Determination of 4-hydroxy-2-nonenal in primary rat hepatocyte cultures by liquid chromatography with laser induced fluorescence detection

Abstract

An HPLC (high performance liquid chromatography) method with laser induced fluorescence (LIF) detection is described for the determination of 4-hydroxy-2-nonenal (HNE) formed from lipid peroxidation in rat hepatocytes. Carbonyl compounds were fluorescently labelled by incubating the hepatocyte samples with a tagging reagent, 4-(2-carbazoylpyrrolidin-1-yl)7-nitro-2, 1,3-benzoxadiazole (NBD-ProCZ), at 60°C for 10 min. The hydrazone derivatives were extracted with a C18 solid phase extraction (SPE) cartridge and separated on a reversed-phase HPLC column. The detection limit was 2.5 fmol or 0.5 nM (5 μL injection) of HNE in the cell homogenate. Method precision (C.V.) was 5% at the 5 nM level. The method has been used to determine free HNE in rat hepatocyte samples treated with several pro-oxidant toxins. A significant HNE increase (from 4 to 27.6 pmol/106 cells) was observed with the samples treated by allyl alcohol. The results were in accordance with those for malondialdehyde formation as measured by a thiobarbituric acid (TEA) assay.