TY - JOUR
T1 - Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography-electrochemical detection coupled with immunoaffinity purification
T2 - Association with cigarette smoking
AU - Sawa, Tomohiro
AU - Tatemichi, Masayuki
AU - Akaike, Takaaki
AU - Barbin, Alain
AU - Ohshima, Hiroshi
PY - 2006/2/15
Y1 - 2006/2/15
N2 - 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.
AB - 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.
KW - 8-Nitroguanine
KW - 8-Nitroxanthine
KW - Cigarette smoking
KW - DNA damage
KW - Free radicals
KW - Human urine
KW - Immunoaffinity purification
KW - Nitric oxide
KW - Peroxynitrite
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UR - http://www.scopus.com/inward/citedby.url?scp=31644447230&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2005.09.035
DO - 10.1016/j.freeradbiomed.2005.09.035
M3 - Article
C2 - 16458202
AN - SCOPUS:31644447230
VL - 40
SP - 711
EP - 720
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
SN - 0891-5849
IS - 4
ER -