Study participants
Healthy experienced EC users (vapers) where recruited for this study. To participate to the study they had to be: (1) former daily smokers of at least 5 years; (2) daily EC users for at least 1 month; (3) between the ages of 18 and 60; (4) clinically healthy, with no history of cardiovascular and lung disease or hematological problems; (5) able to remain abstinent from EC use for at least 8 hours; and (6) willing to provide blood samples. Exclusion criteria were: (1) pregnant or lactating females; (2) history of fainting or feeling faint associated with providing blood samples; and (3) being unwilling to provide written informed consent to participate to the study. The protocol was approved by the ethics committee of our institution and written informed consent was signed by all subjects before participating to the study.
Materials and clinical procedure
Two types of EC devices with the same liquid were used by the participants on two separate days, in a randomized cross-over design (Figure 1). The first-generation device was a typical cigarette-like EC (V2cigs, Miami, Florida, USA). The lithium battery has a capacity of 250 mAh and was fully charged before use. Empty cartomizers were bought by the same company and were filled according to company’s instructions with approximately 1 ml of an 18 mg/ml nicotine containing liquid (Max Blend, Flavourart SRL, Oleggio, Italy). Users were provided with more fully-charged batteries if discharged and new cartomizers if emptied during the 65 minute period. The new-generation device consisted of a large lithium battery part (capacity of 2600 mAh) with an internal electronic circuit which includes a current stabilizer and allows the user to manually adjust the energy applied to the atomizer (EVIC, Joyetech, ShenZhen, China). A new-generation atomizer (EVOD, KangerTech, ShenZhen, China) was used and was filled with approximately 2 ml of liquid. The energy delivery to the atomizer was set to 9 watts.
Participants visited the laboratory after abstaining from EC use, caffeine, alcohol and food intake for at least 8 hours. Carbon monoxide in exhaled breath was measured by a calibrated Bedfont Micro Smokerlyzer. Subsequently, a venous catheter was introduced in an antecubital vein and 8 ml of venous blood was collected in lithium-heparinized vacutainers. Participants were asked to take 10 puffs in 5 minutes, simulating tobacco cigarette use26. After this period, they were asked to use the ECs ad lib for 60 more minutes (total duration of use: 65 minutes). Ten minutes after the end of the 65 minute period, carbon monoxide levels were measured again.
Blood samples and nicotine measurements
Blood samples were taken after the 5 minute period and every 15 minutes during the additional 60 minute period. The samples were stored in ice and were centrifuged within 1 hour. Plasma was separated and stored at −70°C until analyzed. Measurements of nicotine levels were performed in a specialized laboratory by Gas Chromatography with an NPD-80 Specific Detector. The lowest limit of quantification (LOQ) for this method was 0.5 ng/ml. For samples with nicotine levels below the LOQ, a value of LOQ/2 was assigned for statistical analysis.
Questionnaires on smoking and EC dependence, craving assessment and EC use effects
To define their past dependence to tobacco cigarettes, two tests were performed; the Fagerström Test for Cigarette Dependence (FTCD)12 and the Cigarette Dependence Scale (CDS)13. To evaluate their current EC dependence, the previously mentioned tests were also applied for EC use. Since both questionnaires include a question for cigarette consumption, the question was adjusted for EC use based on the results of a survey of 19,441 EC users performed by our group (unpublished data). The consumption was classified according to percentiles (quartiles for FTCD and quintiles for CDS). The results of the two tests after excluding the question on cigarette and EC consumption were also reported. To avoid any interaction between the answers, the questionnaires for smoking and EC use were administered on separate days to the participants.
To assess craving for nicotine before and after EC use, a shortened version of the Cigarette Withdrawal Scale (CWS) was used14,15. It consists of rating the extend of agreement with the following statements, adopted for EC use: (1) “The only thing I can think about is using (vaping) an EC”; (2) “I miss the EC terribly”; and (3) “I feel an irresistible need to vape”. For each question, answers were scored as: 0 = “totally disagree”, 1 = “mostly disagree”, 2 = “more or less agree”, 3 = “mostly agree”, 4 = “totally agree”. The sum of the scores from each question was calculated and reported. Additionally, a simple craving rating was used, by using a 100 mm visual analogue scale and asking: “How much do you crave an EC right now?”. Participants were asked to draw a cross through the horizontal line and the score was calculated by measuring the distance between the cross and the left anchor. Both CWS and simple craving rating were asked at baseline, after 5 minutes and after 65 minutes of use.
To assess the effects of nicotine and EC use, participants responded to 100 mm visual analogue scale questions after the end of the 65 minute period of EC use. Questions were adopted from Vansickel et al8. and from Hutsmuller and Stitzer27. Each word or phrase was centered above the horizontal line that represented a scale from 0 to 100 points; the left anchor was “Not at all” and the right anchor was “Extremely”. The score was assessed in a similar way as described above for the simple craving rating.
Statistical analysis
Categorical variables were expressed as number (percentage) while continuous variables as mean (SEM). To compare CWS and simple nicotine craving scale, repeated measures analysis of variance (ANOVA) was used, with two within-subjects factors: timing (3 levels) and session (2 levels). To compare exhaled carbon monoxide levels, repeated measures ANOVA was also used but timing had 2 levels (baseline and post-65 minute); for nicotine levels, timing had 6 levels. To assess perceived effects of nicotine and EC use, paired student’s t-test was used. A two-tailed P value of ≤ 0.05 was considered statistically significant. All analyses were performed with commercially available statistical software (SPSS v.18, Chicago, Illinois, USA).
