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1.  CYP2A6 and CYP2B6 genetic variation and its association with nicotine metabolism in South Western Alaska Native people 
Pharmacogenetics and Genomics  2012;22(6):429-440.
Objectives
Alaska Native people (AN) have a high prevalence of tobacco use and associated morbidity and mortality when compared to the general U.S. population. Variation in the CYP2A6 and CYP2B6 genes, encoding enzymes responsible for nicotine metabolic inactivation and procarcinogen activation, has not been characterized in AN and may contribute to the increased risk.
Methods
AN people (n = 400) residing in the Bristol Bay region of South Western Alaska were recruited for a cross-sectional study on tobacco use. They were genotyped for CYP2A6*1X2A, *1X2B, *1B, *2, *4, *7, *8, *9, *10, *12, *17, *35 and CYP2B6*4, *6, *9 and provided plasma and urine samples for measurement of nicotine and metabolites.
Results
CYP2A6 and CYP2B6 variant frequencies among the AN Yupik people (n=361) were significantly different from other ethnicities. Nicotine metabolism (as measured by the plasma and urinary ratio of metabolites trans-3’hydroxycotinine to cotinine [(3HC/COT)] was significantly associated with CYP2A6 (P< 0.001) but not CYP2B6 genotype (P = 0.95) when controlling for known covariates. Of note, plasma 3HC/COT ratios were high in the entire Yupik people, and among the Yupik CYP2A6 wild-type participants they were substantially higher than previously characterized racial/ethnic groups (P < 0.001 vs. Caucasians and African Americans).
Conclusions
Yupik AN people have a unique CYP2A6 genetic profile which associated strongly with in vivo nicotine metabolism. More rapid CYP2A6-mediated nicotine and nitrosamine metabolism in the Yupik people may modulate tobacco-related disease risk.
doi:10.1097/FPC.0b013e3283527c1c
PMCID: PMC3349071  PMID: 22569203
CYP2A6; CYP2B6; nicotine; tobacco; smoking; genetic variation; Alaska Native people
2.  Biomonitoring of Urinary Cotinine Concentrations Associated with Plasma Levels of Nicotine Metabolites after Daily Cigarette Smoking in a Male Japanese Population 
Human biomonitoring of plasma and urinary levels of nicotine, cotinine, and 3′-hydroxycotinine was conducted after daily cigarette smoking in a population of 92 male Japanese smokers with a mean age of 37 years who had smoked an average of 23 cigarettes per day for 16 years. Members of the population were genotyped for the nicotine-metabolizing enzyme cytochrome P450 2A6 (CYP2A6). The mean levels of nicotine, the levels of its metabolites cotinine and 3′-hydroxycotinine, and the sum of these three levels in subjects one hour after smoking the first cigarette on the sampling day were 20.1, 158, 27.7, and 198 ng/mL in plasma and 846, 1,020, 1,010, and 2,870 ng/mL in urine under daily smoking conditions. Plasma levels of 3′-hydroxycotinine and urinary levels of nicotine and 3′-hydroxycotinine were dependent on the CYP2A6 phenotype group, which was estimated from the CYP2A6 genotypes of the subjects, including those with whole gene deletion. Plasma cotinine levels were significantly correlated with the number of cigarettes smoked on the day before sampling (r = 0.71), the average number of cigarettes smoked daily (r = 0.58), and the Brinkman index (daily cigarettes × years, r = 0.48) under the present conditions. The sum of nicotine, cotinine, and 3′-hydroxycotinine concentrations in plasma showed a similar relationship to that of the plasma cotinine levels. Urinary concentrations of cotinine and the sum of nicotine metabolite concentrations also showed significant correlations with the plasma levels and the previous day’s and average cigarette consumption. The numbers of cigarettes smoked per day by two subjects with self-reported light smoking habits were predicted by measuring the urinary cotinine concentrations and using linear regression equations derived from above-mentioned data. These results indicate that biomonitoring of the urinary cotinine concentration is a good, easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers independent of genetic polymorphism of CYP2A6.
doi:10.3390/ijerph7072953
PMCID: PMC2922738  PMID: 20717551
cytochrome P450; CYP2A6; 3′-hydroxycotinine; genetic polymorphism; smoking index; biomarker
3.  UGT2B10 genotype influences nicotine glucuronidation, oxidation and consumption 
Background
Tobacco exposure is routinely assessed by quantifying nicotine metabolites in plasma or urine. On average, 80% of nicotine undergoes C-oxidation to cotinine. However, interindividual variation in nicotine glucuronidation is substantial and glucuronidation accounts for from 0 to 40% of total nicotine metabolism. We report here the effect of a polymorphism in a UDP-glucuronsyl transferase, UGT2B10, on nicotine metabolism and consumption.
Methods
Nicotine, cotinine, their N-glucuronide conjugates, and total trans-3'-hydroxycotinine were quantified in the urine (n=327) and plasma (n =115) of smokers. Urinary nicotine N-oxide was quantified in 105 smokers. Nicotine equivalents, the sum of nicotine and all major metabolites, were calculated for each smoker. The relationship of the UGT2B10 Asp67Tyr allele to nicotine equivalents, N-glucuronidation, and C-oxidation was determined.
Results
Individuals heterozygous for the Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than wild-type, resulting in a 60% lower ratio of cotinine glucuronide:cotinine, a 50% lower ratio of nicotine glucuronide:nicotine and increased cotinine and trans-3'-hydroxycotinine. Nicotine equivalents, a robust biomarker of nicotine intake, were lower among Asp67Tyr heterozygotes compared to individuals without this allele; 58.2 nmol/ml (95% CI, 48.9 – 68.2) versus 69.2 nmol/ml (95% CI, 64.3 – 74.5).
Conclusions
Individuals heterozygous for UGT2B10 Asp67Tyr consume less nicotine than do wild type smokers. This striking observation suggests that variations in nicotine N-glucuronidation, as reported for nicotine C-oxidation, may influence smoking behavior.
Impact
UGT2B10 genotype influences nicotine metabolism and should be taken into account when characterizing the role of nicotine metabolism on smoking.
doi:10.1158/1055-9965.EPI-09-0959
PMCID: PMC2882998  PMID: 20501767
Nicotine; glucuronide; UGT2B10; cotinine; smoking; glucuronidation
4.  Biomarkers of Secondhand Smoke Exposure in Automobiles 
Tobacco control  2013;23(1):10.1136/tobaccocontrol-2012-050724.
Objectives
The objectives of this study were: (1) to characterize the exposure of nonsmokers exposed to secondhand smoke (SHS) in a vehicle using biomarkers, (2) to describe the time-course of the biomarkers over 24 h, and (3) to examine the relationship between tobacco biomarkers and airborne concentrations of SHS markers.
Methods
Eight nonsmokers were individually exposed to SHS in cars with fully open front windows and closed back windows over an hour from a smoker who smoked 3 cigarettes at 20 min intervals. The nonsmokers sat in the backseat-passenger side, while the smoker sat in the driver’s seat. Plasma cotinine and urine cotinine, 3-hydroxycotinine (3HC), and 4-(methylnitrosoamino)-(3-pyridyl)-1-butanol (NNAL) were compared in samples taken at baseline and several time-points after exposure. Nicotine, particulate matter (PM2.5), and carbon monoxide (CO) were measured inside and outside the vehicle and ventilation rates in the cars were measured.
Results
Average plasma cotinine and the molar sum of urine cotinine and 3HC (COT+3HC) increased 4-fold, urine cotinine increased 6-fold, and urine NNAL increased ~27 times compared to baseline biomarker levels. Plasma cotinine, urine COT+3HC and NNAL peaked at 4–8 hours post-exposure while urine cotinine peaked within 4 hours. Plasma cotinine was significantly correlated to PM2.5 (Spearman correlation (rs = 0.94) and CO (rs = 0.76) but not to air nicotine. The correlations between urine biomarkers, cotinine, COT+3HC, and NNAL and air nicotine, PM2.5, and CO were moderate but non-significant (rs range, 0.31 – 0.60).
Conclusion
Brief SHS exposure in cars resulted in substantial increases in levels of tobacco biomarkers in nonsmokers. For optimal characterization of SHS exposure, tobacco biomarkers should be measured within 4–8 h post-exposure. Additional studies are needed to better describe the relationship between tobacco biomarkers and environmental markers of SHS.
doi:10.1136/tobaccocontrol-2012-050724
PMCID: PMC3670969  PMID: 23349229
Cigarettes; Secondhand smoke; passive smoking; automobiles; motor vehicles; biomarkers; cotinine; tobacco-specific nitrosamines
5.  Stability of the Nicotine Metabolite Ratio in Ad Libitum and Reducing Smokers 
Background
The ratio of two nicotine metabolites, cotinine (COT) and trans-3′-hydroxycotinine (3-HC), has been validated as a method of phenotyping the activity of the liver enzyme cytochrome P450 (CYP) 2A6, and thus the rate of nicotine metabolism. Our objective was to evaluate the correlates and stability of the 3-HC:COT ratio in ad libitum and reducing smokers, using nicotine replacement therapy (NRT), over a period of months.
Methods
Smokers (N = 123, 94% Caucasian) participated in a smoking reduction study where one-third of the sample smoked ad libitum for 8 weeks (Waitlist phase), before joining the rest of the participants for 12 weeks of cigarette reduction (Reduction phase) using NRT. Urinary nicotine, cotinine, and 3-HC were measured at each visit.
Results
The baseline 3-HC:COT ratio was significantly but weakly correlated with cigarettes/day (r = .19), body mass index (r = -.27), and waking at night to smoke (r = .23). As assessed by repeated measures ANOVA, the 3-HC:COT ratio was stable in the Waitlist phase (coefficient of variation for 3-4 measurements, 38% [Range = 5%-110%]), while minor variation was noted in the Reduction phase (coefficient of variation for 3-5 measurements, 35% [Range = 10%-107%]).
Conclusions
In non-reducing ad libitum smokers, the 3-HC:COT ratio was generally stable, while during smoking reduction using NRT, some small variation was detected. While the current findings are suggestive of the stability of the 3-HC:COT ratio in a predominantly Caucasian sample smoking freely or reducing smoking with NRT, additional research is needed in more diverse populations.
doi:10.1158/1055-9965.EPI-08-0242
PMCID: PMC2765478  PMID: 18559554
6.  Identification of novel CYP2A6*1B variants; the CYP2A6*1B allele is associated with faster in vivo nicotine metabolism 
Background
Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotine’s metabolism. CYP2A6 genetic variants contribute to the inter-individual and inter-ethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotine’s in vivo metabolism.
Methods
Intravenous infusions of deuterium-labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B.
Results
We discovered three novel CYP2A6*1B variants in the 3′-flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B and CYP2A6*1B/*1B groups in total nicotine clearance (17.2±5.2, 19.0±6.4 and 20.4±5.9, P < 0.02), nonrenal nicotine clearance (16.4±5.0, 18.5±6.2 and 19.8±5.7, P < 0.01) and the plasma 3HC/COT ratio (0.26±0.1, 0.26±0.1 and 0.34±0.1, P < 0.001). There were also differences in total nicotine (29.4±12.9, 25.8±0.12.9 and 22.4±12.4, P < 0.01), cotinine (29.2±8.1, 32.2±9.1 and 33.0±6.6, P < 0.01) and trans-3′-hydroxcotinine (32.4±9.1, 34.2±12.3 and 41.3±11.3, P < 0.001) excreted in the urine.
Conclusion
We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.
doi:10.1038/sj.clpt.6100246
PMCID: PMC2921956  PMID: 17522595 CAMSID: cams1335
7.  Utility and relationships of biomarkers of smoking in African-American light smokers 
While expired carbon monoxide (CO) and plasma cotinine (COT) have been validated as biomarkers of self-reported cigarettes per day (CPD) in heavy smoking Caucasians, their utility in light smokers is unknown. Further, variability in CYP2A6, the enzyme that mediates formation of COT from nicotine (NIC) and its metabolism to trans-3′-hydroxycotinine (3HC), may limit the usefulness of COT. We assessed whether CO and COT are correlated with CPD in African-American light smokers (≤10CPD, n=700), a population with known reduced CYP2A6 activity and slow COT metabolism. We also examined whether gender, age, BMI, smoking mentholated cigarettes or rate of CYP2A6 activity, by genotype and phenotype measures (3HC/COT), influence these relationships. At baseline, many participants (42%) exhaled CO ≤10ppm, the traditional cutoff for smoking, while few (3.1%) had COT below the cutoff of ≤14ng/ml; thus COT appears to be a better biomarker of smoking status in this population. CPD was weakly correlated with CO and COT (r = 0.32–0.39, p<0.001), and those reporting fewer CPD had higher CO/cigarette and COT/cigarette, although the correlations coefficients between these variables were also weak (r = −0.33 and −0.08, p < 0.05). The correlation between CPD and CO was not greatly increased when analyzed by CYP2A6 activity, smoking mentholated cigarettes or age, although it appeared stronger in females (r = 0.38 vs.0.21, p<0.05) and obese individuals (r = 0.38 vs.0.24, p<0.05). Together, these results suggest that CO and COT are weakly associated with self-reported cigarette consumption in African-American light smokers, and that these relationships are not substantially improved when variables previously reported to influence these biomarkers are considered.
doi:10.1158/1055-9965.EPI-09-0956
PMCID: PMC2791893  PMID: 19959692 CAMSID: cams1333
biomarkers; smoking; cotinine; carbon monoxide; African-Americans
8.  Utility and relationships of biomarkers of smoking in African-American light smokers 
While expired carbon monoxide (CO) and plasma cotinine (COT) have been validated as biomarkers of self-reported cigarettes per day (CPD) in heavy smoking Caucasians, their utility in light smokers is unknown. Further, variability in CYP2A6, the enzyme that mediates formation of COT from nicotine (NIC) and its metabolism to trans-3′-hydroxycotinine (3HC), may limit the usefulness of COT. We assessed whether CO and COT are correlated with CPD in African-American light smokers (≤10CPD, n=700), a population with known reduced CYP2A6 activity and slow COT metabolism. We also examined whether gender, age, BMI, smoking mentholated cigarettes or rate of CYP2A6 activity, by genotype and phenotype measures (3HC/COT), influence these relationships. At baseline, many participants (42%) exhaled CO ≤10ppm, the traditional cutoff for smoking, while few (3.1%) had COT below the cutoff of ≤14ng/ml; thus COT appears to be a better biomarker of smoking status in this population. CPD was weakly correlated with CO and COT (r = 0.32–0.39, p<0.001), and those reporting fewer CPD had higher CO/cigarette and COT/cigarette, although the correlations coefficients between these variables were also weak (r = −0.33 and −0.08, p < 0.05). The correlation between CPD and CO was not greatly increased when analyzed by CYP2A6 activity, smoking mentholated cigarettes or age, although it appeared stronger in females (r = 0.38 vs.0.21, p<0.05) and obese individuals (r = 0.38 vs.0.24, p<0.05). Together, these results suggest that CO and COT are weakly associated with self-reported cigarette consumption in African-American light smokers, and that these relationships are not substantially improved when variables previously reported to influence these biomarkers are considered.
doi:10.1158/1055-9965.EPI-09-0956
PMCID: PMC2791893  PMID: 19959692
biomarkers; smoking; cotinine; carbon monoxide; African-Americans
9.  NICOTINE METABOLISM IN PREGNANT AND NON-PREGNANT RABBITS 
Smoking remains a major public health concern during pregnancy and is associated with numerous adverse effects. Recently the clearance of nicotine (NIC) and cotinine (COT) was shown to be substantially increased in pregnant women compared to non-pregnant controls. The present study investigated the usefulness of the rabbit for studying the molecular basis for the observed changes in NIC and COT disposition during pregnancy. NIC was largely metabolized to COT in rabbit liver microsomes (approximately 50% of total metabolism) with significant amounts of nicotine-N’-oxide and nornicotine also being detected. The conversion of NIC to COT was also detected in rabbit placental and fetal liver microsomes albeit at only a fraction of the rate in adult rabbit liver microsomes. The major products of COT metabolism in rabbit liver microsomes were 5’-hydroxycotinine, cotinine-N’-oxide and norcotinine. Differences between human and rabbit liver were most apparent for COT, with the major human metabolite 3’-hydroxycotinine, being formed at only low levels in rabbit liver microsomes. Pregnancy had no effect on the metabolism of NIC or on the expression of CYP2A6 immunoreactive proteins in rabbit liver microsomes. These studies provide a complete quantitative assessment of NIC metabolism in rabbit liver microsomes and suggest that the rabbit may not be an appropriate animal model to study the effects of pregnancy on NIC and COT metabolism. However, a molecular understanding of these effects is essential for prediction of the pharmacological and toxicological consequences of smoking during pregnancy.
doi:10.1080/14622200802238878
PMCID: PMC2923225  PMID: 18686186
Nicotine; Pregnancy; Hepatic metabolism; Cotinine
10.  Human metabolic profiles are stably controlled by genetic and environmental variation 
A comprehensive variation map of the human metabolome identifies genetic and stable-environmental sources as major drivers of metabolite concentrations. The data suggest that sample sizes of a few thousand are sufficient to detect metabolite biomarkers predictive of disease.
We designed a longitudinal twin study to characterize the genetic, stable-environmental, and longitudinally fluctuating influences on metabolite concentrations in two human biofluids—urine and plasma—focusing specifically on the representative subset of metabolites detectable by 1H nuclear magnetic resonance (1H NMR) spectroscopy.We identified widespread genetic and stable-environmental influences on the (urine and plasma) metabolomes, with (30 and 42%) attributable on average to familial sources, and (47 and 60%) attributable to longitudinally stable sources.Ten of the metabolites annotated in the study are estimated to have >60% familial contribution to their variation in concentration.Our findings have implications for the design and interpretation of 1H NMR-based molecular epidemiology studies. On the basis of the stable component of variation quantified in the current paper, we specified a model of disease association under which we inferred that sample sizes of a few thousand should be sufficient to detect disease-predictive metabolite biomarkers.
Metabolites are small molecules involved in biochemical processes in living systems. Their concentration in biofluids, such as urine and plasma, can offer insights into the functional status of biological pathways within an organism, and reflect input from multiple levels of biological organization—genetic, epigenetic, transcriptomic, and proteomic—as well as from environmental and lifestyle factors. Metabolite levels have the potential to indicate a broad variety of deviations from the ‘normal' physiological state, such as those that accompany a disease, or an increased susceptibility to disease. A number of recent studies have demonstrated that metabolite concentrations can be used to diagnose disease states accurately. A more ambitious goal is to identify metabolite biomarkers that are predictive of future disease onset, providing the possibility of intervention in susceptible individuals.
If an extreme concentration of a metabolite is to serve as an indicator of disease status, it is usually important to know the distribution of metabolite levels among healthy individuals. It is also useful to characterize the sources of that observed variation in the healthy population. A proportion of that variation—the heritable component—is attributable to genetic differences between individuals, potentially at many genetic loci. An effective, molecular indicator of a heritable, complex disease is likely to have a substantive heritable component. Non-heritable biological variation in metabolite concentrations can arise from a variety of environmental influences, such as dietary intake, lifestyle choices, general physical condition, composition of gut microflora, and use of medication. Variation across a population in stable-environmental influences leads to long-term differences between individuals in their baseline metabolite levels. Dynamic environmental pressures lead to short-term fluctuations within an individual about their baseline level. A metabolite whose concentration changes substantially in response to short-term pressures is relatively unlikely to offer long-term prediction of disease. In summary, the potential suitability of a metabolite to predict disease is reflected by the relative contributions of heritable and stable/unstable-environmental factors to its variation in concentration across the healthy population.
Studies involving twins are an established technique for quantifying the heritable component of phenotypes in human populations. Monozygotic (MZ) twins share the same DNA genome-wide, while dizygotic (DZ) twins share approximately half their inherited DNA, as do ordinary siblings. By comparing the average extent of phenotypic concordance within MZ pairs to that within DZ pairs, it is possible to quantify the heritability of a trait, and also to quantify the familiality, which refers to the combination of heritable and common-environmental effects (i.e., environmental influences shared by twins in a pair). In addition to incorporating twins into the study design, it is useful to quantify the phenotype in some individuals at multiple time points. The longitudinal aspect of such a study allows environmental effects to be decomposed into those that affect the phenotype over the short term and those that exert stable influence.
For the current study, urine and blood samples were collected from a cohort of MZ and DZ twins, with some twins donating samples on two occasions several months apart. Samples were analysed by 1H nuclear magnetic resonance (1H NMR) spectroscopy—an untargeted, discovery-driven technique for quantifying metabolite concentrations in biological samples. The application of 1H NMR to a biological sample creates a spectrum, made up of multiple peaks, with each peak's size quantitatively representing the concentration of its corresponding hydrogen-containing metabolite.
In each biological sample in our study, we extracted a full set of peaks, and thereby quantified the concentrations of all common plasma and urine metabolites detectable by 1H NMR. We developed bespoke statistical methods to decompose the observed concentration variation at each metabolite peak into that originating from familial, individual-environmental, and unstable-environmental sources.
We quantified the variability landscape across all common metabolite peaks in the urine and plasma 1H NMR metabolomes. We annotated a subset of peaks with a total of 65 metabolites; the variance decompositions for these are shown in Figure 1. Ten metabolites' concentrations were estimated to have familial contributions in excess of 60%. The average proportion of stable variation across all extracted metabolite peaks was estimated to be 47% in the urine samples and 60% in the plasma samples; the average estimated familiality was 30% for urine and 42% for plasma. These results comprise the first quantitative variation map of the 1H NMR metabolome. The identification and quantification of substantive widespread stability provides support for the use of these biofluids in molecular epidemiology studies. On the basis of our findings, we performed power calculations for a hypothetical study searching for predictive disease biomarkers among 1H NMR-detectable urine and plasma metabolites. Our calculations suggest that sample sizes of 2000–5000 should allow reliable identification of disease-predictive metabolite concentrations explaining 5–10% of disease risk, while greater sample sizes of 5000–20 000 would be required to identify metabolite concentrations explaining 1–2% of disease risk.
1H Nuclear Magnetic Resonance spectroscopy (1H NMR) is increasingly used to measure metabolite concentrations in sets of biological samples for top-down systems biology and molecular epidemiology. For such purposes, knowledge of the sources of human variation in metabolite concentrations is valuable, but currently sparse. We conducted and analysed a study to create such a resource. In our unique design, identical and non-identical twin pairs donated plasma and urine samples longitudinally. We acquired 1H NMR spectra on the samples, and statistically decomposed variation in metabolite concentration into familial (genetic and common-environmental), individual-environmental, and longitudinally unstable components. We estimate that stable variation, comprising familial and individual-environmental factors, accounts on average for 60% (plasma) and 47% (urine) of biological variation in 1H NMR-detectable metabolite concentrations. Clinically predictive metabolic variation is likely nested within this stable component, so our results have implications for the effective design of biomarker-discovery studies. We provide a power-calculation method which reveals that sample sizes of a few thousand should offer sufficient statistical precision to detect 1H NMR-based biomarkers quantifying predisposition to disease.
doi:10.1038/msb.2011.57
PMCID: PMC3202796  PMID: 21878913
biomarker; 1H nuclear magnetic resonance spectroscopy; metabolome-wide association study; top-down systems biology; variance decomposition
11.  The ability of plasma cotinine to predict nicotine and carcinogen exposure is altered by differences in CYP2A6: the influence of genetics, race and sex 
Background
Cotinine, a nicotine metabolite, is a biomarker of tobacco, nicotine and carcinogen exposure. However a given cotinine level may not represent the same tobacco exposure; for example, African Americans have higher cotinine levels than Caucasians after controlling for exposure.
Methods
Cotinine levels are determined by the amount of cotinine formation and the rate of cotinine removal which are both mediated by the enzyme CYP2A6. Since CYP2A6 activity differs by sex (estrogen induces CYP2A6) and genotype, their effect on cotinine formation and removal were measured in non-smoking Caucasians (Study 1, n=181) infused with labeled nicotine and cotinine. The findings were then extended to ad libitum smokers (Study 2, n=163).
Results
Study 1: Reduced CYP2A6 activity altered cotinine formation less than cotinine removal resulting in ratios of formation to removal of 1.31 and 1.12 in CYP2A6 reduced and normal metabolizers (P=0.01), or 1.39 and 1.12 in males and females (P=0.001), suggesting an overestimation of tobacco exposure in slower metabolizers. Study 2: Cotinine again overestimated tobacco and carcinogen exposure by ≥25% in CYP2A6 reduced metabolizers (≈2 fold between some genotypes) and in males.
Conclusions
In people with slower, relative to faster, CYP2A6 activity cotinine accumulates resulting in substantial differences in cotinine levels for a given tobacco exposure.
Impact
Cotinine levels may be misleading when comparing those with differing CYP2A6 genotypes within a race, between races with differing frequencies of CYP2A6 gene variants (i.e. African Americans have higher frequencies of reduced function variants contributing to their higher cotinine levels) or between the sexes.
doi:10.1158/1055-9965.EPI-12-1234-T
PMCID: PMC3617060  PMID: 23371292
Tobacco; Cotinine; CYP2A6; Polycyclic aromatic hydrocarbons; NNAL
12.  The Contribution of Common UGT2B10 and CYP2A6 Alleles to Variation in Nicotine Glucuronidation among European Americans 
Pharmacogenetics and genomics  2013;23(12):706-716.
UDP-glucuronosytransferase-2B10 (UGT2B10) is the primary catalyst of nicotine glucuronidation. To develop a predictive genetic model of nicotine metabolism, the conversion of deuterated (D2)-nicotine to D2-nicotine-glucuronide, D2-cotinine, D2-cotinine-glucuronide, and D2-trans-3'-hydroxycotinine were quantified in 188 European Americans, and the contribution of UGT2B10 genotype to variability in first-pass nicotine glucuronidation assessed, following a procedure previously applied to nicotine C-oxidation. The proportion of total nicotine converted to nicotine-glucuronide (D2-nicotine-glucuronide/ (D2-nicotine +D2-nicotine-glucuronide +D2-cotinine +D2-cotinine-glucuronide +D2-trans-3'-hydroxycotinine)) was the primary phenotype. The variant, rs61750900T (D67Y) (minor allele frequency (MAF) = 10%), is confirmed to abolish nicotine glucuronidation activity. Another variant, rs112561475G (N397D) (MAF = 2%), is significantly associated with enhanced glucuronidation. rs112561475G is the ancestral allele of a well-conserved amino acid, indicating that the majority of human UGT2B10 alleles are derived hypomorphic alleles. CYP2A6 and UGT2B10 genotype explain 53% of the variance in oral nicotine glucuronidation in this sample. CYP2A6 and UGT2B10 genetic variants are also significantly associated with un-deuterated (D0) nicotine glucuronidation in subjects smoking ad libitum. We find no evidence for further common variation markedly influencing hepatic UGT2B10 expression in European Americans.
doi:10.1097/FPC.0000000000000011
PMCID: PMC3919513  PMID: 24192532
UGT2B10; nicotine; cotinine; metabolism; glucuronidation; CYP2A6
13.  Variation in Trans-3′-Hydroxycotinine Glucuronidation Does Not Alter the Nicotine Metabolite Ratio or Nicotine Intake 
PLoS ONE  2013;8(8):e70938.
Background
CYP2A6 metabolizes nicotine to its primary metabolite cotinine and also mediates the metabolism of cotinine to trans-3′-hydroxycotinine (3HC). The ratio of 3HC to cotinine (the “nicotine metabolite ratio”, NMR) is an in vivo marker for the rate of CYP2A6 mediated nicotine metabolism, and total nicotine clearance, and has been associated with differences in numerous smoking behaviors. The clearance of 3HC, which affects the NMR, occurs via renal excretion and metabolism by UGT2B17, and possibly UGT2B10, to 3HC-glucuronide. We investigated whether slower 3HC glucuronidation alters NMR, altering its ability to predict CYP2A6 activity and reducing its clinical utility.
Methods
Plasma NMR, three urinary NMRs, three urinary 3HC glucuronidation phenotypes and total nicotine equivalents were examined in 540 African American smokers. The UGT2B17 gene deletion and UGT2B10*2 were genotyped.
Results
The UGT2B17 gene deletion, but not UGT2B10*2 genotype, was associated with slower 3HC glucuronidation (indicated by three 3HC-glucuronidation phenotypes), indicating its role in this glucuronidation pathway. However, neither lower rates of 3HC glucuronidation, nor the presence of a UGT2B17 and UGT2B10 reduced function allele, altered plasma or urinary NMRs or levels of smoking.
Conclusions
Variation in 3HC glucuronidation activity, including these caused by UGT2B17 gene deletions, did not significantly alter NMR and is therefore unlikely to affect the clinical utility of NMR in smoking behavior and cessation studies. This study demonstrates that NMR is not altered by differences in the rate of 3HC glucuronidation, providing further support that NMR is a reliable indicator of CYP2A6 mediated nicotine metabolism.
doi:10.1371/journal.pone.0070938
PMCID: PMC3732272  PMID: 23936477
14.  Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography tandem mass spectrometry 
A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β-D-glucuronide (NIC GLUC), cotinine-N-β-D-glucuronide (COT GLUC), nicotine-1′-oxide (NNO), cotinine-N-oxide (CNO), trans-3′-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery® HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1 – 7.5 ng/mg for NIC, COT GLUC and AB; and 0.025 – 7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R2 ≥ 0.99 for all analytes). Extraction recoveries ranged from 64% - 115%, LC-MS/MS matrix effects were ≤ 21%, and overall process efficiency ranged from 57% - 93% at low and high quality control concentrations. Intra- and inter-assay imprecision and accuracy for all analytes were ≤ 12.9% and ≥ 86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (−) NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.
doi:10.1016/j.jchromb.2011.09.026
PMCID: PMC3210385  PMID: 21963483
liquid chromatography mass spectrometry; nicotine; metabolites; brain; rat
15.  Determination of the Nicotine Metabolites Cotinine and Trans-3′-Hydroxycotinine in Biologic fluids of Smokers and Non-Smokers using Liquid Chromatography - Tandem Mass Spectrometry: Biomarkers for Tobacco Smoke Exposure and for Phenotyping Cytochrome P450 2A6 Activity 
The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3′-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by Cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography – tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3′-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1 ng/ mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.
doi:10.1016/j.jchromb.2010.12.012
PMCID: PMC3050598  PMID: 21208832
Nicotine; Cotinine; trans-3′-hydroxycotinine; Cytochrome P450 2A6 (CYP2A6); tobacco; secondhand smoke
16.  Nicotine Metabolism in Three Ethnic/Racial Groups with Different Risks of Lung Cancer 
Previously, we documented that smoking-associated lung cancer (LC) risk is greater in Hawaiians and lower in Japanese, compared to whites. Nicotine metabolism by CYP2A6 varies across ethnicity/race and is hypothesized to affect smoking behavior. We investigated whether higher CYP2A6 activity results in the smoker extracting more nicotine (adjusting for cigarettes per day [CPD]) and being exposed to higher levels of tobacco-specific nitrosamine [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] and pyrene, a representative polycyclic aromatic hydrocarbon. We conducted a cross-sectional study of 585 smokers among the three main ethnic/racial groups in Hawaii, and examined whether differences in CYP2A6 activity correlate with the ethnic/racial differences in LC risk. We assessed CYP2A6 activity by nicotine metabolite ratio [total trans 3′-hydroxycotinine (3-HC) : total cotinine] and caffeine metabolite ratio (1,7-dimethyl uric acid : 1,7-dimethylxanthine) in 12-hour urine. We also measured urinary nicotine equivalents (sum of nicotine, cotinine and 3-HC and their respective glucuronides), a marker of nicotine dose, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide, markers of NNK exposure, and 1-hydroxypyrene (1-OHP), a marker of pyrene exposure. The nicotine metabolite ratio was higher in whites than in Japanese, and intermediate in Hawaiians (p’s <0.05). CPD-adjusted nicotine equivalents were lower in Japanese compared to Hawaiians or whites (p=0.005 and <0.0001, respectively), and greater in men than women (p<0.0001). Nicotine equivalents and total NNAL increased with CYP2A6 activity, indicating that smokers with greater nicotine metabolism smoke more extensively and have a higher internal NNK dose. The particularly low nicotine metabolism of Japanese smokers may contribute to their previously described decreased LC risk.
doi:10.1158/1055-9965.EPI-08-0424
PMCID: PMC2599805  PMID: 19029401
17.  Association of nicotine metabolite ratio and CYP2A6 genotype with smoking cessation treatment in African-American light smokers 
CYP2A6 is the main nicotine metabolizing enzyme in humans. We investigated the relationships between CYP2A6 genotype, baseline plasma 3HC/COT (a phenotypic marker of CYP2A6 activity), and smoking behaviors in African-American light smokers. Cigarette consumption, age of initiation, and dependence scores did not differ between 3HC/COT quartiles or CYP2A6 genotype groups. Slow metabolizers (both genetic and phenotypic) had significantly higher plasma nicotine levels suggesting cigarette consumption was not reduced to adjust for slower rates of nicotine metabolism. Individuals in the slowest 3HC/COT quartile had higher quit rates with both placebo and nicotine gum treatments (OR 1.85, 95% CI 1.08-3.16, p = 0.03). Similarly, the slowest CYP2A6 genotype group had higher quit rates, although this did not reach significance (OR 1.61, 95% CI 0.95-2.72, p = 0.08). 3HC/COT ratio, and possibly CYP2A6 genotype, may be useful in the future for personalizing the choice of smoking cessation treatment for African-American light smokers.
doi:10.1038/clpt.2009.19
PMCID: PMC3698861  PMID: 19279561
Cytochrome P450 2A6; CYP2A6; nicotine; cotinine; trans-3′-hydroxycotinine; African-Americans; smoking; light smokers
18.  Stability of the Nicotine Metabolite Ratio in Smokers of Progressively Reduced Nicotine Content Cigarettes 
Nicotine & Tobacco Research  2013;15(11):1939-1942.
Introduction:
The nicotine metabolite ratio (NMR), the ratio of trans-3′-hydroxycotinine (3-HC) to cotinine, has been used as a biomarker of the rate of CYP2A6-mediated nicotine metabolism. While stable in smokers who maintain constant smoking consumption, since smoking has been shown to inhibit nicotine metabolism and this inhibition could be mediated by the nicotine in the smoke, NMR could change during nicotine reduction. The objective of this study was to determine the reproducibility (or stability) of plasma NMR in smokers of progressively reduced nicotine content (RNC) cigarettes.
Methods:
We analyzed data from subjects in a clinical trial of smoking progressively RNC cigarettes. Plasma NMR in 30 smokers whose plasma cotinine levels had decreased by at least 50% from the use of the first test cigarette (12mg nicotine content) to the final test cigarette (1mg nicotine content) was measured on 4 occasions over a period of 24 weeks.
Results:
Plasma cotinine and 3-HC decreased by an average of 85% and 84%, respectively, following the use of the first type of RNC cigarette to the last type. Plasma NMR had an average absolute change of 28.5% over the same period. Using repeated measures analysis, changes in plasma NMR over time were not significant with or without controlling for the effects of age, body mass index, gender, and race (p = .24 and p = .23, respectively). The reliability coefficient for repeated measurements of plasma NMR was .72. The average within-subject coefficient of variation for plasma NMR was 21.6% (SD = 12.0%).
Conclusion:
The plasma NMR is relatively stable over time as nicotine levels decline in smokers of progressively RNC cigarettes.
doi:10.1093/ntr/ntt065
PMCID: PMC3790625  PMID: 23674838
19.  Reproducibility of the Nicotine Metabolite Ratio in Cigarette Smokers 
Background
The nicotine metabolite ratio (NMR or 3-hydroxycotinine/cotinine) has been used to phenotype CYP2A6-mediated nicotine metabolism. Our objectives were to analyze (a) the stability of NMR in plasma, saliva, and blood in various storage conditions, (b) the relationship between NMRs derived from blood, plasma, saliva, and urine, and (c) the reproducibility of plasma NMR in ad libitum cigarette smokers.
Methods
We analyzed data from four clinical studies. In studies 1 and 2, we assessed NMR stability in saliva and plasma samples at room temperature (~22°C) over 14 days and in blood at 4°C for up to 72 hours. In studies 2 and 3, we used Bland-Altman analysis to assess agreement between blood, plasma, saliva, and urine NMRs. In study 4, plasma NMR was measured on 6 occasions over 44 weeks in 43 ad libitum smokers.
Results
Reliability coefficients for stability tests of NMR in plasma and saliva at room temperature were 0.97 and 0.98, respectively, and 0.92 for blood at 4°C. Blood NMR agreed consistently with saliva and plasma NMRs but showed more variability in relation to urine NMR. The reliability coefficient for repeated plasma NMR measurements in smokers was 0.85.
Conclusion
The NMR is stable in blood, plasma, and saliva at the conditions tested. Blood, plasma, and saliva NMRs are similar while urine NMR is a good proxy for these NMR measures. Plasma NMR was reproducible over time in smokers.
Impact
One measurement may reliably estimate a smoker’s NMR for use as an estimate of the rate of nicotine metabolism.
doi:10.1158/1055-9965.EPI-12-0236
PMCID: PMC3392523  PMID: 22552800
Nicotine metabolite ratio (NMR); cotinine; 3-hydroxycotinine; biological stability; chemical stability
20.  Nicotine Dependence Pharmacogenetics: Role of Genetic Variation in Nicotine-Metabolizing Enzymes 
Journal of neurogenetics  2009;23(3):252-261.
Nicotine-dependence pharmacogenetics research is an emerging field, and a number of studies have begun to characterize the clinical relevance and predictive power of genetic variation in drug-metabolizing enzymes and drug target genes for response to medication. The present paper focuses on evidence for the role of nicotine-metabolizing enzymes in smoking behavior and response to treatment. Nicotine metabolism is mediated primarily by cytochrome P450 2A6 (CYP2A6). Genetic variation in the CYP2A6 gene has been linked with several smoking behavior phenotypes. Individuals who carry null or reduced activity alleles for CYP2A6 smoke fewer cigarettes per day, are less dependent on nicotine, and may have an easier time quitting smoking. A phenotypic measure of CYP2A6 enzyme activity, defined as the ratio of the nicotine metabolites 3′hydroxycotinine/cotinine, also predicts successful quitting with the transdermal nicotine patch, and counseling alone. Faster metabolizers of nicotine respond more poorly to these treatments; however, they may be excellent candidates for non-nicotine therapies, such as bupropion. Inherited variation in the CYP2B6 enzyme is also associated with response to bupropion treatment and counseling alone for smoking cessation. Inhibition of the CYP2A6 enzyme to slow nicotine metabolism is a promising approach to increase nicotine availability and potentially reduce harm from tobacco smoking.
doi:10.1080/01677060802572887
PMCID: PMC3772540  PMID: 19169923
tobacco; nicotine; addiction; genetics; pharmacogenetics
21.  A Link between Adolescent Nicotine Metabolism and Smoking Topography 
Adult slow nicotine metabolizers have lower smoke exposure, carbon monoxide levels, and plasma nicotine levels than normal and fast metabolizers. Emerging evidence suggests nicotine metabolism influences smoking topography. This study investigated the association of nicotine metabolism (the ratio of plasma 3-hydroxycotinine to cotinine; 3OHCOT/COT) with smoking topography in adolescent smokers (N=85, 65% female, 68% European American, mean age 15.3 ± 1.2, mean cigarettes per day (CPD) 18.5 ± 8.5, mean Fagerström Test for Nicotine Dependence (FTND) 7.0 ± 1.2) presenting for a nicotine replacement therapy trial. Measures obtained included puff volume, inter-puff interval, number of puffs, puff duration, and puff velocity. Linear regression analysis controlling for hormonal contraception use showed that 3OHCOT/COT ratios predicted mean puff volume in the overall sample (t = 2.126, p = .037, adjusted R2 = .067). After gender stratification, faster metabolism predicted higher mean puff volume (t = 2.81, p = .009, adjusted R2 = .192) but fewer puffs (t = −3.160, p=0.004, adjusted R2 = .237) and lower mean puff duration (t = −2.06, p = .048, adjusted R2 = .101) among boys only, suggesting that as nicotine metabolism increases, puff volume increases but puffing frequency decreases. No significant relationships were found between nicotine metabolism and total puff volume, mean puff duration, inter-puff interval, or puff velocity. If confirmed in a broader sample of adolescent smokers, these findings suggest that, as among dependent adult smokers, rate of metabolism among adolescent boys is linked to select parameters of puffing behavior that may impact cessation ability.
doi:10.1158/1055-9965.EPI-08-0592
PMCID: PMC2720594  PMID: 19423535
adolescent; metabolism; nicotine; smoking
22.  Estimation of Nicotine Dose after Low Level Exposure Using Plasma and Urine Nicotine Metabolites 
Background
We sought to determine the optimal plasma and urine nicotine metabolites, alone or in combination, to estimate the systemic dose of nicotine after low level exposure.
Methods
We dosed 36 nonsmokers with 100, 200 or 400 μg deuterium-labeled nicotine (doses similar to exposure to secondhand smoke, SHS) by mouth daily for 5 days and then measured plasma and urine nicotine metabolites at various intervals over 24 hours.
Results
The strongest correlations with nicotine dose were seen for the sum of four [cotinine + cotinine-glucuronide + trans-3′-hydroxycotinine + 3HC-glucuronide] or six [ including also nicotine + nicotine-glucuronide] of the major nicotine metabolites in 24 hour urine collection (r = 0.96), with lesser correlations for these metabolites using spot urines corrected for creatinine at various times of day (r = 0.72 – 0.80). Plasma [cotinine + trans 3′ hydroxycotine] was more highly correlated with nicotine dose than plasma cotinine alone (r = 0.82 vs 0.75).
Conclusions
Our results provide guidance for selection of biomarkers to estimate the dose of nicotine taken in low level (SHS) tobacco exposure.
Impact
This is probably relevant to active smoking as well.
doi:10.1158/1055-9965.EPI-09-1303
PMCID: PMC2867075  PMID: 20447913
23.  Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine, Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers' Urine 
PLoS ONE  2014;9(7):e101816.
Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies.
doi:10.1371/journal.pone.0101816
PMCID: PMC4094486  PMID: 25013964
24.  Urine nicotine metabolite concentrations in relation to plasma cotinine during low-level nicotine exposure 
Nicotine & Tobacco Research  2009;11(8):954-960.
Introduction
Plasma or saliva cotinine concentrations are used widely as biomarkers of secondhand smoke (SHS) exposure and have been associated with the risk of SHS-related disease. Concentrations of cotinine and other nicotine metabolites are considerably higher in urine than in plasma or saliva, making chemical analysis easier. In addition, urine is often more convenient to collect in some SHS exposure studies. The optimal use of nicotine metabolites in urine, singly or in combination, with or without correction for urine creatinine concentration, to estimate plasma cotinine concentration with low-level nicotine exposure has not been determined.
Methods
We dosed 36 nonsmokers with 100, 200, or 400 μg deuterium-labeled nicotine (simulating exposure to SHS) by mouth daily for 5 days and then measured plasma and urine cotinine and metabolites at various intervals over 24 hr.
Results
A plasma cotinine concentration of 1 ng/ml corresponds on average to a daily intake of 100 μg nicotine. Cotinine concentrations in urine averaged four to five times those in plasma. Correction of urine cotinine for creatinine concentration improved the correlation between urine and plasma cotinine. Measuring multiple cotinine metabolites in urine did not improve the correlation with plasma cotinine, compared with the use of urine cotinine alone.
Discussion
Measurement of urine cotinine corrected for creatinine concentration appears to be the best predictor of plasma cotinine.
doi:10.1093/ntr/ntp092
PMCID: PMC2734286  PMID: 19525206
25.  Carbamazepine but not valproate induces CYP2A6 activity in smokers with mental illness 
BACKGROUND
Anti-epileptic drugs (AED) are increasingly used in the management of serious mental illness yet their effects on nicotine metabolism have not been studied.
METHODS
This study investigated the effects of 3 AED (carbamazepine, CBZ; oxcarbazepine, OCB or valproic acid, VPA) on nicotine and nicotine metabolite levels in 149 smokers with schizophrenia and bipolar disorder who participated in an afternoon blood draw for nicotine, cotinine, and 3’-hydroxycotinine (3HC). The ratio of 3HC to cotinine was calculated as a marker of CYP2A6 metabolic activity. Eight smokers were taking CBZ, six were taking OCB and 40 were taking VPA.
RESULTS
The 3HC/cotinine ratio was significantly higher in individuals taking CBZ or OCB (combined, n=14) vs. those not taking it (mean 0.993 vs. 0.503; p< 0.001). The cotinine/cigarette per day ratio was significantly lower in individuals taking CBZ or OCB. The 3HC/cotinine ratios were also significantly higher in the subgroup of individuals taking CBZ (n=8) vs. those not taking it. There were no significant differences in nicotine or cotinine levels or 3HC/cotinine ratios in individuals taking VPA vs. those not taking it. We conducted backward stepwise linear regression models to identify predictors of the log transformed 3HC/cotinine ratios. Taking CBZ and number of cigarettes smoked per day were significant determinants of log 3HC/Cotinine.
CONCLUSIONS
CBZ likely induces hepatic metabolism via CYP2A6 and is associated with increased 3HC/Cotinine ratios.
IMPACT
Increased nicotine metabolism in individuals using AED has implications for increased smoking behavior and exposure to more tobacco toxins that warrants further study.
doi:10.1158/1055-9965.EPI-10-0384
PMCID: PMC2952059  PMID: 20719908
nicotine; smoking; carbamazepine; metabolism

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