Association of serum total bilirubin level with severity of coronary atherosclerosis is linked to systemic inflammation

Accepted Manuscript Association of Serum Total Bilirubin Level with Severity of Coronary Atherosclerosis is Linked to Systemic Inflammation Mehmet Kadri Akboga, MD, Ugur Canpolat, MD, Asife Sahinarslan, MD, Yakup Alsancak, MD, Serdar Nurkoc, MD, Dursun Aras, MD, Sinan Aydogdu, MD, Adnan Abaci, MD PII:

S0021-9150(15)00142-2

DOI:

10.1016/j.atherosclerosis.2015.02.051

Reference:

ATH 13972

To appear in:

Atherosclerosis

Received Date: 27 August 2014 Revised Date:

20 January 2015

Accepted Date: 23 February 2015

Please cite this article as: Akboga MK, Canpolat U, Sahinarslan A, Alsancak Y, Nurkoc S, Aras D, Aydogdu S, Abaci A, Association of Serum Total Bilirubin Level with Severity of Coronary Atherosclerosis is Linked to Systemic Inflammation, Atherosclerosis (2015), doi: 10.1016/ j.atherosclerosis.2015.02.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Association of Serum Total Bilirubin Level with Severity of Coronary Atherosclerosis is

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Linked to Systemic Inflammation

Mehmet Kadri Akboga, MDa,*, Ugur Canpolat, MDa, Asife Sahinarslan, MDb, Yakup Alsancak, MDb, Serdar Nurkoc, MDb, Dursun Aras, MDa, Sinan Aydogdu, MDa, Adnan

a

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Abaci, MDb.

Turkiye Yuksek Ihtisas Education and Research Hospital, Department of Cardiology,

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Ankara, Turkey.

Gazi University Medical Faculty, Department of Cardiology, Ankara, Turkey.

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There are found four tables and two figures in the manuscript.

*Corresponding author

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Mehmet Kadri Akboga, MD.

Turkiye Yuksek Ihtisas Training and Research Hospital, Cardiology Clinic, Ankara, Turkey.

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Phone: (+90) 312 3061134

E-mail: [email protected]

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Abstract Objective: Although cardiovascular protective action of bilirubin has been attributed to its antioxidant effect, there was scarce data regarding the anti-inflammatory properties. Herein,

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we aimed to assess the relationship between serum total bilirubin level and severity of coronary artery disease (CAD) in association with the direct inflammatory marker such as Creactive protein (CRP), the other indirect markers included in inflammation process such as

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neutrophil to lymphocyte ratio (NLR) and red cell distribution width (RDW) in patients with stable CAD.

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Methods: Angiographic data of 1501 patients were analyzed in this retrospective crosssectional study. Patients were categorized according to Gensini scores as control, mild CAD and severe CAD groups. The association of clinical and laboratory parameters with the severity of CAD were determined by multivariable linear regression analysis. Results: Total bilirubin level in the control group was significantly higher than those of the

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other groups. After multivariable linear regression analysis total bilirubin [β= -3.131 (-4.481, -1.782), p<0.001] was significantly associated with the severity of CAD. Futhermore, there

was a moderate and significant inverse correlation between serum total bilirubin level and the

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severity of CAD (r= -0.173, p<0.001), CRP (r= -0.112, p<0.001), NLR (r= -0.070, p=0.026)

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and RDW (r= -0.074, p=0.027).

Conclusion: Serum total bilirubin level was independently and inversely associated with the severity of coronary atherosclerosis in patients with stable CAD. In addition, total bilirubin level was inversely correlated with CRP, NLR and RDW. These results suggest that besides its already known effect on the oxidative stress, higher serum total bilirubin level may exhibit an anti-inflammatory effect in the coronary atherosclerotic process. Key words: Bilirubin; Inflammation; CRP; NLR; RDW; Atherosclerosis.

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Association of Serum Total Bilirubin Level with Severity of Coronary Atherosclerosis is

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Linked to Systemic Inflammation

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1. Introduction Coronary atherosclerosis, the plague of the current age, includes a complex interaction between circulation and arterial wall components which leads to firing of very powerful

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weapons such as oxidative stress and inflammation [1,2]. Oxidative stress is fundamentally linked to atherogenesis by free radical production and formation of lipid peroxidation that increase vascular inflammation subsequently [3,4]. Thus, inflammation and oxidative stress

progression and rupture of lipid-rich lesions.

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are milestones of the atherosclerosis and are supposed to take place in the initiation,

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Bilirubin as an effective endogenous antioxidant is known as the scavenger of reactive oxygen radicals and attenuates the uptake of oxidized low density lipoprotein which is a crucial step in the atherosclerotic process [5-7]. Thus, it has been hypothesized that increased physiological concentrations of serum bilirubin level might reduce atherogenic risk. However,

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none of the clinical studies investigating the association of serum bilirubin level with the extent and severity of coronary artery disease (CAD), assessed the relation of serum bilirubin level with inflammatory markers in patients with CAD [8-10].

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Hence, in this retrospective cross-sectional study we aimed to investigate the relationship between serum total bilirubin level and Gensini score as an indicator of the

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severity and extent of CAD in association with the direct inflammatory markers like Creactive protein (CRP) and indirect markers included in inflammation process such as neutrophil to lymphocyte ratio (NLR) and red cell distribution width (RDW) in a large number of patients with stable CAD.

2. Methods

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In this retrospective cross-sectional study, patients who admitted to our outpatient clinic with angina pectoris or angina equivalents and underwent coronary angiography were enrolled. The decision for coronary angiography was made according to a positive

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noninvasive stress test or high clinical suspicion for a severe coronary artery stenosis. The coronary angiography (CAG) data (January 2007- July 2013) of our Cardiology Clinic was reviewed retrospectively and patients with CAD were included in the study. Among 38,506

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patients in whom CAG has been performed; after evaluation according to exclusion criteria and availability of laboratory data including serum total bilirubin level, 1501 patients

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remained for further analysis (Figure 1). The data of 380 subjects who have undergone CAG due to angina equivalents (e.g. dyspnea, diaphoresis, extreme fatigue, or pain at a site other than the chest) with an abnormal non-invasive test results and had normal CAG findings were randomly selected from the same CAG database as the control group. Exclusion criteria were as following: recent acute coronary syndrome either with or

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without ST-segment elevation (≤6 months before enrollment), patients with single serum total bilirubin elevations (particularly for exclusion of possible diagnosis for Gilbert syndrome), previous revascularization history (coronary artery bypass grafting or percutaneous coronary decompensated

heart

failure,

non-ischemic

dilated

cardiomyopathy,

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intervention),

symptomatic peripheral vascular disease (transient ischemic attack, stroke, intermittent

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claudication, peripheral revascularization or amputation), evidence of ongoing infection or inflammation, chronic kidney disease (serum creatinine >1.4mg/dl), history of any liver disease (with liver function parameters >3x upper normal value), chronic obstructive pulmonary disease, autoimmune disease, hematologic disease and previous diagnosis of malignancy. As aforementioned above, we tried but could not be sure to have fully excluded patients with Gilbert Syndrome due to its high prevalence among people living in Western Europe [11]. 3

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The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by Institutional Ethics Committee. 2.1. Study population

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The study population consisted of 1501 patients. Baseline clinical characteristics including history of hypertension, diabetes mellitus, smoking, family history of CAD were recorded. Diabetes mellitus was defined as fasting plasma glucose level over 126 mg/dl or

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glucose level over 200 mg/dl at any measurement or active use of an antidiabetic agent. Hypertension was defined as either active use of antihypertensive medication or

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documentation of a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg. Smoking was defined as current smoking. The family history of CAD was defined as presence of a history of CAD or sudden cardiac death in a first-degree relative before the age of 55 years for men and before the age of 65 years for women.

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2.2. Coronary angiography

Standard Judkins technique was used for coronary artery visualization. At least two orthogonal plane images were taken for each coronary artery. The coronary angiograms were

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reviewed by two physicians to assess the Gensini score [12]. The severity of CAD was scored as 1 for 1-25% narrowing, 2 for 26-50%, 4 for 51-75%, 8 for 76-90%, 16 for 91-99%, and 32

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for a completely occluded artery. The score is then multiplied by a factor according to the importance of the coronary artery. The multiplication factor is 5 for a left main stem (LMS) lesion, 2.5 for proximal left anterior descending artery (LAD) and proximal circumflex artery (Cx) lesions, 1.5 for a mid-LAD lesion, and 1 for distal LAD, mid/distal Cx and right coronary artery lesions. The multiplication factor for any other branch is 0.5 [12]. The patients with coronary stenosis were divided into two groups according to Gensini score; namely cases of mild atherosclerosis (Gensini score 1–20) and severe atherosclerosis (Gensini score >20) 4

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[13]. Stable angina pectoris was diagnosed according to the criteria recommended by the current guidelines [14]. 2.3. Laboratory analysis

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Laboratory parameters (including complete blood count (CBC), biochemistry panel and cholesterol panel that were taken before the index coronary angiography) of all the participitants were recorded. In our instutition, the blood samples are routinely collected after

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12-hour fasting in the morning. Serum bilirubin concentrations were determined by the enzymatic colorimetric method using a clinical chemistry autoanalyzer (Aeroset, Abbott

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Laboratory, Abbott Park, IL, USA) and for CBC analysis, automatic blood counter (A CellDyn 3500, Abbot, IL, USA) was used. The CRP level was analyzed with Beckman Coulter Inc (Image 800, California, USA). 2.4. Statistical analysis

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In all statistical analysis SPSS 17.0 Statistical Package Program for Windows (SPSS Inc., Chicago, IL, USA) was used. Kolmogorov-Smirnov test was used to test normality of distribution. Quantitative variables with a normal distribution were specified as the mean ±

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standard deviation, and those with non-normal distribution were specified with median (minimum and maximum); categorical variables were specified with number and percentage

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values. Kruskal–Wallis test or ANOVA test was used to compare continuous variables according to Gensini score, as appropriate. Categorical variables were compared with Chisquare test. Correlation analysis was performed to examine the relationship between total bilirubin level and Gensini score, NLR, RDW and CRP. To assess the association of clinical and laboratory parameters with the severity of coronary atherosclerosis, multivariable linear regression analysis was performed by including the parameters, which were significantly

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different between the groups and possible confounding factors. A p value of <0.05 was accepted as statistically significant.

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3. Results Baseline characteristics and laboratory parameters of the study groups were presented in Tables 1 and 2. The patients were categorized into 3 groups according to their Gensini score. While 380 patients were in control group (group 1) with normal coronary arteries, the

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remaining 1121 patients constituted the study group, which was further divided into two groups (groups 2 and 3). In the study group, 497 patients were assigned to group 2 (mild

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CAD), and 624 patients were assigned to group 3 (significant CAD). Patients in the study group (groups 2 and 3) were older and more likely to be male, diabetic, hypertensive and higher use of RAS blockers when compared to the control group (p<0.05). The CRP and indirect inflammatory markers like NLR, RDW, neutrophil and mean platelet volume (MPV)

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were significantly higher in the study group, whereas serum total bilirubin (Figure 2) and high-density lipoprotein cholesterol (HDL-C) levels were significantly higher in the control group (p<0.05). Low-density lipoprotein cholesterol (LDL-C) level was also higher in the

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study group, however the difference did not reach to statistical significance (p>0.05). Left ventricular ejection fraction (LVEF) was significantly lower in the study group than in the

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control group (p<0.05). There were no significant differences between the groups with respect to other parameters.

In addition, Gensini score and inflammatory parameters were compared between the

groups set according to serum total bilirubin tertiles (Tertile 1, 2 and 3) (Table 3). As bilirubin tertile increased, the Gensini score, CRP and RDW levels were also increased (p<0.05). Furthermore, there was a moderate and significant inverse correlation between serum total bilirubin level and the severity of CAD determined by Gensini score (r= -0.173, 6

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p<0.001). Moreover, a significant inverse correlation was determined between serum total bilirubin level and direct inflammatory markers like CRP (r= -0.112, p<0.001) and indirect inflammatory markers including NLR (r= -0.070, p=0.026) and RDW (r= -0.074, p=0.027).

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Demographic, clinical and laboratory variables differing among groups (none, mild and severe CAD) were included in multivariable linear regression analysis to delineate the association with Gensini score. Age (β=0.096, p=0.005), LVEF (β= -0.409, p<0.001), serum

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total bilirubin (β= -0.164, p<0.001), CRP (β=0.083, p=0.013), RDW (β=0.084, p=0.012) and LDL-C levels were significantly associated with the Gensini score (Table 4).

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4. Discussion

To the best of our knowledge, this is the first clinical study showing the negative correlation between serum total bilirubin level and systemic inflammatory markers including CRP and indirect markers involved in inflammatory process such as NLR and RDW in

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patients with coronary atherosclerosis. Furthermore, this is the first report showing an inverse correlation between serum total bilirubin level and the severity of CAD assessed by using Gensini score.

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It has been well known that both initiation and progression of atherosclerosis is a multifactorial process which mainly includes a low-grade chronic inflammatory state and

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increased oxidative stress [15,16]. Bilirubin is a natural product of heme oxygenase metabolism which has been recognized as a potent antioxidant whether it is conjugated, unconjugated, free, or albumin bound [7,17]. Bilirubin may protect LDL from oxidation which is an important step in the progression of atherosclerosis due to antioxidant properties [16]. In a recent study, Turfan et al. showed that serum bilirubin levels were independently and inversely associated with the severity of CAD in patients with stable angina pectoris [10]. Schwertner et al. have found that low serum concentration of bilirubin was associated with the 7

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increased risk and prevalence of CAD [18]. Similary, in a meta-analysis, Novotny and colleagues reported that serum bilirubin is a negative risk factor for CAD [19]. Gul et al. demonstrated that higher serum bilirubin levels were independently associated with in-

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hospital adverse outcomes in patients with ST-segment elevation acute myocardial infarction who undergo primary percutaneous coronary intervention [20]. In addition, Gilbert’s syndrome, also known as idiopathic unconjugated hyperbilirubinemia, is a common

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congenital hyperbilirubinemia caused by a mutation is associated with lower risk of cardiovascular disease [21]. In another study by Sung et al., there was a strong inverse and

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independent relation between total, conjugated and unconjugated bilirubin concentrations and coronary artery calcium scores [22]. Our study results are in consistent with the previous study findings. We showed an inverse correlation between serum total bilirubin level and the Gensini score. In multivariable linear regression analysis, serum total bilirubin level was significantly associated with the severity of coronary atherosclerosis. Furthermore, this

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relationship was independent from various confounding factors including clinical risk factors and laboratory parameters. Although we have had an effort to exclude patients with definite and possible diagnosis of Gilbert Syndrome in our study population, it is not fully possible

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due to its prevalence of 5-10% among people living in Western Europe [11]. So, the probability of such a pathological condition in small number of our patients might have an

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impact on the presence and severity of coronary atherosclerosis. In previous studies, CRP, NLR and RDW were shown to be as sensitive markers of

chronic systemic inflammation occurring in the progression of atherosclerosis [23-26]. Moreover, it has been demonstrated that CRP, NLR and RDW are significantly elevated in many cardiovascular diseases and closely related with poor prognosis [27-30]. Furthermore, Tapan et al. found that hs-CRP was negatively correlated with total bilirubin level in patients with Gilbert’s syndrome [31]. Additionally, Yoshino and colleagues demonstrated that total 8

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bilirubin level was inversely correlated with hs-CRP and in multivariate analysis hs-CRP has been found as the independent predictor of total bilirubin level in overweight patients [32]. Our study also showed that total bilirubin level was inversely correlated with inflammatory

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markers including CRP, other indirect markers involved in inflammatory process such as NLR and RDW in patient with coronary atherosclerosis. These results suggest that besides its anti-oxidant effects, higher total bilirubin level may exhibit an anti-inflammatory effect in

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patients with coronary atherosclerosis.

Results of this study should be interpreted in the light of some limitations. First, the

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present study has a retrospective cross-sectional design with a single-center data. Because of the retrospective retrieval of the patient data, our study also lacks the prognostic role of serum bilirubin on cardiovascular outcomes. However, our study population consisted of a largescale real-world patients with stable CAD. Second, our measurements for serum bilirubin and inflammatory markers were based on a single determination and the time-course relationship

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with vascular events can not be extrapolated from the study. Although we excluded patients with single serum total bilirubin elevations, we could not be sure to have fully excluded patients with Gilbert Syndrome due to its high prevalence (5-10%) among people living in

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Western Europe [11].

In conclusion, serum total bilirubin level was independently and inversely associated

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with the severity of coronary atherosclerosis in patients with stable CAD. In addition, total bilirubin level was inversely correlated with direct and indirect markers of inflammation including CRP, NLR and RDW in patient with coronary atherosclerosis. These results suggest that besides its already known effect on the oxidative stress, higher serum total bilirubin level may exhibit an anti-inflammatory effect in the coronary atherosclerotic process. Further studies are needed to clarify our findings.

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Funding Sources: None

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Disclosures: The authors have no conflicts of interest to disclose.

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References [1] Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-43.

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[2] Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005;352:1685-95.

[3] Witztum JL, Berliner JA. Oxidized phospholipids and isoprostanes in atherosclerosis. Curr

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Opin Lipidol 1998;9:441-8.

[4] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol.

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Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915-24.

[5] Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043-6. [6] Dennery PA, McDonagh AF, Spitz DR, Rodgers PA, Stevenson DK. Hyperbilirubinemia

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results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia. Free Radic Biol Med 1995;19:395-404.

[7] Wu TW, Fung KP, Wu J, Yang CC, Weisel RD. Antioxidation of human low density

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lipoprotein by unconjugated and conjugated bilirubins. Biochem Pharmacol 1996;51:859-62. [8] Kang SJ, Kim D, Park HE, Chung GE, Choi SH, Choi SY, et al. Elevated serum bilirubin

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levels are inversely associated with coronary artery atherosclerosis. Atherosclerosis 2013;230:242-8.

[9] Ghem C, Sarmento-Leite RE, de Quadros AS, Rossetto S, Gottschall CA. Serum bilirubin concentration in patients with an established coronary artery disease. Int Heart J 2010;51:8691.

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[10] Turfan M, Duran M, Poyraz F, Yayla C, Akboga MK, Sahinarslan A, et al. Inverse relationship between serum total bilirubin levels and severity of disease in patients with stable coronary artery disease. Coron Artery Dis 2013;24:29-32.

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[11] Claridge LC, Armstrong MJ, Booth C, Gill PS. Gilbert's syndrome. BMJ 2011;342:d2293 [12] Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol 1983;51:606.

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[13] Oishi Y, Wakatsuki T, Nishikado A, Oki T, Ito S. Circulating adhesion molecules and severity of coronary atherosclerosis. Coron Artery Dis 2000;11:77-81.

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[14]. Fihn SD, Gardin JM, Abrams J, Berra K, Blankenship JC, Dallas AP, et al. 2012 ACCF/ AHA/ ACP/ AATS/ PCNA/ SCAI/ STS Guideline for the diagnosis and management of patients with stable ischemic heart disease: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, and the American College of Physicians, American Association for Thoracic Surgery, Preventive Nurses

Association,

Society

for

Cardiovascular

Angiography

and

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Cardiovascular

Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol 2012;60:44-164. [15] Drakopoulou M, Toutouzas K, Stefanadi E, Tsiamis E, Tousoulis D, Stefanadis C.

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Association of inflammatory markers with angiographic severity and extent of coronary artery disease. Atherosclerosis 2009;206:335-9.

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[16] Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1785-92. [17] Neuzil J, Stocker R. Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J Biol Chem. 1994;269:16712–16729 [18] Schwertner HA, Jackson WG, Tolan G. Association of low serum concentration of bilirubin with increased risk of coronary artery disease. Clin Chem 1994;40:18-23. 12

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[19] Novotny L, Vitek L. Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies. Exp Biol Med (Maywood) 2003;228:568-71. [20] Gul M, Uyarel H, Ergelen M, Akgul O, Karaca G, Turen S, et al. Prognostic value of

primary coronary intervention. Am J Cardiol 2013;111:166-71.

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total bilirubin in patients with ST-segment elevation acute myocardial infarction undergoing

[21] Lin JP, O’Donnell CJ, Schwaiger JP, Cupples LA, Lingenhel A, Hunt SC, et al.

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Association between the UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the framingham heart study. Circulation 2006;114:1476–81.

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[22] Sung KC, Shin J, Lim YH, Wild SH, Byrne CD. Relation of conjugated bilirubin concentrations to the presence of coronary artery calcium. Am J Cardiol 2013;112:1873-9. [23] Crisby M, Kublickiene K, Henareh L, Agewall S. Circulating levels of autoantibodies to oxidized low-density lipoprotein and C-reactive protein levels correlate with endothelial function in resistance arteries in men with coronary heart disease. Heart Vessels 2009;24:90-

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5.

[24] Arbel Y, Finkelstein A, Halkin A, Birati EY, Revivo M, Zuzut M, et al. Neutrophil/lymphocyte ratio is related to the severity of coronary artery disease and clinical

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outcome in patients undergoing angiography. Atherosclerosis 2012;225:456-60. [25] Wang X, Zhang G, Jiang X, Zhu H, Lu Z, Xu L. Neutrophil to lymphocyte ratio in

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relation to risk of all-cause mortality and cardiovascular events among patients undergoing angiography or cardiac revascularization: a meta-analysis of observational studies. Atherosclerosis 2014;234:206-13. [26] Tonelli M, Sacks F, Arnold M, Moye L, Davis B, Pfeffer M; for the Cholesterol and Recurrent Events (CARE) Trial Investigators. Relation Between Red Blood Cell Distribution Width and Cardiovascular Event Rate in People With Coronary Disease. Circulation 2008;117:163-8. 13

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[27] Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, et al. Creactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med 2004;350:1387-97.

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[28] Tanındı A, Erkan AF, Ekici B, Alhan A, Töre HF. Neutrophil to lymphocyte ratio is associated with more extensive, severe and complex coronary artery disease and impaired myocardial perfusion. Turk Kardiyol Dern Ars 2014;42:125-30.

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[29] Akboga MK, Akyel A, Sahinarslan A, Yayla C, Alsancak Y, Gokalp G, et al. Neutrophil to lymphocyte ratio is increased in patients with rheumatic mitral valve stenosis? Anadolu

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Kardiyol Derg 2014 Apr 16. [Epub ahead of print]

[30] Isik T, Kurt M, Ayhan E, Tanboga IH, Ergelen M, Uyarel H. The impact of admission red cell distribution width on the development of poor myocardial perfusion after primary percutaneous intervention. Atherosclerosis 2012;224:143-9.

[31] Tapan S, Dogru T, Tasci I, Ercin CN, Ozgurtas T, Erbil MK. Soluble CD40 ligand and

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soluble P-selectin levels in Gilbert's syndrome: a link to protection against atherosclerosis? Clin Biochem 2009;42:791-5.

[32] Yoshino S, Hamasaki S, Ishida S, Kataoka T, Yoshikawa A, Oketani N, et al. between

bilirubin

concentration,

coronary

endothelial

function,

and

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Relationship

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inflammatory stress in overweight patients. J Atheroscler Thromb 2011;18:403-12.

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Figure Legends Figure 1. STROBE diagram showing the flow of screened patients and reasons for missingness of data.

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Figure 2. Serum total bilirubin level according to severity of coronary atherosclerosis by

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using Gensini score.

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Table 1. Baseline characteristics of the study population (n=1501). Control group (n=380)

Mild CAD (n=497)

Severe CAD (n=624)

p value

Age (years)

54.9±10.6

60.6±10.5

63.2±10.5

<0.001

Male, n (%)

171 (45.0)

321 (64.6)

Hypertension, n (%)

136 (35.8)

247 (49.7)

Diabetes mellitus, n (%)

75 (19.7)

113 (22.7)

Smoking, n (%)

126 (33.2)

187 (37.6)

Family history of CAD, n (%)

61 (16.1)

89 (17.9)

124 (19.9)

0.306

RAS blocker, n (%)

87 (22.9)

168 (33.8)

208 (33.3)

0.001

Diuretic, n (%)

40 (10.5)

48 (9.7)

86 (13.8)

0.076

Calcium channel blocker, n (%)

41 (10.8)

65 (13.1)

90 (14.4)

0.253

β-blocker, n (%)

40 (10.5)

59 (11.9)

92 (14.7)

0.118

Statin, n (%)

39 (10.3)

61 (12.3)

76 (12.2)

0.590

Antiaggregant, n (%)

45 (11.8)

64 (12.9)

95 (15.2)

0.269

Oral antidiabetic, n (%)

67 (17.6)

95 (19.1)

128 (20.5)

0.528

61.5±8

55.6 ±11

<0.001

64.6 ±4

440 (70.5)

<0.001

323 (51.8)

<0.001

170 (27.2)

0.020

239 (38.3)

0.232

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LVEF (%)

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Parameters

Data were given as mean ± SD or %. CAD, coronary artery disease; RAS, renin-angiotensin system;

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LVEF, left ventricular ejection fraction.

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Severe CAD (n=624)

p value

14.1±1.2

14.3±1.2

14.2±1.2

0.392

13.1 (11.0-21.0)

13.3 (10.7-19.5)

WBC (x10³/mm³) a

7.4 (3.2-12.0)

7.7 (3.6-12.2)

Neutrophil (x10³/mm³) a

4.4 (1.8-10.1)

4.5 (1.5-9.4)

Lymphocyte (x10³/mm³)a

2.3 (0.7-4.2)

2.2 (0.7-4.3)

Hemoglobin (g/dl) RDW (%) a

Monocyte (x10³/mm³)

0.57 ±0.2

0.59 ±0.2

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Control group (n=380)

13.8 (10.9-21.8)

0.001

7.9 (2.1-12.5)

0.081

4.7 (1.5-10.2)

0.011

2.1 (0.4-4.7)

0.109

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Parameters

0.60 ±0.2

0.301

1.95 (0.6-11.6)

2.04 (0.7-16.1)

2.12 (0.7-30.0)

0.002

Platelet a

235 (202-281.0)

238 (198-280)

240 (200-289)

0.119

MPV (fL) a

8.3 (5.3-14.1)

8.5 (5.7-12.5)

8.7 (5.1-16.4)

0.002

Total bilirubin (mg/dl) a

0.65 (0.1-2.1)

0.60 (0.1-2.6)

0.53 (0.1-2.4)

<0.001

20 (6-57)

21 (9-51)

20 (7-54)

0.128

22 (7-55)

21 (6-61)

21 (9-63)

0.234

0.83±0.18

0.84±0.16

0.85±0.19

0.312

5.2 (1.0-50.0)

7.0 (1.0-71.0)

7.3 (1.2-79.0)

<0.001

M AN U

NLR a

AST (U/l) Creatinine (mg/dl) CRP (mg/dl) a

TE D

ALT (U/l)

Uric acid (mg/dl) a

5.6 (2.4-11.7)

5.5 (2.0-13.6)

0.103

Total cholesterol (mg/dl)a

193.5 (116-335)

192.5 (103-375)

195 (90-386)

0.827

AC C

EP

5.3 (2.7-13.4)

HDL-C (mg/dl) a

44 (21-91)

41 (22-92)

40 (17-94)

<0.001

LDL- C (mg/dl) a

119 (41-249)

120 (40-258)

124 (37-268)

0.123

Triglyceride (mg/dl) a

133 (46-462)

140 (49-444)

140 (45-464)

0.351

Data were given as mean ± SD or median. CAD, coronary artery disease, RDW, red cell distribution width; WBC, White blood cell; NLR, neutrophil to lymphocyte ratio; MPV, mean platelet volume; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. a

Median (minimum-maximum).

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Table 3 Comparison of some laboratory parameters between bilirubin tertiles. Mid bilirubin tertile (n=491)

High bilirubin tertile (n=499)

p#

p*

Gensini score

19.5 (2.5-48)

14 (2-41)

5 (0-32)

<0.001

<0.001

CRP (mg/dl)

7.7 (3.6-14.1)

6.9 (3.6-13.0)

5.6 (2.9-10.6)

0.001

<0.001

13.7 (13.0-14.9)

13.4 (12.8-14.4)

13.3 (12.5-14.5)

0.026

0.091

2.06 (1.6-2.8)

2.04 (1.5-2.6)

2.0 (1.5-2.6)

0.097

0.095

RDW (%)

SC

NLR

a

RI PT

Low bilirubin tertile (n=511)

Parameters

CRP, C-reactive protein; RDW, red cell distribution width; NLR, neutrophil to lymphocyte ratio.

Bilirubin tertile groups were compared by using Kruskal-Wallis test.

*

Comparison of high bilirubin tertile group with other groups by using Mann-Whitney test.

a

Median (interquartile range).

AC C

EP

TE D

M AN U

#

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Table 4 Univariable and multivariable linear regression analysis of Gensini score are associated with potential continuous variables in the coronary artery disease group. Multivariable linear regression analysis β (95% CI) p

Age

0.655 (0.499-0.811)

<0.001

0.338 (0.103-0.573)

LVEF

-1.471 (-1.640, -1.303)

<0.001

Total bilirubin

-4.556 (-5.965, -2.146)

CRP

Standardized

Collinearity statistics

β

RI PT

Variables

Univariable linear regression analysis β (95% CI) p

0.096

VIF 1.063

-1.605 (-1.869, -1.342)

<0.001

-0.409

1.078

<0.001

-3.131 (-4.481, -1.782)

<0.001

-0.164

1.050

0.456 (0.284-0.628)

<0.001

0.245 (0.052-0.438)

0.013

0.083

1.023

RDW

3.037 (1.564-4.509)

<0.001

1.995 (0.443-3.547)

0.012

0.084

1.021

NLR

2.463 (0.739-4.186)

0.005

1.508 (-0.412-3.428)

0.124

0.051

1.024

MPV

0.776 (-0.556-2.108)

0.083

-0.417 (-2.249-1.416)

0.655

-0.015

1.044

LDL-C

0.055 (0.010-0.100)

0.017

0.116 (0.053-0.179)

<0.001

0.123

1.067

HDL-C

-0.226 (-0.392, -0.059)

0.008

-0.163 (-0.396-0.071)

0.172

-0.046

1.049

M AN U

SC

0.005

CI, confidence interval; VIF, variance inflation factor; LVEF, left ventricular ejection fraction; CRP, C-reactive protein; RDW, red

TE D

cell distribution width; NLR, neutrophil to lymphocyte ratio; MPV, mean platelet volume; LDL-C, low-density lipoprotein

AC C

EP

cholesterol; HDL-C, high-density lipoprotein cholesterol.

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

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Highlights *The role of serum bilirubin in cardiovascular protection is attributed to its antioxidant effect. *In this study, we showed an inverse relationship between serum total bilirubin and coronary

RI PT

atherosclerosis confirmatory to previous knowledge. *To our best knowledge, the first time it was reported in this study that there was an inverse association of serum total bilirubin with inflammatory biomarkers including CRP, NLR and

AC C

EP

TE D

M AN U

SC

RDW.