Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: nonspecificity of thiobarbituric acid-reactivity.

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (approximately 20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced approximately 40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA, TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result.