Abstract
Here we present a simple, specific, and sensitive liquid chromatography/mass spectrometry method to measure 4-hydroxy-2(E)-nonenal-glutathione (HNE-GSH), the major stable hepatic metabolite of HNE after GSH conjugation, as a marker of oxidative stress in rat liver and hepatocytes. Commonly employed methods for the measurement of lipid peroxidation-derived free aldehydes or modified proteins suffer from the artificial formation of HNE or HNE adducts to cellular molecules during sample preparation and derivatization, resulting in an overestimation of background levels. Basal levels of HNE-GSH in liver tissue from untreated rats were detected in amounts of 20 pmol/g liver. Rats exposed to a single dose of iron nitrilotriacetate (Fe(III)NTA; 15 mg Fe/kg bw, ip), a model compound for the induction of oxidative stress, revealed a fivefold increase in the hepatic HNE-GSH levels compared to controls 5 h after dosing. Moreover, a significant increase in HNE-mercapturic acid (HNE-MA) and its reduced metabolite DHN-MA was evident at 5 or 24 h after treatment, which was also reflected in increased plasma concentrations of these secondary HNE-GSH metabolites. In agreement with the in vivo data, a time-dependent increase in the levels of HNE-GSH from <1 to 123 +/- 16 pmol/10(6) cells over 5 h was detected in rat hepatocytes treated with Fe(III)NTA (150 microM). An increase in cellular HNE-GSH from <1.0 to 7.2 +/- 0.3 pmol/10(6) cells could be observed in rat hepatocytes treated with allyl alcohol (500 microM, 3 h), known for generation of HNE in hepatocytes. These data suggest that the direct measurement of the stable GSH conjugation product of cellular HNE in rat primary hepatocytes or its secondary metabolites may represent a reliable biomarker of oxidative stress-induced lipid peroxidation in rat liver in vivo.
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