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Am. J. Respir. Crit. Care Med., Volume 156, Number 6, December 1997, 1902-1907

Plasma 3-Nitrotyrosine in Cigarette Smokers

STEFANO PETRUZZELLI, ROBERTO PUNTONI, PAOLO MIMOTTI, NOLITA PULERÁ, FILOMENA BALIVA, EDO FORNAI, and CARLO GIUNTINI

Laboratory of Cell Biology, Division of Respiratory Pathophysiology, Cardiopulmonary Department, University of Pisa; and Institute of Clinical Physiology, National Research Council, Pisa, Italy

Peroxynitrite has been associated with increased oxidative reactions and DNA damage in inflamed tissues as it may cause a reduction of plasma antioxidants as well. Nitration of tyrosine residues of proteins leads to the production of 3-nitrotyrosine (NTYR), which may be considered as a marker of NO·-dependent oxidative damage. We developed a highly sensitive method to detect NTYR in human plasma and tested it in cigarette smokers and in healthy control subjects. Peripheral venous blood (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 nicotine, 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®-equivalent antioxidant capacity (TEAC) was also measured using metmyoglobin as peroxidase and a phenothiazine as a radical donor. NTYR was detectable (detection limit: 0.02 ng/injection) in 11 smokers (mean ± SD: 1.60 ± 1.24 ng/mg protein) and in two nonsmokers (1.10 and 1.20 ng/mg protein, respectively). NTYR was not associated with nicotine and cotinine levels or expired 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 showed a biphasic, negative relationship with NTYR (r = 0.96, p < 0.001). This highly sensitive HPLC/GC-TEA method for detection and quantitation of plasma NTYR may be used for monitoring oxidative reactions associated with tobacco smoking. This assay might be incorporated into molecular epidemiologic studies for lung chronic inflammatory and neoplastic disorders in which exposure to oxidants may be an important risk factor.




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