Reversibility of Propylene Glycol/Glycerol Intake Effects on Cardiorespiratory Function
Phase: NA
Conditions: Lung Injury
Sponsor: Université Libre de Bruxelles

Unique Protocol ID: P2016/466 / B406201629930 / NCT03410511   
Brief Title: Propylene Glycol/Glycerol Intake and Cardiorespiratory 
Function (PGGICF)   
Official Title: Reversibility of Propylene Glycol/Glycerol Intake Effects on 
Cardiorespiratory Function   
 
1. 
Background 
 
1.1. 
State of the art 
 
Electronic cigarettes (e-cigarettes) are increasingly popular worldwide. About 5% of adults in 
the western world are current users. 1 The liquid (e-liquid) carriers vaporized are composed of 
propylene glycol (PG) and glycerol (GLY), which transport nicotine and/or flavorings. 2 Regular 
e-cigarettes users purchase last generation devices delivering high energy level to low coil 
resistance. 3 High energy settings are used to increase heat and vapor production, 4 to 
enhance the throat and nicotine hits, 5 whilst nicotine concentration in the e-liquid is strongly 
reduced through time. E-cigarettes with high output wattage increase the quantity of e-liquid 
consumed per puff, and produce volatile carbonyls by thermal degradation. 4 Daily exposures 
to large amounts of high temperature PG/GLY aerosol may present a hazard to health. 
 
1.2. 
Study of acute vaping effects on cardiorespiratory parameters (2016-2017) 
 
We found that acute PG/GLY mix (50:50) inhalation: 1) induced a sustained transcutaneous 
hypoxia without modifying carbon dioxide (CO2) tension; 2) decreased hemoglobin oxygen 
saturation, as assessed by fingertip pulse oximeter;  3) injured the lower airway, as reflected 
by urine and serum club cell protein 16 (CC16) rises, 6 and small airway constriction during 
lung function tests. 7  
 
1.3. 
Study of acute nicotine free vaping effects on arterial O2 concentration (2017-
2018) 

 
At our knowledge, we are the first to demonstrate that acute high wattage PG/GLY vaping 
induces transcutaneous hypoxia. Transcutaneous hypoxia has been described with tobacco 
smoking, as a consequence of nicotine-induced vasoconstriction, 9 but effects of a nicotine 
free PG/GLY liquid vaporization on this parameter were unknown. Tissue oxygen (O2) and 
CO2 tensions reflect the delivery of O2 and the extraction of CO2, which are both dependent 
on respiratory and microcirculatory functions. 10 The decrease in underlying dermal O2 levels 
could be explained by: 1) a decrease in arterial O2 concentration; 2) changes in hemodynamic 
parameters; and/or 3) microcirculatory endothelial dysfunction. 10 Our current experiments are 
focusing on acute effects of PG/GLY vaping on arterial O2 concentration, namely, 
concentration of hemoglobin, O2 saturation of hemoglobin and its biological determinants and 
arterial O2 partial pressure. 11-2 
 
1.4. 
Studying the reversibility of PG/GLY aerosol effects on lungs in regular e-
cigarettes users 
 
Our new working hypothesis is that high temperature vaping deposits a hot and moist large 
amount nanoparticular PG/GLY aerosol deeply in the lungs, 13 resulting in small airways 
inflammation and constriction,13 rheological surfactant and mucus properties disturbances,14 
small bronchioles and alveoli collapses, 14 arterial oxygenation impairments, which decrease 
hemoglobin-oxygen saturation and induce the tissue hypoxia we observed. 10  
In order to verify the new hypotheses aforementioned on PG/GLY vaping induced-lung gas 
exchanges perturbations and its reversibility, as well as to disentangle respective effects of 
PG/GLY and nicotine, we will test effects of vaping cessation in regular e-cigarettes users by 
means of classical cardiorespiratory parameters assessments, 17-19 lung function tests with 
diffusing capacity analysis and a maximal cycle ergospirometer test. 20 Lastly, quantitative 
discovery serum proteomics 21 and metabolomics 22 in the serum, urine and exhaled breath 

condensate will be performed with the view to highlight biomarkers associated with chronic 
high wattage vaping exposition. 
 
2. 
Goals of the research 
 
This research proposal tests the following hypothesis regarding the reversibility of PG/GLY 
and PG/GLY plus nicotine vaping in regular e-cigarettes users 
 
A. In comparison to pursuit of PG/GLY vaping with or without nicotine, e-cigarette 
cessation for five days: 
• 
Increases transcutaneous O2 tension and O2-haemoglobin saturation 
• 
Decreases respiratory rhythm 
• 
Increases lung diffusing capacities and forced expiratory flows 
• 
Increases maximal rate of oxygen consumption during cycle ergospirometer test 
• 
Modifies heart rate variability 
• 
Decreases arterial stiffness 
• 
Decreases serum PG/GLY and nicotine concentrations 
 
B. In comparison to PG/GLY vaping with nicotine, e-cigarette cessation for five days or 
PG/GLY vaping: 
• 
Increase cutaneous vascular conductance and decrease arterial stiffness 
• 
Increase acetylcholine and heat mediated vasodilation 
 
C. Each of the three experimental sessions (e-cigarette cessation, PG/GLY vaping and 
pursuit regular consumptions): 
• 
Induces specific serum, urine and exhaled breath condensate metabolomics profiles 
• 
Induces specific serum proteomics profile 

 
This research proposal tests also the following hypothesis regarding acute effects of PG/GLY 
vaping with and without nicotine in regular e-cigarettes users: 
 
A. In comparison to baseline, PG/GLY vaping without nicotine: 
• 
Decreases transcutaneous O2 tension and O2-haemoglobin saturation 
• 
Increases respiratory rhythm 
• 
Increases serum PG/GLY concentrations 
• 
Induces specific serum, urine and exhaled breath condensate metabolomics profiles 
 
B. In comparison to baseline and PG/GLY without nicotine, PG/GLY vaping with nicotine: 
• 
Decreases transcutaneous O2 tension and O2-haemoglobin saturation 
• 
Decrease continuous vascular conductance and increase arterial stiffness 
• 
Increases respiratory rhythm 
• 
Increases serum PG/GLY and nicotine concentrations 
• 
Induces specific serum, urine and exhaled breath condensate metabolomics profiles 
 
3. 
Methodology 
 
3.1. 
Participants, interventions, periods and randomization 
 
The participants, who are exclusive vapers since at least one years, will undergo three 
experimental periods in a random order: e-cigarettes cessation for five days (cessation 
session), nicotine free vaping for five days (nicotine free session), regular e-cigarettes 
consumption (regular consumption session). Baseline clinical cardiorespiratory parameters 
will be assessed at the start of each of the 3 experimental sessions. The participants will, then, 
vape sham-puffs (cessation session), nicotine free-puffs (nicotine free session) and 

nicotine puffs (regular consumption session). During acute exposition, we will monitor at 
each puff any feelings of throat irritation, burn taste and Borg perception scale items (cough, 
chest tightness, breathlessness, secretions and wheezing). This acute exposition will be 
followed by clinical cardiorespiratory parameters assessment, lung function tests and maximal 
cycle ergometer test.  
 
3.2. 
Measures to ensure strict compliance of the study protocol 
 
All the participants will sign a declaration on honor that they will respect the study protocol. 
Twelve hours fasting blood samples will be drawn during each experimental session at 
baseline and 1 hour after acute exposition. All blood samples will undergo serum nicotine, PG 
and GLY concentration analysis to confirm compliance of the study protocol. 23-26 In case of a 
protocol violation, the participant will not be allowed to continue the study, and his full dataset 
will be discarded from the study.  
 
3.2.1 Serum nicotine concentration 
 
At each experimental session, participants will be negative for urine cotinine (urine dipstick – 
detection threshold of 50 ng/ml – NarcoCheck©) 23 and concentration of serum nicotine should 
be below 2 ng/ml, a value confirming the status of nicotine free vaping and the lack of 
significant nicotine exposure. 24,27 Serum nicotine will be measured by means of a mass 
spectrometer with a jet stream electrospray ion source. 
 
3.2.2 Serum propylene glycol and glycerol 
 
Since serum PG and GLY normal values in regular vapers are unknown, we will take means 
of PG and GLY serum concentration measured during the regular consumption session 

and nicotine free session for further comparison to the value obtained during the cessation 
session, in order to confirm the compliance to the study protocol. Serum values of PG and 
GLY will be assessed using gas chromatography with a flame ionization detector. 25 
 
3.3 Project plan and various step 
 
The 5h-sessions will take place in the morning at the Hôpital Erasme. During each session, 
participants will be tested  before and after acute exposure at the Cardiology Laboratory (ULB 
– Pr. Philippe van de Borne) for: 1) noninvasive continuous hemodynamic parameters 
(Electrocardiogram monitoring (AdInstrument©) and a beat-to-beat hemodynamic monitoring 
system17 Finapres Medical Systems©) 2) continuous respiratory rhythm, end-tidal carbon 
dioxide and blood oxygen saturation (Capnostream©); 28 3) transcutaneous oxygen and carbon 
dioxide partial pressures (Perimed©); 10 4) skin microcirculatory blood flow by means of laser 
Doppler perfusion monitoring (continuous vascular conductance, heat test, acetylcholine and 
sodium nipride iontophoresis) (Perimed©); 18 5) Fractional exhaled carbon monoxide will be 
assessed at the start of each experimental session using a Smokerlyzer (SineFuma©, Breda, 
Holland);  6) arterial stiffness indices (Sphygmocor©); 29 7) exhaled breath condensate (Viasys 
Ecoscreen©); 19 8) Urine and 9) venous blood sample. Then, the participants will be directed 
to the Laboratory of Exercise Physiology, Faculty of Motor Sciences (ULB – Pr. Vitalie Faoro) 
to perform assessment of: 10) lung function tests plus capillary and membranous lung diffusing 
capacity as well as maximal ergometer cycle test 20 (details in appendix) (figure 2). After 
completion of clinical study in all participants, biological samples will undergo proteomics 21 
(ULB – Pr. David Communi) and metabolomics 22 (UMons – Pr. Jean-Marie Colet) analysis 
(details in appendix). 
 
3.3.1 Noninvasive continuous hemodynamic parameters 
 

After 20 minutes of comfortable rest, in the supine position, humeral blood pressure will be 
determined according to guidelines. 30 Blood pressure measurements will be performed before 
(baseline) and immediately after vaping sessions or sham-vaping. A cuff will be placed on the 
middle phalanx of the right middle finger in order to obtain a finger blood pressure waveform 
with a beat-to-beat hemodynamic monitoring system (Finometer Pro©, FMS, Amsterdam, the 
Netherlands).17 This permitted a continuous monitoring of the humeral systolic and diastolic 
blood pressures throughout all the experimental sessions duration. Pulse rate will be 
monitored throughout the study by the finometer recordings. In addition a continuous 
electrocardiogram monitoring will be performed (AdInstrument©). 
 
3.3.2 Continuous respiratory rhythm, end-tidal carbon dioxide and blood oxygen 
saturation 
 
Continuous, non-invasive, real-time respiratory status monitoring of end-tidal CO2 pressure, 
O2 saturation in arterial blood, respiration rate, and pulse rate will be assessed with the 
Capnostream 35 monitor© (Oridion Medical Ltd, Jerusalem, Israël) throughout all the 
experimental sessions duration. 
 
3.3.3 transcutaneous oxygen and carbon dioxide partial pressures 
 
A PeriFlux system 5000 (Perimed©, Järfälla, Sweden) will explore transcutaneous oxygen (O2) 
and carbon dioxide (CO2) partial pressures by means of a PF 5040 unit and a dual 
transcutaneous O2 and CO2 partial pressures E5280 electrode. After wiping the area with 
alcohol, a TC 550 fixation ring (Perimed©, Järfälla, Sweden) will be applied to the skin in the 
medial side of the right arm (lower third and 5 cm distal to the antecubital fossa) at the level 
of the heart. Four drops of contact liquid (TC 560, Perimed©, Järfälla, Sweden) will be instilled 

inside the TC 550 fixation ring. Avoiding superficial vessels and body hair, the electrode will 
be placed at the same location for each of the two sessions on the basis of multiples pictures 
taken during the experimental session. Transcutaneous O2 and CO2 partial pressures values 
will be considered stable when variations did not exceed ±2 mmHg within 1 min. Electrode 
remembraning will be done every two sessions. 
 
3.3.4 skin microcirculatory blood flow by means of laser Doppler perfusion monitoring 
 
3.3.4.1 Skin continuous vascular conductance 
 
Skin continuous microcirculatory blood flow will be assessed using a PeriFlow system 5000, 
PF 5010/5020 with the thermostatic probe 457 (Perimed©, Järfälla, Sweden) at the level of 
the right arm at least 5 cm from the PF 5040 with combined transcutaneous O2 and CO2 - 
E5280 electrode; and mean arterial pressure will be measured throughout the sessions with 
a beat-to-beat hemodynamic monitoring system (Finometer Pro, FMS©, Amsterdam, the 
Netherlands) on the right middle finger.17 Cutaneous vascular conductance will be then 
calculated as skin continuous microcirculatory blood flow divided by mean arterial pressure. 
 
3.3.4.2 Evaluation of microvascular reactivity to pharmacological stimulation. 
 
“Endothelium-dependent and -independent vasodilatation of the skin microcirculation 
(measured in perfusion units, PU= 10 mV) will  be noninvasively and continuously evaluated 
by laser Doppler fluxmetry technique  in combination with iontophoresis (Periflux 5001 and 
PeriIont, Perimed, Jarfalla, Sweden) of acetylcholine (ACh), after vaping or sham vaping and 
iontophoresis of sodium nitroprusside (SNP), minutes after vaping or sham vaping. The laser 
Doppler flowmetry probes will be constantly heated at throughout the study so as to avoid 
changes in microvascular blood flow due to local skin temperature variations. Drug-delivery 

electrodes (PF 383, Perimed) incorporated into the head of the laser probe (PF 481-1, 
Perimed), will be carefully placed, by the use of specific double sided skin adhesive patches 
on the forearms. The drug-delivery electrodes will be filled with 200 µl ACh dissolved in distilled 
water and will be attached with the laser probe to a standardized site on the right forearm 
(anterior and proximal part) that had no visible veins. The dispersive electrode will be attached 
approximately 15 cm away from the electrophoresis chamber, according to the manufacturer’s 
instructions. A protocol of three fixed doses (current intensity-delivery time) will be employed, 
resulting in an incremental dose±response curve. After measuring the resting flow, three doses 
of 0.1 ACh dissolved in distilled water will be delivered using a cathodal current (0.1 mA for 80 
seconds, with total charges of 8 millicoulombs) at 120-second intervals. The mean values of 
the resting flow will be considered to be the basal flow values for each patient. Using a new 
delivery electrode applied on the leff forearm, three doses of a solution of SNP dissolved in 
distilled water will be delivered using an anodal current (0.1 mA for 80 seconds, with total 
charges of 8 millicoulombs at 120-second intervals). The curves of the microvascular flow 
increases induced by SNP and Ach will always have a total recording time of 20 minutes: three 
times 80-second of iontophoresis at 120-second intervals and 10 minutes to reach the plateau 
response after the last dose. “ 18 (CLINICS 2011;66(4):599-605) 
 
3.3.4.3 Heat test 
 
“Using another laser probe (PF 457, Perimed) at the level of right distal arm (anterior side) 
that will be positioned at the beginning of the recordings. Maximal skin microvascular 
vasodilatation will be measured after vaping or sham vaping using prolonged (20 minutes) 
local heating of the laser probe to 44°C. The use of the area under the blood flow/time curve 
(AUC) for assessing skin microvascular reactivity using Laser Doppler Perfusion Monitoring is 
well validated because it represents the global flow response to different physiological and 
pharmacological stimuli.”33-35 (CLINICS 2011;66(4):599-605) 
 

3.3.5 fractional exhaled carbon dioxide 
 
Fractional exhaled carbon monoxide will be assessed at the start of each experimental 
session using a Smokerlyzer (SineFuma©, Breda, Holland). Fractional exhaled carbon 
monoxide should be less than 5 ppm. 36 
 
3.3.6 arterial stiffness indices 
 
3.3.6.1 Aortic wave reflection assessment 
 
“Using applanation tonometry at the level of left radial pulse, arterial waveforms will be 
recorded during 8-second with a high-fidelity SPC-301 micromanometer (Millar Instrument©, 
Texas, USA) by means of a fully automated and validated system (SphygmoCor, Atcor 
Medical), version 9.0 software (AtCor Medical©, New South Wales, Australia). Three minutes 
before aortic wave reflection assessments, manual blood pressure measurements (Mercury 
sphygmomanometer, WelchAllyn©, New York, USA) will be performed according to 
guidelines29 in order to calibrate radial pulse wave. Aortic pressure waveforms will be derived 
from the peripheral radial pressure waves using a validated generalized transfer function.  This 
has been shown to accurately estimate aortic pressure waveforms based on the assumption 
that mean blood pressure does not change along the arterial tree. In case of decreased aortic 
compliance or increased peripheral resistance, the reflected wave come back faster to the 
aorta. This can be assessed by the augmentation index (AIx; 100× augmentation 
pressure/pulse pressure), representing the pressure boost induced by the return of these 
reflected waves to the aorta, expressed as a percentage of the pulse pressure. Higher values 
of AIx indicate an earlier return of the reflected wave to the aorta. Ventricular ejection time 
determines the aortic pressure waveform. In case of heart rate alterations with ventricular 

ejection time modifications, AIx changes may be masked or overestimated. This is why all the 
AIx presented in the article will be corrected for heart rate (Aix75) according to the linear 
relationship established by Wilkinson et al,37 namely, that for every 10-bpm increase in heart 
rythm, AIx decreases by 4%. These measurements will be made ten minutes before and three 
times after exposure. “ Clinical and Experimental Pharmacology and Physiology (2009) 36, 784–789 
 
3.3.6.2 Pulse wave velocity measurements 
 
“We will assess the carotid-femoral pulse wave velocity (PWV) using a sequential waveform 
measurements approach at carotid and femoral sites with applanation tonometry (SPC-301 
micromanometer) and SphygmoCor software as previously described. 29 The pulse waves 
time period between the carotid and femoral sites will be assessed with an electrocardiograph-
derived R wave as a fixed point. This pulse wave time will be the average of 10 consecutive 
beats. The difference between the path in cm from the left carotid sampling site to the 
suprasternal notch, and the path in cm from the left femoral sampling site to the suprasternal 
notch will be used to define the distance which the pulse wave travels.” Clinical and Experimental 
Pharmacology and Physiology (2009) 36, 784–789 
 
3.3.7 exhaled breath condensate 
 
The participant will close his nose with a nose-clip and approach the mouthpiece. For 
collecting breathing condensate, the participants will breathe via an elbow piece with 
connected mouthpiece through a nonrebreathing valve block in which inspiratory and 
expiratory are separated. During expiration, the breathing air flows through a lamellar 
condenser which is cooled by the refrigerator circuit. This lamellar condenser is cooled down 
to -30°C. In this condenser, the breathing condensate is precipitated on the inner wall and 
drops in the sample collection vessel. The machine which will be used is a ECoScreen 

(VIASYS Healthcare GmbH, Hoechberg, Germany). The exhaled breath condensates will be 
drawn at the start of the experimental and 90 minutes after exposure.   
 
3.3.8 Urine and venous blood samples 
 
Blood will be drawn in the left antecubital vein before and 60 minutes after exposure. Urine 
specimens will be collected one hour before and 100 minutes after exposure. 
 
3.3.9 Lung function and diffusing capacity evaluation 
 
“In order to detect lung diffusing perturbations at a pulmonary capillary or membrane level 
induced by vaping, we will assess effects of e-cigarettes cessation on lung diffusing capacities 
for carbon monoxide (DLCO) and nitric oxide (DLNO) measured with a double nitric 
oxide/carbon monoxide single breath technique at the Laboratory of Exercise Physiology (ULB 
– Faculty of Motor Sciences – Pr. Vitalie Faoro). Briefly, the transit of carbon monoxide through 
the alveolocapillary membrane and into the plasma and intraerythrocytic compartments 
determines the DLCO, as defined by the Roughton and Forster equation. On the other hand, 
the DLNO is thought to represent the true membrane diffusing capacity because of its very 
high affinity for hemoglobin and its independence from pulmonary capillary blood volume. 
Therefore, the DLNO/DLCO ratio can be used to differentiate thickened alveolocapillary 
membranes (both DLNO and DLCO are decreased, and the DLNO/DLCO ratio is normal) and 
decreased perfusion of ventilated alveoli (the DLNO less decreased than the DLCO; therefore, 
the DLNO/DLCO ratio is elevated).” 20 (J Appl Physiol (1985) 2014;116:919-26.) 
 
3.3.10 Maximal ergometer cycle test 

 
“The sessions will consist of an incremental exercise test on a cycle ergometer to 
determine 
. Initial workload will be set at 35 W and workload will increase by 15 W with 
each 1 min stage until volitional fatigue. Breath by breath data will be collected and analysed 
every 5 s using a metabolic system that will be calibrated with room air and standardized gas. 
The slope of ventilation relative to carbon dioxide production (
/
) will be calculated with 
values from rest to maximal exercise. 
 will be considered to be achieved when two of the 
following criteria will be met: an increase in oxygen consumption of less than 100 ml 
min−1 with a further increase in workload, respiratory exchange ratio (RER) greater than 1.1 
or achievement of age-predicted maximal heart rate. Pulse oximetry oxygen saturation (
) 
will be continuously monitored with a finger pulse oximeter.” Med Sci Sports Exerc. 2017 Oct;49(10):2131-
2138. 
 
3.3.11 Serum proteomics analysis 
 
The discovery of differentially expressed protein profiles in e-cigarettes users and its potential 
reversibility after e-cigarette cessation might be helpful to better understand the 
physiopathology of tissue hypoxia after vaping and to better characterize the lung 
inflammation we observed. Serum proteomics will be assessed using recent quantitative 
sequential window acquisition of all theoretical fragment-ion spectra (SWATH) liquid 
chromatography/mass spectrometry (ULB – Faculty of Medicine - Pr David Communi).  It 
allows reproducible identification and accurate quantification of thousands of proteins from 
complex protein mixtures with performance characteristics that approach those of 
multiple/selected reaction monitoring. Fragment ion spectra from all measurable peptides of 
a digested proteome are generated through a data-independent acquisition with a much lower 
ion suppression effect between signals. Moreover, once acquired, the data can be perpetually 

analyzed in silico to test new potential biomarkers. 21 The relative quantification of proteins in 
the serum and the assessment of their potential post-translational modifications between 
experimental sessions and groups (e-cigarettes users and non-smokers-non-e-cigarettes 
users) will allow to discover new biomarkers associated with e-cigarettes vaping. Furthermore, 
the concentrations of these newly discovered biomarkers will be measured in the serum and 
urine by using dedicated commercial ELISA kits (or by Western blotting followed by 
immunodetection). 
 
3.3.12 Serum, urine and breath exhaled condensate metabolomics analysis 
 
In order to better understand effects of regular e-cigarettes consumption on human 
metabolome and to confirm vaping cessation, serum, urine and breath exhaled condensate of 
our participants will undergo metabolomics analysis. This will be done using nuclear magnetic 
resonance spectrometry at the Department of Human Biology & Toxicology (University of 
Mons - Faculty of Medicine – Pr. Jean-Marie Colet) as previously described. 22 
 
3.3.13 Sample size calculation 
 
The primary objective of this study is to determine whether e-cigarette vaping cessation for 
five days increases transcutaneous oxygen partial pressure in regular users. Based on our 
previous researches, we assume that baseline mean transcutaneous oxygen partial pressure 
will be 80 mm Hg with a standard deviation of 7 mm Hg. To demonstrate that e-cigarette vaping 
cessation for five days significantly increases transcutaneous oxygen partial pressure, we 
assume a minimal rise in transcutaneous oxygen partial pressure of 10 mm Hg. We will need 
16 patients, with 80% power to detect a group difference of 10 mm Hg in the change of 
transcutaneous oxygen partial pressure at a two-sided alpha-level of 0.05. We will enroll 21  

participants in order to be sure to achieve a sufficient statistical power. 
 
4. References 
 
1. Kasza KA, Ambrose BK, Conway KP, Borek N, Taylor K, Goniewicz ML, Cummings 
KM, Sharma E, Pearson JL, Green VR, Kaufman AR, Bansal-Travers M, Travers MJ, 
Kwan J, Tworek C, Cheng YC, Yang L, Pharris-Ciurej N, van Bemmel DM, Backinger 
CL, Compton WM, Hyland AJ. Tobacco-Product Use by Adults and Youths in the United 
States in 2013 and 2014. N Engl J Med 2017;376 :342-353 
2. Dinakar C, O'Connor GT. The Health Effects of Electronic Cigarettes. N Engl J Med 
2016;375: 1372-1381 
3. Barrington-Trimis JL, Gibson LA, Halpern-Felsher B, Harrell MB, Kong G, Krishnan-
Sarin S, Leventhal AM, Loukas A, McConnell R, Weaver SR. Type of E-Cigarette 
Device Used among Adolescents and Young Adults: Findings from a pooled analysis 
of 8 studies of 2,166 vapers. Nicotine Tob Res 2017 (doi: 10.1093/ntr/ntx069) 
4. Talih S, Salman R, Karaoghlanian N, El-Hellani A, Saliba N, Eissenberg T, Shihadeh 
A. "Juice Monsters": Sub-Ohm Vaping and Toxic Volatile Aldehyde Emissions. Chem 
Res Toxicol 2017; 30: 1791-1793 
5. Etter JF. Throat hit in users of the electronic cigarette: An exploratory study. Psychol 
Addict Behav. 2016;30:93-100 
6. Hermans C, Bernard A. Lung epithelium-specific proteins: characteristics and potential 
applications as markers. Am J Respir Crit Care Med 1999;159:646-78 
7. Marseglia GL, Cirillo I, Vizzaccaro A, Klersy C, Tosca MA, La Rosa M,Marseglia A, 
Licari A, Leone M, Ciprandi G. Role of forced expiratory flow at 25-75% as an early 

marker of small airways impairment in subjects with allergic rhinitis. Allergy Asthma 
Proc. 2007;28:74-8 
8. Whitsett JA, Wert SE, Weaver TE. Alveolar surfactant homeostasis and the 
pathogenesis of pulmonary disease. Annu Rev Med. 2010;61:105-19 
9. Jensen JA, Goodson WH, Hopf HW, Hunt TK. Cigarette smoking decreases tissue 
oxygen. Arch Surg 1991; 126: 1131-4. 
10. Wattel FE, Mathieu DM, Neviere RR. Transcutaneous Oxygen Pressure 
Measurements: A Useful Technique to Appreciate the Oxygen Delivery to Tissues. J. 
Hyperbaric Med 1991; 6: 269-282 
11. Rittmeyer IC, Nydegger UE. Influence of the cryoprotective agents glycerol and 
hydroxyethyl starch on red blood cell ATP and 2,3-diphosphoglyceric acid levels. Vox 
Sang. 1992;62:141-5 
12. Collins JA, Rudenski A, Gibson J, Howard L, O'Driscoll R. Relating oxygen partial 
pressure, saturation and content: the haemoglobin-oxygen dissociation curve. Breathe 
(Sheff) 2015;11:194-201 
13. Manigrasso M, Buonanno G, Fuoco FC, Stabile L, Avino P. Aerosol deposition doses 
in the human respiratory tree of electronic cigarette smokers. Environ Pollut 2015; 196: 
257–267 
14. Przybyla RJ, Wright J, Parthiban R, Nazemidashtarjandi S, Kaya S, Farnoud AM. 
Electronic cigarette vapor alters the lateral structure but not tensiometric properties of 
calf lung surfactant. Respir Res 2017;18:193. 
15. Fowles JR, Banton MI, Pottenger LH. A toxicological review of the propylene glycols. 
Crit Rev Toxicol 2013;43:363-90. 
16. Robergs RA, Griffin SE. Glycerol. Biochemistry, pharmacokinetics and clinical and 
practical applications. Sports Med. 1998;26:145-67 

17. Guelen I, Westerhof BE, Van Der Sar GL, Van Montfrans GA, Kiemeneij F, Wesseling 
KH, Bos WJ. Finometer, finger pressure measurements with the possibility to 
reconstruct brachial pressure. Blood Press Monit 2003; 8:27-30 
18. Dawson EA, Low DA, Meeuwis IH, Kerstens FG, Atkinson CL, Cable NT, Green DJ, 
Thijssen DH. Reproducibility of Cutaneous Vascular Conductance Responses to Slow 
Local Heating Assessed Using seven-Laser Array Probes. Microcirculation 
2015;22:276-84. 
19. Hoffmeyer F, Raulf-Heimsoth M, Brüning T. Exhaled breath condensate and airway 
inflammation. Curr Opin Allergy Clin Immunol 2009 ;9 :16 22 
20. Faoro V, Huez S, Vanderpool R, Groepenhoff H, de Bisschop C, Martinot JB, Lamotte 
M, Pavelescu A, Guénard H, Naeije R. Pulmonary circulation and gas exchange at 
exercise in Sherpas at high altitude. J Appl Physiol (1985) 2014;116:919-26. 
21. Collins BC, Hunter CL, Liu Y, Schilling B, Rosenberger G, Bader SL, Chan DW,Gibson 
BW, Gingras AC, Held JM, Hirayama-Kurogi M, Hou G, Krisp C, Larsen B, Lin L, Liu 
S, Molloy MP, Moritz RL, Ohtsuki S, Schlapbach R, Selevsek N, Thomas SN, Tzeng 
SC, Zhang H, Aebersold R. Multi-laboratory assessment of reproducibility, qualitative 
and quantitative performance of SWATH-mass spectrometry. Nat Commun 2017; 
8:291 
22. Colet JM. Metabonomics in the preclinical and environmental toxicity field. Drug Discov 
Today Technol 2015;13:3-10 
23. Kim S. Overview of Cotinine Cutoff Values for Smoking Status Classification. Int J 
Environ Res Public Health 2016;13(12) 
24. Russell MAH. Estimation of smoke dosage and mortality of non-smokers from 
environmental tobacco smoke. Toxicology letters 1987; 35, 9-18 
25. Orton DJ, Boyd JM, Affleck D, Duce D, Walsh W, Seiden-Long I. One-step extraction 
and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas 

chromatography (GC) with flame ionization detection (FID). Clin Biochem 2016 
Jan;49(1-2):132-8 
26. Psychogios N, Hau DD, Peng J, Guo AC, Mandal, R., Bouatra, S, Wishart DS. The 
Human Serum Metabolome. PLoS ONE 2011; 6, e16957 
27. Benowitz NL, Burbank AD. Cardiovascular toxicity of nicotine: Implications for 
electronic cigarette use. Trends Cardiovasc Med 2016; 26: 515-23 
28. Ortega R, Connor C, Kim S, Djang R, Patel K. Monitoring ventilation with 
capnography. N Engl J Med 2012;367:e27. 
29. Butlin M, Qasem A. Large Artery Stiffness Assessment Using SphygmoCor 
Technology. Pulse (Basel) 2017;4(4):180-92. 
30. Pickering, T.G. et al; Subcommittee of Professional and Public Education of the 
American Heart Association Council on High Blood Pressure Research. 
Recommendations for blood pressure measurement in humans and experimental 
animals: Part 1: blood pressure measurement in humans: a statement for 
professionals from the Subcommittee of Professional and Public Education of the 
American Heart Association Council on High Blood Pressure Research. Hypertension 
2005;45:142-61. 
31. Nishiyama T, Nakamura S, Yamashita K. Effects of the electrode temperature of a new 
monitor, TCM4, on the measurement of transcutaneous oxygen and carbon dioxide 
tension. J Anesth 2006;20:331-4 
32. Restrepo RD, Hirst KR, Wittnebel L, Wettstein R. AARC clinical practice guideline: 
transcutaneous monitoring of carbon dioxide and oxygen: 2012. Respir Care 
2012;57:1955-62 
33. Opazo Saez AM, Mosel F, Nurnberger J, Rushentsova U, Gossl M, Mitchell A, et al. 
Laser doppler imager (ldi) scanner and intradermal injection for in vivo pharmacology 

in human skin microcirculation: Responses to acetylcholine, endothelin-1 and their 
repeatability. Br J Clin Pharmacol 2005;59:511–9. 
34. Leslie SJ, Affolter J, Denvir MA, Webb DJ. Validation of laser doppler flowmetry 
coupled with intra-dermal injection for investigating effects of vasoactive agents on the 
skin microcirculation in man. Eur J Clin Pharmacol 2003;59:99–102.  
35. Rossi M, Carpi A, Di Maria C, Galetta F, Santoro G. Spectral analysis of laser doppler 
skin blood flow oscillations in human essential arterial hypertension. Microvasc 
Res 2006;72:34–41. 
36. Ferrari M, Zanasi A, Nardi E, Morselli Labate AM, Ceriana P, Balestrino A, Pisani L, 
Corcione N, Nava S. Short-term effects of a nicotine-free e-cigarette compared to a 
traditional cigarette in smokers and non-smokers. BMC Pulm Med 2015;15:120 
37. Wilkinson IB, MacCallum H, Flint L, Cockcroft JR, Newby DE, Webb DJ: The influence 
of heart rate on aug- mentation index and central arterial pressure in humans. J Physiol 
2000; 525 Pt 1: 263-270  
38. Kutsche I, Gildehaus G, Schuller D, Schumpe A. Oxygen solubilities in aqueous 
alcohol solutions. Journal of Chemical and Engineering Data 1984; 29: 286-287 
39. Aschenbrenner O, Styring P. Comparative study of solvent properties for carbon 
dioxide absorption. Energy & Environmental Science 2010; 3: 1106-1113