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Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease
Journal Pre-proofs Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease Yoshimi Kishimoto, Hanako Niki, Emi Saita, Susumu Ibe, Tomohiko Umei, Kotaro Miura, Yukinori Ikegami, Reiko Ohmori, Kazuo Kondo, Yukihiko Momiyama PII: DOI: Reference:
S0009-8981(20)30050-4 https://doi.org/10.1016/j.cca.2020.01.030 CCA 16010
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
13 December 2019 16 January 2020 28 January 2020
Please cite this article as: Y. Kishimoto, H. Niki, E. Saita, S. Ibe, T. Umei, K. Miura, Y. Ikegami, R. Ohmori, K. Kondo, Y. Momiyama, Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease, Clinica Chimica Acta (2020), doi: https://doi.org/10.1016/j.cca.2020.01.030
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CCA-D-19-01825 R1 Research article
Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease
Yoshimi Kishimotoa,*, Hanako Nikib, Emi Saitaa, Susumu Ibeb, Tomohiko Umeib, Kotaro Miurab, Yukinori Ikegamib, Reiko Ohmoric, Kazuo Kondoa,d, Yukihiko Momiyamab
aEndowed
Research Department “Food for Health”, Ochanomizu University, Tokyo,
Japan bDepartment
of Cardiology, National Hospital Organization Tokyo Medical Center,
Tokyo, Japan cFaculty
of Regional Design, Utsunomiya University, Tochigi, Japan
dInstitute
of Life Innovation Studies, Toyo University, Gunma, Japan
Address for Correspondence: Yoshimi Kishimoto, Ph.D. Endowed Research Department “Food for Health”, Ochanomizu University 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan E-mail: [email protected] Tel: +81-3-5978-5810, Fax: +81-3-5978-2694
1
Abstract Objective: Heme oxygenase-1 (HO-1) degrades heme to CO, iron, and biliverdin/bilirubin. Although serum bilirubin levels were often reported in patients with coronary artery disease (CAD), HO-1 levels in patients with CAD and the association between HO-1 and bilirubin levels have not been clarified. Methods: We measured plasma HO-1 and serum total bilirubin levels in 262 patients undergoing coronary angiography. Results: HO-1 levels were higher in patients with CAD than without CAD (median 0.46 vs. 0.35 ng/mL, P<0.01), but bilirubin were lower in patients with CAD than without CAD (0.69 vs. 0.75 mg/dL, P<0.02). Notably, HO-1 levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.35, 0.51, 0.45, and 0.44 ng/mL, and were highest in 1-vessel disease (P<0.05). Bilirubin levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.75, 0.70, 0.68, and 0.66 mg/dL (P=NS). No correlation was found between HO-1 and bilirubin levels. In multivariate analysis, HO-1 levels were a significant factor for CAD independent of atherosclerotic risk factors and bilirulin levels. Odds ratio for CAD was 2.32 (95%CI=1.29-4.17) for high HO-1 (>0.35 ng/mL). Conclusions: Patients with CAD were found to have high HO-1 and low bilirubin levels in blood, but no correlation was found between HO-1 and bilirubin levels.
Keywords: Coronary artery disease; HO-1; Bilirubin
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1. Introduction Heme degradation pathway has been recognized to play a protective role against the development of atherosclerosis [1, 2, 3]. Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to generate carbon monoxide (CO), ferrous iron and biliverdin, and biliverdin is subsequently converted to bilirubin by biliverdin reductase. Bilirubin is recognized as an endogenous anti-oxidant that can inhibit LDL oxidation [4]. In addition, bilirubin has anti-inflammatory property [5]. In LDL receptor-deficient mice, the intraperitoneal administration of bilirubin was shown to reduce atherosclerosis [6]. These findings thus suggest that bilirubin has anti-atherosclerotic effect. In 1994, Schwertner et al. [7] reported serum total bilirubin levels to be lower in patients with coronary artery disease (CAD) than in those without CAD. Since then, many studies have reported bilirubin levels to be lower in patients with CAD than in those without CAD [7, 8, 9, 10, 11] and to negatively correlate with the severity of CAD [9, 10, 11]. Therefore, low bilirubin levels in blood have been considered to be a promotive factor in the development of CAD. Accumulating evidence has recently pointed to HO-1 as an important molecule in modulating the process of atherosclerosis [1, 2, 3]. HO-1 is considered to play a protective role against atherosclerosis, mainly due to the degradation of pro-oxidant heme, the generation of anti-oxidants biliverdin and bilirubin, and the production of vasodilator CO [12]. In apoE-deficient mice, a lack of HO-1 accelerated atherosclerosis [13], whereas HO-1 induction reduced atherosclerosis in LDLreceptor knockout mice [14]. The HO-1 gene (HMOX1) transfer reduced atherosclerosis in apoE-deficient mice [15]. Furthermore, Wang et al. [16] demonstrated HO-1 overexpression in atherosclerotic lesions, which was recognized as a protective response against the progression of atherosclerosis. HO-1, which is a
3
32-kDa heat shock protein, is an intracellular enzyme induced by oxidative stress and inflammation [1, 2] and is released into plasma from leukocytes, macrophage, smooth muscle cells and endothelial cells that are activated or damaged by oxidative stress or inflammation [2, 17]. However, studies showing blood HO-1 levels in patients with atherosclerotic disease, such as CAD, are scarce. Idriss et al. [17] measured plasma HO-1 levels in 70 patients with stable CAD and 50 controls and reported HO-1 levels to be higher in patients with CAD than in controls. Recently, we assessed plasma HO-1 levels in 410 patients undergoing coronary angiography and found HO-1 levels to be higher in patients with CAD than in those without CAD [18]. High HO-1 levels in blood may thus reflect a protective response against the development of CAD. However, the association between blood HO-1 and bilirubin levels in patients with CAD has not yet been clarified. The present study expands upon our previous report [18] by comparing plasma HO-1 levels and serum total bilirubin levels in patients with and without CAD.
2. Materials and methods 2.1. Study patients In our previous study [18], we measured plasma HO-1 levels in 410 consecutive patients undergoing elective coronary angiography for suspected CAD at Tokyo Medical Center. Patients with acute coronary syndrome or those with a history of percutaneous coronary intervention or cardiac surgery were excluded. Of the 410 study patients, 36 with PAD were excluded from the present study, because HO-1 levels were found to be low in patients with PAD [18, 19]. Of the remaining 374 study patients, 262 had their serum total bilirubin levels measured and were thus included in the present study. Hypertension was defined as blood pressures of ≥140/90 mmHg or on drugs, and 154 (59%) patients were taking anti-hypertensive drugs. 4
Hyperlipidemia was defined as LDL-cholesterol level of >140 mg/dL or on drugs, and 88 (34%) patients were taking statins. Diabetes mellitus (DM) (a fasting plasma glucose level of ≥126 mg/dL or on treatment) was present in 64 (24%) patients, and 105 (40%) were smokers (≥10 pack-yrs). Our study was performed following the principles outlined in the Helsinki Declaration and was approved by the Ethics Committee of Tokyo Medical Center (the institutional ethics committee of our hospital) (approval No. R07-054/R15-056). After written informed consent was obtained, overnight-fasting blood samples were taken on the morning of the day when coronary angiography was performed.
2.2. Measurements of plasma HO-1, C-reactive protein (CRP) and serum total bilirubin levels Blood samples were collected in EDTA-containing tubes, and the plasma was stored at –80 ºC. Plasma HO-1 levels were measured by an enzyme-linked immunosorbent assay (ELISA) with a commercially available kit (Human HO-1 ELISA Kit; Enzo Life Sciences Inc., Farmingdale, NY, USA) at Ochanomizu University in accordance with the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were all <10%. Plasma high-sensitivity C-reactive protein (hsCRP) levels were also measured by a BNII nephelometer (Dade Behring, Tokyo, Japan). However, serum total bilirubin levels were measured by an enzymatic method using an autoanalyzer (BioMajesty JCA-BM6070; JEOL, Tokyo, Japan) on the day of blood sampling.
2.3. Coronary angiography Angiograms were recorded on a cineangiogram system (Philips Electronics Japan, Tokyo, Japan). CAD was defined as at least one coronary artery having >50% 5
luminal diameter stenosis on angiograms. The severity of CAD was represented as the numbers of >50% stenotic vessels and stenotic segments and the severity score of stenosis. The degree of coronary stenosis in each segment was scored from 0 to 4 points (0, ≤25%; 1, 26%-50%; 2, 51%-75%; 3, 76%-90%; 4, >90% stenosis), and the severity score was defined as the sum of scores of all segments. Coronary artery segments were defined as 29 segments according to the Coronary Artery Surgery Study (CASS) classification. All angiograms were evaluated by a single cardiologist (Y.M.), blinded to the clinical and laboratory data.
2.4. Statistical analysis Differences between 2 groups were evaluated by unpaired t-test, Mann-Whitney U test, and chi-squared test for normally distributed variables, non-normally distributed variables, and categorical variables, respectively. Differences among ≥3 groups were evaluated by an analysis of variance with Scheffe’s test, Kruskal-Wallis test with Steel-Dwass test, and chi-squared test for normally distributed variables, nonnormally distributed variables, and categorical variables, respectively. Correlations between HO-1, bilirubin or hsCRP levels and the severity of CAD were evaluated by Spearman’s rank correlation test. To determine the cut-off points of HO-1 and bilirubin levels for CAD, a relative cumulative frequency distribution curve was created, and then the optimum cut-off points were determined to be 0.35 ng/mL and 0.70 mg/dL, respectively. The receiver–operating characteristic (ROC) curves were created, and the areas under ROC curves (AUC) were measured to compare the diagnostic abilities of HO-1 and bilirubin levels to predict CAD. Regarding the cut-off point of hsCRP levels, the previously reported cut-off point of 1.0 mg/L for CAD or cardiovascular events was used [20,21]. A multiple logistic regression analysis was used to determine the independent association between HO-1, bilirubin or hsCRP 6
levels and CAD. A P value of <0.05 was considered to be statistically significant. Results are presented as the mean±SD or the median value.
3. Results Among the 262 study patients, CAD was present in 138 patients (53%) (1-vessel disease [1-VD], n=50; 2-vessel disease [2-VD], n=46; 3-vessel disease [3-VD], n=42). Compared with 124 patients without CAD, 138 with CAD were older and had a male predominance; higher prevalence of hypertension, DM and hyperlipidemia; and lower HDL-cholesterol levels (Table 1). Plasma hsCRP levels tended to be higher in patients with CAD than in those without CAD (median 0.63 vs. 0.50 mg/L, P=NS) (Fig. 1). However, a stepwise increase in hsCRP levels was found depending on the number of >50% stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD (P<0.05), and hsCRP levels were highest in 3-VD (P<0.05) (Fig. 1). Plasma hsCRP levels correlated with the number of >50% stenotic segments and the severity score (r=0.13 and r=0.12, respectively, P<0.05). Notably, plasma HO-1 levels were significantly higher in patients with CAD than in those without CAD (median 0.46 vs. 0.35 ng/mL, P<0.01) (Fig. 2). Interestingly, HO-1 levels in the 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively (P<0.01), and HO-1 levels were highest in 1-VD (P<0.05) (Fig. 2). In contrast, serum total bilirubin levels were significantly lower in patients with CAD than in those without CAD (median 0.69 vs. 0.75 mg/dL, P<0.02) (Fig. 3). Bilirubin levels tended to decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among the 4 groups (Fig. 3). HO-1 levels positively correlated with the number of stenotic segments and the severity score (r=0.16 and
7
r=0.15, respectively, P<0.02), whereas bilirubin levels negatively correlated with the number of stenotic segments and the severity score (r=-0.14 and r=-0.15, respectively, P<0.05). However, no significant correlation was found between HO-1 and bilirubin levels (P=NS) (Fig. 4). Bilirubin levels negatively correlated with hsCRP levels (r=-0.14, P<0.05), whereas no significant correlation was found between HO-1 and hsCRP levels. The sensitivity and specificity to predict CAD were 69% and 52% for a HO-1 level of >0.35 ng/mL and 52% and 61% for a bilirubin level of <0.70 mg/dL, respectively (Table 1). The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65) (Fig. 5). To elucidate the independent association between HO-1 or bilirubin levels and CAD, variables (age, gender, hypertension, DM, smoking, hyperlipidemia, statin use, and HDL-cholesterol, hsCRP, HO-1, and bilirubin levels) were entered into a multiple logistic regression model. As a result, HO-1 levels were found to be a significant factor associated with CAD independent of hsCRP and bilirubin levels and atherosclerotic risk factors, but bilirubin levels were not. The odds ratio for CAD was 2.32 (95%CI=1.29-4.17) for a high HO-1 level of >0.35 ng/mL (P<0.01) (Table 2).
4. Discussion In the present study, plasma HO-1 levels were significantly higher in patients with CAD, especially in those with 1-VD, than in those without CAD. In contrast, serum total bilirubin levels were significantly lower in patients with CAD than in those without CAD. However, no significant correlation was found between HO-1 and bilirubin levels. In a multivariate analysis, HO-1 levels were a significant factor associated with CAD independent of bilirubin levels and atherosclerotic risk factors.
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Bilirubin is the end product of breakdown of heme, predominantly originating from hemoglobin in aging red blood cells. Bilirubin is recognized to have anti-oxidant and anti-inflammatory properties [4, 5], suggesting anti-atherosclerotic effect of bilirubin. Regarding serum bilirubin levels in patients with CAD, some studies reported no difference in bilirubin levels between patients with and without CAD [22] or no correlation between bilirubin levels and the severity of CAD [8, 22]. However, most studies reported serum bilirubin levels to be low in patients with CAD [7, 8, 9, 10, 11] and to negatively correlate with the severity of CAD [9, 10, 11]. Our study also found serum total bilirubin levels to be lower in 138 patients with CAD than in 124 without CAD and to negatively, but weaky, correlate with the severity of CAD, defined as the number of stenotic segment and the severity score. Furthermore, bilirubin levels were found to negatively correlate with plasma hsCRP levels, one of inflammatory markers. These findings thus suggest that low bilirubin levels in blood may play a promotive role in the development of CAD. Genetic variations in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) are known to be major determinants of serum bilirubin levels [23, 24]. Recently, Stender et al. [25] investigated the associations between the genotype of UGT1A1 and plasma bilirubin levels and between genetically elevated bilirubin levels and CAD in 67068 subjects and reported that genetically elevated bilirubin levels were not associated with a decreased risk of CAD. They also performed a meta-analysis of 11 studies and showed no association between genetically elevated bilirubin levels and CAD. These findings suggest no causal relationship between elevated bilirubin levels and CAD. Since increased reactive oxygen species (ROS) and oxidative stress are recognized to be involved in the pathogenesis of atherosclerosis, low bilirubin levels
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in patients with CAD may not be a cause of CAD but rather a result of increased oxidative stress, leading to the consumption of endogenous anti-oxidants [26, 27]. HO-1 is considered to play a protective role against atherosclerosis because of its anti-oxidant, anti-inflammatory, anti-apoptotic and anti-proliferative effects [3, 27]. Regarding blood HO-1 levels in patients with CAD, Idriss et al. [17] reported that HO-1 levels were higher in 70 patients with CAD than in 60 controls. In our present study, plasma HO-1 levels were also found to be significantly higher in 138 patients with CAD than in 124 without CAD, in contrast to lower bilirubin levels being noted in patients with CAD. Bilirubin levels tended to decrease depending on the number of stenotic vessels, but HO-1 levels were highest in patients with 1-VD among the 4 groups of CAD(-), 1-VD, 2-VD and 3-VD. Since the capacity to up-regulate HO-1 expression in leukocytes in response to oxidative stress was shown to be reduced in patients with 2-VD or 3-VD [28], patients with severe CAD, such as 2-VD or 3-VD, may have lower plasma HO-1 levels and less-marked protective response against oxidative stress than those with mild CAD, such as 1-VD. These findings thus suggest that patients with CAD, especially those with 1-VD, may have high HO-1 levels, possibly reflecting a protective response against the progression of CAD. Like patients with mild CAD, such as 1-VD, we also reported subjects with carotid plaque to have higher plasma HO-1 levels than those without plaque among 136 subjects with no history of cardiovascular disease undergoing carotid ultrasonography [29]. In contrast, like patients with severe CAD, such as 3-VD, we [18] and others [19] reported that patients with peripheral artery disease (PAD) had low plasma HO-1 levels. Patients with PAD usually have severe atherosclerosis in iliac and femoral arteries and often have CAD, especially 3-VD [30,31]. Although the mechanism of relatively low HO-1 levels in patients with severe atherosclerosis, such as 3-VD and
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PAD, remains unclear, HO-1 defensive response to oxidative stress was reported to be attenuated at advanced age [32] and late stage of DM [33]. HO-1 induction were decreased in aged rats [32]. In diabetic mice, HO-1 activity was increased in early stage of DM while decreased in late stage of DM [33]. Therefore, the long duration of severe stress condition may cause some disruption of HO-1 defense system. HO-1 is an enzyme that degrades heme to CO, iron and biliverdin/bilirubin, but no study has described the correlation between HO-1 and bilirubin levels in patients with stable CAD. Li et al. [34] measured serum HO-1 and total bilirubin levels in 60 patients with stroke and 50 with transient ischemic attack (TIA). They showed that HO-1 levels were higher in patients with stroke than in those with TIA, in contrast to lower bilirubin levels being noted in patients with stroke. In our present study, we found higher HO-1 and lower bilirubin levels in patients with CAD than in those without CAD, but no significant correlation was noted between HO-1 and bilirubin levels. In a multivariate analysis, HO-1 levels were a significant factor for CAD independent of bilirubin levels and atherosclerotic risk factors, but bilirubin levels were not. Hence, the responses to the progression of atherosclerosis would be different between blood HO-1 and bilirubin levels. However, we just measured plasma HO-1 levels and did not evaluate HO-1 activity and protein expression in blood leukocytes. Moreover, heme metabolism and oxygenase pathway is involved in numerous biological processes including oxygen transport, cellular respiration, oxidative biotransformations, and host defence [3]. The lack of correlation between HO-1 and bilirubin levels may thus have been affected by these factors. Further prospective studies will be needed in order to clarify the roles of both HO-1 and bilirubin levels in the process of atherosclerotic disease, such as CAD. Moreover, in the present study, the sensitivity and specificity to predict CAD were 69% and 52% for HO-1 (>0.35 ng/mL) and 52% and 61% for bilirubin level
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(<0.70 mg/dL), and AUC for HO-1 levels tended to be larger than AUC for bilirubin levels. Although bilirubin levels have often been reported to be low in patients with CAD and to be a biomarker for CAD [7-11], our study suggests that HO-1 levels as well as bilirubin levels can be a biomarker associated with CAD. Our study has several limitations. First, the number of study patients was small (n=262), and only 262 of the 374 patients had their bilirubin levels measured, as this measurement was left to the decision of the doctors in charge. Second, angiography was used to evaluate coronary atherosclerosis. Angiography cannot visualize plaques and only shows lumen characteristics. Third, our study did not analyze any HMOX1 gene polymorphisms. Since some HMOX1 gene polymorphisms have been reported to be associated with CAD [35], these polymorphims may have affected plasma HO-1 levels in our patients with CAD. Fourth, our study was cross-sectional in nature and was unable to establish causality, since it only showed some associations and proposed hypotheses. Finally, our study was performed in Japanese patients undergoing coronary angiography, who are generally considered to be a highly select population at high risk for CAD. Our results therefore may not be applicable to the general or other ethnic populations.
5. Conclusion Patients with CAD were found to have high HO-1 levels and low total bilirubin levels in blood. However, no signficant correlation was found between HO-1 and bilirubin levels. HO-1 levels were a significant factor associated with CAD independent of bilirubin levels and atherosclerotic risk factors. Our data suggest the usefulness of HO-1 as a biomarker for CAD and a possible role of HO-1 in the development of CAD.
Funding 12
This study was supported by a grant from Honjo International Scholarship Foundation. Financial funding was also provided by Pfizer Japan Inc., and Bayer Yakuhin Ltd.; however, these sponsors had no role in the design, analysis, or interpretation of our study.
Declaration of interest Our study has no conflicts of interest to disclose.
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Table 1. Clinical characteristics and plasma HO-1 levels and serum total bilirubin levels of patients with and without CAD CAD(-) (n=124)
CAD P value CAD(-)
(n=138)
vs. CAD
Age (years)
66±12
<0.01
Gender (male)
75 (60%)
<0.001
BMI (kg/m2)
24.2±4.8
NS
Hypertension
75 (60%)
<0.005
SBP (mmHg)
130±22
NS
133±21
13 (10%)
<0.001
51 (37%)
HbA1c (%)
5.9±0.7
<0.002
6.3±1.0
Smoking
43 (35%)
NS
Hyperlipidemia
46 (37%)
Statin
2-VD
3-VD
(n=50)
(n=46)
(n=52)
Among 4 groups
72±7
<0.01
33 (72%)
37 (88%)
<0.01
23.9±3.4
23.6±3.4
23.6±2.7
NS
108 (78%) 40 (80%)
34 (74%)
34 (81%)
<0.01
133±21
140±23
130±23
<0.05
13 (26%)
22 (48%)
16 (38%)
<0.001
6.1±0.8
6.5±1.1
6.4±1.1
<0.001
62 (45%)
28 (56%)
18 (39%)
16 (38%)
NS
<0.01
77 (56%)
26 (52%)
26 (57%)
25 (60%)
<0.025
28 (23%)
<0.001
60 (43%)
21 (42%)
19 (41%)
20 (48%)
<0.01
LDL-C (mg/dL)
112±29
NS
114±36
109±36
115±33
121±38
NS
HDL-C (mg/dL)
59±15
<0.001
50±12
51±12
51±11
48±12
<0.001
hsCRP levels
0.50
NS
0.63
0.58
0.58
0.89
<0.05
(mg/L)
[0.27, 1.50]
[0.34, 1.60] [0.31, 0.90]
[0.31, 1.54]
[0.46, 2.72]
>1.0 mg/L
41 (33%)
NS
45 (33%)
17 (37%)
19 (45%)
<0.05
0.35
<0.01
0.46
0.51
0.45
0.44
<0.01
[0.30, 0.61]
[0.38, 0.66]
[0.26, 0.59]
[0.29, 0.57]
HO-1 levels (ng/mL) >0.35 ng/mL Total bilirubin levels
[0.23, 0.53]
68±11
P value
68±10
Diabetes mellitus
69±9
1-VD
111 (80%) 41 (82%) 23.9±3.4
9 (18%)
59 (48%)
<0.001
95 (69%)
39 (78%)
28 (61%)
28 (67%)
<0.005
0.75
<0.02
0.69
0.70
0.68
0.66
NS
[0.57, 0.84]
[0.59, 0.81]
[0.53, 0.88]
[0.56, 0.83]
72 (52%)
24 (48%)
23 (50%)
25 (60%)
(mg/dL)
[0.61, 0.95]
<0.70 mg/dL
48 (39%)
<0.05
Data represent the mean±SD or the number (%) of patients, except for hsCRP, HO-1 and total bilirubin levels which are presented as the median value and interquartile range. BMI = body mass index; SBP = systolic blood pressure; LDL-C = low-density lipoprotein cholesterol; HDL-C = high-density lipoprotein cholesterol. 19
NS
Table 2. Factors associated with CAD (Multiple logistic regression analysis of the 262 study patients) Odds ratio
(95% CI)
P value
Age (per 10-year increase)
1.50
(1.14-1.96)
<0.005
Male gender
3.54
(1.81-6.90)
<0.001
Statin use
2.66
(1.41-5.02)
<0.005
Diabetes mellitus
3.21
(1.56-6.63)
<0.005
Low HDL-C level (<40 mg/dL)
2.49
(1.01-6.22)
<0.05
HO-1 level (>0.35 ng/mL)
2.32
(1.29-4.17)
<0.01
The dependent variable was the presence of CAD. The analysis included age, gender, hypertension, DM, body mass index, smoking, hyperlipidemia, statin use, and HDL-cholesterol (<40 mg/dL), hsCRP (>1.0 mg/L), HO-1 (>0.35 ng/mL) and total bilirubin (<0.70 mg/dL) levels.
20
Figure Legends Figure 1. Plasma hsCRP levels and the presence of CAD or the number of stenotic coronary vessels. Plasma hsCRP levels tended to be higher in CAD than in CAD(-) (p=NS)(left). However, hsCRP levels stepwisely increased depending on the number of stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD, respectively, and were highest in 3-VD (p<0.05 by Kruskal-Wallis test)(right). Figure 2. Plasma HO-1 levels and the presence of CAD or the number of stenotic coronary vessels. Plasma HO-1 levels were significantly higher in CAD than in CAD(-) (p<0.01)(left). Notably, HO-1 levels in 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively, and were highest in 1-VD (p<0.01 by KruskalWallis test)(right). Figure 3. Serum total bilirubin levels and the presence of CAD or the number of stenotic coronary vessels. Serum bilirubin levels were significantly lower in CAD than in CAD(-) (p<0.02)(left). Bilirubin levels tended to stepwisely decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among 4 groups (p=NS by Kruskal-Wallis test)(right). Figure 4. Correlation between HO-1 levels and total bilirubin levels. There was no significant correlation between plasma HO-1 levels and serum total bilirubin levels (P=NS).
21
Figure 5. ROC curves of HO-1 and bilirubin levels for the diagnostic abilitiy of CAD. The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65). However, this difference did not reach statistical significance.
22
Figure 1. Plasma hsCRP levels and the presence of CAD or the number of stenotic coronary vessels. Plasma hsCRP levels tended to be higher in CAD than in CAD(-) (p=NS)(left). However, hsCRP levels stepwisely increased depending on the number of stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD, respectively, and were highest in 3-VD (p<0.05 by Kruskal-Wallis test)(right).
23
Figure 2. Plasma HO-1 levels and the presence of CAD or the number of stenotic coronary vessels. Plasma HO-1 levels were significantly higher in CAD than in CAD(-) (p<0.01)(left). Notably, HO-1 levels in 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively, and were highest in 1-VD (p<0.01 by KruskalWallis test)(right).
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Figure 3. Serum total bilirubin levels and the presence of CAD or the number of stenotic coronary vessels. Serum bilirubin levels were significantly lower in CAD than in CAD(-) (p<0.02)(left). Bilirubin levels tended to stepwisely decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among 4 groups (p=NS by Kruskal-Wallis test)(right).
25
Figure 4. Correlation between HO-1 levels and total bilirubin levels. There was no significant correlation between plasma HO-1 levels and serum total bilirubin levels (P=NS).
26
Figure 5. ROC curves of HO-1 and bilirubin levels for the diagnostic ability of CAD. The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65). However, this difference did not reach statistical significance.
27
Author contributions statement Yoshimi Kishimoto: Investigation, Writing - Original Draft; Emi Saita: Investigation; Hanako Niki, Susumu Ibe, Tomohiko Umei, Kotaro Miura, and Yukinori Ikegami: Data Curation; Reiko Ohmori and Kazuo Kondo: Supervision; Yukihiko Momiyama: Project administration, Formal analysis, Resources, Writing - Review & Editing.
Highlights 1) Plasma HO-1 levels were significantly higher in patients with CAD, especially in 1VD, than in those without CAD. 2) In contrast, serum bilirubin levels were significantly lower in patients with CAD than in those without CAD. 3) There was no correlation between HO-1 and bilirubin levels. 4) In a multivariate analysis, HO-1, but not bilirubin, level was an independent factor for CAD.
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S0009-8981(20)30050-4 https://doi.org/10.1016/j.cca.2020.01.030 CCA 16010
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
13 December 2019 16 January 2020 28 January 2020
Please cite this article as: Y. Kishimoto, H. Niki, E. Saita, S. Ibe, T. Umei, K. Miura, Y. Ikegami, R. Ohmori, K. Kondo, Y. Momiyama, Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease, Clinica Chimica Acta (2020), doi: https://doi.org/10.1016/j.cca.2020.01.030
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.
© 2020 Published by Elsevier B.V.
CCA-D-19-01825 R1 Research article
Blood levels of heme oxygenase-1 versus bilirubin in patients with coronary artery disease
Yoshimi Kishimotoa,*, Hanako Nikib, Emi Saitaa, Susumu Ibeb, Tomohiko Umeib, Kotaro Miurab, Yukinori Ikegamib, Reiko Ohmoric, Kazuo Kondoa,d, Yukihiko Momiyamab
aEndowed
Research Department “Food for Health”, Ochanomizu University, Tokyo,
Japan bDepartment
of Cardiology, National Hospital Organization Tokyo Medical Center,
Tokyo, Japan cFaculty
of Regional Design, Utsunomiya University, Tochigi, Japan
dInstitute
of Life Innovation Studies, Toyo University, Gunma, Japan
Address for Correspondence: Yoshimi Kishimoto, Ph.D. Endowed Research Department “Food for Health”, Ochanomizu University 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan E-mail: [email protected] Tel: +81-3-5978-5810, Fax: +81-3-5978-2694
1
Abstract Objective: Heme oxygenase-1 (HO-1) degrades heme to CO, iron, and biliverdin/bilirubin. Although serum bilirubin levels were often reported in patients with coronary artery disease (CAD), HO-1 levels in patients with CAD and the association between HO-1 and bilirubin levels have not been clarified. Methods: We measured plasma HO-1 and serum total bilirubin levels in 262 patients undergoing coronary angiography. Results: HO-1 levels were higher in patients with CAD than without CAD (median 0.46 vs. 0.35 ng/mL, P<0.01), but bilirubin were lower in patients with CAD than without CAD (0.69 vs. 0.75 mg/dL, P<0.02). Notably, HO-1 levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.35, 0.51, 0.45, and 0.44 ng/mL, and were highest in 1-vessel disease (P<0.05). Bilirubin levels in CAD(-), 1-vessel, 2-vessel, and 3-vessel disease were 0.75, 0.70, 0.68, and 0.66 mg/dL (P=NS). No correlation was found between HO-1 and bilirubin levels. In multivariate analysis, HO-1 levels were a significant factor for CAD independent of atherosclerotic risk factors and bilirulin levels. Odds ratio for CAD was 2.32 (95%CI=1.29-4.17) for high HO-1 (>0.35 ng/mL). Conclusions: Patients with CAD were found to have high HO-1 and low bilirubin levels in blood, but no correlation was found between HO-1 and bilirubin levels.
Keywords: Coronary artery disease; HO-1; Bilirubin
2
1. Introduction Heme degradation pathway has been recognized to play a protective role against the development of atherosclerosis [1, 2, 3]. Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to generate carbon monoxide (CO), ferrous iron and biliverdin, and biliverdin is subsequently converted to bilirubin by biliverdin reductase. Bilirubin is recognized as an endogenous anti-oxidant that can inhibit LDL oxidation [4]. In addition, bilirubin has anti-inflammatory property [5]. In LDL receptor-deficient mice, the intraperitoneal administration of bilirubin was shown to reduce atherosclerosis [6]. These findings thus suggest that bilirubin has anti-atherosclerotic effect. In 1994, Schwertner et al. [7] reported serum total bilirubin levels to be lower in patients with coronary artery disease (CAD) than in those without CAD. Since then, many studies have reported bilirubin levels to be lower in patients with CAD than in those without CAD [7, 8, 9, 10, 11] and to negatively correlate with the severity of CAD [9, 10, 11]. Therefore, low bilirubin levels in blood have been considered to be a promotive factor in the development of CAD. Accumulating evidence has recently pointed to HO-1 as an important molecule in modulating the process of atherosclerosis [1, 2, 3]. HO-1 is considered to play a protective role against atherosclerosis, mainly due to the degradation of pro-oxidant heme, the generation of anti-oxidants biliverdin and bilirubin, and the production of vasodilator CO [12]. In apoE-deficient mice, a lack of HO-1 accelerated atherosclerosis [13], whereas HO-1 induction reduced atherosclerosis in LDLreceptor knockout mice [14]. The HO-1 gene (HMOX1) transfer reduced atherosclerosis in apoE-deficient mice [15]. Furthermore, Wang et al. [16] demonstrated HO-1 overexpression in atherosclerotic lesions, which was recognized as a protective response against the progression of atherosclerosis. HO-1, which is a
3
32-kDa heat shock protein, is an intracellular enzyme induced by oxidative stress and inflammation [1, 2] and is released into plasma from leukocytes, macrophage, smooth muscle cells and endothelial cells that are activated or damaged by oxidative stress or inflammation [2, 17]. However, studies showing blood HO-1 levels in patients with atherosclerotic disease, such as CAD, are scarce. Idriss et al. [17] measured plasma HO-1 levels in 70 patients with stable CAD and 50 controls and reported HO-1 levels to be higher in patients with CAD than in controls. Recently, we assessed plasma HO-1 levels in 410 patients undergoing coronary angiography and found HO-1 levels to be higher in patients with CAD than in those without CAD [18]. High HO-1 levels in blood may thus reflect a protective response against the development of CAD. However, the association between blood HO-1 and bilirubin levels in patients with CAD has not yet been clarified. The present study expands upon our previous report [18] by comparing plasma HO-1 levels and serum total bilirubin levels in patients with and without CAD.
2. Materials and methods 2.1. Study patients In our previous study [18], we measured plasma HO-1 levels in 410 consecutive patients undergoing elective coronary angiography for suspected CAD at Tokyo Medical Center. Patients with acute coronary syndrome or those with a history of percutaneous coronary intervention or cardiac surgery were excluded. Of the 410 study patients, 36 with PAD were excluded from the present study, because HO-1 levels were found to be low in patients with PAD [18, 19]. Of the remaining 374 study patients, 262 had their serum total bilirubin levels measured and were thus included in the present study. Hypertension was defined as blood pressures of ≥140/90 mmHg or on drugs, and 154 (59%) patients were taking anti-hypertensive drugs. 4
Hyperlipidemia was defined as LDL-cholesterol level of >140 mg/dL or on drugs, and 88 (34%) patients were taking statins. Diabetes mellitus (DM) (a fasting plasma glucose level of ≥126 mg/dL or on treatment) was present in 64 (24%) patients, and 105 (40%) were smokers (≥10 pack-yrs). Our study was performed following the principles outlined in the Helsinki Declaration and was approved by the Ethics Committee of Tokyo Medical Center (the institutional ethics committee of our hospital) (approval No. R07-054/R15-056). After written informed consent was obtained, overnight-fasting blood samples were taken on the morning of the day when coronary angiography was performed.
2.2. Measurements of plasma HO-1, C-reactive protein (CRP) and serum total bilirubin levels Blood samples were collected in EDTA-containing tubes, and the plasma was stored at –80 ºC. Plasma HO-1 levels were measured by an enzyme-linked immunosorbent assay (ELISA) with a commercially available kit (Human HO-1 ELISA Kit; Enzo Life Sciences Inc., Farmingdale, NY, USA) at Ochanomizu University in accordance with the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were all <10%. Plasma high-sensitivity C-reactive protein (hsCRP) levels were also measured by a BNII nephelometer (Dade Behring, Tokyo, Japan). However, serum total bilirubin levels were measured by an enzymatic method using an autoanalyzer (BioMajesty JCA-BM6070; JEOL, Tokyo, Japan) on the day of blood sampling.
2.3. Coronary angiography Angiograms were recorded on a cineangiogram system (Philips Electronics Japan, Tokyo, Japan). CAD was defined as at least one coronary artery having >50% 5
luminal diameter stenosis on angiograms. The severity of CAD was represented as the numbers of >50% stenotic vessels and stenotic segments and the severity score of stenosis. The degree of coronary stenosis in each segment was scored from 0 to 4 points (0, ≤25%; 1, 26%-50%; 2, 51%-75%; 3, 76%-90%; 4, >90% stenosis), and the severity score was defined as the sum of scores of all segments. Coronary artery segments were defined as 29 segments according to the Coronary Artery Surgery Study (CASS) classification. All angiograms were evaluated by a single cardiologist (Y.M.), blinded to the clinical and laboratory data.
2.4. Statistical analysis Differences between 2 groups were evaluated by unpaired t-test, Mann-Whitney U test, and chi-squared test for normally distributed variables, non-normally distributed variables, and categorical variables, respectively. Differences among ≥3 groups were evaluated by an analysis of variance with Scheffe’s test, Kruskal-Wallis test with Steel-Dwass test, and chi-squared test for normally distributed variables, nonnormally distributed variables, and categorical variables, respectively. Correlations between HO-1, bilirubin or hsCRP levels and the severity of CAD were evaluated by Spearman’s rank correlation test. To determine the cut-off points of HO-1 and bilirubin levels for CAD, a relative cumulative frequency distribution curve was created, and then the optimum cut-off points were determined to be 0.35 ng/mL and 0.70 mg/dL, respectively. The receiver–operating characteristic (ROC) curves were created, and the areas under ROC curves (AUC) were measured to compare the diagnostic abilities of HO-1 and bilirubin levels to predict CAD. Regarding the cut-off point of hsCRP levels, the previously reported cut-off point of 1.0 mg/L for CAD or cardiovascular events was used [20,21]. A multiple logistic regression analysis was used to determine the independent association between HO-1, bilirubin or hsCRP 6
levels and CAD. A P value of <0.05 was considered to be statistically significant. Results are presented as the mean±SD or the median value.
3. Results Among the 262 study patients, CAD was present in 138 patients (53%) (1-vessel disease [1-VD], n=50; 2-vessel disease [2-VD], n=46; 3-vessel disease [3-VD], n=42). Compared with 124 patients without CAD, 138 with CAD were older and had a male predominance; higher prevalence of hypertension, DM and hyperlipidemia; and lower HDL-cholesterol levels (Table 1). Plasma hsCRP levels tended to be higher in patients with CAD than in those without CAD (median 0.63 vs. 0.50 mg/L, P=NS) (Fig. 1). However, a stepwise increase in hsCRP levels was found depending on the number of >50% stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD (P<0.05), and hsCRP levels were highest in 3-VD (P<0.05) (Fig. 1). Plasma hsCRP levels correlated with the number of >50% stenotic segments and the severity score (r=0.13 and r=0.12, respectively, P<0.05). Notably, plasma HO-1 levels were significantly higher in patients with CAD than in those without CAD (median 0.46 vs. 0.35 ng/mL, P<0.01) (Fig. 2). Interestingly, HO-1 levels in the 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively (P<0.01), and HO-1 levels were highest in 1-VD (P<0.05) (Fig. 2). In contrast, serum total bilirubin levels were significantly lower in patients with CAD than in those without CAD (median 0.69 vs. 0.75 mg/dL, P<0.02) (Fig. 3). Bilirubin levels tended to decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among the 4 groups (Fig. 3). HO-1 levels positively correlated with the number of stenotic segments and the severity score (r=0.16 and
7
r=0.15, respectively, P<0.02), whereas bilirubin levels negatively correlated with the number of stenotic segments and the severity score (r=-0.14 and r=-0.15, respectively, P<0.05). However, no significant correlation was found between HO-1 and bilirubin levels (P=NS) (Fig. 4). Bilirubin levels negatively correlated with hsCRP levels (r=-0.14, P<0.05), whereas no significant correlation was found between HO-1 and hsCRP levels. The sensitivity and specificity to predict CAD were 69% and 52% for a HO-1 level of >0.35 ng/mL and 52% and 61% for a bilirubin level of <0.70 mg/dL, respectively (Table 1). The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65) (Fig. 5). To elucidate the independent association between HO-1 or bilirubin levels and CAD, variables (age, gender, hypertension, DM, smoking, hyperlipidemia, statin use, and HDL-cholesterol, hsCRP, HO-1, and bilirubin levels) were entered into a multiple logistic regression model. As a result, HO-1 levels were found to be a significant factor associated with CAD independent of hsCRP and bilirubin levels and atherosclerotic risk factors, but bilirubin levels were not. The odds ratio for CAD was 2.32 (95%CI=1.29-4.17) for a high HO-1 level of >0.35 ng/mL (P<0.01) (Table 2).
4. Discussion In the present study, plasma HO-1 levels were significantly higher in patients with CAD, especially in those with 1-VD, than in those without CAD. In contrast, serum total bilirubin levels were significantly lower in patients with CAD than in those without CAD. However, no significant correlation was found between HO-1 and bilirubin levels. In a multivariate analysis, HO-1 levels were a significant factor associated with CAD independent of bilirubin levels and atherosclerotic risk factors.
8
Bilirubin is the end product of breakdown of heme, predominantly originating from hemoglobin in aging red blood cells. Bilirubin is recognized to have anti-oxidant and anti-inflammatory properties [4, 5], suggesting anti-atherosclerotic effect of bilirubin. Regarding serum bilirubin levels in patients with CAD, some studies reported no difference in bilirubin levels between patients with and without CAD [22] or no correlation between bilirubin levels and the severity of CAD [8, 22]. However, most studies reported serum bilirubin levels to be low in patients with CAD [7, 8, 9, 10, 11] and to negatively correlate with the severity of CAD [9, 10, 11]. Our study also found serum total bilirubin levels to be lower in 138 patients with CAD than in 124 without CAD and to negatively, but weaky, correlate with the severity of CAD, defined as the number of stenotic segment and the severity score. Furthermore, bilirubin levels were found to negatively correlate with plasma hsCRP levels, one of inflammatory markers. These findings thus suggest that low bilirubin levels in blood may play a promotive role in the development of CAD. Genetic variations in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) are known to be major determinants of serum bilirubin levels [23, 24]. Recently, Stender et al. [25] investigated the associations between the genotype of UGT1A1 and plasma bilirubin levels and between genetically elevated bilirubin levels and CAD in 67068 subjects and reported that genetically elevated bilirubin levels were not associated with a decreased risk of CAD. They also performed a meta-analysis of 11 studies and showed no association between genetically elevated bilirubin levels and CAD. These findings suggest no causal relationship between elevated bilirubin levels and CAD. Since increased reactive oxygen species (ROS) and oxidative stress are recognized to be involved in the pathogenesis of atherosclerosis, low bilirubin levels
9
in patients with CAD may not be a cause of CAD but rather a result of increased oxidative stress, leading to the consumption of endogenous anti-oxidants [26, 27]. HO-1 is considered to play a protective role against atherosclerosis because of its anti-oxidant, anti-inflammatory, anti-apoptotic and anti-proliferative effects [3, 27]. Regarding blood HO-1 levels in patients with CAD, Idriss et al. [17] reported that HO-1 levels were higher in 70 patients with CAD than in 60 controls. In our present study, plasma HO-1 levels were also found to be significantly higher in 138 patients with CAD than in 124 without CAD, in contrast to lower bilirubin levels being noted in patients with CAD. Bilirubin levels tended to decrease depending on the number of stenotic vessels, but HO-1 levels were highest in patients with 1-VD among the 4 groups of CAD(-), 1-VD, 2-VD and 3-VD. Since the capacity to up-regulate HO-1 expression in leukocytes in response to oxidative stress was shown to be reduced in patients with 2-VD or 3-VD [28], patients with severe CAD, such as 2-VD or 3-VD, may have lower plasma HO-1 levels and less-marked protective response against oxidative stress than those with mild CAD, such as 1-VD. These findings thus suggest that patients with CAD, especially those with 1-VD, may have high HO-1 levels, possibly reflecting a protective response against the progression of CAD. Like patients with mild CAD, such as 1-VD, we also reported subjects with carotid plaque to have higher plasma HO-1 levels than those without plaque among 136 subjects with no history of cardiovascular disease undergoing carotid ultrasonography [29]. In contrast, like patients with severe CAD, such as 3-VD, we [18] and others [19] reported that patients with peripheral artery disease (PAD) had low plasma HO-1 levels. Patients with PAD usually have severe atherosclerosis in iliac and femoral arteries and often have CAD, especially 3-VD [30,31]. Although the mechanism of relatively low HO-1 levels in patients with severe atherosclerosis, such as 3-VD and
10
PAD, remains unclear, HO-1 defensive response to oxidative stress was reported to be attenuated at advanced age [32] and late stage of DM [33]. HO-1 induction were decreased in aged rats [32]. In diabetic mice, HO-1 activity was increased in early stage of DM while decreased in late stage of DM [33]. Therefore, the long duration of severe stress condition may cause some disruption of HO-1 defense system. HO-1 is an enzyme that degrades heme to CO, iron and biliverdin/bilirubin, but no study has described the correlation between HO-1 and bilirubin levels in patients with stable CAD. Li et al. [34] measured serum HO-1 and total bilirubin levels in 60 patients with stroke and 50 with transient ischemic attack (TIA). They showed that HO-1 levels were higher in patients with stroke than in those with TIA, in contrast to lower bilirubin levels being noted in patients with stroke. In our present study, we found higher HO-1 and lower bilirubin levels in patients with CAD than in those without CAD, but no significant correlation was noted between HO-1 and bilirubin levels. In a multivariate analysis, HO-1 levels were a significant factor for CAD independent of bilirubin levels and atherosclerotic risk factors, but bilirubin levels were not. Hence, the responses to the progression of atherosclerosis would be different between blood HO-1 and bilirubin levels. However, we just measured plasma HO-1 levels and did not evaluate HO-1 activity and protein expression in blood leukocytes. Moreover, heme metabolism and oxygenase pathway is involved in numerous biological processes including oxygen transport, cellular respiration, oxidative biotransformations, and host defence [3]. The lack of correlation between HO-1 and bilirubin levels may thus have been affected by these factors. Further prospective studies will be needed in order to clarify the roles of both HO-1 and bilirubin levels in the process of atherosclerotic disease, such as CAD. Moreover, in the present study, the sensitivity and specificity to predict CAD were 69% and 52% for HO-1 (>0.35 ng/mL) and 52% and 61% for bilirubin level
11
(<0.70 mg/dL), and AUC for HO-1 levels tended to be larger than AUC for bilirubin levels. Although bilirubin levels have often been reported to be low in patients with CAD and to be a biomarker for CAD [7-11], our study suggests that HO-1 levels as well as bilirubin levels can be a biomarker associated with CAD. Our study has several limitations. First, the number of study patients was small (n=262), and only 262 of the 374 patients had their bilirubin levels measured, as this measurement was left to the decision of the doctors in charge. Second, angiography was used to evaluate coronary atherosclerosis. Angiography cannot visualize plaques and only shows lumen characteristics. Third, our study did not analyze any HMOX1 gene polymorphisms. Since some HMOX1 gene polymorphisms have been reported to be associated with CAD [35], these polymorphims may have affected plasma HO-1 levels in our patients with CAD. Fourth, our study was cross-sectional in nature and was unable to establish causality, since it only showed some associations and proposed hypotheses. Finally, our study was performed in Japanese patients undergoing coronary angiography, who are generally considered to be a highly select population at high risk for CAD. Our results therefore may not be applicable to the general or other ethnic populations.
5. Conclusion Patients with CAD were found to have high HO-1 levels and low total bilirubin levels in blood. However, no signficant correlation was found between HO-1 and bilirubin levels. HO-1 levels were a significant factor associated with CAD independent of bilirubin levels and atherosclerotic risk factors. Our data suggest the usefulness of HO-1 as a biomarker for CAD and a possible role of HO-1 in the development of CAD.
Funding 12
This study was supported by a grant from Honjo International Scholarship Foundation. Financial funding was also provided by Pfizer Japan Inc., and Bayer Yakuhin Ltd.; however, these sponsors had no role in the design, analysis, or interpretation of our study.
Declaration of interest Our study has no conflicts of interest to disclose.
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Table 1. Clinical characteristics and plasma HO-1 levels and serum total bilirubin levels of patients with and without CAD CAD(-) (n=124)
CAD P value CAD(-)
(n=138)
vs. CAD
Age (years)
66±12
<0.01
Gender (male)
75 (60%)
<0.001
BMI (kg/m2)
24.2±4.8
NS
Hypertension
75 (60%)
<0.005
SBP (mmHg)
130±22
NS
133±21
13 (10%)
<0.001
51 (37%)
HbA1c (%)
5.9±0.7
<0.002
6.3±1.0
Smoking
43 (35%)
NS
Hyperlipidemia
46 (37%)
Statin
2-VD
3-VD
(n=50)
(n=46)
(n=52)
Among 4 groups
72±7
<0.01
33 (72%)
37 (88%)
<0.01
23.9±3.4
23.6±3.4
23.6±2.7
NS
108 (78%) 40 (80%)
34 (74%)
34 (81%)
<0.01
133±21
140±23
130±23
<0.05
13 (26%)
22 (48%)
16 (38%)
<0.001
6.1±0.8
6.5±1.1
6.4±1.1
<0.001
62 (45%)
28 (56%)
18 (39%)
16 (38%)
NS
<0.01
77 (56%)
26 (52%)
26 (57%)
25 (60%)
<0.025
28 (23%)
<0.001
60 (43%)
21 (42%)
19 (41%)
20 (48%)
<0.01
LDL-C (mg/dL)
112±29
NS
114±36
109±36
115±33
121±38
NS
HDL-C (mg/dL)
59±15
<0.001
50±12
51±12
51±11
48±12
<0.001
hsCRP levels
0.50
NS
0.63
0.58
0.58
0.89
<0.05
(mg/L)
[0.27, 1.50]
[0.34, 1.60] [0.31, 0.90]
[0.31, 1.54]
[0.46, 2.72]
>1.0 mg/L
41 (33%)
NS
45 (33%)
17 (37%)
19 (45%)
<0.05
0.35
<0.01
0.46
0.51
0.45
0.44
<0.01
[0.30, 0.61]
[0.38, 0.66]
[0.26, 0.59]
[0.29, 0.57]
HO-1 levels (ng/mL) >0.35 ng/mL Total bilirubin levels
[0.23, 0.53]
68±11
P value
68±10
Diabetes mellitus
69±9
1-VD
111 (80%) 41 (82%) 23.9±3.4
9 (18%)
59 (48%)
<0.001
95 (69%)
39 (78%)
28 (61%)
28 (67%)
<0.005
0.75
<0.02
0.69
0.70
0.68
0.66
NS
[0.57, 0.84]
[0.59, 0.81]
[0.53, 0.88]
[0.56, 0.83]
72 (52%)
24 (48%)
23 (50%)
25 (60%)
(mg/dL)
[0.61, 0.95]
<0.70 mg/dL
48 (39%)
<0.05
Data represent the mean±SD or the number (%) of patients, except for hsCRP, HO-1 and total bilirubin levels which are presented as the median value and interquartile range. BMI = body mass index; SBP = systolic blood pressure; LDL-C = low-density lipoprotein cholesterol; HDL-C = high-density lipoprotein cholesterol. 19
NS
Table 2. Factors associated with CAD (Multiple logistic regression analysis of the 262 study patients) Odds ratio
(95% CI)
P value
Age (per 10-year increase)
1.50
(1.14-1.96)
<0.005
Male gender
3.54
(1.81-6.90)
<0.001
Statin use
2.66
(1.41-5.02)
<0.005
Diabetes mellitus
3.21
(1.56-6.63)
<0.005
Low HDL-C level (<40 mg/dL)
2.49
(1.01-6.22)
<0.05
HO-1 level (>0.35 ng/mL)
2.32
(1.29-4.17)
<0.01
The dependent variable was the presence of CAD. The analysis included age, gender, hypertension, DM, body mass index, smoking, hyperlipidemia, statin use, and HDL-cholesterol (<40 mg/dL), hsCRP (>1.0 mg/L), HO-1 (>0.35 ng/mL) and total bilirubin (<0.70 mg/dL) levels.
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Figure Legends Figure 1. Plasma hsCRP levels and the presence of CAD or the number of stenotic coronary vessels. Plasma hsCRP levels tended to be higher in CAD than in CAD(-) (p=NS)(left). However, hsCRP levels stepwisely increased depending on the number of stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD, respectively, and were highest in 3-VD (p<0.05 by Kruskal-Wallis test)(right). Figure 2. Plasma HO-1 levels and the presence of CAD or the number of stenotic coronary vessels. Plasma HO-1 levels were significantly higher in CAD than in CAD(-) (p<0.01)(left). Notably, HO-1 levels in 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively, and were highest in 1-VD (p<0.01 by KruskalWallis test)(right). Figure 3. Serum total bilirubin levels and the presence of CAD or the number of stenotic coronary vessels. Serum bilirubin levels were significantly lower in CAD than in CAD(-) (p<0.02)(left). Bilirubin levels tended to stepwisely decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among 4 groups (p=NS by Kruskal-Wallis test)(right). Figure 4. Correlation between HO-1 levels and total bilirubin levels. There was no significant correlation between plasma HO-1 levels and serum total bilirubin levels (P=NS).
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Figure 5. ROC curves of HO-1 and bilirubin levels for the diagnostic abilitiy of CAD. The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65). However, this difference did not reach statistical significance.
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Figure 1. Plasma hsCRP levels and the presence of CAD or the number of stenotic coronary vessels. Plasma hsCRP levels tended to be higher in CAD than in CAD(-) (p=NS)(left). However, hsCRP levels stepwisely increased depending on the number of stenotic vessels: 0.50 in CAD(-), 0.58 in 1-VD, 0.58 in 2-VD, and 0.89 mg/L in 3-VD, respectively, and were highest in 3-VD (p<0.05 by Kruskal-Wallis test)(right).
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Figure 2. Plasma HO-1 levels and the presence of CAD or the number of stenotic coronary vessels. Plasma HO-1 levels were significantly higher in CAD than in CAD(-) (p<0.01)(left). Notably, HO-1 levels in 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.51, 0.45, and 0.44 ng/mL, respectively, and were highest in 1-VD (p<0.01 by KruskalWallis test)(right).
24
Figure 3. Serum total bilirubin levels and the presence of CAD or the number of stenotic coronary vessels. Serum bilirubin levels were significantly lower in CAD than in CAD(-) (p<0.02)(left). Bilirubin levels tended to stepwisely decrease depending on the number of stenotic vessels: 0.75 in CAD(-), 0.70 in 1-VD, 0.68 in 2-VD, and 0.66 mg/dL in 3-VD, but no significant difference was found among 4 groups (p=NS by Kruskal-Wallis test)(right).
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Figure 4. Correlation between HO-1 levels and total bilirubin levels. There was no significant correlation between plasma HO-1 levels and serum total bilirubin levels (P=NS).
26
Figure 5. ROC curves of HO-1 and bilirubin levels for the diagnostic ability of CAD. The AUC for HO-1 levels was 0.62 (95%CI=0.55–0.68), which tended to be larger than the AUC for bilirubin levels (0.58; 95%CI=0.52–0.65). However, this difference did not reach statistical significance.
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Author contributions statement Yoshimi Kishimoto: Investigation, Writing - Original Draft; Emi Saita: Investigation; Hanako Niki, Susumu Ibe, Tomohiko Umei, Kotaro Miura, and Yukinori Ikegami: Data Curation; Reiko Ohmori and Kazuo Kondo: Supervision; Yukihiko Momiyama: Project administration, Formal analysis, Resources, Writing - Review & Editing.
Highlights 1) Plasma HO-1 levels were significantly higher in patients with CAD, especially in 1VD, than in those without CAD. 2) In contrast, serum bilirubin levels were significantly lower in patients with CAD than in those without CAD. 3) There was no correlation between HO-1 and bilirubin levels. 4) In a multivariate analysis, HO-1, but not bilirubin, level was an independent factor for CAD.
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