• Incubation of Human Liver Microsomes
• Analysis of Smokers Urine

   

Incubation of Human Liver Microsomes -- Hecht et al. (2000) demonstrated that NNK is directly formed from the 2'-hydroxylation pathway of nicotine by incubating human liver P450 2A6 and cofactors with nicotine. The products of the incubation were then analyzed by High Performance Liquid Chromatography (HPLC). Results indicated that aminoketone from the 2'-hydroxylation pathway is produced in the presence of nicotine and cytochrome P450 2A6, but not in incubations lacking the hepatic enzyme. Although the 2'-hydroxylation pathway was previously known to occur in bacterial systems, conclusive evidence of this mechanism in mammals is a novel finding and is still debated in the literature.

To compare the rate of aminoketone and cotinine formation, nicotine was incubated with P450 2A6 and the appearance of products monitored by liquid chromatography-electrospray ionization-MS and HPLC and UV detection. The rate of formation of the NNK precursor aminoketone was found to be only 11% of the rate of cotinine production, confirming the fact that cotinine is the major nicotine metabolite. Incubation mixtures with cotinine substrate did not yield the final metabolic keto acid product of 2'-hydroxylation. This keto acid product was produced in a similar mixture with aminoketone instead of cotinine. Thus, the main source of keto acid in humans was found to be the 2'-hydroxylation pathway. Although the keto and hydroxy acid metabolites can be produced from cotinine as well, the 5'-hydroxylation pathway produces negligible amounts as demonstrated by these experiments (Hecht et al. 2000).

Analysis of Smoker Urine -- Hecht et al. previously reported that keto and hydroxy acid metabolic products comprise 10-15% of nicotine metabolites found in the urine of smokers and smokers using the nicotine patch. Products derived from the 5'-hydroxylation pathway contributed 80% of urine nicotine metabolites. This research could not produce evidence for endogenous production of NNK by ex-smokers on nicotine replacement therapy because urinary analyses cannot detect formation of NNK in specific body tissues. However, the metabolic pathway of nicotine illustrates that NNK is formed from the aminoketone intermediate found in the 2'-hydroxylation route, meaning that NNK could indeed be formed endogenously in people using tobacco products or undergoing nicotine replacement therapies. Although this experiment did not detect NNK formation endogenously does not preclude its synthesis in human tissues that metabolize nicotine. Such formation of NNK would result in a much greater degree of exposure to NNK than the actual amount (80-770ng consumed per cigarette) present in tobacco products (Hecht et al. 2000).

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