Study of cardiovascular disease biomarkers among tobacco consumers, part 1: biomarkers of exposure

Figures & data

Table 1. Urinary biomarkers of exposure and bioanalytical methods.

Biomarker^a	Abbreviation	Chemical Constituent	Method	LLOQ	LOD
Nicotine equivalents	NicEq	Nicotine	NA
Unconjugated nicotine	NIC-U	Nicotine	LC-MS/MS	10 mg mL^−1	5 ng mL^−1
Unconjugated cotinine	COT-U	Nicotine	LC-MS/MS	7.4 ng mL^−1	2.5 ng mL^−1
Unconjugated trans-3′-hydroxycotinine	OHCOT-U	Nicotine	LC-MS/MS	12.3 ng mL^−1	4.1 ng mL^−1
Nicotine-glucuronide	NIC-G	Nicotine	LC-MS/MS	10 ng mL^−1	3 ng mL^−1
Cotinine-glucuronide	COT-G	Nicotine	LC-MS/MS	25 ng mL^−1	10 ng mL^−1
Trans-3′-hydroxycotinine-glucuronide	OHCOT-G	Nicotine	LC-MS/MS	21.6 ng mL^−1	7.2 ng mL^−1
Cotinine-N-oxide	CNO	Nicotine	LC-MS/MS	10 ng mL^−1	4 ng mL^−1
Nicotine-N-oxide	NNO	Nicotine	LC-MS/MS	10 ng mL^−1	5 ng mL^−1
Norcotinine	NCOT	Nicotine	LC-MS/MS	10 ng mL^−1	3 ng mL^−1
Nornicotine	NNIC	Nicotine	LC-MS/MS	10 ng mL^−1	4 ng mL^−1
Total 4-(methylnitrosamino)-1(3-pyridyl)-butanol	NNAL-T	NNK	LC-MS/MS	5 pg mL^−1	2 pg mL^−1
Unconjugated 4-(methylnitrosamino)-  1(3-pyridyl)-butanol	NNAL-U	NNK	LC-MS/MS	5 pg mL^−1	2 pg mL^−1
4-(Methylnitrosamino)-1(3-pyridyl)-butanol  glucuronide	NNAL-G^b	NNK	Calculated	NA	NA
3-Hydroxypropylmercapturic acid	3-HPMA	Acrolein	LC-MS/MS	20 ng mL^−1	6.2 ng mL^−1
S-phenylmercapturic acid	S-PMA	Benzene	LC-MS/MS	0.05 ng mL^−1	0.03 ng mL^−1
Monohydroxybutenylmercapturic acid	MHBMA	1,3-Butadiene	LC-MS/MS	0.10 ng mL^−1	0.03 ng mL^−1
3-Hydroxy-1-methylpropyl-mercapturic acid	HMPMA	Crotonaldehyde	LC-MS/MS	52 ng mL^−1	17 ng mL^−1
total 1-hydroxypyrene	1-OHP-T	Pyrene	GC-MS/MS	0.01 ng mL^−1	0.003 ng mL^−1
o-Toluidine	o-T	o-Toluidine	GC-MS	2.5 ng L^−1	0.8 ng L^−1
2-Aminonaphthalene	2-AN	2-Aminonaphthalene	GC-MS	1.7 ng L^−1	0.6 ng L^−1
3-Aminobiphenyl	3-ABP	3-Aminobiphenyl	GC-MS	1.3 ng L^−1	0.5 ng L^−1
4-Aminobiphenyl	4-ABP	4-Aminobiphenyl	GC-MS	1.5 ng L^−1	0.5 ng L^−1
N-Acetyl-S-(2-carbamoylethyl)cysteine	AAMA	Acrylamide	LC-MS/MS	4.1 ng mL^−1	1.2 ng mL^−1
N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine	GAMA	Acrylamide	LC-MS/MS	1.0 ng mL^−1	0.3 ng mL^−1
Creatinine	CRE	Creatinine	Picric acid assay	13.1 mg dL^−1	NA

^aAnalyzed at Analytisch Biologisches Forschungslabor (ABF, Munich, Germany) except for creatinine, which was analyzed at Celerion (formerly MDS Pharma, Lincoln, NE).

^bCalculated as NNAL-T–NNAL-U

LLOQ, lower limit of quantitation; NA, not applicable; LC-MS/MS, liquid chromatography-tandem mass spectrometry; GC-MS, gas chromatography–mass spectrometry.

Table 2. Blood biomarkers of exposure and analytical methods.

Biomarker^a	Abbreviation	Chemical constituent	Matrix	Method	LLOQ	LOD
Nicotine	NIC-U	Nicotine	Serum	LC-MS/MS	2.00 ng mL^−1	NA
Cotinine	COT-U	Nicotine	Serum	LC-MS/MS	20.00 ng mL^−1	NA
Carboxyhemoglobin	%COHb	Carbon monoxide	Whole blood	Spectrophotometric	0.1 mg dL^−1	NA

^aAnalyzed at Celerion (formerly MDS Pharma, Lincoln, NE).

LLOQ, lower limit of quantitation; NA, not applicable; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Table 3. Demographic summary statistics, SMK, MSC and NTC.

	SMK	MSC	NTC
n	60	48	60
Race
African-American	2	1	6
Caucasian	57	47	45
Hispanic	0	0	5
Other^a	1	0	4
Age
Mean (SD)	37.2 (6.9)	36.5 (6.3)	36.9 (7.0)
Min, max	26, 48	26, 49	26, 48
Weight (lbs)
Mean (SD)	197.0 (35.9)	214.1 (37.5)	194.8 (28.8)
Min, max	124.0, 300.4	149.0, 333.6	138.6, 284.6
Height (inch)
Mean (SD)	70.6 ()	70.7 (2.2)	70.3 (2.3)
Min, max	65.5, 77.0	64.5, 75.0	65.8, 75.0
BMI (kg/m^2)
Mean (SD)	27.8 (4.9)	30.1 (5.0)	27.8 (4.5)
Min, max	19.1, 39.3	21.5, 49.3	19.9, 46.3
COPD status (n)
FEV1/FVC <70	8	1	4
FEV1/FVC ≥70	52	47	56

^aAmerican Indian/Alaskan Native, Asian/Pacific Islander and European/Middle Eastern. Min, minimum; max, maximum; FEV1/FVC, the forced expiratory volume in 1 s/the forced vital capacity; SD, standard deviation.

Table 4. Demographic summary statistics by age stratum, SMK, MSC and NTC.

	SMK				MSC				NTC
Age (years)	26–31	32–37	38–43	44–49	26–31	32–37	38–43	44–49	26–31	32–37	38–43	44–49
n	15	15	15	15	14	12	15	7	15	15	15	15
Race
African-American	1	0	0	1	1	0	0	0	2	1	2	1
Caucasian	14	14	15	14	13	12	15	7	7	12	12	14
Hispanic	0	0	0	0	0	0	0	0	4	1	0	0
Other^a	0	1	0	0	0	0	0	0	2	1	1	0
Age
Mean (SD)	28 (1.4)	34.3 (1.9)	40.7 (1.5)	45.7 (1.4)	28.7 (1.8)	35 (1.6)	40.7 (1.7)	45.9 (2.2)	27.6 (1.4)	34.3 (1.7)	39.5 (1.5)	46.1 (1.3)
Min, max	26, 31	32, 37	38, 43	44, 48	26, 31	32, 37	38, 43	44, 49	26, 31	32, 37	38, 42	44, 48
Weight (lbs)
Mean (SD)	197.2 (32.0)	196.7 (26.1)	205.7 (44.8)	188.4 (39.5)	214.0 (39.7)	217.7 (34.0)	216.5 (43.3)	203.2 (30.2)	187.2 (31.4)	200.2 (35.2)	191.7 (14.8)	200.1 (30.4)
Min, max	136.8, 251.6	158.0, 259.2	124.0, 269.0	127.4, 300.4	149.0, 273.2	158.0, 259.8	152.0, 333.6	153.8, 244.2	138.6, 232.8	156.4, 284.6	163.8, 225.2	155.2, 252.4
Height (inch)
Mean (SD)	70.4 (2.3)	71.2 (2.4)	69.9 (2.3)	71.1 (2.4)	70.8 (2.0)	69.8 (2.7)	71.1 (2.4)	70.9 (1.6)	70.6 (2.4)	70.2 (2.3)	70.6 (2.0)	70.0 (2.6)
Min, max	66.5, 73.3	68.0,76.8	65.5,74.0	68.5,77.0	67.5,73.8	64.5,73.3	67.8,75.0	69.0,73.0	67.0,75.0	65.8,74.8	67.5,73.8	66.0,73.8
BMI (kg/m^2)
Mean (SD)	28.0 (4.9)	27.4 (4.3)	29.6 (6.1)	26.1 (4.2)	29.9 (4.4)	31.2 (3.4)	30.3 (6.8)	28.4 (3.8)	26.4 (4.1)	28.8 (6.3)	27.1 (2.5)	28.7 (4.1)
Min, max	19.9,39.1	22.6,38.3	19.7,39.3	19.1,35.7	23.0,35.7	24.5,35.3	22.5,49.3	21.5,32.2	19.9,33.0	22.3,46.3	22.7,31.1	22.2,36.8
COPD status (n)
FEV1/FVC <70	1	1	4	2	0	0	1	0	0	0	2	2
FEV1/FVC ≥70	14	14	11	13	14	12	14	7	15	15	13	13

^aAmerican Indian/Alaskan Native, Asian/Pacific Islander and European/Middle Eastern. SD, standard deviation.

Table 5. Summary of tobacco product use, SMK and MSC.

	SMK				MSC
Age (years)	26–31	32–37	38–43	44–49	26–31	32–37	38–43	44–49
n	15	15	15	15	14	12	15	7
Years of product use
Mean (SD)	10.9 (2.1)	17.5 (3.8)	21.1 (3.9)	27.0 (5.1)	12.0 (3.2)	18.3 (5.8)	24.3 (2.9)	26.3 (3.3)
Min, max	7, 15	10, 24	14, 27	11, 33	7, 18	4, 28	18, 29	20, 29
Product use^a
Mean (SD)	20.5 (5.7)	19.3 (4.8)	19.2 (4.2)	24.3 (8.4)	4.6 (1.9)	4.4 (1.7)	3.8 (1.2)	3.9 (2.0)
Min, max	15, 40	10.5, 30	15, 30	15, 40	2, 7	2, 7	2, 7	2, 7
Product use, last 24 h^b
Mean (SD)	18.0 (3.2)	15.1 (5.4)	19.8 (5.5)	21.9 (9.1)	1.0 (0.3)	1.0 (0.1)	1.1 (0.3)	0.9 (0.2)
Min, max	11.5, 24	2, 21	10, 30	10, 40	0.5, 2	0.5, 1	0.5, 2	0.5, 1
FTND^c score
Mean (SD)	4.5 (1.8)	5.3 (1.6)	4.7 (1.6)	5.9 (2.1)	5.4 (2.0)	5.8 (1.9)	5.3 (2.2)	5.6 (1.2)
Min, max	2, 8	2, 8	2, 7	3, 10	2, 9	3, 9	2, 9	4, 7

^aCigarettes per day for SMK, tins per week for MSC.

^bNumber of cigarettes for SMK and number of tins for MSC reported on 1-day diary.

^cSMK completed the Fagerström test for nicotine dependence, MSC completed the Fagerström test for nicotine dependence-ST. SD, standard deviation.

Table 6. Summary of urine BoE comparisons.

			LS means ± SE^a			Group comparison p value^b
Chemical constituent	Biomarker	Age (years)	SMK	MSC	NTC	SMK versus MSC	SMK versus NTC	MSC versus NTC
Nicotine	NicEq (µg mg^−1 CRE)^c	26–31	8.22 ± 1.27	7.92 ± 1.36	0.06 ± 1.27	1.0000	<0.0001	<0.0001
		32–37	8.03 ± 1.27	11.6 ± 1.42	0.04 ± 1.31	0.1908	<0.0001	<0.0001
		38–43	7.73 ± 1.27	10.49 ± 1.31	0.06 ± 1.27	0.3957	<0.0001	<0.0001
		44–49	9.53 ± 1.31	18.74 ± 1.86	0.04 ± 1.27	0.0003	<0.0001	<0.0001
NNK	NNAL-T (pg mg^−1 CRE)		656 ± 72.9	1594 ± 85.6	7.94 ± 72.9	<0.0001	<0.0001	<0.0001
Benzene	S-PMA (ng mg^−1 CRE)		2.63 ± 0.19	0.51 ± 0.23	0.46 ± 0.19	<0.0001	<0.0001	1.0000
Acrolein	3-HPMA (ng mg^−1 CRE)		1824 ± 73.7	160 ± 85.7	145 ± 74.3	<0.0001	<0.0001	1.0000
1-Hydroxypyrene	1-OHP-T (ng mg^−1 CRE)		0.22 ± 0.01	0.08 ± 0.02	0.05 ± 0.01	<0.0001	<0.0001	0.5439
1,3-Butadiene	MHBMA (ng mg^−1 CRE)		4.37 ± 0.29	0.45 ± 0.34	0.72 ± 0.29	<0.0001	<0.0001	1.0000
Crotonaldehyde	HMPMA (ng mg^−1 CRE)		7229 ± 263	598 ± 309	660 ± 263	<0.0001	<0.0001	0.8787
o-Toluidine	o-T (pg mg^−1 CRE)		193 ± 7.15	43.3 ± 8.84	41.2 ± 7.16	<0.0001	<0.0001	1.0000
2-Aminonaphthalene	2-AN (pg mg^−1 CRE)		31.2 ± 1.16	1.99 ± 1.44	1.23 ± 1.16	<0.0001	<0.0001	0.6813
4-Aminobiphenyl	4-ABP (pg mg^−1 CRE)		21.4 ± 0.93	2.74 ± 1.15	2.43 ± 0.93	<0.0001	<0.0001	0.8307
3-Aminobyphenyl	3-ABP (pg mg^−1 CRE)		11.1 ± 0.50	0.6 ± 0.62	0.66 ± 0.50	<0.0001	<0.0001	0.9412
Acrylamide	AAMA (ng mg^−1 CRE)		221 ± 8.65	106 ± 10.1	78.0 ± 8.73	<0.0001	<0.0001	0.1199
Acrylamide	GAMA (ng mg^−1 CRE)		35.1 ± 1.08	16.1 ± 1.26	15.8 ± 1.09	<0.0001	<0.0001	1.0000

^aLeast squares means ± SE.

^bBonferroni-adjusted. p values that exceeded 1.000 after adjustment are reported as 1.000.

^cSignificant group-by-age interaction (p < 0.05).

Table 7. Summary of blood BoE comparisons.

			LS Means ± SE^a			Group comparison p value^b
Chemical constituent	Biomarker	Age (years)	SMK	MSC	NTC	SMK versus MSC	SMK versus NTC	MSC versus NTC
Carbon monoxide	COHb (% saturated)		7.88 ± 0.17	1.12 ± 0.20	1.06 ± 0.17	<0.0001	<0.0001	0.7960
Nicotine	NIC-U (ng mL^−1)		31.5 ± 0.99	28.7 ± 1.15	0.27 ± 0.99	0.0709	<0.0001	<0.0001
Nicotine	COT-U (ng mL^−1)^c	26–31	323 ± 45.3	466 ± 46.9	1.20 ± 45.3	0.0870	<0.0001	<0.0001
		32–37	352 ± 45.3	466 ± 50.6	0.11 ± 45.3	0.2826	<0.0001	<0.0001
		38–43	363 ± 45.3	554 ± 45.3	0.25 ± 45.3	0.0102	<0.0001	<0.0001
		44–49	339 ± 45.3	773 ± 66.3	0.11 ± 45.3	<0.0001	<0.0001	<0.0001

^aLeast squares means ± SE.

^bBonferroni-adjusted. p values that exceeded 1.000 after adjustment are reported as 1.000.

^cSignificant group-by-age interaction (p < 0.05).