Free Radical Biology & Medicine, Vol. 9, pp. 465-471, 1990 0891-5849/90 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1990 PergamonPressplc - • " Original Contribution CARDIOPROTECTION BY Cu,Zn-SUPEROXIDE DISMUTASE IS LOST AT HIGH DOSES IN THE REOXYGENATED HEART BASSAM A. OMAR,t~ NABIL M. GAD,* MARIA C. JORDAN,* SCOTT P. STRIPLIN,* WILLIAM J. RUSSELL, JAMES M. DOWNEY,* and JOE M. MCCORDt Departments of Biochemistry and *Physiology, University of South Alabama, Mobile, AL 36688, USA (Received 26 April 1990; Revised and Accepted 10 August 1990) Abstract--Limited dose-response curves for superoxide dismutase (SOD) were assessed in isolated and in vivo hearts. SOD at 2.3, 7, 20, or 50 mg/L suppressed CK release in Langendorff rat hearts by 61%, 63%, 72%, and 30%, respectively. SOD at 0.5, 1, 5, and 50 mg/L suppressed LDH release in Langendofff rabbit hearts by 32%, 48%, 54%, and - 12%, respectively. In rabbit hearts subjected to coronary artery ligation and reperfusion in vivo, SOD at 2, 5, or 15 mg/kg reduced infarct size by 10%, 30% or 19%, respectively, while 50 mg/kg increased infarct size by 28%. In conclusion, while SOD was protective at low doses in all models, protection was lost at higher doses in the isolated rat and rabbit hearts, and exacerbation of damage was seen in the in vivo rabbit hearts. Keywords--Myocardial ischemia, Reperfusion injury, Free radicals, Superoxide dismutase, Dose-response, Rat, Rabbit INTRODUCTION Myocardial reperfusion injury is a complex and contro- versial phenomenon. The apparent free radical compo- nent of this injury varies considerably from model to model. The enzyme superoxide dismutase (SOD) has been at the center of this controversy in that there is lit- fie agreement as to whether SOD does ~-4 or does not 5-7 protect the reperfused or reoxygenated heart. The models of myocardial injury differ in many important ways. While some models are perfused with blood which contains both antioxidants and neutrophils, others have been perfused with buffer. The end points employed for assessing ischemic damage have included release of biochemical markers of cell injury, preservation of mechanical function, reduction in the incidence of arrhythmias, infarct size by tetrazolium staining or histology, and production of oxidation products. All of these indicators have specific limitations and none can be absolutely equated with injury per se. The species is also critical. Rat and dog have higher levels of xanthine oxidase in their hearts 3 than rabbits 8 and humans, 9 suggesting a fundamental difference in their response to tPresent Address: Webb-Waring Lung Institute, University of Colo- rado, 4200 E. Ninth Ave., Box C-321, Denver, CO 80262. ~:Author to whom correspondence should be addressed. ischemia/reperfusion. Rabbits also have very high levels of extracellular (EC) SOD l° which might provide sub- stantial protection to the heart in blood-perfused mod- els. The dose and schedule of SOD administration has differed widely among the studies. Many published studies have employed a single SOD dose, and usually quite a high one. 6 It is possible that SOD may have been nonoptimally administered in some of the negative studies. In the present study we obtained limited dose- response curves for SOD in two widely employed mod- els, the hypoxic/reoxygenated isolated heart, and the in vivo model of regional coronary artery occlusion and reperfusion. Furthermore, both rabbit and rat hearts were examined in the former protocol. The most significant finding is that while SOD was protective to the hearts in all models at relatively low doses, it lost its protec- tive effect at higher doses. MATERIALS AND METHODS Enzyme analysis Lactate dehydrogenase (LDH) was assayed by moni- toring the rate of oxidation of NADH according to Sigma procedure #340-UV (Sigma Chemicals, St. Louis, MO). Creatine kinase (CK) was assayed by the dephosphory- lation of phosphocreatine and the subsequent formation 465