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

✍ Scribed by Yi-Ming Liu; Hideto Jinno; Masaaki Kurihara; Naoki Miyata; Toshimasa Toyo’oka

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 82 KB
• Volume: 13
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/(sici)1099-0801(199902)13:1<75::aid-bmc817>3.0.co;2-6

✦ Synopsis

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 C 18 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 mL 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/10 6 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 (TBA) assay.