The Clarification of Mechanisms for Electron Transfer and Identification of Radicals in Myocardium

本研究通过自由基清除剂的心肌保护作用间接显示氧自由基(O2-FR)参与再灌注损伤，并用电子自旋共振(ESR)波谱直接显示冻存心肌中的自由基。用双荧光法测定心肌坏死区与灌注区的比值来评价心肌损伤的严重程度。经超氧化物歧化酶、过氧化氢酶和甘露醇处理的犬出现局限性坏死(34.1±12.0%比66.0±11.3%)，间接证实O_2-FR在缺血/再灌注损伤的发生中起重要作用。为了直接证明自由基假说，用电子自旋共振波谱(ESR)测定了冰冻心肌中的自由基。ESR谱显示了四个自由基，其中两个被其他研究者称为O_2-FR和N-中心的自由基被证明是机械产生的人工自由基。在无伪影的样品中，未检出O2-FR。心肌固有的一种自由基被鉴定为辅酶Q_(10)自由基，这可能为再灌注损伤的严重程度提供一个指标。ESR自旋捕捉法是一种直接测量氧自由基的最佳方法。

This study indirectly showed the involvement of oxygen derived free radicals (O_2-FR) in reperfusion injury by the myocardial protection of radical scavengers and directly demonstrated free radicals in the frozem myocardium using electron spin resonance (ESR) spectroscopy.In 19 open chest dogs, the left anterior descending coronary artery was occluded for 90 min and subsequently reperfused for 60 min. The severity of myocardial injury was evaluated by ratios of necrotic to perfused areas determined by dual fluorescence methods. Dogs treated with SOD, catalase and mannitol disclosed significantly limited necrosis than that in control (34.1 12.0 vs. 66.0 11.3%); indirectly veraifying that the O_2-FR play an important role in the genesis of ischemia/reperfusion injury.For direct proof of free radical hypothesis, radicals in the frozen myocardium were measured using electron spin resonance spectroscopy (ESR). The ESR spectra exhibited four radicals, however, two radicals which had been previously claimed as O_2-FR and N-centered radical by other investigators were shown as mechanically yielded artifactual radicals. In artifact-free samples, O_2-FR could not be detected. One radical native to the myocardium was identified as coenzyme Q_<10> radical which might provide an index of severity of reperfusion injury. Spin trapping method with ESR can be an optimal method for the direct measurement of oxygen derived radicals.