Interstitial equilibration of superoxide dismutase correlates with its protective effect in the isolated rabbit heart

We examined the effect of preischemic equilibration of the rabbit heart with superoxide dismutase (SOD) on the extent of recovery of contractile function following an episode of ischemia. First, hearts were perfused with Krebs-Henseleit buffer. The pulmonary artery was cannulated and its flow diverted as the vascular effluent, and all other orifices were tied off. The fluid seeping from the epicardial surface represented the interstitial outflow. SOD was added to the perfusate and the interstitial and vascular effluents were assayed for SOD at regular intervals. Second, hearts were perfused in the Langendorff mode. SOD was included in the perfusate at all times at 20,000 U/l. After either 15 or 50 min of equilibration the hearts were subjected to 1 h of ischemia followed by 1 h of reperfusion. The developed tension was measured via a balloon in the left ventricle. Control hearts showed a recovery of developed tension of 63 +/- 12%. Human recombinant (h) Cu,Zn-SOD, which equilibrated with the interstitial fluid by 20 +/- 10% and 92 +/- 7% after 15 and 60 min of perfusion respectively, caused a recovery of 68 +/- 29% (non-significant) and 92 +/- 18% (P less than 0.01) with 15 and 50 min of equilibration respectively. The positively charged hrMn-SOD and sheep Cu,Zn-SOD, however, equilibrated much faster reaching 84 +/- 13% and 95 +/- 11% at 15 min respectively, which correlated with a recovery of 99 +/- 11% and 96 +/- 10% (P less than 0.01) respectively. HrCu,Zn-SOD conjugated to polyethylene glycol equilibrated much slower reaching 38 +/- 10% after 1 h, which correlated with lack of protection even after 50 min of equilibration. Therefore, the protection afforded by SOD to the isolated rabbit heart correlates with the concentration of SOD in the interstitial fluid. The rate of equilibration depends on the charge as well as the size of the enzyme.