In this study we compared two methods, electron spin resonance (ESR) spectroscopy and high performance liquid chromatography (HPLC), which are currently used to detect directly hydroxyl radical (OH.) formation in the ischemic and reperfused heart. Isolated buffer-perfused rat hearts were subjected to 30 min of normothermic global ischemia followed by 30 min of reperfusion. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) was used as a spin-trap agent to detect OH. radicals by ESR and HPLC. In additional HPLC studies, salicylic acid was infused into the heart for the detection of OH. radicals. In all studies, the effects of Superoxide dismutase (SOD) and catalase (CAT) on the OH. generation were examined. The results of our studies indicate that, irrespective of the method, OH. was always detected when an ischemic heart was reperfused and showed ventricular fibrillation. The OH. concentration increased dramatically between 60 and 90 sec of reperfusion, peaked between 180 and 210 sec, and then progressively decreased. In all cases, both SOD and CAT were able to reduce the formation of OH. radicals, with SOD being relatively more effective. Our results indicate that OH. was produced only in the fibrillating hearts that peaked between 180 and 210sec (1.64 ± 0.09 nmol/ mL measured by ESR), but not in the non-fibrillating hearts. Although SOD or CAT reduced the OH. formation, they had no effects on the incidence of reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT). However, when SOD (5 × 104 IU/L) was coadministered with CAT (5 × 104 IU±L), the incidence of reperfusion-induced VF (total) and VT was reduced from their control value of 92 and 100 to 33 (P < 0.05) and 50% (P < 0.05), respectively. The results of this study indicate that the HPLC method, as well as ESR, can be used to detect OH. formation in ischemic/ reperfused hearts. Because of the convenience, reproduribility and greater sensitivity, the HPLC technique may be more suitable for OH. detection. Our results further suggest the potential therapeutic value of the combination therapy of SOD and CAT for the reduction of reperfusion-induced VF and VT.
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