DIRECT-DETECTION AND QUANTIFICATION OF SINGLET OXYGEN DURING ISCHEMIA AND REPERFUSION IN RAT HEARTS

To detect singlet oxygen (O-1(2)) in postischemic reperfused hearts, 5,8-endoperoxide, an oxidation product of beta-carotene, was used as a marker for O-1(2) 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 mu M 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 O-1(2) 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 O-1(2) 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 O-1(2) in the heart, 2) O-1(2) production during reperfusion is dependent on the duration of initial ischemia, and 3) O-1(2) is one of the major factors in postischemic reperfusion injury.