Direct detection and quantification of singlet oxygen during ischemia and reperfusion in rat hearts

To detect singlet oxygen (1O2) in postischemic reperfused hearts, 5,8-endoperoxide, an oxidation product of beta-carotene, was used as a marker for 1O2 generation and was quantified using high-performance liquid chromatography (HPLC). Isolated rat hearts were subjected to ischemia for 5, 10, 20, 30, and 60 min followed by 10 min of reperfusion with buffer containing 25 microM beta-carotene. The coronary effluent was collected, extracted, and injected into the HPLC unit. The production of 5,8-endoperoxide was maximum during the first 2 min of reperfusion. Maximal accumulated amount of 1O2 was observed in hearts subjected to 60-min ischemia (36.2 +/- 1.7 nmol.10 min-1.g-1) as compared with 10-min ischemia (6.2 +/- 1.0 nmol.10 min-1.g-1). There was a good correlation between the amount of 1O2 production and cardiac function. Treatment with 25 mM histidine significantly decreased 5,8-endoperoxide from 7.02 +/- 0.47 to 0.98 +/- 0.11 nmol.min-1.g-1 (P < 0.01) and improved cardiac function in the group with 60-min ischemia. This study demonstrates that 1) the present method is useful and reliable for the measurement of 1O2 in the heart, 2) 1O2 production during reperfusion is dependent on the duration of initial ischemia, and 3) 1O2 is one of the major factors in postischemic reperfusion injury.