Detection of hydroxyl radicals in the post-ischemic reperfused heart using salicylate as a trapping agent

The formation of hydroxyl radical in the post-ischemic reperfused heart was measured with high performance liquid chromatography and ultraviolet detection using salicylic acid. Hydroxyl radicals react with salicylic acid yielding 2,3- and 2,5-dihydroxybenzoic acid, which can be separated by the liquid chromatography. Isolated rat hearts were perfused with 1 mM salicylic acid and were subjected to 30 mins of global ischemia followed by aerobic or anaerobic reperfusion at 37 degrees C. The effluent from the hearts was collected at various intervals, extracted with ether, and injected into the high performance liquid chromatography unit. 2,5-dihydroxybenzoic acid was present only after aerobic reperfusion and was not detected before ischemia. The liquid chromatography peak of 2,3-dihydroxybenzoic acid was too small for quantitation. The concentration of 2,5-dihydroxybenzoic acid was the highest within 300 s of reperfusion. 2,5-dihydroxybenzoic acid was not detected in the ischemic hearts during anaerobic reperfusion. In ischemic hearts perfused with mannitol, the amount of 2,5-dihydroxybenzoic acid after reperfusion was reduced. These data suggest that hydroxyl radicals are produced in the post-ischemic reperfused heart and that the present method is useful and reliable for the measurement of hydroxyl radicals in the heart.