REDUCTION OF HYDROXYL RADICAL GENERATION IN A RAT HINDLIMB MODEL OF ISCHEMIA-REPERFUSION INJURY USING CROSS-LINKED HEMOGLOBIN-SUPEROXIDE DISMUTASE-CATALASE

The effects of PolyHb (intermolecularly crosslinked hemoglobin) and Po lyHb-SOD-CAT (intermolecularly crosslinked hemoglobin, superoxide dism utase and catalase) on the production of hydroxyl radical was studied using a rat hindlimb model of ischemia-reperfusion. Hydroxyl radical g eneration was assessed by an indirect assay based on the hydroxylation of 4-hydroxybenzoate. The hydroxylation product, 3,4 dihydroxybenzoat e (3,4 DHBA), was analyzed by high performance liquid chromatography a nd electrochemical detection. The identification of 3,4 DHBA was confi rmed by analysis of authentic standard and an in vitro hydroxyl radica l generation system. Ischemia was induced in both hindlimbs by ligatio n of the infrarenal aorta. After a 4hr ischemic period, hindlimbs were simultaneously perfused with PolyHb-SOD-CAT (5 g/dl) into one limb an d PolyHb (5 g/dl) into the other limb via femoral arterial catheters. Each perfusate also contained the hydroxyl radical trap, 4-hydroxbenzo ate (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 prelimina ry results show that perfusion with PolyHb-SOD-CAT may alleviate oxida tive stress in a model of ischemia-reperfusion.