Reduction of hydroxyl radical generation in a rat hindlimb model of ischemia-reperfusion injury using crosslinked hemoglobin-superoxide dismutase-catalase

The effects of PolyHb (intermolecularly crosslinked hemoglobin) and PolyHb-SOD-CAT (intermolecularly crosslinked hemoglobin, superoxide dismutase and catalase) on the production of hydroxyl radical was studied using a rat hindlimb model of ischemia-reperfusion. Hydroxyl radical generation was assessed by an indirect assay based on the hydroxylation of 4-hydroxybenzoate. The hydroxylation product, 3,4 dihydroxybenzoate (3,4 DHBA), was analyzed by high performance liquid chromatography and electrochemical detection. The identification of 3,4 DHBA was confirmed by analysis of authentic standard and an in vitro hydroxyl radical generation system. Ischemia was induced in both hindlimbs by ligation of the infrarenal aorta. After a 4hr ischemic period, hindlimbs were simultaneously perfused with PolyHb-SOD-CAT (5 g/dl) into one limb and PolyHb (5 g/dl) into the other limb via femoral arterial catheters. Each perfusate also contained the hydroxyl radical trap, 4-hydroxbenzoate (5 mM). Femoral venous effluents were analyzed for the presence of the 3,4 DHBA. Data indicates that greater 3,4 DHBA production occurs during PolyHb perfusion as compared to PolyHb-SOD-CAT. These preliminary results show that perfusion with PolyHb-SOD-CAT may alleviate oxidative stress in a model of ischemia-reperfusion.