Monitoring of lipoprotein oxidation by gas chromatographic analysis of hydroxy fatty acids

Abstract

We describe a method developed for the quantitative analysis of hydroxy fatty acids derived from fatty acid monohydroperoxides formed during lipoprotein oxidation. The procedure starts with catalytic hydrogenation of the lipid extract, whereby hydroperoxyl groups are converted to hydroxyl groups and double bonds are eliminated, and the risk for lipid oxidation during the rest of the procedure is eliminated. The fatty acids are converted to methyl esters, which are fractionated by gas chromatography on a nonpolar column. The major differences to existing methods are that a mass spectrometer is not required and that the specificity thus lost is replaced by gas chromatography before and after acetylation of the hydroxyl groups. This changes the retention times of the hydroxyacids with respect to the unsubstituted fatty acids moving them to positions usually occupied by trace components only. The method allows quantification of monohydroxy fatty acids derived from 18-, 20- and 22-carbon polyunsaturated fatty acids. Positional isomers are separated from each other to some extent. The method has been mainly used for analysis of hydroperoxides in human low density lipoprotein preparations and for following lipoprotein oxidation in vitro.