Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples

Abstract

A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n−6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37°C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic, analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate andtert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20–40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were, effective inducers n−6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were signficantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n−6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation.