Increased 4–Hydroxynonenal Levels in Experimental Alcoholic Liver Disease: Association of Lipid Peroxidation With Liver Fibrogenesis

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Abstract
The precise role of lipid peroxidation in the pathogenesis of alcoholic liver disease is still being debated. To explore the issue, this study was undertaken to investigate the status of lipid peroxidation, antioxidants and prooxidants at two discrete stages of experimental alcoholic liver disease. Male Wistar rats were intragastrically fed a high–fat diet plus ethanol for 5 or 16 wk (the duration that resulted in initiation of centrilobular liver necrosis or liver fibrosis, respectively). Lipid peroxidation was assessed in isolated microsomes and mitochondria with three parameters: malondialdehyde equivalents as determined by thiobarbituric acid assay, conjugated diene formation and 4–hydroxynonenal as a 2,4–dinitrophenylhydrazone derivative. To assess antioxidant systems, hepatic concentrations of glutathione, methionine and α–tocopherol were determined. The concentration of nonheme iron, a known prooxidant, was also measured. At wk 5, centrilobular liver necrosis was already evident in the ethanol–fed animals, with two– or threefold increases in plasma AST and ALT levels. At this stage, neither malondialdehyde equivalents nor conjugated diene values were elevated, and the 4–hydroxynonemal level was below 0.2 nmol/mg protein. Hepatic concentrations of methionine and α–tocopherol in these animals were increased two–and threefold, respectively, whereas the reduced glutathione level remained unchanged. When alcoholic liver disease had progressed to perivenular or bridging fibrosis at wk 16, all three parameters of lipid peroxidation showed consistent increases that were accompanied by significant reductions in the hepatic glutathione and methionine levels. Interestingly, the control animals pair–fed with the high–fat diet also had significantly elevated 4–hydroxynonenal levels at wk 16 compared to the wk 5 level. The liver concentration of nonheme iron was not increased at wk 5 or wk 16. These results demonstrate (a) dissociation between the initiation of alcoholic liver necrosis and enhanced lipid peroxidation, (b) association of enhanced lipid peroxidation with liver fibrogenesis and depressed antioxidant system, (c) the first demonstration of increased 4–hydroxynonenal level in experimental alcoholic liver disease and (d) possible implication of a high–fat diet in hepatic 4–hydroxynonenal generation. (Hepatology 1992;16:448-453.)