Reoxygenation injury in isolated rat hepatocytes: relation to oxygen free radicals and lipid peroxidation

Abstract

Oxygen free radical (OFR) formation and lipid peroxidation (LP) were measured in freshly isolated perfused rat hepatocytes during 2-h reoxygenation after 2.5 h of anoxia. Superoxide anions and hydrogen peroxide (H2O2) were detected by enhanced chemiluminescence. LP and cell damage were assessed by measuring malondialdehyde (MDA) and lactic dehydrogenase (LDH) release, respectively. During anoxia, the chemiluminescence decreased to background levels and MDA remained constant, whereas LDH release increased progressively to 168 +/- 22 mU/min in 2.5 h. During reoxygenation after a 2.5-h period of anoxia, superoxide formation increased rapidly to 125 +/- 16 nA and then it declined progressively toward 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 rate 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 before the peak rise in LDH. Moreover, after shorter periods of anoxia (1-2 h), hepatocytes produced measurable amount of OFR but without a significant increase in LDH release. These results demonstrate that 1) isolated liver parenchymal cells generate measurable amounts of superoxide anions and of H2O2 during reoxygenation after 1-2.5 h of anoxia, 2) lipid peroxidation follows the formation of OFR, and 3) reoxygenation injury is correlated to OFR generation but not to lipid peroxidation.