Nitration of γ-tocopherol prevents its oxidative metabolism by HepG2 cells

γ-tocopherol (γT) is one of the major forms of vitamin E consumed in the diet. Previous reports have suggested increased levels of nitrated γ-tocopherol (5-NO2-γT) in smokers and individuals with conditions associated with elevated nitrative stress. The monitoring of 5-NO2-γT and its possible metabolite(s) may be a useful marker of reactive nitrogen species generation in vivo. The major pathway for the metabolism of γT is the cytochrome P450 dependent oxidation to its water-soluble metabolite γ-CEHC, which is excreted in urine. In order to determine if 5-NO2-γT could be metabolised via the same route and detected in urine we developed a sensitive gas chromatography–mass spectrometry assay for 5-NO2-γ-CEHC. 5-NO2-γ-CEHC was synthesised and its structure confirmed by proton nuclear magnetic resonance and mass spectrometry. While γ-CEHC was abundant in urine from healthy volunteers, as well as patients with coronary heart disease and type 2 diabetes, 5-NO2-γ-CEHC was undetectable (limit of detection of 5 nM). To understand this observation we examined the uptake and metabolism of γT and 5-NO2-γT by HepG2 cells. γT was readily incorporated into cells and metabolised to γ-CEHC over a period of 48 hours. In contrast, 5-NO2-γT was poorly incorporated into HepG2 cells and not metabolised to 5-NO2-γ-CEHC over the same time period. We conclude that nitration of γT prevents its incorporation into liver cells and therefore its metabolism to the water-soluble metabolite. Whether 5-NO2-γT could be metabolised via other pathways in vivo requires further investigation.