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Measurement of 3OH‐cotinine:cotinine ratio in saliva using HPLC with UV detection as a marker of CYP2A6 expression in human cigarette smokers
Author(s) -
Javors Martin,
Koek Wouter,
Lamb Richard
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.655
Subject(s) - cotinine , cyp2a6 , chemistry , chromatography , nicotine , high performance liquid chromatography , saliva , biochemistry , medicine , metabolism , cytochrome p450 , cyp1a2
The metabolism of nicotine to cotinine to 3OH‐cotinine is mediated by CYP2A6 enzyme. Genetic variation in human CYP2A6 can alter nicotine metabolism, resulting in altered smoking behaviors (Schoedel et al, 2004; Malaiyandi et al, 2006). Determination of the ratio of 3OH‐cotinine:cotinine serves as an index of CYP2A6 activity. For this preliminary study, saliva 3OH‐cotinine and cotinine were quantified using HPLC with UV detection. Saliva was collected using salivette tubes and stored at −80 o C. Extraction of 3OH‐cotinine and cotinine was achieved using Bond Elute Certify prep columns. The HPLC‐UV system included an Alltima C18 column (5μ, 4.6 × 150 mm), mobile phase containing 5 mM phosphate, 0.1% IPCC‐MS2 ion pairing agent, 11%(v/v) acetonitrile (pH 4.5). The flow rate was 1 ml/min and UV wavelength was 260 nm. Concentrations were expressed in ng/ml. Results show that quantification of 3OH‐cotinine and cotinine by this HPLC procedure compared favorably with results using an ELISA kit. 3OH‐cotinine:cotinine ratios ranged from 0.055 to 0.978 in 28 subjects with a mean ± SD of 0.442 ± 0.23. The data appeared to be continuous and not bimodal and passed a statistical test for normal distribution. This HPLC‐UV procedure is suitable for identifying variable levels of liver CYP2A6 in cigarette smokers as a potential genetic marker. Supported by a NIDA grant 13304 to RJL.