LACK OF MYOCARDIAL LIPID-PEROXIDATION DURING ACUTE REPERFUSION INJURY IN PERFUSED GUINEA-PIG HEARTS

Objective: The aim was to test the hypothesis that free radical induced lipid peroxidation contributes to the arrhythmias and contractile changes associated with reperfusion. Evidence of oxidative stress in the membrane lipids was sought using four different approaches: (1) determination of lipid peroxidation metabolites; (2) direct detection of free radical formation using a lipophilic spin trap; (3) assessment of lipid peroxidation substrate; and (4) evaluation of antioxidant levels in the heart. Methods: Electrical and contractile functions were monitored in isolated perfused guinea pig hearts subjected to ischaemia for 20 or 60 min followed by reperfusion for 1 or 20 min. The reperfused hearts were flushed with cold phosphate buffer with EDTA and the ventricles weighed and homogenised. Malonaldehyde content was determined using both a thiobarbituric acid spectrophotometric assay and a p-aminobenzoate spectrofluorescence assay. A lipophilic spin trap, PBN, was injected into guinea pigs 2 h before they were killed. Hearts containing spin traps were perfused for 5 min, then subjected to ischaemia followed by 1 min reperfusion. Lipid extracts containing PBN were examined using electron paramagnetic resonance spectroscopy. The content of polyunsaturated fatty acids and alpha tocopherol were determined by GLC and HPLC, respectively. Results: Malonaldehyde content of the heart tissues did not increase during any combination of ischaemic and reperfusion episodes. The assays were effective at detecting raised malonaldehyde in hearts perfused with either 0.5 mM malonaldehyde standard or 0.5 mM cumene hydroperoxide. No spin adducts were found in either the extracted membrane lipids or the aqueous layer. Spin adducts were detected in myocardial lipids from animals exposed to non-lethal doses of gamma irradiation, confirming the presence of the spin trap in the heart tissue and the ability to detect spin adducts when formed. There were no differences in the percentage of total fatty acids that were polyunsaturated between reperfused hearts and continously perfused controls. Furthermore, alpha tocopherol content of the hearts exposed to ischaemia and reperfusion was not decreased relative to hearts perfused without an ischaemic episode. Conclusions: The results suggest that the early functional disturbances during reperfusion are not related to peroxidation of membrane lipids.