Evaluation of purine nucleotide loss, lipid peroxidation and ultrastructural alterations in post-hypoxic hepatocytes

1. Hypoxic alterations in isolated rat hepatocytes were demonstrated by a 90% ATP loss during 60 min of ischaemia and temporary increases of nucleotide degradation products. 2. The oxidative stress during reoxygenation was demonstrated in these cells by a decrease in reduced glutathione (GSH) concentration (30%) and a threefold increase in lipid peroxidation products such as 4-hydroxynonenal and thiobarbituric acid-reactive substances (TBA-RSs). The tremendous GSH loss could not be balanced by the slight oxidized glutathione (GSSG) increase during reoxygenation. 3. For the first time the involvement of free radicals was directly demonstrated using electron spin resonance (ESR) spectroscopy in reoxygenated liver cells. Using the spin trap 5,5-dimethylpyrroline-1-oxide (DMPO), a carbon-centred radical and the adduct of the hydroxyl radical could be detected during early reoxygenation. 4. Morphological alteration of cells was observed, beginning during hypoxia and increasing during post-hypoxic reoxygenation. Electron microscopic findings of hypoxic and posthypoxic cell damage included pyknosis of nuclei, spherical transformation of mitochondria and increased number of vesicles.