Reductions in biomarkers of exposure (BoE) to harmful or potentially harmful constituents (HPHCs) following partial or complete substitution of cigarettes with electronic cigarettes in adult smokers

Figures & data

Table 1. Urine, blood and inhalation biomarkers of tobacco smoke exposure.

URINE: Biomarkers of exposure analyzed
Nicotine equivalents^a	NNN	NNAL	1-OHP	3-HPMA	S-PMA	MHBMA	HMPMA	CEMA
Nicotine +5 major metabolites
^aNicotine equivalents measured: included nicotine and five major nicotine metabolites: nicotine gluc; cotinine; cotinine-gluc; trans-3’-hydroxycotinine; and trans-3’-hydroxycotinine-gluc. NNAL: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; 1-HOP: 1-hydroxypyrene; 3-HPMA: 3-hydroxypropylmercapturic acid; S-PMA: S-phenylmercapturic acid; MHBMA: Monohydroxy-3-butenyl mercapturic acid; HMPMA: 3-hydroxy-1-ethylpropylmercapturic acid; CEMA: 2-cyanoethylmercapturic acid.
HPHCs-associated with urine biomarkers
Nicotine	Tobacco Specific Nitrosamines (TSNA)		PAH	Volatile Organic Compounds (VOC’s)
Nicotine	NNN	NNK	Pyrene	Acrolein	Benzene	1,3-Butadiene	Crotonaldehyde	Acrylonitrile
PAH: Polycyclic Aromatic Hydrocarbons; NNN: N-Nitrosonornicotine; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Clinical endpoints of urine HPHCs
Nicotine Exposure	Cancer	Cancer	Cancer	Cancer	Cancer	Cancer	Cancer	Cancer
Analysis method and Lower Limit of Quantification (LLOQ)
LC-MS-MS 50–200 ng/mL^b	LC-MS-MS 0.2 pg/mL	LC-MS-MS 5 pg/mL	LC-MS-MS 10 pg/mL	LC-MS-MS 20 ng/mL	LC-MS-MS 25 pg/mL	LC-MS-MS 0.1 ng/mL	LC-MS-MS 20 ng/mL	LC-MS-MS 0.275 ng/mL
^aNicotine equivalents: calculated as the molar sum of nicotine and five major nicotine metabolites excreted in urine over 24 h (mg nicotine equivalents/24 h). ^bLLOQ for Nicotine equivalents: 50 ng/mL for Nicotine, Nicotine gluc, Cotinine and Trans-3’-hydroxycotinine; 200 ng/mL for Cotinine-gluc and Trans-3’-hydroxycotinine-gluc.
BLOOD: Biomarkers of exposure analyzed
Blood CarboxyHemoglobin (COHb)			Plasma Nicotine		Plasma Cotinine		Plasma Trans-3’hydroxycotinine
Chemical constituents associated with blood biomarkers
Carbon Oxide			Nicotine
Clinical endpoints associated with blood biomarkers
Carbon Monoxide (CO) exposure			Nicotine exposure		Nicotine exposure		Nicotine exposure
Analysis method and Lower Limit of Quantification (LLOQ)
Spectrophotometric 0.50%			LC-MS-MS 0.2 ng/mL		LC-MS-MS 1.0 ng/mL		LC-MS-MS 1.0 ng/mL
INHALATION: Biomarkers of exposure analyzed
Exhaled Carbon Monoxide (CO)					Exhaled Nitric Oxide (NO)
Analysis method and Lower Limit of Quantification (LLOQ)
Bedfont Micro + Smokerlyzer: 1 ppm (0–500 ppm)					Niox Mino: 5 ppb (5–300 ppb)

Table 2. Summary of study demographics and FTCD scores by product use groups and overall.

	Exclusive E-Cigarette use groups			Dual use groups
Trait/Test	Tobacco rechargeable N = 15	Cherry rechargeable N = 15	Cherry disposable N = 15	Tobacco rechargeable N = 15	Cherry rechargeable N = 15	Cherry disposable N = 15	Nicotine cessation N = 15	Overall N = 105
Gender
Female	6 (40%)	3 (20%)	9 (60%)	6 (40%)	3 (20%)	7 (47%)	3 (20%)	37 (35%)
Male	9 (60%)	12 (80%)	6 (40%)	9 (60%)	12 (80%)	8 (53%)	12 (80%)	68 (65%)
Race
American Indian/Alaska Native	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (7%)	0 (0%)	1 (1%)
Black or African American	2 (13%)	6 (40%)	1 (7%)	2 (13%)	4 (27%)	1 (7%)	1 (7%)	17 (16%)
Black or African American, American Indian/Alaska	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (7%)	1 (1%)
White	13 (87%)	9 (60%)	14 (93%)	13 (87%)	11 (73%)	13 (87%)	13 (87%)	86 (82%)
Age (years)
Mean	37.1	40.1	33.9	36.6	36.8	39.3	41.1	37.8
SD	11.4	10.6	11.8	10.8	11.6	10.6	11.2	11.1
BMI (kg/m^2)
Mean	28.2	26.2	28.7	28.9	27.2	27.8	27.8	27.8
SD	5.5	6.5	5.7	5.5	4.7	5.1	4.9	5.4
Cigarettes per Day
Mean	18.4	17.3	15.4	18.7	20.5	21.1	20.4	18.8
SD	7.1	6.2	3.3	6.6	7.3	5.8	7.5	6.5
Years Smoked
Mean	19.2	20.3	15.0	19.3	14.6	21.7	21.3	18.8
SD	12.9	10.5	10.9	10.1	11.6	8.7	10.6	10.8
Usual brand cigarette flavor
Menthol	6 (40%)	7 (47%)	8 (53%)	3 (20%)	7 (47%)	5 (33%)	3 (20%)	39 (37%)
Non-Menthol	9 (60%)	8 (53%)	7 (47%)	12 (80%)	8 (53%)	10 (67%)	12 (80%)	66 (63%)
FTCD Score
Mean	5.3	5.1	5.3	5.5	5.7	5.2	5.6	5.4
SD	1.5	2.0	1.5	2.0	1.1	1.7	2.0	1.7

Figure 1. Urine biomarkers – Day 5 percent change from Day −1.

Figure 2. Blood biomarkers – Day 5 percent change from Day −1.

Table 3. Urine biomarker concentration summary and statistical comparisons.

	Exclusive E-Cigarette use groups			Dual use groups
Biomarker	Classic tobacco rechargeable N = 15	Cherry rechargeable N = 13	TCherry disposable N = 13	Classic tobacco rechargeable N = 14	Cherry rechargeable N = 15	Cherry disposable N = 13	Nicotine cessation N = 13
NNAL (ng/24 h)
Day −1	427.6 ± 218.8	383.7 ± 178.8	299.1 ± 165.0	430.8 ± 217.1	422.0 ± 257.5	343.3 ± 123.3	481.6 ± 377.5
Day 5	174.3 ± 144.6	149.2 ± 80.3	111.1 ± 68.9	328.6 ± 178.9	321.1 ± 177.3	269.2 ± 96.3	175.1 ± 140.8
p value Day −1 vs Day 5	<0.0001	<0.0001	<0.0001	0.0063	0.0042	0.0028	0.0004
p value Day 5 vs Cessation	0.1940	0.2456	0.2593	<0.0001	<0.0001	<0.0001	NA
3-HPMA (μg/24 h)
Day −1	1521.7 ± 820.0	1903.0 ± 1132.7	1353.7 ± 598.8	1644.1 ± 501.3	1474.6 ± 519.9	1489.5 ± 567.1	2004.1 ± 1137.8
Day 5	214.4 ± 94.3	263.1 ± 64.7	246.7 ± 101.5	1046.2 ± 360.6	1070.7 ± 342.2	1155.4 ± 368.5	228.8 ± 84.2
p value Day −1 vs Day 5	<0.0001	0.0001	<0.0001	<0.0001	0.0009	0.0062	<.0001
p value Day 5 vs Cessation	0.5137	0.6099	0.3194	<0.0001	<0.0001	<0.0001	NA
HMPMA (μg/24 h)
Day −1	523.8 ± 225.3	657.2 ± 328.9	533.4 ± 208.3	590.7 ± 178.7	597.5 ± 198.0	504.5 ± 167.1	797.7 ± 429.4
Day 5	71.3 ± 33.1	83.2 ± 32.3	78.0 ± 20.7	391.8 ± 151.2	394.6 ± 119.3	386.8 ± 94.1	78.1 ± 18.6
p-value Day −1 vs Day 5	<0.0001	<0.0001	<0.0001	0.0001	0.0001	0.0094	<.0001
p-value Day 5 vs Cessation	0.4785	0.5206	0.4211	<0.0001	<0.0001	<0.0001	NA
CEMA (μg/24 h)
Day −1	219.7 ± 98.5	266.1 ± 140.9	201.0 ± 72.8	256.0 ± 97.9	246.2 ± 109.8	223.5 ± 61.6	289.7 ± 132.2
Day 5	33.4 ± 21.8	41.3 ± 30.4	25.9 ± 11.2	172.8 ± 72.1	168.3 ± 50.9	173.0 ± 63.7	41.0 ± 19.7
p value Day −1 vs Day 5	<0.0001	<0.0001	<0.0001	0.0002	0.0010	0.0002	<.0001
p value Day 5 vs Cessation	0.2902	0.6357	0.4549	<0.0001	<0.0001	<0.0001	NA
1-OHP (ng/24 h)
Day −1	317.4 ± 138.7	302.9 ± 171.5	260.9 ± 166.8	363.6 ± 174.1	294.5 ± 145.5	304.1 ± 122.7	364.0 ± 200.7
Day 5	93.7 ± 52.9	85.9 ± 32.2	90.6 ± 38.4	235.1 ± 121.1	206.3 ± 90.9	224.1 ± 89.5	108.2 ± 55.0
p value Day −1 vs Day 5	<.0001	0.0002	0.0007	0.0006	0.0004	0.0007	<.0001
p value Day 5 vs Cessation	0.8331	0.7524	0.4115	<0.0001	<0.0001	<0.0001	NA
NNN (ng/24 h)
Day −1	18.6 ± 12.1	13.7 ± 11.5	13.9 ± 12.5	14.3 ± 8.6	12.3 ± 8.1	11.3 ± 5.4	16.2 ± 12.1
Day 5	1.2 ± 2.4	0.7 ± 0.9	1.2 ± 3.3	8.9 ± 7.7	7.6 ± 5.4	7.1 ± 3.1	0.2 ± 0.1
p value Day −1 vs Day 5	<.0001	0.0011	0.0045	0.0001	0.0019	0.0032	0.0005
p value Day 5 vs Cessation	0.6402	0.6223	0.3974	<0.0001	<0.0001	<0.0001	NA
MHBMA (μg/24 h)
Day −1	4.9 ± 3.5	5.9 ± 3.8	4.6 ± 3.2	5.0 ± 2.9	3.4 ± 2.5	4.5 ± 2.8	5.6 ± 3.6
Day 5	0.3 ± 0.1	0.3 ± 0.1	0.3 ± 0.1	3.5 ± 2.1	2.8 ± 1.8	4.3 ± 2.3	0.4 ± 0.1
p value Day −1 vs Day 5	0.0002	0.0002	0.0006	0.0024	0.0320	0.1539	0.0002
p value Day 5 vs Cessation	0.8548	0.8106	0.7313	<0.0001	<0.0001	<0.0001	NA
S-PMA (μg/24 h)
Day −1	6.3 ± 3.7	8.1 ± 4.9	6.3 ± 3.9	7.0 ± 4.4	4.9 ± 3.5	6.9 ± 5.5	7.6 ± 4.3
Day 5	0.3 ± 0.1	0.3 ± 0.3	0.4 ± 0.2	4.9 ± 3.3	3.6 ± 2.3	6.0 ± 4.6	0.3 ± 0.2
p value Day −1 vs Day 5	<.0001	<.0001	0.0001	0.0031	0.0055	0.0072	<0.0001
p value Day 5 vs Cessation	0.5274	0.7602	0.4430	<0.0001	<0.0001	<0.0001	NA
Nicotine equivalents^a (mg/24 h)
Day −1	17.0 ± 6.6	17.6 ± 8.7	14.5 ± 4.4	16.6 ± 5.5	16.0 ± 6.2	15.7 ± 3.9	20.0 ± 8.9
Day 5	10.7 ± 9.1	12.7 ± 9.7	10.5 ± 9.6	18.4 ± 7.2	15.9 ± 5.5	15.8 ± 5.0	0.5 ± 0.2
p value Day −1 vs Day 5	0.0115	0.0415	0.0033	0.4188	0.9103	0.8519	<.0001
p value Day 5 vs Cessation	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	NA

Values are presented as mean ± SD.

^a Nicotine equivalents: calculated as the molar sum of nicotine and five major nicotine metabolites (nicotine gluc, cotinine, cotinine-gluc, trans-3’-hydroxycotinine; trans-3’-hydroxycotinine-gluc) excreted in urine over 24 h and reported as nicotine equivalents (mg/24 h). Nicotine equivalents (mg/24 h) = [nicotine (mg/162.23 (mg/mmol) + nicotine-gluc (mg/338.36 (mg/mmol) + cotinine (mg/176.22 (mg/mmol) + cotinine-gluc (mg/352.34 (mg/mmol) + trans-3'-hydroxycotinine (mg/192.22 (mg/mmol) + trans-3'-hydroxycotinine-gluc (mg/368.34 (mg/mmol)] × 162.23 (mg/mmol)].

Table 4. Blood biomarker concentration summary and statistical comparisons.

	Exclusive E-Cigarette use groups			Dual use groups
Biomarker	Classic tobacco rechargeable N = 15	Cherry rechargeable N = 13	Cherry disposable N = 13	Classic tobacco rechargeable N = 14	Cherry rechargeable N = 15	Cherry disposable N = 13	Nicotine cessation N = 13
Blood COHb (%)
Day −1	6.3 ± 2.0	6.3 ± 2.0	6.0 ± 1.5	5.2 ± 2.0	4.8 ± 1.4	5.6 ± 1.9	7.4 ± 2.3
Day 5	1.2 ± 0.6	1.2 ± 0.5	1.0 ± 0.6	4.0 ± 1.5	3.8 ± 0.9	4.3 ± 1.3	1.6 ± 0.4
p value Day −1 vs Day 5	0.0001	<0.0001	<0.0001	0.0179	0.0775	0.0170	0.0001
p value Day 5 vs Cessation	0.5011	0.6009	0.4794	<0.0001	<0.0001	<0.0001	NA
Plasma Nicotine (ng/mL)
Day −1	13.0 ± 6.1	14.7 ± 5.2	13.3 ± 6.5	12.5 ± 5.9	8.8 ± 3.2	11.1 ± 4.1	16.0 ± 7.0
Day 5	6.9 ± 6.3	8.4 ± 5.9	6.6 ± 5.6	9.5 ± 6.2	8.1 ± 5.2	7.9 ± 4.4	0.1 ± 0.0
p value Day −1 vs Day 5	0.0033	0.0035	0.0053	0.0518	0.6197	0.0112	<0.0001
p value Day 5 vs Cessation	<0.0001	<0.0001	0.0002	<0.0001	<0.0001	<0.0001	NA
Plasma Cotinine (ng/mL)
Day −1	260.1 ± 128.1	299.9 ± 93.7	250.1 ± 92.4	247.6 ± 99.0	213.6 ± 62.8	218.4 ± 58.3	282.2 ± 135.9
Day 5	164.5 ± 167.4	202.1 ± 103.2	149.2 ± 116.1	261.5 ± 119.4	211.5 ± 70.2	212.9 ± 89.3	5.49 ± 6.7
p value Day −1 vs Day 5	0.0438	0.0160	0.0112	0.6554	0.8935	0.7474	<0.0001
p value Day 5 vs Cessation	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	NA
Plasma Trans-3’ hydroxycotinine (ng/mL)
Day −1	164.5 ± 167.4	202.1 ± 103.2	149.2 ± 116.1	261.5 ± 119.4	211.5 ± 70.2	212.9 ± 89.3	5.49 ± 6.7
Day 5	70.4 ± 59.0	85.0 ± 55.7	69.4 ± 56.5	102.2 ± 46.8	107.8 ± 50.7	98.5 ± 29.3	3.8 ± 2.7
p value Day −1 vs Day 5	0.1626	0.3316	0.2073	0.0821	0.0051	0.1082	<0.0001
p value Day 5 vs Cessation	<0.0001	<0.0001	0.0002	<0.0001	<0.0001	<0.0001	NA

Values are presented as mean ± SD.

Table 5. Regression analyzes of the Day −1 to Day 5% change in the amount of urine biomarker amount excreted against the % change in cigarettes per day (CPD).

Urine biomarker	Slope	R-square	p Value
NNAL	0.4154	0.1518	0.0108
3-HPMA	0.6940	0.4105	<0.0001
HMPMA	0.7878	0.4289	<0.0001
CEMA	0.7096	0.4891	<0.0001
1-OHP	0.6297	0.4227	<0.0001
NNN	0.7766	0.3181	0.0001
MHBMA	0.7469	0.2874	0.0003
S-PMA	0.7259	0.4636	<0.0001
Nicotine Equivalents	−0.0296	0.0002	0.9316

Table 6. Regression analyzes of nicotine equivalents excretion and Day −1 and Day 5 product use.

Relationship assessed	Slope	R-square	p Value
Day −1 nicotine equivalents amount excreted against cigarettes smoked (all Groups)	0.7485	0.2137	<0.0001
Day 5 nicotine equivalents amount excreted against cigarettes smoked (dual use Groups)	0.3598	0.0289	0.2814
Day 5 nicotine equivalents amount excreted against estimated nicotine from e-cigarettes (dual use Groups)	0.3970	0.4502	<0.0001
Day 5 nicotine equivalents amount excreted against estimated nicotine from e-cigarettes (exclusive use Groups)	0.4794	0.8538	<0.0001

Table 7. Summary of regression analyzes of nicotine equivalents excretion and Day −1 and Day 5 product use.

Relationship assessed	Slope	p Value	R-square
Day −1 nicotine equivalents amount excreted against cigarettes smoked (All Cohorts)	0.7485	<0.0001	0.2137
Day 5 nicotine equivalents amount excreted against cigarettes smoked (Dual Use Cohorts)	0.3598	0.2814	0.0289
Day 5 nicotine equivalents amount excreted against estimated nicotine from blu products (Dual use Cohorts)	0.3970	<0.0001	0.4502
Day 5 nicotine equivalents amount excreted against estimated nicotine from blu products (Exclusive Use Cohorts)	0.4794	<0.0001	0.8538

Figure 3. Exhaled breath biomarkers – Day 5 percent change from Day −1.

Table 8. Exhaled breath biomarker summary and statistical comparisons.

	Exclusive E-Cigarette use groups			Dual use groups
Biomarker	Classic tobacco rechargeable N = 15	Cherry rechargeable N = 13	Cherry disposable N = 13	Classic tobacco rechargeable N = 14	Cherry rechargeable N = 15	Cherry disposable N = 13	Nicotine cessation N = 13
CO (ppm)
Day −1	27.2 ± 10.5	27.3 ± 6.9	26.9 ± 6.4	25.1 ± 7.3	25.4 ± 7.7	24.7 ± 5.5	29.3 ± 10.4
Day 5	2.9 ± 0.8	2.9 ± 0.8	2.7 ± 0.9	17.3 ± 5.7	16.1 ± 3.3	18.2 ± 5.7	2.8 ± 0.7
p value Day −1 vs Day 5	<0.0001	<0.0001	<0.0001	0.0021	<0.0001	0.0002	<0.0001
p value Day 5 vs Cessation	0.7990	0.8033	0.9109	<0.0001	<0.0001	<0.0001	NA
NO (ppb)
Day −1	14.8 ± 12.8	11.5 ± 4.8	10.0 ± 4.0	14.9 ± 11.1	10.6 ± 4.6	14.3 ± 13.5	11.3 ± 4.0
Day 5	23.3 ± 21.6	15.5 ± 9.0	14.3 ± 6.5	12.9 ± 6.3	10.7 ± 4.4	11.4 ± 6.0	16.8 ± 10.1
p value Day −1 vs Day 5	0.0075	0.1325	0.0053	0.3118	0.9415	0.3287	0.0321
p value Day 5 vs Cessation	0.2370	0.6031	0.5674	0.0313	0.0615	0.0119	NA

Values are presented as mean ± SD. A paired t-test was used to make the Day −1 vs Day 5 within group comparisons, a linear mixed model was used to make the Day 5 comparisons to the Cessation group.