PLASMA 3-NITROTYROSINE IN CIGARETTE SMOKERS

Peroxjrnitrite has been associated with increased oxidative reactions and DNA damage in inflamed tissues as it may cause a reduction of plas ma antioxidants as well. Nitration of tyrosine residues of proteins le ads to the production of 3-nitrotyrosine (NTYR), which may be consider ed as a marker of NO .-dependent oxidative damage. We developed a high ly sensitive method to detect NTYR in human plasma and tested it in ci garette smokers and in healthy control subjects. Peripheral venous blo od (10 ml) was obtained in 20 healthy, asymptomatic cigarette smokers (13 males, 7 females; age: 49 +/- 11 yr) and in 18 healthy nonsmokers (10 males and 8 females; age: 36 +/- 6 yr). In smokers, plasma nicotin e, cotinine, and expired CO levels were measured. NTYR was determined with a sequential HPLC/gas chromatography-thermal energy analysis (GC- TEA) technique. The total plasma Trolox(R)-equivalent antioxidant capa city (TEAC) was also measured using metmyoglobin as peroxidase and a p henothiazine as a radical donor. NTYR was detectable (detection limit: 0.02 ng/injection) in 11 smokers (mean +/- SD: 1.60 +/- 1.24 ng/mg pr otein) and in two nonsmokers (1.10 and 1.20 ng/mg protein, respectivel y). NTYR was not associated with nicotine and cotinine levels or expir ed CO in smokers. Plasma TEAC in smokers was significantly lower (0.43 +/- 0.38 mM) than in nonsmokers (1.42 +/- 0.3 mM; p < 0.001) and show ed a biphasic, negative relationship with NTYR (r = 0.96, p < 0.001). This highly sensitive HPLC/GC-TEA method for detection and quantitatio n of plasma NTYR may be used for monitoring oxidative reactions associ ated with tobacco smoking. This assay might be incorporated into molec ular epidemiologic studies for lung chronic inflammatory and neoplasti c disorders in which exposure to oxidants may be an important risk fac tor.