title: 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 degrees 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 microL 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.
authors: Department of Chemistry, Jackson State University, MS 39217, USA.
authors: Y M
authors: YM
authors: Liu
authors: H
authors: H
authors: Jinno
authors: M
authors: M
authors: Kurihara
authors: N
authors: N
authors: Miyata
authors: T
authors: T
authors: Toyo'oka
mesh_terms: D000447:Aldehydes / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007834:Lasers; D015227:Lipid Peroxidation; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008297:Male; D008315:Malondialdehyde / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000447:Aldehydes; D008315:Malondialdehyde; C027576:4-hydroxy-2-nonenal