Direct measurement of myocardial free radical generation in an in vivo model: Effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase

Objectives. The purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow. Background. This phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts. Methods. Thirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 °K. Results. Three radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with g_I = 2.033 and g_I = 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and a_N = 24 G suggestive of a nitrogencentered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 ± 0.8 μmol compared with 4.4 ± 0.9 μmol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 ± 2.5 μmol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 ± 1.2 μmol (p < 0.05 vs. Group III). Conclusions. This study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.