title: A LC-MS/MS method for concurrent determination of nicotine metabolites and role of CYP2A6 in nicotine metabolism in U937 macrophages: implications in oxidative stress in HIV + smokers.
abstract: Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3-1028.1 ng/ml for nicotine and 0.3-652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2-3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30-60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV + smokers.
authors.affiliation: Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas, MO 64108, USA.
authors.forename: Mengyao
authors.initials: M
authors.lastname: Jin
authors.forename: Ravinder
authors.initials: R
authors.lastname: Earla
authors.forename: Ankit
authors.initials: A
authors.lastname: Shah
authors.forename: Rajya L
authors.initials: RL
authors.lastname: Earla
authors.forename: Raeesa
authors.initials: R
authors.lastname: Gupte
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000032:analysis* / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D009538:Nicotine / Q000032:analysis* / Q000378:metabolism; D009602:Nitrosamines / Q000032:analysis* / Q000378:metabolism; D018384:Oxidative Stress; D012907:Smoking; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009602:Nitrosamines; D009538:Nicotine; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D003367:Cotinine