Measurement of hydroxy-conjugated dienes after ischemia-reperfusion in canine skeletal muscle

Recent studies have suggested that oxygen-derived free radicals are involved in the reperfusion injury of ischemic skeletal muscle. Although postischemic necrosis and increased vascular permeability have been attenuated with the addition of free radical scavengers, no unequivocal chemical evidence for free radical injury in skeletal muscle is available. The purpose of this study was to identify products of free radical-mediated membrane injury by isolation of lipid oxidation products (hydroxy-conjugated dienes) from postischemic skeletal muscle. The bilateral canine gracilis muscle model was used, and in each pair one muscle was exposed to 3 and the other to 5 h of normothermic ischemia. Muscle biopsies were taken before and at end ischemia, as well as during the first 3 h of reperfusion. Phospholipids were extracted from the muscle biopsies and the fatty acids hydrolyzed from the 2 position. After methylation, the oxidized fatty acid esters were separated by high-performance liquid chromatography. Hydroxy diene peaks absorbing at 235 nm were collected and subjected to gas chromatography-mass spectrometry (GC-MS) for positive structural identification. No significant increase in the level of conjugated dienes occurred during ischemia. Significant increases, however, were detected during the period of reperfusion, although the time when peak levels were achieved varied between animals. The cumulative sum of dienes produced during reperfusion in both 3- and 5-h muscles was significantly increased over pre- and end-ischemic values. The hydroxy-conjugated diene isomers of 18:2 and 20:4 were positively identified in reperfusion biopsies by GC-MS. These studies provide chemical evidence of free radical-mediated lipid oxidation during reperfusion of ischemic skeletal muscle.