Real‐time continuous‐flow spin trapping of hydroxyl free radical in the ischemic and post‐ischemic myocardium

Abstract

Real‐time monitoring of spin‐trapped oxygen‐derived free radicals released by the isolated ischemic and reperfused rat heart has been achieved by ESR analysis of the coronary effluents using continuous flow detection and high‐speed acquisition techniques. Two nitrone spin traps 5,5‐dimethyl pyrroline 1‐oxide (Me2PnO) and 3,3,5,5‐tetramethyl pyrroline 1‐oxide (Me4PnO) have been separately perfused at a concentration of 40 mM during a sequence of 50 min of low‐flow ischemia (1 ml/min) followed by 30 min of global ischemia and subsequent reperfusion at the control flow rate (14 ml/min). ESR spectra were sequentially obtained in 5‐min or 30‐s blocks during low‐flow ischemia and reperfusion, respectively. The results show the formation of OH′ free radicals in the ischemic and reperfused heart, as demonstrated by the observation of Me2PnO‐OH (aN=aH= 14.9 G; g= 2.0053) and Me4PnO‐OH (aN= 15.2 G, aH= 16.8 G; g= 2.0055) spin adducts. There is no evidence of significant biological carbon‐centered or peroxyl free radicals spin‐adduct formation in the coronary effluents or in lipid extracts analyzed after reflow. The OH′ generation began 15–20 min after the onset of ischemia and was moderate, peaking at 30–40 min. During reperfusion, an intense formation of OH′ spin adducts was observed, with a maximum at 30–60 s and a further gradual decrease over the following 2 min. Cumulative integrated values of the amount of spin adducts released during the ischemic period show a Me2PnO‐OH level fourfold greater than that of Me4PnO‐OH. It was 2.5 times greater during reflow, reflecting slower kinetics with the more stable Me4PnO. The original ESR detection technique developed in this study allows accurate real‐time quantitative monitoring of the oxygen‐derived free radicals generated during myocardial injury. It might provide a quick and reliable new means for assessing the efficacy of free‐radical inhibitors. Copyright © 1989, Wiley Blackwell. All rights reserved