32] The measurement of ω- and ω-1 hydroxylation of fatty acids by mixed-function oxidase systems

Abstract

Summary Several methods are described in the literature 2,6,11 for the separation, identification, and quantitation of the products of fatty acid hydroxylation catalyzed by either bacterial or mammalian monooxygenases. The free fatty acid and its hydroxylated metabolites can be separated using silicic and chromatography, but the possible ω and ω-1 hydroxy fatty acids cannot be resolved using this technique. However, the methyl esters of the two major hydroxylated metabolites can be resolved by either thin-layer, gas, or high-pressure liquid chromatography. Thin-layer chromatography of ω and ω-1 hydroxy fatty acid methyl esters usually requires repeated solvent development to adequately separate the two metabolites. Although radio-gas chromatography is sometimes limited by CO 2 quenching, complete separation and quantitation of a number of hydroxy fatty acid methyl esters can also be achieved using this method. High-pressure liquid chromatography (HPLC) can be used to completely resolve the two methylated hydroxy metabolites, and this separation can be achieved easily using an isocratic HPLC instrument consisting of a solvent delivery system and a micro-particle octodecylsilane column. This system, coupled with a refractive index detector, allows the collection of the methyl esters of the metabolites and lauric acid in minimum volume.