Biomarkers

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Korean male active smokers: quantifying their

smoking habits and the transformation factor
among biomarkers in urine and blood

Jiyeon Yang , Shervin Hashemi , Wonseok Han , Chaelin Lee , Younseok Kang
& Youngwook Lim

To cite this article: Jiyeon Yang , Shervin Hashemi , Wonseok Han , Chaelin Lee , Younseok
Kang & Youngwook Lim (2020): Korean male active smokers: quantifying their smoking
habits and the transformation factor among biomarkers in urine and blood, Biomarkers, DOI:
10.1080/1354750X.2020.1797879

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Published online: 11 Sep 2020.

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BIOMARKERS
https://doi.org/10.1080/1354750X.2020.1797879

ORIGINAL ARTICLE

Korean male active smokers: quantifying their smoking habits and the
transformation factor among biomarkers in urine and blood
Jiyeon Yanga#, Shervin Hashemia, Wonseok Hana, Chaelin Leea, Younseok Kangb and Youngwook Lima#
a
Institute for Environmental Research, Yonsei University College of Medicine, Seoul, Republic of Korea; bEnvironment Testing Division,
Eurofins Korea Analytic Service Co., Ltd, Anyang, Republic of Korea

ABSTRACT ARTICLE HISTORY

Objectives: The aim of the study was to investigate the correlations within the levels of biomarkers in Received 9 April 2020
different biological matrices, along with smoking topography variables, among active male smokers in Accepted 12 July 2020
Korea. Accordingly, we defined a transformation factor to convert level of tobacco smoke exposure
KEYWORDS
and impact biomarkers from different biometrics.
Biological matrices;
Methods: We examined smoking topography of recruited volunteers using a self-reporting survey. biomarker analysis; NMR;
The level of tobacco smoke exposure and impact biomarkers in subjects’ urine and blood were ana- smoking topography;
lysed. Results were used to assess the correlations between the topography survey items with bio- tobacco science;
markers in biological matrices. The relationship between the biomarkers in urine and blood was transformation factor
analysed. Accordingly, we defined a transformation factor as the ratio of different biomarkers in urine
and blood matrices.
Results: Significant correlations among smoking topography variables and biomarkers were found.
Besides, a strong significant association was found among urine and blood cotinine (q ¼ 0.817) and
NMR (q ¼ 0.905). Urine vs blood cotinine and NMR transformation factors were calculated to be 6.17 L-
Blood/g-Creatinine and 10.2, respectively.
Conclusions: The validated transformation factor connects epidemiological cohort studies with
tobacco smoking exposure risk assessment. Hence, this study might be beneficial for further habit-
based smoking risk assessments to obtain successful regional cession policies.

Introduction responses to smoking cessation treatments (Dempsey et al.

The World Health Organization (WHO) states that consuming
tobacco causes nearly eight million deaths a year worldwide
(World Health Organization 2019). Jha et al. (2013) suggest
that smokers may live ten years less than non-smokers.
Smoking cessation is a significant solution for preventing fur-
ther damages in smokers. Adults who quit smoking are esti-
mated to live at least six years longer than those who
continue smoking (Jha et al. 2013). Accordingly, several
ongoing comprehensive programmes and studies are aiming
for smoking cessation (Zhu et al. 2012, Jha et al. 2013,
Golechha 2016, Shaik et al. 2016, Patrick et al. 2018).
Nicotine is the primary component of tobacco, which can
cause dependency and make cessation difficult for many
smokers. Ceasing the habit successfully requires an under-
standing of smokers’ behaviours and their attitudes towards
nicotine uptake. Nicotine metabolizes into cotinine and trans-
30 -hydroxycotinine via the liver enzyme CYP2A6 (Benowitz
et al. 2009, Fix et al. 2017). Consequently, the level of nicotine
metabolites, along with the nicotine metabolite ratio (NMR),
are being used in biological matrices as biomarkers for expos-
ure to tobacco smoke and its impact on humans. They are also
indicators for the CYP2A6 activities in predicting smokers’
2004, Lerman et al. 2006, St. Helen et al. 2013).
The tobacco-specific carcinogen 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanol (NNAL) is a metabolite of the tobacco-
specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyr-
idyl)-1-butanone (NNK). NNK is probably the most common
systemic lung carcinogen, causing lung cancer, which is only
found in tobacco and tobacco products (Xia et al. 2011).
NNAL is primarily defaecated through urination (Xia et al.
2005). Therefore, urinary NNAL is an important biomarker for
determining a subject’s exposure to NNK.
Accordingly, several studies have been conducted on
investigating the status of the mentioned biomarkers in dif-
ferent biological matrices and connecting them to smoking
topography (Benowitz et al. 2003, Kandel et al. 2007, Strasser
et al. 2011, Schnoll et al. 2014).
Tobacco smoke exposure biomarkers in the blood are
broadly used in prospective epidemiological cohort studies
by storing blood samples in biobanks, to assess the effect of
smoking on contracting diseases like lung cancer or Chronic
Obstructive Pulmonary Disease (COPD) in regular active
smokers. However, in order to assess the tobacco smoke
exposure risk in active or passive smokers, it is crucial to
understand the efficiency of CYP2A6 in metabolizing the

CONTACT Youngwook Lim envlim@yuhs.ac Institute for Environmental Research, Yonsei University College of Medicine, Seoul, Republic of Korea
#
Jiyeon Yang and Youngwook Lim are responsible for statistical design and analysis. E-mail: JYYANG67@yuhs.ac (J. Yang), envlim@yuhs.ac (Y. Lim)
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 J. YANG ET AL.
tobacco smoke exposure biomarkers. Accordingly, these
studies are mostly based on results from measuring the lev-
els of biomarkers in a urine sample (Torres et al. 2018).
Therefore, studies on evaluating the concentration correla-
tions and conversions of different biomarkers from one bio-
logical matrix to another (e.g. from urine to blood) are
essential (St. Helen et al. 2012).
On the other hand, each country has its specific smoking
topography. Therefore, there is a demand for conducting
regional studies. In this case, too few literature contributions
are made in the Republic of Korea. Most of the studies in
Korean communities are limited to focussing on smoking
patterns and topography (Gunter et al. 2020). In particular,
Kim and Yu (2018) conducted a study on investigating smok-
ing topography among Korean smokers. Jung et al. (2012)
analysed the urinary cotinine level as an indicator for assess-
ing tobacco smoke exposure in Korea. There is a knowledge
gap for investigating the effects of smoking topography on
significant biomarkers in different biological matrices in the
Korean community. Besides the definition of transformation
factor as the ratio of tobacco smoke exposure (cotinine and
NNAL) and impact (NMR), biomarkers in different matrices
are useful for epidemical studies, when the available meas-
ured data are limited to only one biological matrix, in pre-
dicting the level of biomarkers in other matrices.
Consequently, for a sample of Korean active smokers, this
study aims to (1) evaluate the correlations within the levels of
biomarkers in different biological matrices, along with the
smoking topography, and (2) to calculate and assess the trans-
formation factor to convert the level of tobacco smoke expos-
ure, and impact biomarkers from urine to blood biometrics,
and vice versa, to make a connection between concepts of epi-
demiological cohort studies and the effect of tobacco smoke
on the human body, as well as its exposure risk assessments.
Clinical significance
 An Individual’s smoking behavior significantly correlates
with impact biomarkers for tobacco exposure.
 The impact biomarkers found in urine are significantly
associated with those found in the blood.
 Transformation factors enable the prediction of blood bio-
markers from known urinary biomarkers.
Materials and methods
Subjects
Local adult smokers between the age group of 20 and 60,
who had been daily smokers for at least one month, were
Table 1. Demographic and Basic Smoking Habit Data.
Parameter (Unit)
Age (years)
Age at first smoking experience (years)
Total smoking duration (years)
Cigarette consumption per day – CPD (cigarette/day)
Nominal Nicotine Yield (mg/cigarette)a
a
Based on the brand of the cigarette product.
eligible to participate in this study. Volunteers enrolled in
the study, which was conducted by the Macromill Embrain
(Seoul, Rep. of Korea), by responding to online and offline
recruiting invitations. Among the enrolled volunteers who
were excluded were the users of nicotine replacement ther-
apy, electronic cigarettes, and chewing tobacco, along with
individuals who were officially diagnosed with asthma,
chronic obstructive pulmonary disease (COPD), and
lung cancer.
Accordingly, a total of 34 local smokers (all male) were
recruited. Participants’ ages ranged from 20 to 50 (20 s: 11
people, 30 s: 11 people, and 40 s: 12 people). Subjects were
invited to the Institute for Environmental Research at Yonsei
University (IERY) located in Seoul. Participants signed a letter
of consent, and were paid for participating after being
informed about the objectives and procedure of the study.
They also had full authority to stop their participation at any
time for any reason.
Everyone was then assigned a unique ID code. Thereafter,
all participants were interviewed, and were asked to fill a
self-reporting smoking topography survey containing ques-
tions regarding their demographic status and smok-
ing topography.
Immediately after the interview and participation in the
smoking topography survey, subjects were asked to provide
urine and blood samples for the analysis of biomarker levels.
Each participant individually collected at least 150 ml of their
urine sample in a sterile urine specimen collection cup with
leak-proof lid, labelled with the subject’s ID code. Then, two
expert medical personnel from the Severance Hospital of the
Yonsei University Health System (Seoul, Rep. of Korea) per-
formed the blood sampling for each subject. Blood samples
were stored in a 6 ml blood BD VacutainerV blood collection
R
tube, labelled with the subject’s ID code.
The objectives of this study were comprehensively
explained to all participants during the interview. The
Severance Hospital of the Yonsei University Health System
Institutional Review Board (IRB) approved the study design.
Procedure
Participants provided information on demographic data,
tobacco use patterns and habits, the brand and cigarette
product they most frequently smoke and completed the
Fagerstro€m Test for Nicotine Dependence (FTND)
(Fagerstrom and Schneider 1989). As presented in Table 1,
questions regarding demographic data were restricted to
the age of the participant at the time of participating in the
study, the age at which they got initiated into smoking, the
Value (Mean ± SD) Range
(n ¼ 34) (Min–MAX)
35.2 ± 8.7 22–49
20.8 ± 3.2 14–32
14.7 ± 8.3 1.5–30
15.8 ± 5.7 8–30
0.36 ± 0.19 0.1–0.6

total smoking duration in terms of years or months, cigarette
consumption per day (CPD), and the smoking brand and
product of the cigarette they had been continually smoking
for at least one month. The tobacco use patterns and habit
section of the survey included multiple-choice questions
regarding smoking two or more cigarettes in a row, puff
count, smoking proportion, mean of smoking time, and
inhalation depth per puff. The FTND included the items
regarding the Time to First Cigarette (TTFC) and the
Heaviness of Smoking Index (HSI).
Collected urine and blood samples were respectively
stored in a freezer (approximately 15  C) and a medical
refrigerator (2–4  C) for 18 hours. After that, all samples were
located in one cooler box and (1–2  C) and sent to the
Eurofins Korea Analytic Service Co. (Anyang, Rep. of Korea)
for analysing the levels of biomarkers. The levels of cotinine
and trans-30 -hydroxycotinine in both urine and blood sam-
ples, including the total of unconjugated and conjugated
metabolites, along with urinary creatinine and NNAL, were
determined using the high-performance liquid chromatog-
raphy-tandem mass spectroscopy (HPLC-MS/MS) technique
as described by Jacob et al. (2011). The detection limit of
this analytical technique is 0.073 lg/L and 0.092 lg/L for coti-
nine and trans-30 -hydroxycotinine in both urine and blood
matrices. The detection limit of the method for urinary and
blood NNAL is 1.0 and 5.0 ng/L.
Data analysis
According to the information on the label of the presented
smoking brand and product of the cigarette, the nominal
nicotine yield was calculated. Moreover, based on the infor-
mation gathered from the survey, the minimum and max-
imum of daily puff count for everyone was calculated by
multiplying the number of cigarette consumption per day,
and the minimum and maximum amount of puff count
respectively. The arithmetic mean of these numbers was con-
sidered as average daily puff count.
All urinary biochemical levels were expressed as a ratio to
the urinary creatinine concentrating and then adjusted for
urinary creatinine concentration to take into account varia-
tions in urinary dilution between participants (Thompson
et al. 1990).
The levels of biomarkers in all biological matrices within
the 95% prediction interval have been recognized as statis-
tically valid ones. Due to the relatively low number of sub-
jects, the statistical analysis was applied to investigate the
nonparametric Spearman’s correlation between the bio-
markers data and data obtained from the topography survey.
Moreover, the association of biomarkers in urine and
blood has been analysed to determine the urine vs blood
transformation factor for each biomarker. All analyses were
performed with IBMV SPSSV Statistics version 25 (IBM
R R
Company, Armonk, NY, USA). The significance level (a) was
set at 0.05.
BIOMARKERS 3
Urine vs blood transformation factors
With regard to tobacco smoking exposure and impact bio-
markers, the levels of urinary cotinine, NNAL, and NMR were
observed in both urine and blood. Following this, a ratio was
calculated mapping the relationship of the levels of each
impact biomarker in urine with each corresponding impact
biomarker in blood. When the distribution of the measured
data was close to log-normal, ratios were first calculated as
the difference of the log-scaled values. Then the achieved
log-scaled value was back-transformed. For each biomarker,
the arithmetic mean of the ratios was considered to be the
urine vs blood tobacco smoke exposure (cotinine and NNAL)
and impact (NMR) transformation factor.
After that, to validate the application of the measured
transformation factor, the values of blood biomarkers were
predicted using the measured level of urinary biomarkers,
and the defined transformation factor. The results of these
calculations were statistically compared with the measured
levels of biomarkers in blood, through a paired sample
t-test (a ¼ 0.05).
Results
Analysis of topography survey and level of biomarkers
in biological matrices
Table 2 presents the results of the self-reporting topography
survey and determination of the biomarkers in urine and
blood. The mean of the nominal nicotine yield was approxi-
mately 0.4 mg/cigarette. The result of the tobacco consump-
tion pattern indicates that above 80% of the participants
were smoking more than one cigarette in one smoking
event. The average of smoked cigarettes in row was 2.3.
More than 82% of the participants reported smoking a cigar-
ette in less than 11–14 puffs, while less than 18% of partici-
pants reported smoking a cigarette in more than 15 puffs.
Moreover, approximately 53% of the subjects reported
taking about 1–1.5 minutes to smoke one single cigarette.
These facts indicate fast smoking habits in the majority of
the subjects. Besides, the majority of participants (around
62%) smoke 50–70% of one cigarette in one smoking event.
Around 56% of subjects smoke with full inhalation and
draw the smoke deep inside their lungs. The participants
were asked to rate their inhalation depth during smoking
from one (keeping the smoke in mouth) to ten (inhaling the
smoke down to the lung). Results gave a mean value of
7.26 ± 1.66, which indicates a smoking habit between half
and full inhalation.
The mean time interval between smoking events was
around 68 minutes. Approximately 32% of participants
reported smoking their first cigarette of the day in less than
5 minutes after they wake up in the morning. The results of
measuring the HSI reflect a mean of 2.6 out of 6. Meanwhile,
the average of the FTND score was 3.9 out of 10. These
results indicate a relatively low nicotine dependency among
the subjects of this study.
The results of measuring urinary biomarkers indicate
means of cotinine, trans-30 -hydroxycotinine, and NNAL to be
4 J. YANG ET AL.

Table 2. The Results of the self-reporting topography survey and level of biomarkers in urine and blood.
Multiple Value Range
Section Parameter (Unit) choice response n (%n or Mean ± SD) (Min–Max)
Tobacco Use Pattern Smoking several Yes 28 82.4% –
cigarettes in a row No 6 17.6% –
Number of smoked cigarettes in a row 28 2.3 ± 0.6 2–4
Puff count (times per 5–10 10 29.5% –
unit of cigarette) 11–14 18 52.9% –
15–17 6 17.6% –
18–20 0 0.0% –
>20 0 0.0% –
Average daily puff count (times per day) 34 190.7 ± 94.5 60–480
Smoking proportion (% <30 4 11.7% –
of the length per 30–50 0 0.0% –
unit of cigarette) 50–70 21 61.8% –
70–100 9 26.5% –
Mean of smoking time <0.5 1 3.0% –
(minute per unit 0.5–1 11 32.3% –
of cigarette) 1–1.5 18 52.9% –
>2 4 11.8% –
Inhalation depth No inhalation (keeping 2 5.9% –
per puff the smoke in mouth)
Half inhalation (inhaling 13 38.2% –
the smoke down
to throat)
Full inhalation (inhaling 19 55.9% –
the smoke down to
the lung)
Time interval between smoking events (min) 34 67.6 ± 28.6 20–120
FTND Time to first 5 min 11 32.4% –
cigarette (TTFC) 6 min 23 67.6 –
Heaviness of smoking index (HSI) 34 2.56 ± 1.79 0–6
Fagerstr€om test for nicotine dependence (FTND) score 34 3.97 ± 2.56 0–9
Urine Panel Creatinine (g/L-Urine) 34 0.99 ± 0.80 0.04–3.93
Cotinine (lg/g-Creatinine) 25 97.2 ± 54.8 0.4–177.2
Trans-30 -hydroxycotinine (lg/g-creatinine) 30 206.1 ± 170.5 0.7  592.2
Nicotine metabolite ratio (NMR) 28 1.62 ± 1.19 0.07–5.48
Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3- 18 2.27 ± 1.20 0.50–5.23
pyridyl)-1-butanol (NNAL) (ng/g-creatinine)
Blood Panel Cotinine (lg/L-Blood) 27 15.3 ± 9.0 0.5–34.6
Trans-30 -hydroxycotinine (lg/L-Blood) 27 2.79 ± 1.97 0.29–7.25
NMR 26 0.15 ± 0.08 0.02–0.33

approximately 97 lg/g-Creatinine, 206 lg/g-Creatinine, and
2.3 ng/g-Creatinine, respectively. For biomarkers in the blood,
the mean levels of cotinine and trans-30 -hydroxycotinine
were about 15.3 lg/L-Blood and 2.8 lg/L-Blood, respectively.
The level of blood NNAL for all subjects was under the
detection limit (5.0 ng/L-Blood).
For each participant, urinary and blood NMR was calcu-
lated based on the measured levels of cotinine and trans-30 -
hydroxycotinine in each biological matrix. The results reflect
values of 1.6 and 0.2 as the mean of urinary and blood NMR.
Correlations between self-reported smoking habits and
biomarkers concentrations
Table 3 presents the Spearman’s correlation coefficient (q)
between the topography items and the level of biomarkers
in urine and blood matrices. Of our sample, the mean age
and age at which subjects started smoking was consistent
with other prior studies such as those conducted by
Benowitz et al. (2003), Schnoll et al. (2014), and Liakoni et al.
(2019). However, results showed no significant correlation
between the ages of the subjects with regards to any of the
investigated parameters. Also, puff count, mean of smoking
time, and inhalation depth per puff had no significant rela-
tionship with any parameters of the smoking habit and levels
of biomarkers.
On the other hand, our results illustrated that there was a
significantly strong negative correlation between CPD and
the interval time between smoking events (q ¼ 0.751,
p < 0.001). Meanwhile, CPD had a significant positive associ-
ation with TTFC (q ¼ 0.593, p < 0.001), HSI score (q ¼ 0.736,
p < 0.001), FTND score (q ¼ 0.787, p < 0.001), urinary cotinine
(q ¼ 0.451, p ¼ 0.023), urinary trans-30 -hydroxycotinine
(q ¼ 0.508, p ¼ 0.004), urinary NNAL (q ¼ 0.677, p < 0.001),
and blood cotinine (q ¼ 0.505, p ¼ 0.007). Besides, the correl-
ation between CPD and urinary NMR was close to being sig-
nificant (q ¼ 0.331, p ¼ 0.056), which is similar to the results
presented by Benowitz et al. (2003).
The estimated average daily puff count had a significantly
strong negative correlation with the time interval between
smoking events (q ¼ 0.668, p < 0.001). Meanwhile, it
showed significant correlation with TTFC (q ¼ 0.488,
p ¼ 0.003), HSI (q ¼ 0.617, p < 0.001), FTND (q ¼ 0.664,
p < 0.001), urinary cotinine (q ¼ 0.505, p ¼ 0.010), urinary
trans-30 -hydroxycotinine (q ¼ 0.436, p ¼ 0.016), urinary NNAL
(q ¼ 0.625, p ¼ 0.006), and blood cotinine (q ¼ 0.417,
 BIOMARKERS 5

p ¼ 0.031). No significant relationship was observed

−0.053
0.312
0.024
−0.037

0.318

0.139

0.235

−0.146

0.202
0.188
0.196

CPD: cigarette consumption per day; TTFC: Time to First Cigarette; HSI: Heaviness of Smoking Index; FTND: Fagerström Test for Nicotine Dependence; NMR: nicotine metabolite ratio; NNAL: 4-(methylnitrosamino)-1-(3-
Cotinine hydroxycotinine NMR
between the average daily puff count and NMR in any bio-
logical matrices.
Smoking proportion per unit of cigarette had a signifi-
Blood panel

Trans-3′-

0.418*

0.400*

0.383*
0.040
0.304
−0.103
0.204

−0.055

0.123

−0.026

0.376
cant correlation with TTFC (q ¼ 0.346, p ¼ 0.045), HSI score
(q ¼ 0.380, p ¼ 0.027), FTND score (q ¼ 0.395, p ¼ 0.021),
urinary trans-30 -hydroxycotinine (q ¼ 0.487, p ¼ 0.006), urin-
ary NMR (q ¼ 0.506, p ¼ 0.002), and blood trans-30 -hydroxy-
0.677** 0.505**

0.568**
0.521**
0.687**
0.625** 0.417*
−0.035

0.119

0.350

0.004

−0.106

−0.212
cotinine (q ¼ 0.418, p ¼ 0.030).
The time interval between smoking events had a signifi-

0.554**
cant negative association with parameters such as TTFC

−0.356*

0.379*
0.430*
NNAL
0.220

0.199

0.076

−0.116

0.067
(q ¼ 0.519, p ¼ 0.002), HSI score (q ¼ 0.687, p < 0.001),
FTND score (q ¼ 0.655, p < 0.001), and urinary NNAL
0.506**

(q ¼ 0.356, p ¼ 0.039).
−0.023
0.331
0.156
0.028

0.169

0.301

−0.297

0.270
0.313
0.245
NMR

Moreover, the FTND score reflected a significant relation-

Urine panel

ship with urinary cotinine (q ¼ 0.564, p ¼ 0.003), urinary

Cotinine hydroxycotinine

trans-30 -hydroxycotinine (q ¼ 0.449, p ¼ 0.013), urinary NNAL

Trans-3′-

0.508**

0.487**
0.436*

0.376*
0.449*
0.047

0.095

0.071

0.084

−0.145

0.339

(q ¼ 0.554, p ¼ 0.001), blood cotinine (q ¼ 0.687, p < 0.001),

and blood trans-30 -hydroxycotinine (q ¼ 0.383, p ¼ 0.049).
These results are consistent with the study conducted previ-
0.519**

0.564**

ously by Benowitz et al. (2003).

0.787** 0.451*

0.664** 0.505*

0.457*
−0.095

0.254

0.219

0.006

0.155

−0.519** −0.687** −0.655** −0.115

Table 3. Spearman’s correlation coefficient (ρ) for the self-reporting topography survey and levels of biomarkers in urine and blood.

Correlations between levels of biomarkers in urine

0.895**
0.940**
0.395*
FTND
0.062

0.264

−0.127

0.231

1.000

and blood
Table 4 presents the Spearman’s correlation coefficient (q)
0.736**

0.617**

0.952**
0.380*
0.013

0.232

−0.199

0.221

1.000

between different biomarkers in urine and blood. The

HSI

results yielded significantly strong positive correlations

between urinary cotinine and urinary trans-30 -hydroxycoti-
0.593**

0.488**

0.346*
0.041

0.155

−0.212

0.195

1.000
TTFC

nine (q ¼ 0.645, p ¼ 0.001), urinary NNAL (q ¼ 0.571,

p ¼ 0.021), blood cotinine (q ¼ 0.817, p < 0.001), and blood
trans-30 -hydroxycotinine (q ¼ 0.537, p ¼ 0.010).
Time interval

−0.751**

−0.668**
between
smoking

Urinary trans-30 -hydroxycotinine had a significant associ-

events
0.024

−0.317

−0.208

0.055

−0.079

1.000

ation with urine NMR (q ¼ 0.619, p < 0.001), blood cotinine

(q ¼ 0.622, p ¼ 0.001), blood trans-30 -hydroxycotinine
(q ¼ 0.927, p < 0.001), and blood NMR (q ¼ 0.684, p < 0.001).
Mean of Inhalation

per puff
0.252
0.238
0.203
0.252

0.123

−0.031

1.000
depth

Urinary NMR had a significant correlation with blood

trans-30 -hydroxycotinine (q ¼ 0.673, p < 0.001), and blood
NMR (q ¼ 0.905, p < 0.001). No significant correlation was
Smoking smoking

−0.030
0.013
0.317
0.186

0.175

1.000
time

found between urinary NNAL and any biomarkers in blood.

Blood cotinine had a significant correlation with blood
count proportion

trans-30 -hydroxycotinine (q ¼ 0.570, p ¼ 0.003). There was

−0.126
0.296
0.171
0.297

1.000

also a significant association between blood trans-30 -hydrox-

ycotinine and blood NMR (q ¼ 0.747, p < 0.001).
1.000 0.287 0.843**
1.000 0.739**
Average

1.000 0.118 −0.272 −0.064

1.000
daily
puff

Urine vs blood transformation factors for

different biomarkers
Age CPD count
Puff

The distribution of the raw data of urine and blood bio-

markers within the 95% prediction interval was approxi-
mately log-normal, which is consistent with prior literature
(Levi et al. 2007, St. Helen et al. 2012). Hence, to analyse
**p < 0.01; *p < 0.05.

pyridyl)-1-butanol.

the urine vs blood transformation factor for biomarkers, the

smoking events
depth per puff

aggregate data of cotinine and NMR were transformed to

smoking time
Average daily

Time interval
proportion
puff count
Puff count

Inhalation

the logarithmic scale. Because the level of blood NNAL for

between
Smoking

Mean of

all subjects was under the detection limit, calculating the

FTND
TTFC
CPD
Age

HSI

transformation factor for this biomarker was not possible.

6 J. YANG ET AL.
Table 4. Spearman’s correlation coefficient (ρ) for the levels of different biomarkers in urine and blood.
Urine panel
Cotinine Trans-3′-hydroxycotinine
Urine panel
Cotinine 1.000 0.645**
Trans-3′-hydroxycotinine 1.000
NMR
NNAL
Blood panel
Cotinine
Trans-3′-hydroxycotinine
NMR
**p < 0.01; * p < 0.05.
NMR: nicotine metabolite ratio; NNAL: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.
Figure 1(a,b) illustrates the relationship between the levels
of cotinine and NMR in urine and blood matrices. Our results
suggest a strong positive correlation between urine and
blood cotinine (r ¼ 0.88), trans-30 -hydroxycotinine (r ¼ 0.94),
and NMR (r ¼ 0.88). In comparison with the study conducted
by St. Helen et al. (2012), which involved a similar number of
subjects, our results for NMR reflect a higher correlation
coefficient.
For each biomarker, the mean difference between meas-
urements in urine and blood in the log-scale was calculated.
Then the antilogarithm of these results was considered as
the urine vs blood transformation factor for each biomarker.
Our results showed a value of 6.17 L-Blood/g-Creatinine (95%
confidence interval ¼ 4.32–8.82) and 10.2 (95% confidence
interval ¼ 8.90–11.7) for tobacco smoking exposure (coti-
nine) and impact (NMR) transformation factor, respectively.
As presented in Figure 1(c), the correlation coefficient for
urinary and blood NMR has been increased (r ¼ 0.93) after
adjusting urinary cotinine and trans-30 -hydroxycotinine con-
centrations for urinary creatinine concentration, as suggested
by Thompson et al. (1990). However, the value of the NMR
transformation factor after this adjustment became 10.1
(95% confidence interval ¼ 8.97–11.4), which were not
changed significantly comparing to the results of prior
calculations.
Validation of urine vs blood transformation factors
For investigating the applicability of the defined transform-
ation factors for each biomarker in epidemical studies, suffi-
cient validation was required. Accordingly, the levels of
blood biomarkers were calculated using the values for the
same biomarker in urine and the transformation factor. The
results of these calculations were named the ‘predicted
value’ for the blood biomarker. After that, the predicted lev-
els of the blood biomarker were compared with the meas-
ured values obtained from the blood biomarker analyses
through the t-test.
Figure 2 illustrates the results of this comparison. The
results of the t-test show a p-value of 0.64 and 0.98, respect-
ively, for blood cotinine and NMR, which means there is no
statistically significant difference between the predicted and
measured sets of data. Therefore, in a local study, when the
available biological matrix is limited to either urine or blood
for biomarker analysis, the calculated transformation factor
Blood panel
NMR NNAL Cotinine Trans-3′-hydroxycotinine NMR
0.109 0.571* 0.817** 0.537* 0.161
0.619** 0.343 0.622** 0.927** 0.684**
1.000 0.024 0.51 0.673** 0.905**
1.000 0.429 0.321 0.118
1.000 0.570** 0.024
1.000 0.747**
1.000
can be used to estimate the levels of the biomarkers in the
other matrix with acceptable accuracy.
Discussion
Our study investigated the smoking topography and the lev-
els of tobacco exposure and impact biomarkers in urine and
blood of Korean male active smokers. The results are com-
parable with prior investigations and studies. The results of
the topography survey regarding puff times and FTND are in
sync with the results from a smoking topography study on
Korean participants conducted by Kim and Yu (2018). This
study states a mean of 15 puffs per cigarette per capita.
Additionally, most of the participants (around 42%) had an
FTND score of 3 to 5. However, the majority of participants
in the mentioned study reported a CPD of 6 to 10, which is
relatively lower than the ones our sample subjects stated in
their self-reporting smoking topography survey. Our results
regarding both the FTND and CPD scores are consistent with
the ones presented by Chung et al. (2015) in an investigation
of the Korean American smoking topography. Meanwhile,
the FTND and CPD scores were about 30% and 27.5% lower
respectively than the results presented by Benowitz
et al. (2003).
Our results regarding the urinary cotinine and NNAL are
within the ranges of those measured and presented by the
seventh Korea National Health and Nutrition Examination
Survey for the year 2017 regarding native male smokers
aged between 22 and 49, which were, respectively,
0.38–6345.2 lg/g-Creatinine (n ¼ 428), and 0.46–868.64 ng/g-
Creatinine (n ¼ 202) (Korea Centers for Disease Control and
Prevention 2018). Comparing our results for biomarkers in
both urine and blood with the ones reported by Torres et al.
(2018) shows that the level of cotinine in urine and blood of
our samples were within the concentration ranges for smok-
ers. Nevertheless, the levels of the rest of the biomarkers
were lower than the ranges mentioned by Torres et al.
(2018). This might be because most of the conducted stud-
ies, which are reviewed by Torres et al. (2018), presented
results from non-Asian subjects.
Although both NMR and FTND are indicators for nicotine
dependency, there was no significant correlation between
them in our study. In this case, our results are consistent
with previous research conducted by Benowitz et al. (2003).
The FTND is more an indicator for the behavioural aspects of
 BIOMARKERS 7

Figure 1. Correlation Analysis between Levels of (a) Cotinine, (b) NMR, and (c) adjusted NMR to urinary creatinine concentration in Urine and Blood.

tobacco consumption and addiction while NMR is a bio-
marker to indicate the impact of tobacco use on humans,
and therefore represents both behavioural and physiological
aspects of tobacco consumption and addiction(West et al.
2011, Schnoll et al. 2014).
Our results for the urine vs blood cotinine and NMR trans-
formation factor were within the ranges of the agreement
discussed by St. Helen et al. (2012), which are 1.94–11.9 and
2.89–24.8, respectively. In epidemical studies on the effect of
smoking tobacco on the human body, the blood NMR is
8 J. YANG ET AL.
Figure 2. Correlation Analysis between Predicted and Measured Levels of (a) Cotinine and (b) NMR in Blood.
known as a reliable biomarker to indicate the speed of nico-
tine metabolism(Hamilton et al. 2015). According to several
previous studies, a blood NMR value lower than a range of
0.2–0.3 indicates slow nicotine metabolism (Schnoll et al.
2014, Hamilton et al. 2015, Fix et al. 2017, Shahab et al. 2017,
Liakoni et al. 2019). As illustrated in Figure 3(a), our results
show that approximately 86% of the participants have rela-
tively slow nicotine metabolism. Gold and Lerman (2012)
state that nicotine metabolism, in correlation with genetic
polymorphisms in CYP2A6, is relatively slower among Asians
in comparison with Caucasians and Hispanics.
We have also calculated and assessed the transformation
factor for both tobacco smoke exposure, and impact bio-
marker in urine and blood. The transformation factor is valu-
able for making a connection between the concepts of
epidemiological cohort studies and the impact of tobacco
smoke on the human body, and risk assessment of tobacco
smoke exposure. Furthermore, it is a useful factor to estimate
the level of the biomarker when the available measurement
is run for only one specific biological matrix in an epidemical
study. As a practical example for the applicability of the
transformation factor for Korean male active smokers, when
measuring blood biomarkers is impossible or limited, a value
of urine NMR lower than a range of 2.04–3.06, which is cal-
culated by the transformation factor through our results,
ought to be considered as an indicator of slow nicotine
metabolism. Figure 3(b) shows that the same percentage of
the participants having slow nicotine metabolism also have a
urinary NMR value smaller than the mentioned range. Such
results are useful for further enquiries in assessing the risk of

Figure 3. Distributions of the NMR in (a) Blood, and (b) Urine Indicating Nicotine Metabolism Speed.
tobacco exposure as well as in defining local policies for suc-
cessful smoking cessation programmes. According to Fix
et al. (2017), people with slower nicotine metabolism tend to
have better success in smoking cessation. Additionally,
according to Benowitz et al. (2003), people who metabolize
nicotine rapidly are expected to smoke more since they may
need more nicotine.
Our findings should be considered keeping in mind the
several limitations that arose mostly due to the scope of our
finances. The sample size of this study was relatively smaller
than usual. Although the recruiting invitation was announced
on a broad national scale through different online and offline
media, subjects were limited to the active male smokers
BIOMARKERS 9
living in the Seoul metropolitan city and Gyeonggi province,
possibly because of the high population density of these
areas. On the other hand, social and cultural considerations
may be some of the reasons why women showed less inter-
est in responding to the recruiting invitation. Besides, investi-
gation on smoking topography through self-reporting survey
heavily relies on the participant’s understanding and memory
of their smoking behaviours, while using smoking topog-
raphy data loggers can provide more accurate information
on smoking habits by directly measuring smoking topog-
raphy parameters.
However, these limitations may not have significantly
impacted our results. The social and cultural factors affecting
10 J. YANG ET AL.
the smoking habits of Koreans and Korean Americans are
monolithic (Gunter et al. 2020). Thus, neither residency area
nor gender of smokers caused significant differences in
smoking topography of native Koreans in the study con-
ducted by Kim and Yu (2018). According to Peters et al.
(2014), differences in smoking behaviour between women
and men have decreased over time. Peters et al. (2014) also
mentioned that in the Asia-Pacific region, women relatively
smoke a fewer number of cigarettes than men.
Consequently, for local studies in this region, male smokers
may be able to provide more realistic data for further
investigations.
Another limitation of this study was the analysis of blood
NNAL. For all samples, the concentration of blood NNAL was
lower than the detection limit. Studies of Carmella et al.
(2005) show a range of 17–88 fmol/L-Blood for blood NNAL.
The detection of urinary NNAL can not only indicate the
uptake of the lung carcinogen NNK but also has a relatively
long half-life when compared with the blood NNAL (Yuan
et al. 2014). Accordingly, analysing urinary biomarkers like
NNAL might be more accurate with regards to epidemi-
cal studies.
Conclusions
This case study aimed to evaluate the correlation between
smoking topography and the levels of biomarkers as well as
the relationship between different biomarkers in different
biological matrices for Korean male active smokers.
Accordingly, the smoking habits of the subjects were investi-
gated through a self-reported survey and interview. The level
of significant tobacco exposure and impact biomarkers,
including cotinine, trans-30 -hydroxycotinine, NNAL, and NMR
in urine and blood samples of participants, were analysed.
The level of blood NNAL for all participants was lower than
the detection limit. Obtained data were used to investigate
the correlation between smoking habits and the levels of
biomarkers. Moreover, transformation factors for biomarkers
in different biological matrices were calculated and validated.
Our achieved results might be beneficial for local
researchers to estimate the levels of biomarkers in the miss-
ing biological matrix when the available samples are limited
to either urine or blood. Moreover, the presented data gives
a good vision of the smoking attitudes of Korean male active
smokers. Accordingly, the reported results can be utilized for
further smoking risk assessment studies to find efficient solu-
tions for reducing the risks of tobacco smoke exposure for
both active and second-hand smokers. More national-level
studies with a broader range of samples, including both men
and women, would be required to support further action
towards making new policies regarding smoking cessation.
Disclosure statement
The authors report no conflict of interest.
Funding
This work was supported by the Research Program funded by the Korea
Centres for Disease Control and Prevention [fund code 2019-E6704-00].
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