneous assay of the activities of two key regulatory enzymes in cholesterol metabolism, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), and cholesterol 7α-hydroxylase (EC 1.14.13.7), is described. The assay is based on the measurement of [ 2H 3]mevalonolactone and 7α-hydroxycholesterol produced by the incubation of [ 2H 3]HMG-CoA and endogenous cholesterol with hamster liver microsomes using isotope dilution mass spectrometry. The incubation mixture was purified by means of solid extraction cartridges, and the extract was treated with benzylamine followed by dimethylethylsilyl imidazole. The resulting ether derivatives of the mevalonylbenzylamide and 7α-hydroxycholesterol were quantified by gas chromatography—mass spectrometry with selected-ion monitoring in a high resolution mode. The method made it possible to assay simultaneously the activities of HMG-CoA reductase and cholesterol 7α-hydroxylase in hamster liver microsomes with high sensitivity and accuracy.