Evaluation of a weighted genetic risk score for the prediction of biomarkers of CYP2A6 activity.

The nicotine metabolite ratio (NMR; 3-hydroxycotinine/cotinine) is an index of CYP2A6 activity. CYP2A6 is responsible for nicotine's metabolic inactivation and variation in the NMR/CYP2A6 is associated with several smoking behaviors. Our aim was to integrate established alleles and novel genome-wide association studies (GWAS) signals to create a weighted genetic risk score (wGRS) for the CYP2A6 gene for European-ancestry populations. The wGRS was compared with a previous CYP2A6 gene scoring approach designed for an alternative phenotype (C2/N2; cotinine-d2/(nicotine-d2 + cotinine-d2)). CYP2A6 genotypes and the NMR were assessed in European-ancestry participants. The wGRS training set included N = 933 smokers recruited to the Pharmacogenetics of Nicotine Addiction and Treatment clinical trial [NCT01314001]. The replication cohort included N = 196 smokers recruited to the Quit 2 Live clinical trial [NCT01836276]. Comparisons between the two CYP2A6 phenotypes and with fractional clearance were made in a laboratory-based pharmacokinetic study (N = 92 participants). In both the training and replication sets, the wGRS, which included seven CYP2A6 variants, explained 33.8% (P < 0.001) of the variance in NMR, providing improved predictive power to the NMR phenotype when compared with other CYP2A6 gene scoring approaches. NMR and C2/N2 were strongly correlated to nicotine clearance (ρ = 0.70 and ρ = 0.79, respectively; P < 0.001), and to one another (ρ = 0.82; P < 0.001); however reduced function genotypes occurred in slow NMR but throughout C2/N2. The wGRS was able to predict smoking quantity and nicotine intake, to discriminate between NMR slow and normal metabolizers (AUC = 0.79; P < 0.001), and to replicate previous NMR-stratified cessation outcomes showing unique treatment outcomes between metabolizer groups.

Pregnancy-Induced Increases in the Nicotine Metabolite Ratio: Examining Changes During Antepartum and Postpartum.

INTRODUCTION: Pregnancy-induced increases in nicotine metabolism may contribute to difficulties in quitting smoking during pregnancy. However, the time course of changes in nicotine metabolism during early and late pregnancy is unclear. This study investigated how pregnancy alters the nicotine metabolite ratio (NMR), a common biomarker of nicotine metabolism among nonpregnant smokers. METHODS: Urinary NMR (trans-3'-hydroxycotinine [3HC]/cotinine [COT]) was assessed using total (free + glucuronide) and free compounds among women (N = 47) from a randomized controlled trial for smoking cessation who self-reported smoking and provided a urine sample during early pregnancy (M ± SD = 12.5 ± 4.5 weeks' gestation), late pregnancy (28.9 ± 2.0 weeks' gestation), and 6 months postpartum (24.7 ± 1.2 weeks since childbirth). Urine samples were analyzed using liquid chromatography-tandem mass spectrometry and NMR were calculated as Total 3HC/Free COT, Free 3HC/Free COT, and Total 3HC/Total COT. RESULTS: NMR was significantly higher during early and late pregnancy compared to postpartum and significantly increased from early to late pregnancy as measured by Total 3HC/Free COT (0.76, 0.89, 0.60; all p's < .05) and Free 3HC/Free COT (0.68, 0.80, 0.51; all p's < .05). Total 3HC/Total COT did not vary over time (p = .81). CONCLUSIONS: Total 3HC/Free COT and Free 3HC/Free COT increased in the first trimester and continued to increase throughout pregnancy, suggesting a considerable increase in nicotine metabolism over gestation. Future analyses are needed to interpret the changes in NMR in the context of nicotine pharmacokinetics, as well as its impact on changes in smoking behavior and cessation outcomes. IMPLICATIONS: We observed that the NMR was significantly higher as early as 12 weeks' gestation and increased further as a function of gestational age. Among nonpregnant smokers, elevated NMR is associated with smoking phenotypes such as smoking more cigarettes per day and poorer response to nicotine patch; therefore, pregnancy-induced increases in the NMR may contribute to smoking during the first trimester of pregnancy and reducing or quitting smoking may become more challenging as the rate of nicotine metabolism accelerates over the course of pregnancy.

A Comparison of Direct and Indirect Analytical Approaches to Measuring Total Nicotine Equivalents in Urine.

Background: Total nicotine equivalents (TNE), the sum of nicotine and metabolites in urine, is a valuable tool for evaluating nicotine exposure. Most methods for measuring TNE involve two-step enzymatic hydrolysis for indirect quantification of glucuronide metabolites. Here, we describe a rapid, low-cost direct LC/MS assay.Methods: In 139 smokers' urine samples, Bland-Altman, correlation, and regression analyses were used to investigate differences in quantification of nicotine and metabolites, TNE, and nicotine metabolite ratio (NMR) between direct and indirect LC/MS methods. DNA from a subset (n = 97 smokers) was genotyped for UGT2B10*2 and UGT2B17*2, and the known impact of these variants was evaluated using urinary ratios determined by the direct versus indirect method.Results: The direct method showed high accuracy (0%-9% bias) and precision (3%-14% coefficient of variation) with similar distribution of nicotine metabolites to literary estimates and good agreement between the direct and indirect methods for nicotine, cotinine, and 3-hydroxycotinine (ratios 0.99-1.07), but less agreement for their respective glucuronides (ratios 1.16-4.17). The direct method identified urinary 3HC+3HC-GLUC/COT as having the highest concordance with plasma NMR and provided substantially better estimations of the established genetic impact of glucuronidation variants compared with the indirect method.Conclusions: Direct quantification of nicotine and metabolites is less time-consuming and less costly, and provides accurate estimates of nicotine intake, metabolism rate, and the impact of genetic variation in smokers.Impact: Lower cost and maintenance combined with high accuracy and reproducibility make the direct method ideal for smoking biomarker, NMR, and pharmacogenomics studies. Cancer Epidemiol Biomarkers Prev; 27(8); 882-91. ©2018 AACR.

Cigarette consumption and biomarkers of nicotine exposure during pregnancy and postpartum.

BACKGROUND AND AIMS: Smokers can regulate their nicotine intake by altering the number of cigarettes smoked per day (CPD) and their smoking intensity. The current study aimed to compare the utility of self-reported CPD, total nicotine equivalents (TNE) and urinary cotinine to estimate nicotine intake during pregnancy. DESIGN: Longitudinal smoking behavior and biomarker data were collected at early pregnancy, late pregnancy and at postpartum as part of a smoking cessation trial to examine voucher-based incentives for decreasing smoking. SETTING: Obstetric practices in Burlington, Vermont, United States. PARTICIPANTS: A subset of participants (n = 47) from the parent trial, recruited between December 2006 and June 2012, who provided a urine sample at each assessment during early pregnancy, late pregnancy and postpartum. MEASUREMENTS: Smoking was assessed using self-reported CPD, TNE, TNE/CPD and urinary cotinine. FINDINGS: Pregnant smokers reported smoking 10.4 CPD at early pregnancy, 7.2 CPD at late pregnancy (a 31% reduction at late pregnancy, P = 0.001) and 8.6 CPD at postpartum (a 19% increase from late pregnancy, P = 0.08). TNE exposure was 41% (P = 0.07) and 48% (P = 0.03) lower at early and late pregnancy, respectively, compared to postpartum. TNE/CPD was on average 167% higher at late pregnancy compared to early pregnancy (P = 0.01) and remained high at postpartum, where it was 111% higher compared to early pregnancy (P = 0.007). Uriniary cotinine underestimated nicotine intake by 55% during early pregnancy and by 65% during late pregnancy compared to postpartum (Pinteraction  < 0.001); the underestimation was greater in slower (Pinteraction  < 0.001) versus faster (Pinteraction  = 0.04) nicotine metabolizers. CONCLUSIONS: Neither cigarettes smoked per day (CPD) nor cotinine provides an accurate estimate of nicotine exposure during pregnancy. CPD underestimates nicotine intake substantially due to under-reporting and/or higher intensity of smoking, while cotinine underestimates nicotine intake markedly due to accelerated nicotine (and cotinine) metabolism during pregnancy.

Longitudinal Influence of Pregnancy on Nicotine Metabolic Pathways.

Nicotine metabolism increases in pregnancy, which may contribute to the difficulties that pregnant women have in quitting smoking. We aimed to determine the extent and timing of changes in nicotine metabolic pathways, including C-oxidation, N-glucuronidation, and the pregnancy-induced influences on the activity of enzymes mediating these pathways (CYP2A6 and UGT2B10, respectively). Current smoking pregnant women (n = 47) provided a urine sample during early pregnancy (12.5 weeks), late pregnancy (28.9 weeks), and 6 months postpartum. Concentrations of urinary nicotine and metabolites were analyzed using liquid chromatography tandem mass spectrometry and compared using general linear repeated measures analyses. Nicotine C-oxidation was 1.07-fold (P = 0.12) and 1.11-fold (P < 0.001) higher at early and late pregnancy, respectively, compared with postpartum. Nicotine N-glucuronidation was 1.33-fold (P = 0.06) and 1.67-fold (P = 0.003) higher at early and late pregnancy, respectively, compared with postpartum. The CYP2A6 phenotype ratio (total 3'-hydroxycotinine/cotinine) was significantly higher at early and late pregnancy compared with postpartum (all P < 0.05) and correlated with nicotine C-oxidation (all P < 0.001), suggesting CYP2A6 activity is induced during pregnancy. The UGT2B10 phenotype ratio (nicotine glucuronide/nicotine) was higher at early and late pregnancy compared with postpartum (P = 0.07 and P < 0.05, respectively) and correlated with a second UGT2B10 phenotype ratio (cotinine glucuronide/cotinine) (all P < 0.001), suggesting UGT2B10 activity is induced during pregnancy. In conclusion, pregnancy-induced increases in nicotine metabolism start by 12 weeks gestation and continue as pregnancy progresses most likely due to induction of CYP2A6 and UGT2B10, resulting in potential reductions in the effectiveness of nicotine replacement therapies and an increase in metabolism of other CYP2A6 and UGT2B10 substrates during pregnancy.

Effect of UGT2B10, UGT2B17, FMO3, and OCT2 genetic variation on nicotine and cotinine pharmacokinetics and smoking in African Americans.

OBJECTIVES: Nicotine metabolism rates differ considerably among individuals, even after controlling for variation in the major nicotine-metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine (COT) pharmacokinetics and smoking was investigated. METHODS: We examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography) before and after stratifying by CYP2A6 genotype in 60 African American (AA) smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and COT. RESULTS: Variants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in COT and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or COT pharmacokinetics among all individuals (n=60) or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and COT Cmax (P=0.005, 0.02, respectively) and decreased nicotine clearance (P=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3'-hydroxycotinine/COT ratio, commonly used as a biomarker of CYP2A6 activity. CONCLUSION: We found that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in AAs. The change in COT pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in COT as a biomarker of tobacco exposure among AA smokers.