title: Nitration of gamma-tocopherol prevents its oxidative metabolism by HepG2 cells.
abstract: Gamma-tocopherol (gammaT) is one of the major forms of vitamin E consumed in the diet. Previous reports have suggested increased levels of nitrated gamma-tocopherol (5-NO2-gammaT) in smokers and individuals with conditions associated with elevated nitrative stress. The monitoring of 5-NO2-gammaT and its possible metabolite(s) may be a useful marker of reactive nitrogen species generation in vivo. The major pathway for the metabolism of gammaT is the cytochrome P450 dependent oxidation to its water-soluble metabolite gamma-CEHC, which is excreted in urine. In order to determine if 5-NO2-gammaT could be metabolised via the same route and detected in urine we developed a sensitive gas chromatography-mass spectrometry assay for 5-NO2-gamma-CEHC. 5-NO2-gamma-CEHC was synthesised and its structure confirmed by proton nuclear magnetic resonance and mass spectrometry. While gamma-CEHC was abundant in urine from healthy volunteers, as well as patients with coronary heart disease and type 2 diabetes, 5-NO2-gamma-CEHC was undetectable (limit of detection of 5 nM). To understand this observation we examined the uptake and metabolism of gammaT and 5-NO2-gammaT by HepG2 cells. gammaT was readily incorporated into cells and metabolised to gamma-CEHC over a period of 48 hours. In contrast, 5-NO2-gammaT was poorly incorporated into HepG2 cells and not metabolised to 5-NO2-gamma-CEHC over the same time period. We conclude that nitration of gammaT prevents its incorporation into liver cells and therefore its metabolism to the water-soluble metabolite. Whether 5-NO2-gammaT could be metabolised via other pathways in vivo requires further investigation.
authors.affiliation: School of Medicine and Pharmacology, University of Western Australia, PO Box X2213, GPO Perth 6847, West Australia.
authors.forename: Jason H Y
authors.initials: JH
authors.lastname: Wu
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Hodgson
authors.forename: Natalie C
authors.initials: NC
authors.lastname: Ward
authors.forename: Michael W
authors.initials: MW
authors.lastname: Clarke
authors.forename: Ian B
authors.initials: IB
authors.lastname: Puddey
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Croft
mesh_terms: D000328:Adult; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D002839:Chromans / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D011422:Propionates / Q000652:urine; D026361:Reactive Nitrogen Species / Q000378:metabolism; D024504:gamma-Tocopherol / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000652:urine
chemical_list: C406664:5-nitro-gamma-tocopherol; D002839:Chromans; D065607:Cytochrome P-450 Enzyme Inhibitors; D011422:Propionates; D026361:Reactive Nitrogen Species; C107690:2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman; D024504:gamma-Tocopherol; D003577:Cytochrome P-450 Enzyme System