Separation of unsaturated fatty acids and their hydroperoxides and hydroxides was carried out easily and quickly by high-performance liquid chromatography on the porous polymer gel (TSK-Gel LS-140) using n-hexaneiethanol as an eluent. This method was proved to be useful for the quantitative analysis of fatty acids. About 95% of the fatty acid and its derivatives could be extracted from aqueous solution by n-hexane after acidification by HCl solution to pH 1.3. These methods were successfully applied to the analysis of the product in the reaction of bovine erythrocyte glutathione peroxidase with 13-hydroperoxylinoleic acid.
The production of lipid hydroperoxide (1,2) and its reduction (3,4) in various cells of the body are interesting and important problems, since the free radicals which may be produced from the destruction of lipid hydroperoxides cause oxidative damage to various cells and may be one of factors in animal aging (5). Christopherson and other workers (6-8) have suggested that the glutathione peroxidase system may remove lipid peroxides, in this way preventing lipid degradation and membrane disordering (i.e., protecting the membrane from oxidative damage). McCay et al. ( 9), however, have denied this function based on analysis of the fatty acids hydroxides in membranes by thin-layer chromatography.
For study of lipid peroxidation and reduction of the lipid hydroperoxides, a rapid, convenient, sensitive, and quantitative method of analyzing unsaturated fatty acids and their hydroperoxy and hydroxy derivatives is desired. This method must be done under mild conditions, at low temperatures, since the hydroperoxides are heat labile ;;"d;. Recently, progress has been made in the development of highperformance liquid chromatography, and this technique has been successfully applied to the analysis of many compounds (11). Methyl esters of the isomers of hydroperoxylinoleic acids produced by soybean and potato lipoxygenase have been separated by high-performance