Evidence That a Hemoglobin Adduct of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Is a 4-(3-Pyridyl)-4-oxobutyl Carboxylic Acid Ester

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of human hemoglobin and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with Na¹⁸OH which provide strong evidence that the globin adduct which releases HPB upon base hydrolysis is a carboxylic acid ester. Globin was isolated from rats treated with NNK. This globin was reacted with NaCNBH₃, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol (7), but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with Na¹⁸OH, under conditions which would have resulted in incorporation of ¹⁸O into 7 if nucleophilic displacement at carbon 4 of the adduct had occurred. Analysis of the products by GC-MS showed no detectable incorporation of 180 into 7. These results demonstrate that the globin adduct which releases HPB upon base hydrolysis is a 4-(3-pyridyl)-4-oxobutyl carboxylic ester. Consistent with this conclusion, a model ester, α-methyl β-[4-(3-pyridyl)-4-oxobutyl] N-(carbobenzyloxy)-L-aspartate (13), hydrolyzed in base and acid in a manner similar to that observed with globin from NNK-treated rats.