We have developed a simple and reproducible method employing chemiluminescence to measure oxygen-derived free radicals generated in oxygenated myocardium. Isolated perfused rat hearts were frozen in liquid nitrogen during the control perfusion (non-ischemia), after 30 min of global ischemia (ischemia), or after 20 min of reperfusion following 30-min global ischemia (reperfusion). The frozen hearts were ground to a fine powder and then oxygenated by mixing with phosphate-buffered saline (PBS) (pH 7.4 and PO2 183-194 mmHg) containing lucigenin. The mixed solution was injected into a chemiluminescence detection flow cell. The chemiluminescence intensity increased in relation to the lucigenin and myocardium contents of the test solution. However, it was not affected by the temperature of PBS in the range of 25 degrees C to 50 degrees C. The chemiluminescence intensity of ischemic myocardium was 2.5 times larger than that of non-ischemic or reperfused myocardium (P < 0.01). Recombinant human superoxide dismutase (r-h-SOD) reduced dose-dependently the chemiluminescence intensity induced by oxygenation of ischemic myocardium, while chemically inactivated r-h-SOD did not.
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