Tobacco-Specific Nitrosamines and Their Pyridine-N-glucuronides in the Urine of Smokers and Smokeless Tobacco Users

The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, MN 55455, USA.
Cancer Epidemiology Biomarkers & Prevention (Impact Factor: 4.32). 05/2005; 14(4):885-91. DOI: 10.1158/1055-9965.EPI-04-0753
Source: PubMed

ABSTRACT Tobacco-specific nitrosamines are believed to play a significant role as causes of cancer in people who use tobacco products. Whereas the uptake of one tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, has been shown by analysis of its metabolites in urine, there are no published studies on urinary levels of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB) or their metabolites in human urine. We developed a method for quantitation of NNN, NAT, NAB, and their pyridine-N-glucuronides NNN-N-Gluc, NAT-N-Gluc, and NAB-N-Gluc in human urine. Total NNN (NNN plus NNN-N-Gluc) was assayed using 5-methyl-N'-nitrosonornicotine as internal standard. Urine was treated with beta-glucuronidase. Following solvent partitioning and solid-phase extraction, total NNN was determined using gas chromatography with nitrosamine-selective detection. Total NAT and total NAB were quantified in the same samples. Separate quantitation of NNN and NNN-N-Gluc was accomplished by extraction of the urine with ethyl acetate before beta-glucuronidase hydrolysis; NNN was analyzed in the ethyl acetate extract, and after enzyme treatment, NNN released from NNN-N-Gluc was quantified in the extracted urine. Separate analyses of NAT, NAT-N-Gluc, NAB, and NAB-N-Gluc proceeded similarly. Analyte identities were confirmed by gas chromatography-tandem mass spectrometry. Mean levels of total NNN, NAT, and NAB in the urine of 14 smokers were (pmol/mg creatinine) 0.18 +/- 0.22, 0.19 +/- 0.20, and 0.040 +/- 0.039, respectively, whereas the corresponding amounts in the urine of 11 smokeless tobacco users were 0.64 +/- 0.44, 1.43 +/- 1.10, and 0.23 +/- 0.19, respectively. Pyridine-N-glucuronides accounted for 59% to 90% of total NNN, NAT, and NAB. The results of this study show the presence of NNN, NAT, NAB, and their pyridine-N-glucuronides in human urine and provide a quantitative method for application in mechanistic and epidemiologic studies of the role of tobacco-specific nitrosamines in human cancer.