Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.