21

Bull Yamaguchi Med Sch 68（1-2）:21-29, 2021

Altered Levels of Plasma Free Amino Acids in Hyperuricemia among Male Smokers
MH Mahbub,1 Natsu Yamaguchi,1 Ryosuke Hase,1 Hiroshi Sunagawa,1Yasutaka Ishimaru,1 Hiroshi Yamamoto,2 Shinya Kikuchi2 and Tsuyoshi Tanabe1
1Department of Public Health and Preventive Medicine, Graduate School of Medicine,
Yamaguchi University, 1-1-1 Minami Kogushi, Ube , Yamaguchi 755-8505, Japan.
2
Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc.,
1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.
(Received January 28, 2020, accepted December 3, 2020)
Correspondence to MH Mahbub, M.D., M.P.H., Ph.D. E-mail: hossain@yamaguchi-u.ac.jp

Abstract Purpose: The purpose was to explore the existence of distinct patterns of alterations in plasma free amino acids (PFAAs) among smokers with hyperuricemia.
Methods: Venous blood samples from 169 male subjects (95 smokers and 74 nonsmokers) were analyzed for the measurements of 19 PFAAs and uric acid. Mann-Whitney
U-test was applied for comparison of PFAA levels between two groups. Results:
Evaluations of PFAAs made separately in smokers and non-smokers show that compared to the corresponding values for the subjects without hyperuricemia, subjects
with it had significantly higher levels of isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine and valine (P<0.05 to 0.01) and significantly lower levelsof glutamine, glycine and serine (P<0.05 to 0.001) in the smoker group, and a significantly higher level of phenylalanine (P<0.05) in the nonsmoker group. Conclusions:
The findings suggest that hyperuricemia may have remarkable effects on the PFAA
profiles in smokers, which underscores the importance of investigations into themechanisms underlying the effects of cigarette smoking on the possible interactive
relationship between the levels of plasma uric acid and PFAAs.
Key words: Amino acids, plasma profile, cigarette smoking, Brinkman index,
hyperuricemia

Introduction
Cigarette smoking is an established causal
risk factor for multiple diseases of the lungand many other chronic and degenerative diseases affecting various organ systems whichcause enormous burdens and challenges to thehealth care system globally.1 It is also considered to be the single most significant causeof preventable deaths in the world.2 On theother hand, the prevalence of hyperuricemia(HU) has been increasing worldwide.3 A growing number of published studies reported astrong association of elevated uric acid (UA)as an independent risk factor and predictor
for the development and progression of various lifestyle-related diseases.4,5 Despite someconflicting reports, published literature suggests the existence of an association between
smoking and an elevated blood UA level or increased prevalence of HU.6,7
In recent years, plasma free amino acids(PFAAs) profiles are increasingly being usedin the evaluation of various diseases. It has
been observed that cigarette smoking cansignificantly influence the PFAAs and cause

22

MH Mahbub et al.

alterations in the levels of the amino acids.8,9On the other hand, some published studies
reported a clear association between HU and
PFAAs among patients with various lifestyle-related diseases, and suggested the possibility of interplay between PFAAs and UA
in the pathophysiology of those diseases.10,11
It is understandable from the available literature that both cigarette smoking and HU
can cause alterations in PFAAs, and distinct
patterns of differences in the concentrationsof the latter might exist among smokers withand without HU. A better understanding of
the specific patterns of changes in PFAA levels among smokers with HU might contributetowards a better understanding of any associated pathophysiology, and provide useful
information for the prevention and/or early
detection and treatment of diseases causedby the presence of HU among smokers. However, any such potential changes in the concentrations of PFAAs remain unknown as no
study, to the best of our knowledge has hitherto investigated it among smokers with andwithout HU. Therefore, one purpose of thisstudy was to examine the differences in theconcentrations of PFAAs and UA amongst
smokers versus non-smokers. Another purpose was to explore the possible differences in
PFAAs with and without HU in both groupsof subjects in an attempt to clarify the existence of any distinct differences in alterationsof PFAAs among smokers with and without
HU.
Materials and methods

and July 2012 at different health examination centers in Shimane Prefecture, Japan
were initially considered for this study. As
described elsewhere, the health examination
included physical examination, clinical andlaboratory tests, and a self-administered questionnaire containing personal and medical
history.10 A total of 158 subjects aged over 75
years were excluded from the study as they
did not have the data on UA (not measured),leaving 169 subjects for final inclusion in thisstudy. The subjects were free from any serious health problems such as heart diseases,
cancer or renal failure etc.
Measurement of PFAAs
In the current study, we measured theabsolute concentrations (in μmol/L) of 19
PFAAs: alanine (Ala), arginine (Arg), asparagine (Asn), Citrulline (Cit), glutamine (Gln),glycine (Gly), histidine (His), isoleucine (Ile),leucine (Leu), lysine (Lys), methionine (Met),
ornithine (Orn), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
For this purpose, 5 ml of blood samples werecollected and analyzed following the protocol
previously described elsewhere.11 Briefly, after overnight fasting, cubital venous blood
samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA;
Terumo, Tokyo, Japan). The tubes with blood
samples were immediately placed on ice. Thenthe tubes were centrifuged at 3,000 rpm under 4℃ for a period of 15 min and stored at－80℃. The tubes were kept there until thedesired analysis. Before the measurementsof PFAAs, the plasma samples were deproteinized using acetonitrile at a final concentration of 80%. The concentrations of PFAAs
were measured by high-performance liquid
chromatography-electrospray ionization-mass
spectrometry followed by precolumn derivatization.

Study design and ethical issues
For this cross-sectional study, approval of
the protocol was obtained from the relevant
institutional review board of Yamaguchi
University (H25-26-2) and Shimane University (20100129-3). The study was conducted inaccordance with the Declaration of Helsinki
and the study participants were briefed verbally about the detailed protocol. The partici- Measurement of other laboratory variables
pants provided written informed consent to
We determined fasting plasma glucose
participate in this study.
(FPG) and hemoglobin A1c (HbA1c) using thehexokinase method and latex agglutination
Study population
immunoassay, respectively. Also, we meaA total of 327 male subjects undergoing sured high-density lipoprotein cholesterol
their annual health check-up between June (HDLC), low-density lipoprotein cholesterol

Amino Acids in Hyperuricemia among Male Smokers

23

the median and interquartile range (IQR),
and the categorical variables as number andpercent. The differences were assessed by
the Mann-Whitney U-test for the continuous variables and Chi-square (χ2) test, for the
categorical variables. The statistical analyses were performed with the software package SPSS version 22 for Windows (SPSS Inc.,
Chicago, IL, USA). For all statistical tests,
the significance level was set at a two-tailed
P<0.05.

(LDLC), and triglyceride (TG) in the serum
enzymatically. To measure the plasma UA,
we used the the uricase-HMMPS method by
L-type UA.M kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Smoking history
For collection of data on smoking history
from the study subjects, the original questionnaire used Brinkman Index/BI (numberof cigarettes smoked per day x number ofyears of smoking) as described by Brinkman
and Coates.12 Based on the collected data, weclassified all subjects with a BI=0 as nonsmokers (n=74) and the rest subjects as smokers (n=95, including current and past smokers; median and IQR values for BI, 640 and
540, respectively).

Results
Table 1 shows the demographic and clinical characteristics of the study subjects forboth smoker and non-smoker groups. Most
subjects in both groups were the elderly
males. As the results show, no significant
differences in terms of the demographic andclinical variables existed between the twogroups except for the plasma level of UA
which showed a significantly higher value
for the smokers (median and IQR values forthe smoker and non-smoker groups were 6.0
mg/dl and 1.4 mg/dl, and 5.7 mg/dl and 1.4
mg/dl, respectively; P=0.009). Also, no statistically significant differences between the

Data analyses and statistical methods
As our study included only male subjects,
we defined HU as a plasma UA ≥7 mg/dL[11]. The continuous variables in this study
showed a non-normal distribution by Kolmogorov-Smirnov and Shapiro-Wilk tests.
Hence, nonparametric statistical analyses ofthe study data were performed. The continuous variables of this study were presented as

Table 1 Demographic and clinical characteristics of the study populations including smokers
and nonsmokers and further divided according to the presence or absence of hyperuricemia(HU). Values are expressed as median and interquartile range (IQR) for all continuous variables
Variables
Age (Years)
BMI (kg/m2)
FPG (mg/dL)
HbA1c (%)
HDLC (mg/dL)
LDLC (mg/dL)
TG (mg/dL)
SBP (mmHg)
DBP (mmHg)
UA (mg/dL)

Smokers(n = 95)

All
Nonsmokers(n = 74)

MedianIQRMedian

IQR

68.0
23.0
88.0
5.3
58.0
111.0
93.0
125.0
76.0
6.0

8.0
3.5
13.0
0.5
21.0
34.0
73.0
19.0
13.0
1.4

68.0
23.4
88.0
5.3
56.5
113.0
91.5
121.5
75.5
5.7

7.3
4.2
12.0
0.5
20.3
32.5
62.0
17.3
12.0
1.4

P1value
0.855
0.739
0.453
0.386
0.610
0.624
0.169
0.105
0.617
0.009

Smokers (n = 95)
Without HUWith HU(n = 61)(n = 34)MedianIQRMedian

IQR

68.0
22.6
88.0
5.3
60.0
106.0
89.0
124.0
75.0
─

8.5
3.4
12.5
0.6
25.0
33.5
54.5
21.5
12.0
─

68.5
23.6
87.0
5.3
55.5
119.0
125.0
127.0
76.0
─

8.3
4.7
10.5
0.5
19.0
41.5
97.5
15.3
14.5
─

P2value
0.560
0.080
0.638
0.876
0.197
0.049
0.049
0.263
0.310
─

Nonsmokers (n = 74)
Without HUWith HU(n = 58)(n = 16)
MedianIQRMedian

IQR

69.0
23.0
88.0
5.2
57.5
108.5
88.5
122.5
75.0
─

7.5
4.3
12.3
0.6
20.5
30.5
61.5
17.3
11.5
─

66.0
23.6
88.0
5.5
54.5
124.5
104.0
121.0
78.5
─

8.3
3.4
12.5
0.3
17.5
25.5
74.3
27.0
12.5
─

P3value
0.519
0.287
0.921
0.045
0.278
0.039
0.196
0.895
0.411
─

BMI, body mass index; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HbA1C, haemoglobin
A1C; HDLC, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol; SBP, systolicblood pressure; TG, triglycerides; UA, uric acid.
P-values indicate statistically significant difference between groups by Mann-Whitney U Test: P1, smokers
versus nonsmokers; P2, without HU versus with HU in smokers; P3, without HU versus with HU in nonsmokers.

24

MH Mahbub et al.

smokers and nonsmokers were observed foralcohol drinking status (χ2=0.035, P=0.874).
The medications commonly used by the study
subjects were prescribed for the treatment ofhypertension (smokers, 26/95 or 27.4%; nonsmokers, 21/74 or 28.4%), diabetes mellitus(smokers, 14/95 or 14.7%; non-smokers, 8/74
or 10.8%), and dyslipidemia (smokers, 21/95
or 22.1%; non-smokers, 15/74 or 20.3%). However, the smoker and non-smoker groups did
not differ significantly with respect to the useof medications for any of the mentioned diseases (hypertension, χ2=0.021, P=0.884; diabetes mellitus, χ2=0.566, P=0.452; dyslipidemia,2χ =0.084, P=0.773).
As we observed, the prevalence of HU amongsmokers and nonsmokers were 35.8% and21.6%, respectively. When the demographic

and clinical characteristics of the study subjects were compared between subjects without and with HU separately in both smoker
and non-smoker groups, no statistically significant differences were observed except for
marginally significant differences (P=0.049)
for LDLC and TG among the smokers, andsignificant differences for HbA1c (P=0.045)
and LDLC (P=0.039) among the nonsmokers(Table 1).
For the individual amino acids, at first, weinvestigated the existence of any significant
differences in their concentrations between
the smoker and non-smoker groups, as presented in Table 2. However, no significant differences between these two groups could be
revealed for the level of any PFAAs except for
Lys, the plasma concentration of which was

Table 2 Plasma amino acid concentrations (μmol/L) in smokers and nonsmokers
Amino
acids
Ala
Arg
Asn
Cit
Gln
Gly
His
Ile
Leu
Lys
Met
Orn
Phe
Pro
Ser
Thr
Trp
Tyr
Val

Smokers (n = 95)
Median
IQR
360.7
113.6
97.6
23.3
48.2
7.8
36.2
10.9
605.4
89.1
212.7
63.4
80.6
13.4
62.4
19.2
125.3
30.4
184.7
36.5
27.6
5.3
54.4
16.0
62.3
9.2
138.7
49.7
111.4
28.7
123.8
27.3
56.7
14.1
68.5
20.6
226.9
58.6

Nonsmokers (n = 74)
Median
IQR
360.8
110.2
100.5
27.8
49.6
8.7
33.3
10.5
628.6
86.5
211.3
44.6
82.3
11.9
65.7
17.3
125.2
23.0
196.6
41.5
27.0
5.9
52.3
14.6
62.8
12.7
146.9
52.6
110.2
25.1
127.6
37.9
59.1
12.0
67.3
17.7
231.9
52.3

P-value
0.882
0.217
0.270
0.101
0.155
0.761
0.464
0.632
0.874
0.017
0.566
0.255
0.864
0.235
0.584
0.498
0.152
0.314
0.678

Ala, alanine; Arg, arginine; Asn, asparagine; Cit, citrulline; Gln, glutamine; Gly, glycine;His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Orn, ornithine; Phe,phenylalanine; Pro, proline; Ser, serine; Thr, threonine, Trp, tryptophan, Tyr, tyrosine; Val,valine.
P-values indicate statistically significant difference between smoker and nonsmoker groups
by Mann-Whitney U Test.

Amino Acids in Hyperuricemia among Male Smokers

significantly lower among the smokers compared to that for the nonsmokers (P=0.017).
Next, to investigate the differences between
the subjects with and without HU, the statistical tests were performed in both smokers
and nonsmokers separately (Table 3). For thesmokers, the concentrations of 10 out of 19
PFAAs demonstrated significant differences
between the subjects without and with HU.
Among those 10 PFAAs, compared to thecorresponding values for the subjects without HU, subjects with HU had significantly
higher levels of Ile, Leu, Met, Phe, Trp, Tyr
and Val (P<0.05 to 0.01), and significantly
lower levels for Gln, Gly and Ser (P<0.05 to

25

0.001). In contrast, in the nonsmoker group,no individual PFAAs showed any significant
difference in their concentration between thesubjects with and without HU except for Phe,the plasma concentration of which was foundto be higher among the subjects with HU(P=0.039).
Discussion
To our knowledge, this is the first study inthe literature that examined the differences
in PFAAs among smokers with and without HU. We also included nonsmoker subjects with and without HU and examined theTable 3 Differences in plasma concentrations of amino acids (μmol/L) between the subjects
without and with hyperuricemia (HU) in smokers and nonsmokers
Smokers (n = 95)
Amino Without HUWith HU
acids(n = 61)(n = 34)Median IQR Median IQR
Ala
356.3
105.0
366.0
133.2
Arg
102.2
22.7
91.5
17.9
Asn
33.7
7.5
48.0
11.5
Cit
36.2
9.0
35.7
13.7
Gln
626.1
88.7
573.6
80.9
Gly
224.8
74.2
186.1
45.6
His
80.4
12.5
82.2
14.8
Ile
57.7
19.6
70.3
14.6
Leu
116.9
29.4
135.1
28.4
Lys
181.8
40.4
185.1
27.0
Met
26.9
5.0
28.7
4.4
Orn
55.8
15.5
53.2
19.8
Phe
61.5
9.5
64.9
16.5
Pro
134.2
48.1
155.7
49.9
Ser
113.8
29.3
103.7
20.5
Thr
125.7
25.7
120.1
35.0
Trp
55.9
9.4
60.8
14.3
Tyr
64.4
17.2
77.2
20.4
Val
210.4
54.5
247.5
56.0

P1value
0.302
0.101
0.305
0.981
0.007
0.000
0.368
0.004
0.009
0.499
0.026
0.424
0.013
0.126
0.027
0.364
0.007
0.002
0.006

Nonsmokers (n = 74)
Without HUWith HU(n =58)(n = 16)
Median IQR Median IQR
359.4
110.7
377.9
181.2
97.3
27.5
106.3
27.6
49.1
8.8
50.6
12.1
33.3
9.9
32.7
12.4
626.5
85.7
631.5
104.2
211.6
59.1
211.3
27.0
81.4
12.2
84.8
6.6
64.7
16.2
69.6
23.0
122.5
25.7
132.7
17.7
195.5
47.4
212.7
44.1
27.0
5.7
28.2
7.3
51.9
14.6
53.1
18.9
62.2
11.1
71.7
15.7
141.2
57.0
165.5
30.7
109.8
25.2
110.8
28.4
127.3
39.1
135.6
45.2
58.5
12.3
61.4
12.3
67.2
17.4
69.6
20.8
226.2
56.2
238.0
39.9

P2value
0.128
0.684
0.288
0.609
0.743
0.416
0.115
0.243
0.066
0.106
0.276
0.990
0.039
0.085
0.854
0.581
0.282
0.627
0.217

Ala, alanine; Arg, arginine; Asn, asparagine; Cit, citrulline; Gln, glutamine; Gly, glycine;His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Orn, ornithine; Phe,phenylalanine; Pro, proline; Ser, serine; Thr, threonine, Trp, tryptophan, Tyr, tyrosine; Val,valine.
P1 and P2-values indicate statistically significant differences between subjects without andwith HU by Mann-Whitney U Test in the smoker and nonsmoker groups, respectively.

26

MH Mahbub et al.

differences in PFAAs amongst them.
Overall, in our study, the smoker and nonsmoker study populations (also, considering
the presence or absence of HU) were similar
with respect to the demographic and clinicalvariables. The only difference between thesmokers and nonsmokers was for the concentration of UA, which was detected to be significantly higher among smokers comparedwith the nonsmokers. This latter finding does
not support the inverse relationship between
smoking and plasma level of UA reported bya number of studies in the literature.13,14 Conversely, our findings are consistent with thefindings of other previously published studies
which showed higher blood UA levels among
the smokers.6,7 The presence of higher prevalence of HU among smokers observed in ourstudy is probably due to the fact that bothsmoking and ageing is individually associated with increased UA levels especially inmen, and that the elderly subjects were predominant in this study which comprised exclusively male subjects.15,16
Little is known about the changes in PFAA
profiles among smokers as a limited numberof research works has been conducted on thisissue. Furthermore, those studies performed
measurements of various PFAAs that differ
from PFAAs measured in our study. This
makes the comparison of our study findings
with those of others difficult. In a cohort
study, it has been demonstrated that compared with the former smokers and neversmokers, the concentrations of Arg, Orn, andSer were higher among the current smokers.9
In contrast, Tomoda et al. (2014) in an animal
study, exposed rats to cigarette smoke for aperiod of four-weeks and observed a significant reduction in the levels of branched chainamino acids/BCAAs (Ile, Leu, Val) levels in
the rats.17 However, in our study, only theplasma concentration of Lys was significantly lower among the smokers compared tothat for the nonsmokers. Differences in theamount of alcohol consumption by the study
populations,9 the selection of study subjects(human or animal),17 and the differing doses
and duration of exposure to cigarette smoking or cigarette smoke etc might have contributed to the observed inconsistency between the study findings.

The patterns of changes in PFAA profiles
in subjects with and without HU amongsmokers remain uncertain as, to the best of
our knowledge, no other study has hitherto
investigated it. As observed in our study, theconcentration of Phe differed significantly
between the subjects without and with HU(higher in HU group) in both smokers andnonsmokers. This finding is consistent withthe finding of our recently published study,
where the healthy control subjects and patients with 4 types of lifestyle-related diseases showed significantly higher level of
Phe among the subjects with HU comparedwith the subjects without it.11 On the other
hand, among the smokers compared with thesubjects without HU, subjects with HU hadsignificantly altered levels in a number of
PFAAs: subjects with HU had significantly
higher levels of Ile, Leu, Met, Trp, Tyr andVal, and significantly lower levels of Gln, Gly
and Ser than those without HU. Based on
our observed findings of the similarities anddissimilarities in the patterns of changes in
PFAA concentrations amongst the smokers
and nonsmokers with and without HU andalso the findings from previously publishedstudies considered together, it can be assumed
that a close relationship probably exists between smoking and plasma UA and their interaction probably induces further changes in
the plasma PFAAs. We found a lower concentration of several PFAAs (Gln, Gly and Ser)in the smokers with HU compared with those
without it, which might have been caused by
the fact that these amino acids, particularly
of Gly and Ser, play important roles in thebiosynthesis of purine and are increasingly
being utilized in the formation of UA among
the smokers with HU.
Furthermore, we found elevated plasma
concentrations of BCAAs (Ile, Leu, Val) andaromatic amino acids/AAAs (Phe, Tyr, Trp)among the smokers with HU. In a nested
case-control study Wang et al. (2011) observed that that a cluster of BCAAs (Ile, Leu,
Val) and AAAs (Phe, Tyr) predicted the future development of type 2 diabetes up to 12
years prior to onset of it.18 The authors ofthat study suggested that increased levels of
circulating BCAAs may promote insulin resistance possibly via the disruption of insulin

Amino Acids in Hyperuricemia among Male Smokers

signalling in skeletal muscles through activation of the mTOR, JUN and IRS1 signalling pathways. Furthermore, higher levelsof BCAAs in the blood may lead to increased
levels of AAAs as both BCAAs and AAAs
compete for transport into mammalian cellsby the common large neutral amino acidtransporter (LAT1).19 Also, in our previously
published study, the patients with lifestylerelated diseases including DM demonstrated
significantly higher concentration of Trp
and Tyr compared with those for apparently
healthy controls; the concentrations were alsosignificantly higher among the subjects with
HU when the comparison was made between
the latter subjects and subjects without HU.11
Moreover, in the current study, the concentration of Met showed an elevation amongst
the smokers with HU. Met is a precursor ofhomocysteine which has been found to correlate with insulin resistance.20 Although
the findings of our study cannot be directlycompared with those of other studies mentioned here, however, our findings of elevatedplasma concentrations of PFAAs among thesmokers with HU are consistent with the existing published literature as epidemiologic
studies demonstrated a clear association ofcigarette smoking and HU with an increased
risk of diabetes and effects on insulin sensitivity.21,22 However, future studies investigating the effects of smoking on PFAA profiles
among subjects with HU should include themeasurements of insulin sensitivity/resistance in the study populations.
Limitations to the current study
The findings of this study should be interpreted in light of several potential methodological limitations. First, we did not collectdata on dietary habits of the study populations. However, the existence of any such effects on our study findings should be limited
as we included a nonsmoker control groupfor comparison of results. Second, we included current and past smokers as the smoker
population and the question may arise on
the interpretation of current results as it
is unlikely that past smoking might influence amino acid metabolism for years. Sucha classification of smokers was unavoidable
as the original questionnaire included BI for

27

collection of data on the smoking status of
the study subjects. Also, we firmly believethat this does not influence the outcome ofour study because inclusion of past smokers
as smokers means an underestimation of thecurrent results. Third, we did not collect dataon the medications such as those blocking UA
production, lowering the blood UA level and/or promoting the excretion of uric acid However, we believe that the findings of our study
were not influenced by the intake of any such
medications as it would mean an underestimation of the observed results. Fourth, only
male Japanese subjects were included in thisstudy which limits the generalization of thestudy findings to some extent. Fifth, thisstudy was cross-sectional in nature which
does not allow us to speculate any causality
or temporality of the association between altered levels of PFAAs with HU among thecigarette smokers.
Conclusions
The findings of our study demonstrated
that among the smokers with HU comparedwith those without HU, there was an elevation in BCAAs (Ile, Leu, Val) and AAAs (Phe,Trp, Tyr) and Met, and a reduction in the levels of Gln, Gly and Ser. The findings suggestthat HU among smokers may have remarkable effects on the PFAA profiles. The findings may provide new insights into the biological mechanisms of cigarette smoking and
HU-related diseases. However, for validationof the current study findings and a comprehensive understanding of the causal association between the alterations in the levels of
PFAAs with HU among smokers and theunderlying mechanisms, prospective cohort
studies should be conducted in future withinclusion of age-matched large and diverse
populations.
Conflict of Interest
HY and SK are employees of AjinomotoCo., Inc. TT received the research grants
from Ajinomoto Co., Inc. However, this does
not alter the authorsʼ adherences to all of thejournal policies. The remaining authors declare that they have no conflict of interest.

28

MH Mahbub et al.

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