The hepatocellular metabolism of 4-hydroxynonenal by alcohol dehydrogenase, aldehyde dehydrogenase, and glutathione S-transferase.

It has previously been reported that isolated rat hepatocytes rapidly and completely metabolize high concentrations of 4-hydroxy-2,3-(E)-nonenal (4-HNE). However, until this report, the degree to which oxidative-reductive and nonoxidative metabolic pathways function in the depletion of 4-HNE by isolated rat hepatocytes has been speculative. The objective of the present study was to quantitate the extent to which cellular aldehyde dehydrogenases (ALDH; EC 1.2.1.3.), alcohol dehydrogenase (ADH; EC 1.1.1.1.), and glutathione S-transferases (GST; EC 2.5.1.18) function simultaneously during hepatocellular metabolism of 4-HNE. Hepatocytes were incubated with varying concentrations of 4-HNE (50, 100, 250 microM) and reversed-phase HPLC was used to quantitate 4-HNE and the oxidative and reductive metabolites, 4-hydroxy-2-nonenoic acid and 1,4-dihydroxy-2-nonene, respectively. Conjugative metabolism of 4-HNE was determined from the depletion of cellular reduced glutathione (GSH) and concomitant formation of a GSH-4-HNE adduct detected as 2,4-dinitrofluorobenzene derivatives measured by reversed-phase HPLC. Hepatocellular elimination of 4-HNE was estimated at rates of 1.666, 0.902, and 0.219 nmol min-1 10(6) hepatocytes-1 for 50, 100, and 250 microM aldehyde, respectively. At aldehyde concentrations of 50, 100, and 250 microM the maximal concentrations of oxidative (acid) metabolites formed were 5.9, 12.7, and 28.9 nmoles 10(6) hepatocytes-1, whereas the concentrations of the reductive (diol) metabolite were 0.4, 12.6, and 42.3 nmoles 10(6) hepatocytes-1, respectively. The presence of 4-methylpyrazole or cyanamide abolished formation of the reductive metabolite 1,4-dihydroxy-2-nonene or the oxidative metabolite 4-hydroxy-2-nonenoic acid in hepatocyte suspensions. At all 4-HNE concentrations evaluated, hepatocellular glutathione was not completely depleted by the aldehyde and the depletion of cellular reduced GSH corresponded to the production of the GSH-4-HNE conjugate. Metabolism by the alcohol/aldehyde dehydrogenase pathways accounted for approximately 10% of the 4-HNE elimination, while bioconversion by GST represent 50-60% of the total 4-HNE removal by hepatocytes. The enzymatic pathways responsible for the remaining 40% of 4-HNE metabolism remain to be identified. Taken together these results describe the quantitative and dynamic importance of oxidative, reductive, and nonoxidative routes in the metabolism and detoxification of 4-HNE.