Participants were recruited from the Collaborative Genetic Study of Nicotine Dependence (COGEND), a multi-site project in the United States [1 (link)]. Potential subjects were excluded from participation if they reported regularly using psychotropic medications (e.g., paroxetine, fluoxetine, nortriptyline, sertraline, heloperidol, olanzepine, risperidone, quetiapine, diazepam, valproate, lithium), systemic steroids, antihistamines, or cimitidene. Oral contraceptive use was not determined. All subjects were self-identified as being of European ancestry and between 27 and 44 years of age. Self-reported race was previously verified using EIGENSTRAT [2 (link)]. Current smoking status was defined by a mean non-deuterated cotinine measurement of >2 ng/ml. The relatively low cut-point was chosen to exclude all individuals who reported smoking in the previous week. One male subject currently using a nicotine patch was excluded from all analyses of smoking status. Results for all analyses of smoking status did not differ using a more stringent definition (mean D0-cotinine > 20 ng/ml). Subject characteristics are summarized in Supplemental Table 1 . All subjects were requested not to consume food, and especially grapefruit and grapefruit juice, for 12h prior to their visit. Subjects were not asked to abstain from smoking except between their arrival and the first blood draw, 30 minutes after D2-nicotine administration. The time of last cigarette smoked was recorded for each subject.
Subjects were given 2 mg of [3′,3′-D2] nicotine (synthesized and purified to >99.4 % as described previously [21 ]) in 4 oz of juice, and blood was drawn approximately 30, 150, and 240 minutes later. This dose is well tolerated by non-smokers and results in measurable plasma nicotine concentrations[14 (link)]. All but 9 subjects were administered D2-nicotine prior to noon, the majority between 8 AM and 10 AM. The observed difference in plasma D2-cotinine at 30 min (Table 2 ) was not affected by the time of D2-nicotine administration. Plasma was collected and frozen at −20°C until analysis. The study complies with the Code of Ethics of the World Medical Association and obtained informed consent from participants and approval from the appropriate institutional review boards.
Subjects were given 2 mg of [3′,3′-D2] nicotine (synthesized and purified to >99.4 % as described previously [21 ]) in 4 oz of juice, and blood was drawn approximately 30, 150, and 240 minutes later. This dose is well tolerated by non-smokers and results in measurable plasma nicotine concentrations[14 (link)]. All but 9 subjects were administered D2-nicotine prior to noon, the majority between 8 AM and 10 AM. The observed difference in plasma D2-cotinine at 30 min (
