Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N′-nitrosonornicotine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N′-nitrosonornicotine releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluorobenzoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 ± 538 (SD) in snuff dippers, 79.6 ± 189 in smokers, and 29.3 ± 25.9 in nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N′-nitrosonornicotine in humans.

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This study was supported by Grant CA-29580 from the National Cancer Institute. Paper 1 in the series, “Hemoglobin Adducts as Carcinogen Dosimeters.”

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