This is the first study in which urinary biomarkers of the nicotine-derived carcinogen NNN were measured in people who had stopped smoking and used the nicotine patch for 6 months. For the majority of our subjects, urinary total NNN declined considerably after smoking cessation. However, in some subjects, total NNN at one or more timepoints after smoking cessation was comparable with or higher than baseline levels. The overall decline of urinary total NNN was less drastic than that of total NNAL. These results support the hypothesis that endogenous NNN formation occurs in some nicotine patch users.
Urinary total NNN is a relatively new biomarker. To strengthen the reliability of the results obtained here for total NNN, we also measured total NNAL—an established and commonly measured urinary metabolite of the related nicotine-derived carcinogen NNK. Interindividual variation in total NNN and total NNAL observed in the baseline urine samples (see ) was consistent with that reported previously (Carmella, Akerkar, Richie, & Hecht, 1995
; Stepanov & Hecht, 2005
). The levels of total NNN and correlation between total NNN and total NNAL in baseline urine also were similar to those reported earlier (Stepanov & Hecht, 2005
). The initial decrease in urinary total NNN was similar to that observed for total NNAL in our subjects (see ), which was consistent with the results of our previous study that dealt with the excretion of total NNAL after smoking cessation (Hecht et al., 1999
). After this initial decrease, however, mean total NNN fluctuated around the value reached 4 weeks after smoking cessation, while the decline in mean total NNAL stopped after 8 weeks of abstinence from smoking. Four subjects had levels of urinary total NNN that were elevated over or comparable with baseline at one or more timepoints after smoking cessation, indicating that endogenous nitrosation of nicotine does take place.
Different rates of decline in urinary total NNN and total NNAL during nicotine patch use are reflected in the ratio of total NNN to total NNAL in the same urine samples (see ). In the baseline urine samples, total NNN was an average 14% of total NNAL, which is consistent with the previously reported ratio between these biomarkers (Stepanov & Hecht, 2005
). This value increased in samples collected after smoking cessation, and after 24 weeks of nicotine patch use it averaged 38%. This increase in total NNN to total NNAL ratio also was observed when the four subjects who had elevated urinary total NNN after smoking cessation were not included in the data analysis (see ). When expressed as a percentage of the baseline level, total NNN in the urine of patch users was more persistent than total NNAL. Thus, 24 weeks after smoking cessation, urinary total NNN in all our subjects was an average 22% of baseline NNN, whereas this value for total NNAL was 7.3% (see ); this difference was statistically significant (p
= .02). Calculations made without inclusion of Subjects 6, 12, 13, and 16 produced similar results (see ); however, the statistical power of this difference decreased (p
= .06). We further excluded Subjects 2, 7, 11, 14, 19, and 20, who, at any timepoint starting with 8 weeks of patch use, had in their urine 20% or more of the baseline total NNN. Exclusion of these subjects left 10 (50% of our participants) who demonstrated a decrease in total NNN similar to that of total NNAL over the study period (see ). This stratified analysis indicated that endogenous formation of NNN is more or less extensive in some patch users and is virtually nonexistent in others.
The difference in the average rates of decrease of total NNN and total NNAL in the urine of some long-term patch users is consistent with the results of kinetic studies that show that nitrosation of nicotine produces more NNN than NNK (Hecht et al., 1978
) and that nornicotine is nitrosated more readily than is nicotine (Mirvish et al., 1977
), supporting endogenous NNN formation in patch users via nitrosation of nicotine or metabolically formed nornicotine. Ascorbic acid is an effective inhibitor of endogenous nitrosation (Mirvish, 1986
; Mirvish, Wallcave, Eagen, & Shubik, 1972
), and we have demonstrated that it inhibits endogenous NNN formation in rats treated with nornicotine and sodium nitrite (Porubin et al., 2007
). Therefore, a simple approach to block possible NNN formation in nicotine patch users could be supplementation with ascorbic acid.
The absence of a control group in which subjects did not use any NRT product after they quit smoking is the major limitation of the present study. Factors other than endogenous formation, such as low rate of NNN clearance from the body and secondhand smoke exposure, also could contribute to the total NNN levels observed here. However, the kinetics of NNN clearance from the body is unknown, and there are no published studies on total NNN levels in nonsmokers exposed to secondhand smoke. Comparison with a placebo patch group would allow us to address these issues. Also, users of other kinds of NRT products should be studied. In nicotine gum chewers and users of nicotine lozenges, for example, nicotine goes directly to the stomach, where conditions are highly favorable for nitrosation reactions (Mirvish, 1975
; Shepard et al., 1987
In summary, the results of the present study demonstrate that endogenous formation of NNN may occur in some nicotine patch users. However, our findings should not call into question the use of nicotine patch as a smoking cessation product. NRT is an effective tool in the treatment of nicotine dependence. Smoking cessation and use of NRT products significantly decrease exposure to a wide range of carcinogens and toxicants present in cigarette smoke, and the levels of urinary total NNN and total NNAL during patch use were generally extremely low in our study. In the future, supplementation with ascorbic acid could be a simple approach to block possible endogenous NNN formation in nicotine patch users. Similar studies involving other types of NRT products should be conducted.