J.Jpn. Surg. Soc.. 93(4): 369-376, 1992

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Original article

MEASUREMENT OF FREE RADICAL GENERATION FROM ENDOTHELIAL CELLS AND OBSERVATION OF CELL INJURY EXPOSED TO ANOXIA-REOXYGENATION

Oxygen free radicals have been demonstrated to be important mediators in postischemic reperfusion injury. In this study, I determined the superoxide and the hydrogen peroxide generation from human umbilical endothelial cells on reoxygenation following anoxic incubation (1% O₂, 5% CO₂, 94% N₂). The superoxide generation, detected by the reduction of cytochrome,c,was at its maximum 3 minutes after reoxygenation in any anoxic interval. The hydrogen peroxide production, detected by the fluorometric analysis, was observed later than that of superoxide. Treatment of EC with superoxide dismutase and allopurinol attenuated the superoxide production, and catalase attenuated the hydrogen peroxide. Cell injury was assessed by both fura-2 release assay and trypan blue dye exclusion methods. Although cell injury was less than 20% in anoxic condition, it was remarkably increased after reoxygenation. However this cell injury was not completely prevented in the presence of free radical scavengers. Allopurinol was more effective than superoxide dismutase or catalase. In conclusion, EC are the major source of free radicals in postischemic reperfusion which are originated mainly from xanthinene oxidase system and these radicals may also contribute, at least in part, to the EC injury.

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