IJC Metabolic & Endocrine 5 (2014) 24–27

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IJC Metabolic & Endocrine

journal homepage: http://www.journals.elsevier.com/ijc-metabolic-and-endocrine

Impact of serum bilirubin levels on carotid atherosclerosis in patients

with coronary artery disease
Yosuke Tatami a,1, Susumu Suzuki a,b,⁎,1, Hideki Ishii a,1, Yohei Shibata a,1, Naohiro Osugi a,1, Tomoyuki Ota a,1,
Yoshihiro Kawamura a,1, Akihito Tanaka a,1, Kyosuke Takeshita c,1, Toyoaki Murohara a,1
a
Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
b
Department of CKD Initiatives Internal Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
c
Department of Clinical Laboratory, Nagoya University Hospital, Nagoya University Graduate School of Medicine, Nagoya, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background/objectives: Bilirubin protects against oxidative stress-mediated diseases, especially atherosclerotic
Received 2 May 2014 diseases. On the other hand, subjects with carotid atherosclerosis have a high incidence of adverse cardiovascular
Received in revised form 11 August 2014 events. The aim of this study was to evaluate the possible relationship between serum bilirubin levels and carotid
Accepted 17 August 2014 atherosclerosis in patients with coronary artery disease (CAD).
Available online 27 August 2014
Methods: We evaluated a total of 394 patients with chronic CAD, defined as stable angina pectoris or a previous myo-
cardial infarction. They were divided into four groups according to serum bilirubin level. Carotid intima–media
Keywords:
Bilirubin
thickness and plaque score (PS) in the common carotid artery were measured using an ultrasound system. Severe
Carotid atherosclerosis carotid atherosclerosis was defined as PS N 10.
Coronary artery disease Results: With increasing quartiles of serum bilirubin levels, the prevalence of severe carotid atherosclerosis signifi-
cantly decreased (48.2%, 39.6%, 30.3%, and 27.0%, respectively, p for trend = 0.007). After adjusting for other risk
factors, low serum bilirubin levels were independently correlated with severe carotid atherosclerosis in CAD
patients (odds ratio 0.89, 95% confidence interval, 0.81–0.99, p = 0.027).
Conclusion: We demonstrated that low serum bilirubin levels were associated with severe carotid atherosclerosis in
CAD patients. Our data suggest that serum bilirubin levels might be an independent, useful, and cost-effective tool
for evaluating atherosclerotic status in CAD patients.
© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction atherosclerosis could be helpful in predicting future CVD events in

It is commonly accepted that bilirubin, the major product of heme ca-
tabolism, has strong antioxidant properties that enable it to scavenge
peroxyl radicals and to inhibit oxidation of low-density lipoprotein-
derived lipids [1]. Several recent reports have demonstrated that elevated
serum bilirubin levels provide important protective effects against oxida-
tive stress-mediated diseases, especially atherosclerotic diseases [2–4].
Increased carotid intima–media thickness (IMT) is a well-established
index of subclinical atherosclerosis and a risk factor for subsequent
cardiovascular disease (CVD) events [5–7]. It was recently reported
that greater carotid atherosclerosis is also associated with future
CVD events in high-risk patients [8,9]. Therefore, evaluation of carotid
⁎ Corresponding author at: Department of Cardiology, Nagoya University Graduate
School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel.: +81 52
744 2147; fax: +81 52 744 2210.
E-mail address: sususu0531@yahoo.co.jp (S. Suzuki).
1
All authors take responsibility for all aspects of the reliability and freedom from bias of
the data presented and their discussed interpretation.
patients with chronic ischemic heart disease. However, few studies
have investigated the relationship between carotid atherosclerosis and
serum bilirubin levels in CAD patients. In the present study, we exam-
ined the association between serum bilirubin levels and carotid athero-
sclerosis in CAD patients.
2. Methods
2.1. Study subjects
This observational study included a total of 394 patients who were
treated for chronic CAD at Nagoya University Hospital between October
2011 and December 2013. Chronic CAD was defined as stable angina
pectoris or a previous myocardial infarction. Patients were divided
into four groups according to quartiles of total bilirubin levels. The
exclusion criteria were as follows: acute cardiovascular events within
6 months before screening, previous carotid endarterectomy or carotid
artery stenting, chronic liver disease, acute heart failure, and hemodialy-
sis. Institutional ethics committee approval was obtained, and all patients

http://dx.doi.org/10.1016/j.ijcme.2014.08.006
2214-7624/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
 Y. Tatami et al. / IJC Metabolic & Endocrine 5 (2014) 24–27 25

Table 1
Baseline characteristics.

Variables Quartiles of serum total bilirubin levels p, Trend

Quartile 1 Quartile 2 Quartile 3 Quartile 4

Male gender 105 (77.8) 79 (82.3) 71 (79.8) 60 (81.1) 0.85

Age (years) 70.7 ± 9.2 69.9 ± 9.7 69.2 ± 9.0 68.0 ± 9.5 0.19
BMI (kg/m2) 23.5 ± 3.6 24.0 ± 3.9 23.1 ± 4.1 23.6 ± 3.8 0.90
Hypertension 108 (80.0) 74 (77.1) 72 (80.1) 53 (71.6) 0.47
Diabetes 58 (43.0) 37 (38.5) 44 (49.4) 27 (36.5) 0.33
Dyslipidemia 108 (80.0) 79 (82.3) 76 (85.4) 56 (75.7) 0.45
Current smoker 39 (29.5) 21 (23.6) 23 (28.0) 13 (17.6) 0.26
Multi vessel disease 105 (77.8) 69 (71.9) 57 (64.0) 54 (72.9) 0.17
Previous MI 27 (20.0) 23 (24.0) 20 (22.7) 13 (17.6) 0.74
Previous CI 21 (15.6) 12 (12.5) 11 (12.4) 10 (13.5) 0.89
AST (IU/l) 20.5 ± 5.6 21.1 ± 5.1 21.1 ± 6.2 21.2 ± 5.3 0.53
ALT (IU/l) 18.4 ± 7.6 19.1 ± 8.0 19.8 ± 8.9 18.9 ± 8.0 0.62
HDL (mg/dl) 46.9 ± 15.4 49.2 ± 13.5 46.6 ± 14.1 46.9 ± 11.2 0.39
LDL (mg/dl) 98.6 ± 30.7 104.7 ± 35.1 104.3 ± 29.3 103.6 ± 28.4 0.36
Triglycerides (mg/dl) 123 (87–176) 118 (83–163) 116 (90–168) 115 (73–149) 0.73
Fasting glucose (mg/dl) 109 (95–139) 108 (95–146) 109 (96–135) 101 (89–122) 0.12
Hemoglobin A1c (%) 6.2 ± 1.2 6.2 ± 1.1 6.2 ± 1.0 6.0 ± 1.1 0.52
Estimated GFR (ml/min/1.73 m2) 62.3 ± 22.0 66.5 ± 19.1 69.4 ± 17.9 69.4 ± 16.1 0.01
Proteinuria 33 (25.2) 12 (12.9) 15 (17.2) 9 (12.9) 0.06
C-reactive protein (mg/dl) 0.07 (0.03–0.14) 0.06 (0.03–0.12) 0.06 (0.04–0.11) 0.05 (0.03–0.10) 0.10
ACE-I or ARB 77 (57.0) 57 (59.3) 53 (59.6) 43 (59.0) 0.98
β-Blockers 40 (29.6) 30 (31.2) 30 (34.1) 21 (28.4) 0.86
Statins 84 (62.2) 66 (68.8) 60 (67.4) 49 (66.2) 0.74
Anti-diabetes drugs 57 (42.2) 34 (35.4) 44 (49.4) 24 (32.4) 0.11

Data are presented as mean ± SD, median (interquartile range), or n (%).

BMI, body mass index; MI, myocardial infarction; CI, cerebral infarction; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL, high-density lipoprotein; LDL, low-density
lipoprotein; GFR, glomerular filtration rate; ACE-I, angiotensin-converting enzyme inhibitors; ARB, angiotension receptor blocker.

provided written informed consent. Body mass index was calculated as
body weight divided by height squared (kg/m2). After an overnight fast
of 12 h, blood samples were obtained from all patients. Serum total biliru-
bin levels were measured by enzymatic methods using an autoanalyzer.
Hypertension was defined as systolic blood pressure ≥140 mmHg,
diastolic blood pressure ≥90 mmHg, and/or current antihypertensive
medication use. Diabetes mellitus was defined as the use of any anti-
hyperglycemic medication or a current diagnosis of diabetes or having a
fasting plasma glucose concentration N 126 mg/dl or a glycosylated
hemoglobin concentration ≥ 6.5% (National Glycohemoglobin Stan-
dardization Program). Dyslipidemia was defined as low-density
lipoprotein (LDL) cholesterol ≥ 140 mg/dl, high-density lipoprotein
cholesterol b40 mg/dl, triglycerides ≥ 150 mg/dl, or receiving lipid-
lowering therapy. Current smokers were defined as those who
declared active smoking at all available examinations. The estimated
glomerular filtration rate (eGFR) was calculated according to the
new Japanese equation: eGFR (ml/min/1.73 m 2 ) = 194 × serum
creatinine − 1.094 × age − 0.287 × 0.739 (in females) [10]. Pro-
teinuria was defined as a dipstick urinalysis score of 1 + or higher.
2.2. Measurement of carotid atherosclerosis
Ultrasonography of the bilateral common carotid artery, carotid
bifurcation, and internal carotid artery was performed using a high-
resolution carotid ultrasound system with a 7.5-MHz linear array trans-
ducer (Prosound α10; Hitachi ALOKA Medical, Tokyo, Japan). IMT is
defined as the distance from the leading edge of the first echogenic
line to that of the second. The first line represents the lumen–intima
interface, and the second line represents the collagen-containing upper
layer of the adventitia. Max IMT is defined as maximal IMT of several
sites in the bilateral carotid arteries [11,12]. To assess the severity of
carotid atherosclerosis, plaque score (PS) was calculated by summing
all plaque thicknesses in eight segments, as proposed by Handa et al. [7,
12]. The severity of PS was classified as normal, mild, moderate, or severe
if the total PS was b 1.1, 1.1–5.0, 5.1–10.0, or N10.0, respectively. All mea-
surements were performed by a trained physician who was blinded to
the patients' clinical information. A total of 30 randomly selected persons
were measured for evaluation of inter- and intra-observer agreement.
The inter- and intra-observer variability of PS and max IMT were well

Table 2
Data regarding carotid ultrasonography.

Variables Quartiles of serum total bilirubin levels p, Trend

Quartile 1 Quartile 2 Quartile 3 Quartile 4

Max IMT (mm) 2.67 ± 0.95 2.33 ± 0.95 2.36 ± 1.05 2.27 ± 0.98 0.003
PS 9.9 ± 5.0 8.3 ± 5.1 7.8 ± 4.9 7.9 ± 5.9 0.002
PS severity 0.004
Normal (b1.1) 2 (1.5) 6 (6.3) 3 (3.4) 7 (9.5)
Mild (1.1–5) 18 (13.3) 18 (18.8) 28 (31.5) 18 (24.3)
Moderate (5.1–10) 50 (37.0) 34 (35.4) 31 (34.8) 29 (39.2)
Severe (N10) 65 (48.2) 38 (39.6) 27 (30.3) 20 (27.0)

Data are presented as mean ± SD or n (%).

IMT, intima–media thickness; PS, plaque score.
26 Y. Tatami et al. / IJC Metabolic & Endocrine 5 (2014) 24–27

correlated (r = 0.92 [p b 0.001] and r = 0.94 [p = 0.001], and r = 0.97
[p b 0.001] and r = 0.97 [p = 0.001], respectively).
2.3. Statistical methods
Statistical analyses were performed using SPSS, version 18.0 for
Windows (SPSS, Inc., Chicago, IL, USA). Data for normally distributed
continuous variables were expressed as mean ± standard deviation
(SD). Continuous variables that were not normally distributed were
expressed as median (interquartile range). Categorical variables were
expressed as numbers (percentages). Continuous variables were com-
pared using one-way ANOVA or the Kruskal–Wallis test. Categorical
variables were compared using the chi-square test or Fisher's exact
test. To obtain independent predictors of severe carotid atherosclerosis,
multivariate logistic regression analysis was performed for each param-
eter as a dependent variable. A two-sided p value of b0.05 was consid-
ered to indicate statistical significance.
3. Results
A total of 394 patients were analyzed in the present study. They
were divided into four groups according to quartiles of total bilirubin
levels as follows: quartile 1, total bilirubin of ≤ 0.5 mg/dl in 135 pa-
tients; quartile 2, total bilirubin of 0.6 mg/dl in 96 patients; quartile 3,
total bilirubin of 0.7–0.8 mg/dl in 89 patients; and quartile 4, total
bilirubin of ≥0.9 mg/dl in 74 patients. Clinical variables for this popula-
tion, subdivided according to quartiles of total bilirubin levels, are
shown in Table 1. A significant association was observed between
increased quartiles of total bilirubin and eGFR levels. Patients with low
serum bilirubin levels tended to be older, exhibited higher C-reactive
protein levels, and had a higher prevalence of proteinuria than those
with high bilirubin levels. However, other variables, such as body
mass index, lipid profiles, and the prevalences of hypertension and
diabetes mellitus, were comparable among the four groups.
Carotid ultrasonography findings in the enrolled patients are given
in Table 2. We observed that max IMT and PS significantly decreased
across quartiles of total bilirubin levels (p for trend = 0.003 and p for
trend = 0.002, respectively). Moreover, we observed that the preva-
lence of severe carotid atherosclerosis significantly decreased across
quartiles of serum bilirubin levels as follows: quartile 1, 48.2%; quartile
2, 39.6%; quartile 3, 30.3%; and quartile 4, 27.0%; p for trend = 0.007
(Fig. 1).
Even after multivariate adjusting for confounding factors, serum
bilirubin levels were independently associated with severe carotid ath-
erosclerosis in CAD patients [odds ratio 0.89, 95% confidence interval
(CI), 0.81–0.99, p = 0.027, Table 3]. In a receiver operating curve analy-
sis, total bilirubin value b0.55 mg/dl was identified as an effective cutoff
value for predicting severe carotid atherosclerosis (area under the
curve = 0.60, 95% CI, 0.55–0.66, p = 0.001).
4. Discussion
The main finding of the present study was that reduced bilirubin
levels are associated with an increased risk of carotid atherosclerosis
among patients with chronic CAD. These associations were independent
after adjustment for traditional risk factors. Thus, bilirubin could serve
as a useful clinical biomarker for evaluating atherosclerotic status in
high-risk populations.
The relationship between bilirubin and the protective effects of
atherosclerosis is well known, but its underlying mechanisms are not
fully understood and some mechanisms have been proposed. It is well
established that bilirubin has antioxidant and anti-inflammatory activi-
ties in vitro [13,14]. This is supported by human studies reporting that
individuals with elevation in bilirubin levels due to Gilbert's syndrome
have higher antioxidant capacity and lower inflammatory markers
than control subjects [15–17]. Furthermore, several studies have fo-
cused on the association between bilirubin and endothelial function.
Erdogan et al. reported that lower serum bilirubin levels were related
to worse endothelial function in healthy patients [18]. Ishizaka et al.
reported that high serum bilirubin level is inversely associated with
carotid plaque among the population in general health screening tests
[19]. Mashitani et al. reported that serum bilirubin levels were negative-
ly correlated with the progression of diabetic nephropathy [20]. Our
findings showed a significant correlation between serum bilirubin
levels and carotid atherosclerosis. We speculate that elevated bilirubin
levels would inhibit oxidative stress and inflammation, resulting in
the prevention of atherosclerosis progression.
Previous studies have reported a negative correlation between
serum bilirubin levels and CVD in various populations [21–24]. More-
over, an association between serum bilirubin levels and adverse cardio-
vascular events has been reported. Huang et al. examined patients with
cardiac syndrome X and reported that those in the lowest bilirubin
tertile (≤ 0.6 mg/dl) had poorer clinical outcomes [25]. Chan et al.
reported that patients with type 2 diabetes who had lower bilirubin
levels had a higher incidence of lower limb amputation [26]. However,

Table 3
Logistic regression analyses for severe carotid atherosclerosis.

Variables Simple regression Multiple regression

OR 95% CI p OR 95% CI p

Male 0.94 0.57–1.56 0.81

Age 1.07 1.04–1.09 b0.001 1.06 1.03–1.08 b0.001
Hypertension 1.83 1.08–3.08 0.02 1.71 0.99–2.96 0.055
HDL (mg/dl) 1.00 0.98–1.01 0.79
LDL (mg/dl) 1.00 0.99–1.00 0.32
Diabetes mellitus 1.13 0.75–1.70 0.55
Current smoking 1.35 0.84–2.16 0.21
Estimated GFR 0.98 0.97–0.99 0.001 0.99 0.98–1.00 0.14
(ml/mi/1.732)
Proteinuria 1.24 0.73–2.11 0.42
Log-transformed 0.78 0.45–1.34 0.36
C-reactive protein
Statin use 0.80 0.53–1.23 0.31
Total bilirubin 0.86 0.79–0.95 0.002 0.89 0.81–0.98 0.02
(per 0.1 mg increase)
Fig. 1. Association of total bilirubin levels with the prevalence of severe carotid atheroscle-
rosis, defined as a plaque score of N10. The prevalence of severe carotid atherosclerosis sig- Multivariate model includes all variables with p b 0.05 by univariate analysis.
nificantly decreased across quartiles of serum bilirubin levels as follows: quartile 1, 48.2%; OR, odds ratio; CI, confidence interval; BMI, body mass index; HDL, high-density lipopro-
quartile 2, 39.6%; quartile 3, 30.3%; and quartile 4, 27.0%; p for trend = 0.007. tein; LDL, low-density lipoprotein; GFR, glomerular filtration rate.
 Y. Tatami et al. / IJC Metabolic & Endocrine 5 (2014) 24–27
there are limited data regarding atherosclerotic status in patients with
ischemic heart disease. Our findings might also provide an additional
explanation of the correlation between serum bilirubin levels and
worse clinical outcomes in patients in high-risk populations.
Several recent reports showed that the inverse association between
serum bilirubin levels and protective effects against atherogenesis was
stronger in men than in women. Moreover several reports showed the
relationship between low serum bilirubin levels and the risk of CVD in
men, but it was not clear in women [21,27,28]. The presence of estrogen
has been proposed as one of the mechanisms. Estrogen has anti-
atherosclerotic effects in itself, such as preventing the oxidative proper-
ties of LDL cholesterol, and producing the nitric oxide in the vascular
endothelial cell [29]. In this study, we also showed that lower serum
bilirubin levels had strong association with severe carotid atherosclero-
sis in men, but it was not shown in women [odds ratio 0.841, 95% con-
fidence interval (CI) 0.75–0.94, p = 0.003 and odds ratio 1.144, 95% CI
0.90–1.45, p = 0.272, respectively] (data are not shown).
4.1. Study limitations
Several limitations of this study should be considered. First, it was an
observational analysis conducted in a single center and included a
relatively small sample. Second, we did not examine markers of oxida-
tive stress and endothelial function. Third, we had no information of
serum estrogen levels and hormone replacement therapy in female
patients. Finally, we had no data regarding changes in bilirubin levels
and the future progression of carotid atherosclerosis during the patients'
hospital stay.
4.2. Conclusions
In CAD patients, low serum bilirubin levels were independently
associated with severe carotid atherosclerosis. Our data suggest serum
bilirubin levels to be an independent, useful, and cost-effective tool for
evaluating atherosclerotic status in CAD patients.
Disclosures
S.S. has received developmental endowment from Chugai, Dainippon
Sumitomo, Kowa, Kyowa Hakko Kirin, MSD, Nihon Medi-Physics, and
Nippon Boehringer Ingelheim. H.I. has received lecture fees from Astellas
and Otsuka. T.M. has received lecture fees from Bayer, Daiichi Sankyo,
Dainippon Sumitomo, Kowa, MSD, Mitsubishi Tanabe, Nippon Boehringer
Ingelheim, Novartis, Pfizer Japan, Sanofi-aventis, and Takeda. T.M. has
received an unrestricted research grant for the Department of Cardiology,
Nagoya University Graduate School of Medicine from Astellas, Daiichi
Sankyo, Dainippon Sumitomo, Kowa, MSD, Mitsubishi Tanabe, Nippon
Boehringer Ingelheim, Novartis, Otsuka, Pfizer Japan, Sanofi-aventis,
Takeda, and Teijin.
Conflict of interest
Dr. Murohara has received an unrestricted research grant for the
Department of Cardiology, Nagoya University Graduate School of
Medicine from Astellas, Daiichi Sankyo, Dainippon Sumitomo, Kowa,
MSD, Mitsubishi Tanabe, Nippon Boehringer Ingelheim, Novartis,
Otsuka, Pfizer Japan, Sanofi-aventis, Takeda, and Teijin.
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