Phospholipid Peroxidation after 60 min of Global Ischaemia and 10 min of Reperfusion. A Study in the Isolated Rat Heart

Abstract

Peroxidation of polyunsaturated fatty acids in cell membranes is thought to be a crucial factor in the cascade leading to reperfusion damage in the myocardium. However, some studies also describe increased lipid peroxidation in ischaemic tissue. The present study therefore examines phospholipid peroxidation after 60 min of global ischaemia and during the initial phase of reperfusion in isolated Langendorff-perfused rat hearts. Lipids were extracted from these hearts and separated into phospholipid, triglyceride and non-esterified fatty acid fractions. The phospholipid fraction was hydrolysed with phospholipase A 2, and reverse-phase high performance liquid chromatography of the fatty acids derived from the phospholipids was performed. Peroxidized polyunsaturated fatty acids were separated from unchanged fatty acids and amounts of monohydroxy or monophydroperoxy isomers were quantified by measuring conjugated dienes by UV absorption (235 nm). Phospholipids from ischaemic as well as free-radical-exposed tissue contained increased levels of peroxidized polyunsaturated fatty acids (20.7 ± 2.4 and 20.5 ± 2.3 respectively, v 118. ± 1.4 units/mg dry weight in controls). After 2-10 min of reperfusion, a significant increase in phospholipid peroxidation was no longer detected (12.5±1.2 units/mg). The amount and the composition of non-esterified fatty acids were examined by gas chromatography. Ischaemia significantly increased both the amount of non-esterified fatty acids (1.5±0.8 v 4.9±1.8 nmol/mg dry wt) as well as the percentage composed of arachidonic acid (3.4±3.2% v 7.4%±1.4%). Fatty acids levels remained elevated during reperfusion (5.5±1.9 nmol/mg and 7.0±1.4%). In conclusion, our results have demonstrated that prolonged ischaemia alone caused phospholipid peroxidation as well as accumulation of non-esterified arachidonic acid. There was no sign of further phospholipid peroxidation during reperfusion.