It was the goal of this study to assay the potential of inhaled cigarette smoke for endogenous N-nitrosation of amines in smokers by means of measuring urinary excretion of N-nitrosoproline (NPRO). For 12 days, nonsmoking and smoking men were placed on a controlled diet which was relatively low in proline and in ascorbic acid. On Days 1 through 3, the volunteers received the controlled diet alone (Group 1); on Days 4 through 6, the diet was supplemented by a single daily dose of 300 mg of proline (Group 2); on Days 7 through 9, the diet was supplemented by a single daily dose of 1 g of ascorbic acid followed by 300 mg of proline (Group 3); and for the last 3 days, a single daily dose of 1 g ascorbic acid was given (Group 4). Collections of 24-hr urine were made on Days 3, 6, 9, and 12 of the study. The urine was analyzed for NPRO and creatinine and for cotinine, the major metabolite of nicotine. The mean 24-hr NPRO excretion for 13 nonsmokers in Group 1 was 3.6 µg. The NPRO excretion in 13 smokers in Group 1 was found to be 5.9 µg/24 hr, which is significantly higher than that of the nonsmokers (p < 0.05). Urinary NPRO in 14 nonsmokers of Group 2 was significantly lower than that of the 14 smoking volunteers (p < 0.05). Data for Group 3 indicated that those smokers who had shown elevated NPRO excretion in Group 2 had reduced urinary levels of NPRO as a consequence of ascorbic acid intake. Differences in NPRO excretion by smokers and nonsmokers on controlled diet with ascorbic acid but without proline supplements (Group 4) were also insignificant. These findings suggest that the documented endogenous N-nitrosation of proline which occurs as a result of cigarette smoke inhalation may also apply to other N-nitrosatable amines including nicotine and thus lead to in vivo formation of carcinogenic N-nitrosamines.


This study was supported by National Cancer Institute Grant P01-CA-29580. This is No. XXVI of the series, “A Study of Tobacco Carcinogenesis.”

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