Cross-sectional and longitudinal study of association between circulating thiobarbituric acid-reacting substance levels and clinicobiochemical parameters in 1,178 middle-aged Japanese men - the Amagasaki Visceral Fat Study
Nutrition & Metabolism
Cross-sectional and longitudinal study of association between circulating thiobarbituric acid-reacting substance levels and clinicobiochemical parameters in 1,178 middle-aged Japanese men - the Amagasaki Visceral Fat Study
Yukiyoshi Okauchi 0
Ken Kishida 0 2
Tohru Funahashi 0 2
Midori Noguchi 1
Tomoko Ogawa 1
Kohei Okita 0
Hiromi Iwahashi 0
Tetsuya Ohira 3
Akihisa Imagawa 0
Tadashi Nakamura 0
Iichiro Shimomura 0
0 Department of Metabolic Medicine, Graduate School of Medicine, Osaka University , Suita, Osaka 565-0871 , Japan
1 Amagasaki City Office, General Affairs Bureau, Personal Department, Payroll Section, Employee Health Promotion Section , Amagasaki, Hyogo 660-8501 , Japan
2 Department of Metabolism and Atherosclerosis, Graduate School of Medicine, Osaka University , Suita, Osaka 565-0871 , Japan
3 Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University , Suita, Osaka 565-0871 , Japan
Background: Circulating thiobarbituric acid-reacting substance (TBARS) levels, a marker of systemic oxidative stress, are predictive of cardiovascular events. However, they has not been evaluated in Japanese, especially with regard to the factors that contribute to the changes in circulating TBARS levels. We investigated the cross-sectional and longitudinal relationships between circulating TBARS levels and various clinicobiochemical parameters in middleaged men. Methods: In this population-based study (The Amagasaki Visceral Fat Study), 1,178 Japanese male urban workers who had undergone health check-ups in 2006, 2007 and 2008 and were not on medications for metabolic disorders during the follow-up period, were enrolled. Serum TBARS levels were measured by the method of Yagi. The estimated visceral fat area (eVFA) by bioelectrical impedance was measured annually. After health check-ups, subjects received health education with lifestyle modification by medical personnel. Results: The number of cardiovascular risk factors (hypertension, hyperglycemia, low HDL-C, hypertriglyceridemia, hyperuricemia, hyper-LDL-C and impaired renal function) augmented with the increases in log-eVFA (p < 0.0001) and log-TBARS (p < 0.0001). The combination of TBARS and eVFA had a multiplicative effect on risk factor accumulation (F value = 79.1, p = 0.0065). Stepwise multiple regression analysis identified log-eVFA, as well as age, log-body mass index (BMI), LDL-C, log-adiponectin, g-glutamyl transpeptidase (g-GTP) and uric acid as significant determinants of logTBARS. Stepwise multiple regression analysis identified one-year changes in eVFA as well as BMI, g-GTP and estimated glomerular filtration rate (eGFR) as significant determinants of one-year change in TBARS, and biennial changes in eVFA as well as BMI and g-GTP, eGFR as significant determinants of biennial change in TBARS. Conclusions: The present study showed a significant cross-sectional and longitudinal correlation between TBARS and eVFA, as well as BMI and g- GTP, eGFR. Visceral fat reduction may independently associate with the improvement in systemic ROS in middle-aged Japanese men. Trial Registration: The Amagasaki Visceral Fat Study UMIN000002391.
visceral fat accumulation; systemic reactive oxidative stress; visceral fat reduction
Oxidative stress results from an imbalance between
reactive oxygen species (ROS) and antioxidants, and is
influenced by genetic and environmental factors. In the
general population, serum levels of thiobarbituric
acidreacting substance (TBARS), an important biomarker of
systemic oxidative stress, were reported to be associated
with various factors, such as age, body mass index (BMI),
glucose metabolism, lipid metabolism , g-glutamyl
transpeptidas (g-GTP) , uric acid (UA)  and
smoking, and the levels correlated with atherosclerogenesis .
Serum levels of TBARS were strongly predictive of
cardiovascular events in patients with stable coronary artery
disease, independently of traditional risk factors [5,6].
We reported previously that adipose tissue is the
major source of ROS (called FatROS), and that systemic
oxidative stress is closely associated with fat
accumulation in obese animals . These animals also exhibited
selective overproduction of ROS in adipose tissue, as
well as over-expression of NADPH oxidase and
underexpression of antioxidative enzymes . We and others
have demonstrated that increased FatROS and systemic
oxidative stress are the underlying causes of
dysregulated production of adipocytokines (e.g.,
hypoadiponectinemia and high levels of plasminogen activator
inhibitor-1, interleukin-6, and monocyte
chemoattractant protein-1), which might lead to the development of
lifestyle-related diseases [7,8]. In obese mice, treatment
with NADPH oxidase inhibitors reduced ROS
production by the adipose tissue, attenuated the dysregulation
of adipocytokines, and improved diabetes and
hyperlipidemia . However, the level of systemic ROS
production has not been evaluated properly in the general
population, especially with regard to the factors that
contribute to the changes in systemic ROS production.
The present cross-sectional and longitudinal study
investigated the relationship between circulating TBARS
levels and various clinicobiochemical parameters in
middle-aged Japanese male urban workers.
Methods and Procedures
The study subjects were 1,178 Japanese men [age; mean
SD 45 10 (range, 20-68) years], who were employees
of the Amagasaki city office and had undergone annual
health check-ups in three consecutive years (2006, 2007
and 2008). To avoid the influence of medications, we
excluded subjects who were on treatment for diabetes,
hypertension and dyslipidemia during the 2-year
followup period. Table 1 left summarizes the profiles of all
participants at baseline. After the health check-up, the
medical staff, especially health nurses, provided the
individual with annual health promotion program to
promote voluntary lifestyle changes (2006 and 2007) .
In brief, the program emphasized hazards of visceral fat
accumulation and multiple cardiovascular risk factors,
with the aim of encouraging a scientific understanding
of the concept of metabolic syndrome from visceral fat
accumulation to the development of atherosclerotic
cardiovascular diseases. We reported previously that this
program should be useful in reducing visceral fat
accumulation and consequently decrease number of
cardiovascular risks, such as glucose tolerance, dyslipidemia,
high blood pressure , hypoadiponectinemia , and
a prevalence of the metabolic syndrome , leading to
prevention of cardiovascular events [12,13].
Anthropometry and laboratory measurements
Body weight (kg), height (cm), and waist circumference
at umbilical level (cm) were measured in standing
position, and systolic and diastolic blood pressures (SBP,
DBP, respectively) were measured with a standard
mercury sphygmomanometer after rest in sitting position
for at least 5 minutes. BMI was calculated using the
formula [weight (kg)/height (m) 2]. Visceral fat area was
estimated by bioelectrical impedance analysis (eVFA), as
we reported previously . After overnight fasting or
at least 5 hours-fasting, venous blood samples were
collected for measurements of blood glucose (BS),
hemoglobin A1c (HbA1c, National Glycohemoglobin
Standardization Program), triglyceride (TG),
high-density lipoprotein-cholesterol (HDL-C), high-density
lipoprotein-cholesterol (LDL-C), g-glutamyl transpeptidase
(g-GTP), creatinine, uric acid (UA) and adiponectin
(Otsuka Pharmaceutical Co., Tokushima, Japan), while
the subject was in sitting position. For the purpose of
the present study, serum samples that were obtained at
baseline from each participant and stored promptly at
-20C without the addition of exogenous antioxidants
before TBARS assay. After thawing the samples, serum
levels of malondialdehyde in terms of TBARS, a marker
of systemic ROS production, were measured in duplicate
in each of 3,534 samples by the method of Yagi (Japan
Institute for the Control of Aging, Nikken SEIL Co.,
Shizuoka, Japan) , as we [16,17] and other 
previously reported. The selection of this parameter was
based on the fact that malondialdehyde can be
generated from oxidative mechanisms other than lipid
peroxidation, and that TBARS is assumed to represent a
composite of systemic oxidative damage products,
including malondialdehyde . Glomerular filtration
rate was estimated by the following equation (eGFR =
194 serum creatinine -1.094 age -0.287).
We investigated the presence of seven cardiovascular
risk factors: 1) hypertension (SBP 130 and/or DBP 80
mmHg) , 2) hyperglycemia (fasting or postprandial
Data are mean SD (range) or numbers (n) of subjects.
g-glutamyl transpeptidase (g-GTP), eGFR; estimated glomerular filtration rate, TBARS; thiobarbituric acid reactive substances.
year 2006 (n = 1,178)
year 2007 (n = 1,178)
year 2008 (n = 1,178)
BS of 6.10 or 7.77 mmol/L, respectively) , 3) low
HDL-C (HDL-C < 1.04 mmol/L) , 4)
hypertriglyceridemia (fasting or postprandial TG of 1.69 or 2.27
mmol/L, respectively) , 5) hyperuricemia (UA 416
mol/L), 6) hyper-LDL-cholesterolemia (LDL-C 3.64
mmol/L) and 7) impaired renal function (eGFR < 60 mL/
min/1.73 m ).
Data of TBARS, eVFA and adiponectin levels showed
skewed distribution, and were thus log-transformed
before analysis. Pearsons correlation coefficient was
used to examine the relationship between log-TBARS
and various clinicometabolic parameters, and between
one-year and biennial changes in TBARS and various
clinicometabolic parameters. Significant level was set at
p < 0.05. Stepwise multiple regression analysis was
conducted to identify the parameters that significantly
contributed to log-TBARS or one-year and biennial changes
in TBARS. Parameters with F value > 4.0 were
subsequently entered into regression analysis as independent
variables. Differences in the mean number of
obesityrelated cardiovascular risk factors between eVFA and
TBARS were analyzed by the Kruskal-Wallis test (Figure
1). Differences among groups were compared by
unpaired Students t-test for experiments involving only
two groups (Figure 2). Continuous variables were
expressed as mean SEM (Figure 2) or SD (Table 1).
All statistical analyses were performed with the
Statistical Package for Social Sciences (vesion 11.0.1J; SPSS,
The study was approved by the human ethics committee
of Osaka University and a signed informed consent was
obtained from each participant, based on the ethical
guideline of the 2000 Declaration of Helsinki of the
World Medical Association.
The cross-sectional study
First, we compared the mean number of cardiovascular
risk factors (hypertension, hyperglycemia, low HDL-C,
hypertriglyceridemia, hyperuricemia, hyper-LDL-C and
impaired renal function) after classifying the subjects
according to the log-eVFA and log-TBARS values. The
number of risk factors increased significantly with the
increase in eVFA (p < 0.0001 for trend, the
KruskalWallis test). Moreover, the number of cardiovascular
risk factors increased significantly with increased levels
of circulating TBARS (p < 0.0001 for trend, the
KruskalWallis test) (Figure 1). We investigated the
additive effect of TBARS and eVFA on cardiovascular
risk factor accumulation using age-adjusted
multiplicative interaction term (log TBARS log eVFA) in the
linear regression model, and log TBARS and log-eVFA
individually. Multiple linear regression analysis (adopted
factors; age, log TBARS, log-eVFA, log TBARS log
eVFA) identified interaction term (log TBARS log
eVFA) as a significant determinant of cardiovascular risk
factor accumulation (F value = 79.1, p = 0.0065). These
results indicated that the combination of TBARS and
eVFA had a multiplicative effect on cardiovascular risk
factor accumulation (Figure 1).
Second, analysis of data collected in year 2006 showed
positive relationships between log-TBARS and the
following parameters: age, BMI, waist circumference,
logeVFA, SBP, DBP, LDL-C, HbA1c, g-GTP,
ex-/currentsmoking and UA, and a negative relationship between
log-TBARS and log-adiponectin and eGFR (Table 2).
Stepwise multiple regression analysis identified age,
LDL-C, log adiponectin, g-GTP, UA and log-eVFA
[multivariate 1 (eVFA); adopted factors: age, eVFA SBP,
LDL-C, HbA1c, log-adiponectin, g-GTP, eGFR, UA,
smoking status] or log-BMI [multivariate 2 (BMI);
adopted factors; age, BMI SBP, LDL-C, HbA1c,
log-adiponectin, g-GTP, eGFR, UA, smoking status], as
significant determinants of log-TBARS (Table 2).
Next, to clarify whether the degree of obesity or body
fat distribution relates more strongly with circulating
TBARS levels, we divided subjects into four groups
according to BMI (cutoff value 25 kg/m2) and eVFA
(cutoff value 100 cm2), based on Japanese criteria of
obesity and visceral fat accumulation  (Figure 2).
Subjects with eVFA 100 cm2 had significantly higher
levels of circulating TBARS than those with eVFA
<100 cm2, irrespective of BMI (p < 0.0001,
respectively). Interestingly, subjects with visceral fat
accumulation but without overall obesity (eVFA 100 cm2
plus BMI < 25 kg/m2) had significantly higher levels of
circulating TBARS than those without visceral fat
accumulation but with overall obesity (eVFA <100 cm2 plus
BMI 25 kg/m2) (p = 0.026). These results indicate
that body fat distribution, rather than with the degree
of obesity, should be more associated with circulating
Figure 2 Relationship between circulating TBARS levels and body fat distribution. Subjects were divided according to body mass index
(BMI) using a cutoff value of 25 kg/m2 and estimated visceral fat area (eVFA) using a cutoff value of 100 cm2, measured in 2006. Data are mean
SEM. TBARS: thiobarbituric acid-reacting substance.
The longitudinal study
We also investigated the relationship between one-year
changes in TBARS (one-year TBARS) and various
parameters (Table 3A). The one-year TBARS
correlated positively with one-year BMI, eVFA, g-GTP,
eGFR, and UA. Stepwise multiple regression analysis
identified one-year g-GTP, eGFR, log-eVFA
[multivariate 1 (one-year eVFA); adopted factors; one-year
eVFA, g-GTP, eGFR, UA] and log-BMI
[multivariate 2 (one-year BMI); adopted factors; one-year BMI,
g-GTP, eGFR, UA] as significant determinants of
Moreover, we also investigated the relationships
between biennial changes in TBARS (biennial TBARS)
and various parameters (Table 3B). Biennial TBARS
correlated with biennial BMI, waist circumference,
eVFA, LDL-C, g-GTP, eGFR, and UA. Stepwise
multiple regression analysis identified biennial g-GTP,
eGFR, log-eVFA [multivariate 1 (biennial eVFA);
adopted factors; biennial eVFA, LDL-C, g-GTP,
eGFR, UA] and log-BMI [multivariate 2 (biennial
DBMI); adopted factors; biennial BMI, LDL-C,
gGTP, eGFR, UA], as significant determinants of
biennial TBARS (Table 3B).
The cross-sectional study is the first to report that body
fat distribution, i.e. visceral fat accumulation, as well as
Table 2 Cross-sectional results of correlation between log-TBARS and various parameters by uni- and multi-variate
Univariate: Pearsons correlation analysis, Multivariate: Stepwise multiple regression analysis.
Parameters with F value > 4.0 were subsequently entered into the regression analysis as independent variables.
BW: Body weight, BMI: body mass index, WC: waist circumference, Log-eVFA: estimated visceral fat area,
SBP: systolic blood pressure, DBP: diastolic blood pressure, HDL-C: high-density lipoprotein-cholesterol,
LDL-C: low-density lipoprotein-cholesterol, HbA1c: Hemoglobin A1c, eGFR: estimated glomerular filtration rate,
UA: uric acid.
age, BMI, LDL-C, g-GTP and UA as reported previously
[1-3], is important determinant of circulating TBARS
levels in Japanese men (Table 2). Moreover, subjects
with visceral fat accumulation had significantly higher
levels of circulating TBARS than those without visceral
fat accumulation, both in those with and without overall
obesity (Figure 2), as previous report demonstrated that
visceral fat thickness and serum TBARS levels . The
results also emphasize the importance of visceral
adipose tissue per se as the major source of ROS in the
whole body in the general population, as we reported
previously in obese subjects  and in subjects at high
risk for cardiovascular diseases . This present study
demonstrated that visceral fat may be the major source
of systemic oxidative stress, and that visceral fat
accumulation increased systemic oxidative stress with the
underlying causes of cardiovascular risk factor
accumulation (Figure 1) and dysregulated production of
adipocytokines (e.g., hypoadiponectinemia) (Table 2), which
might lead to the development of lifestyle-related disease
including coronary artery disease.
Another aim of the present study was to clarify
whether reduction of visceral fat associated with
reductions in systemic oxidative stress. We reported
previously that visceral fat reduction through lifestyle
modification reduced the number of atherosclerotic
cardiovascular events . The present longitudinal study
indicated that weight loss and reduction of visceral fat
per se through lifestyle modification was associated with
reductions in systemic oxidative stress (Table 3)
accompanied by improvement in cardiovascular risk
accumulation, which probably lessens the risk of atherosclerotic
cardiovascular diseases. Life-style modification designed
to effectively reduce the amount of visceral fat, which is
associated with improvement in liver and renal
dysfunction, is probably beneficial in reducing systemic ROS
overload, leading to combat cardiovascular disease
events in human. There is no doubt that other factors
also influence the status of systemic oxidative stress,
such as physical activity, alcohol consumption,
socioeconomic status, sleep status, psychogenic stress status
and dietary habits. These lifestyle changes themselves
may also reduce circulating TBARS levels, although
these effects were not analyzed in the present study.
The present study also showed a significant
cross-sectional and longitudinal correlation between TBARS and
markers of both liver and renal functions; g-GTP and
eGFR, respectively. g-GTP is the enzyme responsible for
extracellular catabolism of glutathione , and it has
been used as a marker of excessive alcohol intake or of
hepatic diseases . g-GTP, as a scavenger of oxygen
free radicals, plays a key role in rather protecting against
both intracellular and extracellular oxidative stress .
However, many epidemiological studies have suggested
that increased g-GTP levels may be a precocious and
sensitive marker of oxidative stress, identifying persons
Table 3 Longitudinal results of uni- and multi-variate analyses
Correlation between one-year TBARS and values of various parameters
One-year TBARS Univariate Multivariate1 (eVFA)
with low, but persistent, augmentation of nonalcoholic
fatty liver disease . Serum TBARS levels are elevated
in chronic kidney disease patients [25,26]. TBARS
interferes with nitric oxide generation through competitive
inhibition of nitric oxide (NO)-synthase enzyme, and
therefore may lead to impairent in endothelium NO
pathway in kidney [25-27]. Endothelial dysfunction is a
common event described both in chronic and acute
renal failure . A possible direct association between
TBARS and g-GTP or eGFR should be examined by
further experimental studies.
Our study has several limitations. First, the results
may not be applicable to females or non-Japanese
populations. Second, blood samples were collected at random
rather than at fixed daytime. Multiple measurements on
a like-for-like basis should be preformed for better
assessment of TBARS. Third, during the 2-year
followup (from 2006 to 2008), there was only one first-ever
cardiovascular disease event. Monitoring the long-term
effects of visceral fat reduction with lifestyle
modification on the cumulative incidence of
cardiovascular events is required. It is required to clarify the
effects of the health promotion program on compliance
and response and their potential influence on circulating
TBARS. Further controlled studies of blind and
randomized design should be also required. Fourth, smoking
status was collected through a self-questionnaireis
(non, ex- or current-) without Brinkman index (daily number
of cigarettes years). Therefore, the present study could
not investigate longitudinal study for smoking with
reliability. Fifth, the current study did not include the
effects of other important determinants of oxidative
status, such as physical activity, alcohol consumption,
smoking habits, socio-economic status, marital status,
sleep status, dietary habit, and intake of antioxidants,
such as ascorbic acid (vitamin C) and a-tocopherol
(vitamine E), polyphenol and resveratrol (a red wine
constituent) and pharmacological agents. Finally, the
measurement of TBARS using the method of Yagi ,
particularly without an HPLC/GCMS clean-up to isolate
the specific adduct, is limiting. This assay has limited
sensitivity and specificity because TBA reacts with a
variety of compounds (sugars, amino acids, bilirubin and
albumin). More specific markers need to be measured
In conclusion, the absolute value and sequential changes
in systemic oxidative stress correlated significantly with
those of adiposity, especially the amount of visceral fat,
gGTP, and eGFR, in middle-aged Japanese men.
List of Abbreviations
DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate;
eVFA: estimated visceral fat area; -GTP: -glutamyl transpeptidase; HbA1c:
hemoglobin A1c; HDL-C: high-density lipoprotein-cholesterol; LDL-C:
lowdensity lipoprotein-cholesterol; ROS: reactive oxygen species; SBP: systolic
blood pressure; TBARS: thiobarbituric acid-reacting substances; UA: uric acid
Acknowledgements and funding
We thank all members of the Amagasaki Study Group in the Department of
Metabolic Medicine, Osaka University and Amagasaki medical stuffs. This
research was supported in part by grants from the Japan Heart Foundation,
Astellas/Pfizer Grant for Research on Atherosclerosis Update (to K.K.), the
Manpei Suzuki Diabetes Foundation (to T. N.), and a Grant-in-Aid for
Scientific Research on Innovative Areas (Research in a proposed research
area) Molecular Basis and Disorders of Control of Appetite and Fat
Accumulation (to T.F. and K.K.)
YO and KK conducted the research, analyzed data, and wrote the
manuscript. KK reviewed/edited the manuscript. MN and TO conducted the
research. K, HI AI and TN contributed to the discussion. TO provided advice
on statistical analysis. TF and IS contributed to the discussion and wrote the
manuscript. All authors read and approved the final version of the
The authors declare that they have no competing interests.
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