Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography–electrochemical detection coupled with immunoaffinity purification: Association with cigarette smoking

• Published in: Free radical biology & medicine 2006-02, Vol.40 (4), p.711-720
• Main Authors: Sawa, Tomohiro, Tatemichi, Masayuki, Akaike, Takaaki, Barbin, Alain, Ohshima, Hiroshi
• Format: Article
• Language: English
• Subjects: 8-Nitroguanine; 8-Nitroxanthine; Adult; Antibodies, Monoclonal - immunology; Ascites; Biological Assay; Biomarkers; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cigarette smoking; DNA Damage; Electrochemistry; Female; Free radicals; Guanine - analogs & derivatives; Guanine - immunology; Guanine - urine; Human urine; Humans; Immunoaffinity purification; Male; Nitric oxide; Nitrogen Dioxide - metabolism; Nucleic Acids - metabolism; Peroxynitrite; Peroxynitrous Acid - metabolism; Reactive Nitrogen Species - metabolism; Smoking; Spectrometry, Mass, Electrospray Ionization; Xanthines - metabolism
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2005.09.035

We have developed an analytical method to quantitate urinary 8-nitroguanine, a product of nitrative nucleic acid damage caused by reactive nitrogen species such as peroxynitrite and nitrogen dioxide. 8-Nitroguanine was purified from human urine using immunoaffinity columns with an anti-8-nitroguanine antibody, followed by quantitation by high-performance liquid chromatography-electrochemical detection. Four sequential electrodes were used to (a) oxidize interfering compounds (+250 mV), (b) reduce nitrated bases (two online electrodes at −1000 mV), and (c) quantitate reduced derivatives (+150 mV). Using this system 8-nitroxanthine can also be detected, with the detection limits being 25 and 50 fmol/injection for 8-nitroguanine and 8-nitroxanthine, respectively. The method was used to analyze both adducts in the urine of smokers ( n = 12) and nonsmokers ( n = 17). We found that smokers excrete more 8-nitroguanine [median, 6.1 fmol/mg creatinine; interquartile range (IQR), 23.8] than nonsmokers (0; IQR, 0.90) ( p = 0.018), and although 8-nitroxanthine was detected in human urine, its level was not related to smoking status. This is the first report to show that 8-nitroguanine is present in human urine and the methodology developed can be used to study the pathogenic roles of this adduct in the etiology of cancers associated with cigarette smoking and inflammation.