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 (MePnO) 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. 1. 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. 2. 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. 3. 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. 4. 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.