Nitrotyrosine as a new marker for endogenous nitrosation and nitration

A sensitive and selective method has been developed for analysing 3-nitrotyrosine (NTTYR), an exposure marker for exogenous and endogenous nitrosating or nitrating agents, in tissue and blood proteins by gas chromatography-thermal energy analysis. Using this method, a number of kinetic studies were carried out. Free and protein-bound tyrosine were reacted easily to yield NTTYR. The method was also applied to the study of NTTYR formation in vivo; a dose-dependent increase in NTTYR was seen in both plasma proteins and haemoglobin obtained from rats 24 h after intraperitoneal injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid. About 44% and 5% of the oral dose of NTTYR (100 micrograms/rat), respectively, was excreted as these metabolites. Some human urine samples were analysed for NHPA by gas chromatography-thermal energy analysis after ethyl acetate extraction and high-performance liquid chromatography purification; 2.8 +/- 2.3 (mean +/- SD; n = 11) micrograms/24 h, ranging from 0-7.9 micrograms/24 h, were detected (detection limit, 0.2 micrograms/l). In conclusion, NTTYR in proteins or its metabolites in urine could be readily analysed by gas chromatography-thermal energy analysis as a new, additional marker for endogenous nitrosation and nitration.