All smokers were asked to use one commercially-available tobacco cigarette of the same nicotine (1.0 mg), tar (10 mg) and carbon monoxide (10 mg) yields. Electronic cigarette users were asked to use a commercially-available device with liquid containing 11 mg/ml nicotine concentration. The device used was an eGo-T battery (Nobacco, Athens, Greece) with an eGo-C atomiser (Alter Ego, Athens, Greece). It is considered a “second-generation” device. Unlike cigarette-like devices which consist of a small battery and a polyfil-containing atomiser (commonly called “cartomiser”), the electronic cigarette used in this study is a multi-piece system (Figure 1). It consists of a 650 mAh rechargeable lithium battery, delivering 3.5 volts to the atomiser (measured by a volt-meter), and an atomiser consisting of 4 parts: the tank which stores the liquid (capacity of approximately 1.1 ml), the atomiser body, the atomiser head which includes the resistance, and the atomiser cap. It is a manually-activated device, by pressing a button; it does not produce any vapour when not activated by the user.

Figure 1 Electronic cigarette device and liquid used in the study. Full size image

The electronic cigarette liquid used in the study contained 11 mg/ml nicotine and is considered “medium strength” according to manufacturer’s report (USA Mix Med, formerly known as MLB-Med, Nobacco, Athens, Greece). It is sold in 20 ml bottles. It was the only liquid tested by an independent laboratory (National Center for Scientific Research “Demokritos”, mass spectrometry and dioxin analysis laboratory) at the time of study initiation [9]. According to the laboratory report, the contents were: propylene glycol (α -propylene glycol or 1,2-propanediol) in a concentration > 60%, linalool (3,7-dimethylocta-1,6-dien-3-ol) in a concentration < 5%, nicotine (<10%), tobacco essence (<5%), and methyl vanillin (4-hydroxy-3-methoxybenzaldehyde) at < 1%. No tobacco-specific nitrosamines or polycyclic aromatic hydrocarbons were detected.

For every participant, a new cartridge and atomiser head was used. One of the researchers filled the cartridge with 1 ml of liquid; subsequently it was positioned in the atomiser and the participant started using it. The battery was fully charged before being used by each subject.

Study protocol

Participants presented to the echocardiographic laboratory after fasting and refraining from alcohol and caffeine consumption for 4 hours; they were also asked to refrain from smoking and electronic cigarette use for 4 hours before the study.

Participants were allowed to rest for 5 minutes before initiating the echocardiographic examination. A baseline echocardiographic examination was performed in smokers, who were then transferred to a room next to the echocardiography laboratory and smoked 1 tobacco cigarette. For electronic cigarette users, after the baseline echocardiogram they were asked to use the electronic cigarette device ad lib for 7 minutes in another room which was not used by smokers, to avoid environmental exposure to smoke. Subsequently, all participants returned to the echocardiography laboratory and, after 5 minutes of rest, a second echocardiogram was performed in both groups.

Heart rate and BP were measured before and during each echocardiographic examination. The Brinkman index was calculated (product of number of cigarettes smoked daily and years of smoking) according to participants’ self-report. Echocardiograms were performed using a commercially available system (Vivid 7, GE Vingmed, Horten, Norway). Studies were digitally recorded on hard disk for offline analysis using dedicated software (Echopac, GE Medical Systems, Horten, Norway) by a single, blinded to the protocol, experienced echocardiographer. Reported values represent the average of 3 consecutive beats.

Two-dimensional echocardiographic measurements

The echocardiographic examinations were performed according to recent guidelines [9]. LV dimensions, septal and posterior wall thickness were measured from standard 2-dimensional images at parasternal long-axis view. LV mass was indexed to body-surface area. Ejection fraction was evaluated from the apical four and two-chamber views using the Simpson’s rule [10]. Left atrial (LA) antero-posterior diameter was also measured.

Doppler flow and tissue Doppler velocity measurements

From transmitral flow measurements, peak early (E) and late (A) velocities, their ratio (E/A) and E wave deceleration time (DT) were estimated. Ejection time was estimated by recording LV outflow tract velocity. By simultaneously recording aortic and mitral flows using continuous-wave Doppler the isovolumetric relaxation time (IVRT) was measured, and was then corrected to heart rate by dividing it with the square root of R-R interval (IVRTc).

Pulsed-wave Doppler tissue velocities were measured by placing a 1.5 mm sample volume at the lateral, septal, anterior and inferior insertion sites of the mitral leaflets. Systolic (Sm), early diastolic (Em) and late diastolic (Am) peak velocities were measured and averaged from the 4 sites. The ratio of early-to-late annular velocity (Em/Am) and early mitral flow to early diastolic mitral annular velocity (E/Em) were also determined.

Myocardial performance index was measured by two methods (Figure 2): using Doppler flow velocity measurements as described by Tei et al. [11] (MPI) and using pulsed-wave tissue Doppler measurements of mitral annulus velocities (MPIt) [12].

Figure 2 Myocardial performance index, measured by two methods: (1) Doppler flow velocity measurements of mitral inflow and left ventricular outflow tract; the index was derived by the formula: MPI = (a-b)/b, and (2) Pulsed-wave tissue Doppler measurements of mitral annulus velocity; the index was derived from the formula: MPIt = (a’-b’)/b’. Full size image

To check for reproducibility of measurements, the intraobserver mean percent error (the absolute difference between two measurements divided by their mean) was calculated from 10 randomly selected studies 15 days later, analyzed by the same blinded echocardiographer who performed all measurements. The results were 5.1 ± 2.9% for IVRT, 3.5 ± 2.5% for MPI, 3.6 ± 2.2% for MPIt and 2.6 ± 1.9% for Em.

Longitudinal deformation measurements

Longitudinal deformation measurements were performed by analyzing two-dimensional echocardiographic images using the method of speckle tracking echocardiography [13]. End-diastole was defined as the peak of the R wave on the electrocardiographic trace; end-systole (aortic valve closure) was defined from pulsed-wave Doppler tracing at the LV outflow tract as the end of systolic forward flow. Subjects with inadequate tracking of more than one LV segment in each view were excluded from the analysis. By averaging segmental values in all views, end-systolic global strain (GS) was measured. Global peak longitudinal systolic (SRs), early diastolic (SRe) and late diastolic (SRa) strain rate were measured. The intraobserver mean percent error of longitudinal deformation measurements in our laboratory was 3.1 ± 1.5% for GS, 3.6 ± 1.8% for SRs, 3.9 ± 1.9% for SRe and 3.6 ± 2.0% for SRa.

Statistical analysis

The Kolmogorov-Smirnov tests were applied to assess the normality of data; all parameters were normally distributed except from daily cigarette consumption. Continuous variables were expressed as mean ± SD or median (interquartile range). Categorical variables were expressed as number (percentage). Inter-group comparisons of baseline characteristics data were made by unpaired Student’s t-test and Mann–Whitney test; Fisher’s exact test was used for categorical variables.

Repeated measurements analysis of variance (ANOVA) was used in order to evaluate changes in parameters before and after smoking one cigarette or using the electronic cigarette device (before-use and after-use measurements). Changes in echocardiographic and deformation parameters that were significantly different between the two study groups from analysis of variance were further analyzed using linear regression analyses, in order to find if the effect of smoking was significant after adjusting for changes in heart rate and systolic BP. For every parameter, a different linear regression analysis was performed. Change (Δ) in parameter was the dependent variable; group (SM vs. ECIG) and change in heart rate and systolic BP were the independent variables. All P values reported are two-tailed. Statistical significance was set at 0.05 and analyses were conducted using SPSS statistical software (version 18.0, SPSS Inc., Chicago, USA).

A repeated measures ANOVA power analysis was conducted. For this design, 76 participants (40 in the smokers group and 36 in the electronic cigarette users group) achieved a power of 0.90 for the between-subjects main effect at an effect size of 0.30; a power of 0.90 for the within-subjects main effect at an effect size of 0.15; and a power of 0.90 for the interaction effect at an effect size of 0.15.