Real-time continuous-flow spin trapping of hydroxyl free radical in the ischemic and post-ischemic myocardium.
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.
Pietri
S
S
Unité de Recherche Associée 1186 du Centre National de la Recherche Scientifique, Faculté de Médecine de la Timone, Marseille, France.
Culcasi
M
M
Cozzone
P J
PJ
D000818:Animals; D003327:Coronary Disease / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D003497:Cyclic N-Oxides; D005609:Free Radicals; C051831:3,3,5,5-tetramethyl-1-pyrroline N-oxide; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D010100:Oxygen