Analysis of Nicotine Metabolites in Three Ethnic Groups

Abstract

Nicotine is the primary addictive agent in tobacco. Nicotine metabolism influences smoking intensity, and therefore disease risk. A key enzyme in nicotine metabolism is P450 2A6. Smokers with low P450 2A6 activity tend to smoke less than those with higher activity.The goal of this thesis research was to explore the relationship between P450 2A6 metabolism and the nicotine metabolite profile of three ethnic groups that have different frequency of CYP2A6 variant alleles. We also investigated the effect of P450 2A6 activity on the formation of 4-hydroxy-4-(3-pyridyl) butanoic acid (hydroxy acid) relative to the metabolites of other nicotine metabolite pathways. Nicotine is metabolized primarily by CYP2A6 catalyzed 5’-oxidation to cotinine and trans-3'-hydroxycotinine. Two additional pathways generate nicotine-N-oxide, nicotine-N-glucuronide. In addition, nicotine is metabolized by 2’-oxidation which generates hydroxy acid. We were able to add cotinine-N-oxide to a well-established LC-MS/MS analysis by collecting the methanol wash and to analyze hydroxy acid by inserting a strata X-A SPE column prior to the MCX. This newly developed LC-MS/MS method allow us to analyze nicotine and seven metabolites from a single urine sample. Specially, we quantified the distribution of hydroxy acid and cotinine-N-oxide which are measured for the first time in the three ethnic groups and determined their formation did not rely on P450 2A6 activity.