BACKGROUND: Surgically induced ischemia and reperfusion is frequently accompanied by local and remote organ injury. It was hypothesized that this procedure may produce injurious oxidants such as hydrogen peroxide (H2O2), which, if unscavenged, will generate the highly toxic hydroxyl radical (.OH). Accordingly, it was proposed that tourniquet-induced exsanguination for limb surgery may be a useful ischemia-reperfusion model to investigate the presence of oxidants, particularly H2O2. METHODS: In ten patients undergoing knee surgery, catheters were placed in the femoral vein of the limb operated on for collection of local blood and in a vein of the arm for sampling of systemic blood. Tourniquet-induced limb exsanguination was induced for about 2 h. After tourniquet release (reperfusion), blood samples were collected during a 2-h period for measurement of H2O2, xanthine oxidase activity, xanthine, uric acid (UA), glutathione, and glutathione disulfide. RESULTS: At 30 s of reperfusion, H2O2 concentrations increased (approximately 90%) from 133 +/- 5 to 248 +/- 8 nmol.ml-1 (P < 0.05) in local blood samples, but no change was evident in systemic blood. However, in both local and systemic blood, xanthine oxidase activity increased approximately 90% (1.91 +/- 0.07 to 3.93 +/- 0.41 and 2.19 +/- 0.07 to 3.57 +/- 0.12 nmol UA.ml-1.min-1, respectively) as did glutathione concentrations (1.27 +/- 0.04 to 2.69 +/- 0.14 and 1.27 +/- 0.03 to 2.43 +/- 0.13 mumol.ml-1, respectively). At 5 min reperfusion, in local blood, H2O2 concentrations and xanthine oxidase activity peaked at 796 +/- 38 nmol.ml-1 (approximately 500%) and 11.69 +/- 1.46 nmol UA.ml-1.min-1 (approximately 520%), respectively. In local blood, xanthine and UA increased from 1.49 +/- 0.07 to 8.36 +/- 0.33 nmol.ml-1 and 2.69 +/- 0.16 to 3.90 +/- 0.18 mumol.ml-1, respectively, whereas glutathione and glutathione disulfide increased to 5.13 +/- 0.36 mumol.ml-1 and 0.514 +/- 0.092 nmol.ml-1, respectively. In systemic blood, xanthine oxidase activity peaked at 4.75 +/- 0.20 UA nmol.ml-1.min-1. At 10 min reperfusion, local blood glutathione and UA peaked at 7.08 +/- 0.46 mumol.ml-1 and 4.67 +/- 0.26 mumol.ml-1, respectively, while the other metabolites decreased significantly toward pretourniquet levels. From 20 to 120 min, most metabolites returned to pretourniquet levels; however, local and systemic blood xanthine oxidase activity remained increased 3.76 +/- 0.29 and 3.57 +/- 0.37 nmol UA.ml-1.min-1, respectively. Systemic blood H2O2 was never increased during the study. During the burst period (approximately 5-10 min), local blood H2O2 concentrations and xanthine oxidase activities were highly correlated (r = 0.999). CONCLUSIONS: These studies suggest that tourniquet-induced exsanguination for limb surgery is a significant source for toxic oxygen production in the form of H2O2 and that xanthine oxidase is probably the H2O2-generating enzyme that is formed during the ischemia-reperfusion event. In contrast to the reperfused leg, the absence of H2O2 in arm blood demonstrated a balanced oxidant scavenging in the systemic circulation, despite the persistent increase in systemic xanthine oxidase activity.
BACKGROUND: Surgically induced ischemia and reperfusion is frequently accompanied by local and remote organ injury. It was hypothesized that this procedure may produce injurious oxidants such as hydrogen peroxide (H2O2), which, if unscavenged, will generate the highly toxic hydroxyl radical (.OH). Accordingly, it was proposed that tourniquet-induced exsanguination for limb surgery may be a useful ischemia-reperfusion model to investigate the presence of oxidants, particularly H2O2. METHODS: In ten patients undergoing knee surgery, catheters were placed in the femoral vein of the limb operated on for collection of local blood and in a vein of the arm for sampling of systemic blood. Tourniquet-induced limb exsanguination was induced for about 2 h. After tourniquet release (reperfusion), blood samples were collected during a 2-h period for measurement of H2O2, xanthine oxidase activity, xanthine, uric acid (UA), glutathione, and glutathione disulfide. RESULTS: At 30 s of reperfusion, H2O2 concentrations increased (approximately 90%) from 133 +/- 5 to 248 +/- 8 nmol.ml-1 (P < 0.05) in local blood samples, but no change was evident in systemic blood. However, in both local and systemic blood, xanthine oxidase activity increased approximately 90% (1.91 +/- 0.07 to 3.93 +/- 0.41 and 2.19 +/- 0.07 to 3.57 +/- 0.12 nmol UA.ml-1.min-1, respectively) as did glutathione concentrations (1.27 +/- 0.04 to 2.69 +/- 0.14 and 1.27 +/- 0.03 to 2.43 +/- 0.13 mumol.ml-1, respectively). At 5 min reperfusion, in local blood, H2O2 concentrations and xanthine oxidase activity peaked at 796 +/- 38 nmol.ml-1 (approximately 500%) and 11.69 +/- 1.46 nmol UA.ml-1.min-1 (approximately 520%), respectively. In local blood, xanthine and UA increased from 1.49 +/- 0.07 to 8.36 +/- 0.33 nmol.ml-1 and 2.69 +/- 0.16 to 3.90 +/- 0.18 mumol.ml-1, respectively, whereas glutathione and glutathione disulfide increased to 5.13 +/- 0.36 mumol.ml-1 and 0.514 +/- 0.092 nmol.ml-1, respectively. In systemic blood, xanthine oxidase activity peaked at 4.75 +/- 0.20 UA nmol.ml-1.min-1. At 10 min reperfusion, local blood glutathione and UA peaked at 7.08 +/- 0.46 mumol.ml-1 and 4.67 +/- 0.26 mumol.ml-1, respectively, while the other metabolites decreased significantly toward pretourniquet levels. From 20 to 120 min, most metabolites returned to pretourniquet levels; however, local and systemic blood xanthine oxidase activity remained increased 3.76 +/- 0.29 and 3.57 +/- 0.37 nmol UA.ml-1.min-1, respectively. Systemic blood H2O2 was never increased during the study. During the burst period (approximately 5-10 min), local blood H2O2 concentrations and xanthine oxidase activities were highly correlated (r = 0.999). CONCLUSIONS: These studies suggest that tourniquet-induced exsanguination for limb surgery is a significant source for toxic oxygen production in the form of H2O2 and that xanthine oxidase is probably the H2O2-generating enzyme that is formed during the ischemia-reperfusion event. In contrast to the reperfused leg, the absence of H2O2 in arm blood demonstrated a balanced oxidant scavenging in the systemic circulation, despite the persistent increase in systemic xanthine oxidase activity.