Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins

3-Nitrotyrosine (NTYR) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analysing NTYR by gas chromatography with a thermal energy analyser (GC-TEA) was developed. Using this method, a number of kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTYR. NTYR formation in vivo showed a dose-dependent increase in NTYR in both plasma proteins and haemoglobin obtained from rats 24 hr after ip injection of various doses (0.5-2.5 μmol/rat) of tetranitromethane. Major urinary metabolites of NTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44% and 5% of the oral dose of NTYR (100 μg/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analysed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 μg/24 hr, mean ± SD 2.8 ± 2.3 (n = 11) were detected (detection limit 0.2 μg/litre). NTYR in proteins or its metabolites in urine can be readily analysed by GC-TEA as a new/additional marker for endogenous nitrosation and nitration.