Coronary artery disease (CAD), one of the major forms of cardiovascular disease, is the most common cause of human death worldwide 1. Previous studies have evidenced that vulnerable plaques play a critical role in the development of acute coronary syndrome (ACS), which may possibly cause sudden cardiac death 2–4. The pathophysiological mechanism of vulnerable plaques is sudden rupture, swiftly forming a clot that blocks the flow of blood through a coronary artery. Therefore, it is necessary and helpful to seek biomarkers associated with plaque vulnerability in CAD.
Bilirubin, an effective endogenous antioxidant, is considered as the scavenger of reactive oxygen radicals, and it alleviates the amplification of oxidized low-density lipoprotein, which is a major step in the atherosclerotic process 5–7. Several studies have reported that low serum total bilirubin levels were correlated with an increased risk for CAD 8,9. However, the relationship between serum levels of bilirubin and plaque vulnerability has not been investigated yet. The purpose of this study was to examine whether serum bilirubin levels are associated with coronary plaque characteristics in patients with CAD using intravascular ultrasound (IVUS).
Participants and definitions
From January 2014 to June 2015, 85 consecutive patients [45 with ACS and 40 with stable angina pectoris (SAP)] from Hangzhou First People’s Hospital (Zhejiang, China) were enrolled into this study. The patients were divided into SAP and ACS groups according to symptoms and clinical examinations. The ACS group included 19 patients with unstable angina pectoris (UAP) and 26 patients with acute myocardial infarction (AMI). Patients with typical invariable chest pain over 3 months (with typical invariable meaning the same degree of exertion and excitation provocation and the same location, quality, and 3–5-min duration) were categorized into the SAP group 10. Patients with ACS were identified according to the guidelines of the European Society of Cardiology and the American College of Cardiology/American Heart Association 11. The diagnosis of UAP was based on thoracic discomfort of typical character, depressed ST of 0.1 mV or higher, and/or T-wave inversion on ECG, but normal troponin levels (in at least two blood samples 0 and 6 h after admission). UAP symptoms were defined as new onset of severe, progressive, or resting angina. AMI was diagnosed as chest pain, elevation of troponin I (0.10 ng/ml), and new ST-segment change or new left-bundle branch block on ECG. Another 45 participants who were age-matched and had no evidence of cardiovascular disease – defined as no typical chest pain on exertion, no myocardial infarction or UAP, a negative exercise test, and normal coronary artery findings on coronary angiography – were selected as the control group.
Exclusion criteria were as follows: (i) congenital heart disease, cardiomyopathy, primary valvular disease, infectious endocarditis, and secondary cardiac muscle disease; (ii) symptomatic peripheral vascular disease (transient ischemic attack, stroke, intermittent claudication, peripheral revascularization, or amputation); (iii) thyroid disease, autoimmune disease, hematologic disease, malignant tumor, history of any liver disease, digestive system disorders, end-stage renal disease with maintenance hemodialysis, and evidence of ongoing infection or inflammation; (iv) severe stenotic left main coronary artery lesions. The study protocol was approved by the ethics committee of Hangzhou First People’s Hospital, and written informed consent was obtained from each participant.
The age, sex, BMI, clinical presentations, medical history, and medications used were recorded for each participant upon admission to the hospital. BMI was calculated as weight (kg) divided by the square of height (m2).
In AMI patients, blood samples were obtained from the patients upon arrival at the emergency unit. Fasting blood samples were collected the morning after admission for the remaining participants. The levels of total bilirubin and direct bilirubin were determined by the vanadate oxidase method using a clinical automated biochemistry analyzer (Hitachi 7600; Hitachi High-Technologies, Tokyo, Japan). Indirect bilirubin level was calculated as total bilirubin minus direct bilirubin. All of the samples were measured in duplicate.
The standard Judkins technique was used for coronary angiography. Each angiogram was read jointly by two experienced interventional cardiologists who were blinded to the participants’ clinical information. Coronary artery lesions were evaluated using calipers to determine the extent of artery narrowing. CAD was defined as the presence of more than 50% diameter stenosis in at least one major coronary artery. The reference segment diameter was averaged from 5-mm-long angiographically normal segments proximal to the lesion; if a normal proximal segment could not be identified, a distal angiographically normal segment was analyzed as described previously 12. Characteristics of coronary arterial plaques were assessed using a grayscale IVUS combined with an integrated backscatter (IB)-IVUS.
Grayscale intravascular ultrasound
IVUS examination was performed before any intervention and after the intracoronary administration of 200 μg nitroglycerine. The conventional IVUS images and IB signals were acquired using an IVUS system (Clear View; Boston Scientific, Natick, Massachusetts, USA) and a 40 MHz 2.5 Fr intravascular catheter. The imaging catheter was advanced more than 10 mm beyond the lesion, and an automated pullback system was applied to a point greater than 10 mm proximal to the lesion, at a standard pullback speed of 0.5 mm/s. All IVUS data were analyzed by two independent observers.
Images were quantified for external elastic membrane (EEM) cross-sectional area (CSA), lumen area, and plaque+media CSA (P+M CSA) using the software included in the IVUS system. P+M CSA was defined as EEM CSA−lumen area. The percent plaque burden was calculated as follows: [(EEM CSA–lumen area)/EEM CSA]×100. The remodeling index was calculated as the ratio of the EEM CSA at the measured lesion (minimum luminal site) to the reference EEM CSA (the average of the proximal and distal reference segments).
Integrated backscatter-intravascular ultrasound
IB values for three histological categories (calcification, fibrosis, and lipid pool) were calculated as the average power of the ultrasound backscattered signal from a small volume of tissue using a fast Fourier transform, measured in decibels. The calcified volume, fibrous volume, and lipid volume were calculated from three-dimensional IVUS images as the sum of the calcified, fibrous, and lipid areas in each CSA at 1-mm axis intervals, respectively. The percentages of calcified, fibrous, and lipid plaques [calcified, fibrous, and lipid (volume)/plaque area (volume)×100] were calculated automatically.
Numerical data were presented as mean±SD or median (interquartile range), and analyzed using Student’s t-test, one-way analysis of variance, the Mann–Whitney U-test, or the Kruskal–Wallis test, as appropriate. The normality assumption was evaluated using the Shapiro–Wilk test. Categorical data were expressed as number (%) and compared among study groups using the χ2-test or Fisher’s exact test. Pearson’s correlation coefficient was calculated to assess the association between serum concentrations of total bilirubin and characteristics of the coronary plaque. The relationship between the presence of a coronary lipid-rich plaque and serum levels of total bilirubin and other clinical parameters was evaluated using univariate and multivariate logistic regression analyses. Statistical analyses were carried out on SPSS version 20 (IBM-SPSS Inc., Armonk, New York, USA), and the associated results were plotted using GraphPad Prism 6 (GraphPad, San Diego, California, USA). A P-value of less than 0.05 was accepted as statistically significant.
Anthropometric, clinical, and laboratory data of the study population
The anthropometric, clinical, and laboratory data of the patients with ACS (n=45) and SAP (n=40), and of controls (n=40) are summarized in Table 1. The mean age of the ACS group was 63.1±9.6 years and of the SAP group was 63.0±9.7 years, with the control group sharing a similar mean age (61.7±10.1 years). The majority of patients in the ACS and SAP groups were male and in the overweight to obesity range, with mean BMI values of 26.1±4.1 kg/m2 and 25.6±3.4 kg/m2, respectively. No significant differences were observed between these three groups in terms of medical history, such as hypertension, type 2 diabetes mellitus, and family history of CAD, and the current smoking or drinking status. In terms of biochemical characteristics, patients of the ACS group had significantly higher levels of low-density lipoprotein cholesterol compared with the SAP and control groups (P<0.05). Furthermore, the frequencies of patients with ACS and SAP under treatment with aspirin were higher compared with controls (P<0.01 and 0.001).
Serum bilirubin levels
Comparisons of serum levels of total bilirubin, direct bilirubin, and indirect bilirubin are shown in Fig. 1. Serum total bilirubin levels in the ACS group were significantly lower than those in the SAP and control groups (10.42±3.12 vs. 12.31±3.30 μmol, P<0.01; 10.42±3.12 vs. 13.74±2.69 μmol, P<0.001); however, the serum total bilirubin levels were in the normal range among these three groups. The ACS group also had the lowest level of serum direct bilirubin when compared with the SAP and control groups (2.68±0.92 vs. 3.41±1.10 μmol, P<0.01; 2.68±0.92 vs. 3.77±0.89 μmol, P<0.001). Nevertheless, no significant differences were observed in the distribution of serum indirect bilirubin levels between the ACS and SAP groups (7.74±2.79 vs. 8.90±3.29 μmol, P>0.05).
Coronary angiographic and IVUS findings
Table 2 summarizes the angiographic and IVUS findings in patients with CAD. There were no significant differences in target vessel and lesion location between the ACS and SAP groups on angiographic analysis. On analyzing the data from grayscale IVUS images, the results demonstrated that the ratio of the plaque area to the minimum luminal area and the remodeling index were significantly higher among ACS patients than among SAP patients (10.97±4.29 vs. 8.74±4.61 mm2, P<0.05; 1.09±0.19 vs. 1.01±0.14, P<0.05). Furthermore, the IB-IVUS results showed that the ACS group had a significantly higher ratio of percent lipid plaque (51.07±12.38 vs. 43.37±14.13%, P<0.01) and a lower ratio of percent fibrous plaque (43.10±11.13 vs. 50.21±12.89%, P<0.01) than SAP patients.
Relationship between serum total bilirubin concentrations and characteristics of coronary plaque
In all CAD patients, Pearson’s correlation analyses evidenced that serum levels of total bilirubin were negatively correlated with the remodeling index (ρ=−0.275, P<0.05), plaque burden (ρ=−0.413, P<0.001), and lipid plaque (ρ=−0.419, P<0.001), whereas they were found to be positively correlated with fibrous plaque (ρ=0.386, P<0.001) on IVUS (Fig. 2). Multivariate regression analysis showed that serum total bilirubin concentrations were independently associated with plaque burden (β=−7.403, P<0.01), percentage of lipid plaque (β=−0.084, P<0.01), and percentage of fibrous plaque (β=0.061, P<0.05; Table 3).
In addition, over 33% of lipid area was defined as lipid-rich plaque in a lesion on the basis of a previous study 13. Univariate logistic regression analysis identified that total bilirubin levels were negatively correlated with lipid-rich plaques. Multivariate logistic regression analysis indicated estimated glomerular filtration rate [odds ratio, 1.06; 95% confidence interval (CI), 1.01–1.12] and total bilirubin level (odds ratio, 0.78; 95% CI, 0.64–0.95) as independent factors correlated with lipid-rich plaques (Table 4).
To the best of our knowledge, this is the first study to evaluate the relationship between serum levels of total bilirubin and coronary arterial plaque characteristics in patients with CAD using IVUS. Our research showed that serum total bilirubin levels in the ACS group were significantly lower than those in the SAP and control groups, although both groups had serum total bilirubin levels in the normal range. Further IVUS data demonstrated that serum total bilirubin levels were inversely correlated with the percentage of lipid plaques and plaque burden, as well as with the remodeling index, in CAD patients. In addition, the association between serum total bilirubin levels and coronary lipid-rich plaques was found to be statistically significant in both univariate analyses and multivariate analyses after adjusting for recognized risk factors. Collectively, these findings suggested that serum levels of total bilirubin might serve as a biomarker for monitoring CAD, especially for coronary atherosclerotic plaque vulnerability in the pathogenesis of CAD.
It is well known that coronary plaque composition is an important determinant of clinical progression and outcomes of CAD. Large lipid-rich plaques are considered to be histologic markers of plaque vulnerability 14,15. Those vulnerable plaques that do not produce severe stenosis can undergo sudden rupture with acute occlusive thrombosis, leading to ACS and other relevant acute clinical events 16,17. In previous pathologic studies, vulnerable plaque of the culprit lesions was found in approximately 70% of patients with CAD who had sudden cardiac death 18–20. Previous IVUS studies have found the incidence rates of vulnerable plaques to be 16–66% in the culprit lesions of ACS patients 21–23 and 22–32% in the target lesions of SAP patients 21,22,24, respectively. Therefore, more attention must be paid to vulnerable plaques, especially the lipid-rich plaques other than coronary stenosis, in clinical practice.
Several studies have found that serum total bilirubin concentrations are inversely associated with the risk for cardiovascular disease 8,25. Early in 1994, Schwertner et al.8 reported that low serum levels of total bilirubin exhibited a strong association with higher risk for CAD. Subsequently, a meta-analysis of 11 relevant studies showed an inverse relation between serum bilirubin levels and the severity of CAD 26. Interestingly, Gilbert’s syndrome, known as a common idiopathic hyperbilirubinemia caused by a mutation, was also associated with a lower risk for CAD 27. Moreover, a study from Turkey reported that serum bilirubin levels were significantly associated with the presence, severity, and the noncalcified morphology of atherosclerotic plaques detected by computed tomography angiography 28. Gullu et al.29 reported that greater serum bilirubin levels had a protective effect against impaired coronary flow reserve and coronary microvascular dysfunction. Erdogan et al.30 found that greater bilirubin concentrations were related to favorable coronary collateral flow in patients with chronic total occlusion when undergoing coronary angiography. Through our research, we found an inverse correlation between serum total bilirubin levels and coronary plaque vulnerability in CAD patients using IVUS, which was consistent with the findings of previous studies.
Unfortunately, the exact pathophysiological mechanisms underlying this association have not been completely elucidated. It is commonly acknowledged by both in-vitro and in-vivo studies that serum bilirubin has potent antioxidant properties, besides its role in assessing liver function 5,7. This natural product of heme metabolism makes up to 10% of the total antioxidant capacity in normobilirubinemic adults 31,32 and has the ability to scavenge peroxyl radicals and to inhibit peroxidation of LDL-derived lipids, which is known to prevent plaque formation and atherosclerosis 7. Bilirubin can also prolong the survival of human ventricular myocytes against in-situ generated oxyradicals in response to MI 33, and direct application of bilirubin to vascular endothelial tissue appears to improve markers of oxidative stress and cellular dysfunction 34. Further, bilirubin also has anticomplement properties, providing defense against inflammatory processes 35. Overall, the anti-inflammatory and anticomplement effects of bilirubin make it an important protection factor or enable it to slow the progression of atherosclerosis in CAD 36.
Several limitations of the present study should be taken into consideration. First, the relatively small number of participants with and without CAD in this single-center trial limited the power to prove an inverse relationship between total bilirubin level and plaque vulnerability. Second, because racial differences existed in the prevalence of CAD, as well as in the extent and morphology of coronary atherosclerosis, our findings could not be generalized to all populations. Thus, a large-scale multicenter clinical study is required to confirm our results. Finally, repeat intracoronary IVUS imaging was not performed, which might decrease the diagnostic accuracy and affect the investigation of the dynamic nature of coronary artery lesions.
The present study showed that serum total bilirubin levels in the ACS group were lower than those in SAP and control groups. IVUS data indicated that serum total bilirubin levels were inversely associated with coronary atherosclerotic plaque vulnerability. Decreased serum total bilirubin levels may play a role in the development of lipid plaques, which contributes to the pathogenesis of CAD. Thus, serum total bilirubin levels will serve as a sensitive biomarker for the evaluation of coronary atherosclerotic plaque vulnerability.
This study was funded by the Project of Science Technology Department of Zhejiang Province, China (No. 2012C33127).
Conflicts of interest
There are no conflicts of interest.
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