To test this hypothesis, 109 smokers of ≥10 cigarettes per day and ages 18–65 years were recruited from participants in a nicotine replacement therapy trial (for exclusion criteria see 15). The study protocol was approved by the Institutional Review Board of the University of Pennsylvania and all other analytical sites.
Participants were recruited from April 2005 to February 2006. Following informed consent and prior to initiating treatment, they completed measures of demographics, smoking history, current cigarette brand, and nicotine dependence level (Fagerstrom Test of Nicotine Dependence; FTND;16). They provided a 15ml blood sample for analysis of nicotine metabolites, assayed at the University of California, San Francisco via liquid chromatography with tandem mass spectrometry (9 (link)). Genotyping for CYP2A6 variants was performed at the Centre for Addiction and Mental Health (Toronto, Canada) as previously described (17 ). A 30ml urine sample was provided and assayed for total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the sum of NNAL and its glucuronides, at the University of Minnesota per standard procedures (18 ).
In a smoking-approved ventilated room, participants smoked one of their own brand cigarettes under ad libitum conditions, using a smoking topography device (Clinical Research Support System; Borgwaldt, Richmond VA) validated in prior research (19 , 20 (link)). Participants were asked to refrain from smoking for one hour prior to their laboratory smoking session, and were generally compliant (mean 64.3 minutes; SD=35.4, range 42–190). Total puff volume, defined as the sum of all puffs taken, was a priori selected as the outcome measure for analysis (21 (link), 22 ).
NMR values were positively skewed (+1.8) and have positive kurtosis (+4.8) consistent with previously results, and was therefore log-transformed (15 , 17 ). NMR quartiles were created (15 , 23 (link)) and CYP2A6 genotypes were coded as described previously (17 ). NMR quartiles, previously determined from Received Operator Characteristic analyses, have been used to characterize smokers' response to transdermal nicotine treatment and bupropion (15 ; 17 ; 24 (link)), and therefore were used in this study to assist comparisons to previous research that utilized NMR. Hypotheses were tested using analysis of covariance (ANCOVA) where total puff volume and total NNAL were the outcome measures and NMR quartile was the between group factor. Fisher's post-hoc analyses were used to identify quartile differences. Regression analysis was used to examine the association between log-transformed NMR and total puff volume, as well as with NNAL. Stepwise regression analysis was used to examine the association between smoking behavior (daily cigarette consumption, total puff volume, and their product as an index of daily puff volume) and total NNAL levels, retaining covariates at p<.2.