REOXYGENATION INJURY IN ISOLATED RAT HEPATOCYTES - RELATION TO OXYGEN-FREE RADICALS AND LIPID-PEROXIDATION

Oxygen free radical (OFR) formation and lipid peroxidation (LP) were m easured in freshly isolated perfused rat hepatocytes during 2-h reoxyg enation after 2.5 h of anoxia. Superoxide anions and hydrogen peroxide (H2O2) were detected by enhanced chemiluminescence. LP and cell damag e were assessed by measuring malondialdehyde (MDA) and lactic dehydrog enase (LDH) release, respectively. During anoxia, the chemiluminescenc e decreased to background levels and MDA remained constant, whereas LD H release increased progressively to 168 +/- 22 mU/min in 2.5 h. Durin g reoxygenation after a 2.5-h period of anoxia, superoxide formation i ncreased rapidly to 125 +/- 16 nA and then it declined progressively t oward the control level. At the same time, H2O2 production exhibited a biphasic pattern with an initial peak reaching 78 +/- 16 nA at 15.5 /- 1 min, followed by a slower increase to 92 +/- 14 nA during the 2nd h. LDH release increased from 168 +/- 22 to 286 +/- 32 mU/min in the first 30 min of reoxygenation and then declined toward the control rat e during the 2nd h. MDA release increased continuously from 1.16 +/- 0 .18 to 7.75 +/- 0.74 pmol/min. OFR generation occurred 15-30 min befor e the peak rise in LDH. Moreover, after shorter periods of anoxia (1-2 h), hepatocytes produced measurable amount of OFR but without a signi ficant increase in LDH release. These results demonstrate that 1) isol ated liver parenchymal cells generate measurable amounts of superoxide anions and of H2O2 during reoxygenation after 1-2.5 h of anoxia, 2) l ipid peroxidation follows the formation of OFR, and 3) reoxygenation i njury is correlated to OFR generation but not to lipid peroxidation.