Correspondence: Song-lin Du, Department of Thoracic and Cardiovascular Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, 510515, Guangzhou, P. R China. Tel/Fax: � 86 020 61641114. E-mail: gzdusonglin@126.com

(Received 16 September 2012 ; accepted 27 February 2013 )

ORIGINAL ARTICLE

Serum salusin- a levels are inversely correlated with the presence and severity of coronary artery disease

SONG-LIN DU 1 , WU-JUN WANG 1 , JUN WAN 1 , YUE-GANG WANG 2 , ZHEN-KANG WANG 1 & ZHEN ZHANG 1

Departments of 1 Thoracic and Cardiovascular Surgery and 2 Cardiology , Nanfang Hospital of Southern Medical University , Guangzhou , Guangdong , P. R. China

Abstract We sought to measure serum salusin- α levels in patients with coronary artery disease (CAD) and to assess their correlation with the severity of the disease. We enrolled 172 patients with CAD and 91 controls. We assessed the angiographic severity of CAD by coronary atherosclerosis index (CAI) and detected serum salusin- α levels by enzyme-linked immunosorbent assay (ELISA). We demonstrated that CAD patients had signifi cantly lower serum salusin- α levels compared to controls. Moreover, serum salusin- α levels were independently and negatively correlated with the presence and severity of CAD. These fi ndings indicated that salusin- α might serve as a potential biomarker for predicting the development and progression of CAD.

Key Words: Disease , cardiovascular , coronary artery disease , biological factors , salusin- α

Introduction

Coronary artery disease (CAD) is one of the pre-dominant causes of death and imposes a great bur-den on healthcare expenditure worldwide [1]. Therefore, it is of great importance to determine valuable tools for predicting the presence and the severity of CAD earlier. In recent years, biomarkers have increasingly emerged as important alternatives to traditional methods for diagnosis and risk stratifi -cation in cardiovascular diseases [2].

Many bioactive peptides such as cardiac troponin I and T, B-type natriuretic peptide (BNP) and C-reactive protein (CRP) have been well-established as important biomarkers for providing independent diagnostic and/or prognostic value in the fi eld of cardiovascular medi-cine. Salusins are a class of newly identifi ed bioactive peptides discovered by bioinformatics analyses of a full-length cDNA library [3].There are two highly homolo-gous isoforms of salusins identifi ed and characterized by Shichiri et al., which were designated as salusin- α and salusin- β , respectively [3]. It has been reported that salusins are involved in the pathophysiologic pro-cesses of CAD and the two salusin isoforms have oppo-site effects on the pathogenesis of atherosclerosis [4].

The salusin- α suppresses human foam cell formation and thus have anti-atherosclerotic effects. However, salusin- β stimulates the formation of human mac-rophage foam cell and accelerates the development of atherosclerosis [4].

Salusin- α is the major circulating form of salusin in human plasma [4,5]. Previous studies have demon-strated that serum salusin- α levels were decreased in acute coronary syndrome (ACS) patients [4] and cor-related negatively with maximum intima-media thick-ness (IMT) and carotid atherosclerosis in essential hypertension patients [6]. However, the relationship between serum salusin- α levels and the development and progression of CAD has never been fully eluci-dated. Therefore, the aim of the present study was to clarify the association between serum salusin- α levels and the angiographic severity of CAD.

Materials and methods

Participants

This study included 263 consecutive patients under-going coronary angiography from July 2011 to June

Scandinavian Journal of Clinical & Laboratory Investigation, 2013; 73: 339–343

ISSN 0036-5513 print/ISSN 1502-7686 online © 2013 Informa HealthcareDOI: 10.3109/00365513.2013.783227

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2012 for the diagnosis and interventional treatment of CAD in Nanfang Hospital of Southern Medical University, Guangzhou, Guangdong, P. R. China. Conventional invasive coronary angiography was performed according to standard protocols. Angio-graphic CAD was defi ned as stenosis of 50% or greater narrowing of the diameter in coronary arter-ies. Patients were divided into two groups according to the presence (CAD group, n � 172) or absence (Control group, n � 91) of angiographic CAD. Dia-betes was defi ned as fasting plasma glucose (FBG) � 7.0 mmol/L, 2-hour postprandial plasma glucose � 11.1 mmol/L, or use of hypoglycemic agents or insulin shots.

Patients were excluded on the basis of having recent acute coronary syndromes ( � 3 months), previous coronary by-pass surgery or percutane-ous coronary intervention (PCI) ( � 6 months), suspected myocarditis or pericarditis, systemic infl ammatory diseases, advanced hepatic or renal disease and known malignant disease. The study protocol was approved by the ethics committee of Nanfang Hospital, and all patients provided informed consent.

Coronary angiography analyses

Angiographic analyses were carried out by two expe-rienced interventional cardiologists blinded to the study protocol. The severity of CAD was assessed by coronary atherosclerosis index (CAI), which was defi ned as the sum of the following scores by assigning points to each lesion as follows: No sig-nifi cant stenosis of the luminal diameter, 0; stenosis of less than 25%, 1; 25 – 49% stenosis, 2; 50 – 74%, 3; 75 – 100%, 4 [8].

Blood chemistry measurement

Peripheral blood samples were drawn from the ante-cubital vein after overnight fasting and before angio-graphic procedures. After clotting, all the blood samples were centrifuged at 3000 g for 10 min and stored at � 80 ° C until use. Serum lipid profi les, including total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-c) and density lipoprotein cholesterol (HDL-c) were deter-mined by enzymatic procedures using an autoanaly-ser (Olympus AU5400 Automatic Biochemical Analyzer, Olympus Co., Japan). N-terminal pro BNP (NT-proBNP) levels were detected in Roche COBAS6000 automatic electrochemiluminescence immunoassay analyzer (Roche Ltd, Basel, Switzer-land). High-sensitivity CRP (hs-CRP) levels were measured on an Immage 800 Immunochemistry Sys-tem (Beckman Coulter, CA, USA). Serum salusin- α levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit

with high sensitivity and excellent specifi city for detection of human salusin- α (Bachem, Bubendorf, Switzerland) [5].

Statistical analysis

All statistical analyses were performed using 13.0 (SPSS Inc, IL, USA). Kolmogorov-Smirnov test was used to analyze data normality. Data were reported as mean value and SD or median and the inter-quartile range (IQR) if continuous and as n if categorical. Characteristics of subjects between the two groups were analyzed using unpaired t -test, Mann-Whitney U test or Chi-square test when appropriate. Simple/multivariate logistic regression was performed to identify independent predictors for CAD. Spearman rank correlation coeffi cient was employed to determine the correlation between serum salusin- α levels and other parameters (NT-proBNP levels, hs-CRP levels and CAI scores). Mul-tivariate stepwise regression analysis was performed to determine the independent parameters correlated with CAI. A two-tailed probability ( p ) level � 0.05 was considered statistically signifi cant.

Results

Baseline clinical characteristics

The baseline characteristics of the two groups are shown in Table I. The patients with CAD were older and had signifi cantly lower HDL-c levels and higher hs-CRP levels compared to controls. In addition, cardiovascular risk factors such as diabetes and smoking seemed to be more prevalent in CAD patients.

Serum salusin- α levels

Our study demonstrated that CAD patients had signifi cantly lower serum salusin- α levels com-pared to controls (1.46 � 0.46 vs. 2.03 � 0.55 ng/ml, p � 0.01).

Relationship between serum salusin- α levels and the presence of CAD

As demonstrated in Table II, simple logistic regres-sion analysis revealed that age, systolic blood pres-sure (SBP), HDL-c levels, the presence of diabetes and smoking as well as serum salusin- α levels showed a trend ( p � 0.20) toward an association with the presence of angiographic CAD (Table II). Multi-variate logistic regression including these variables was run stepwise to assess their independent contri-bution to the presence of CAD. In the stepwise anal-ysis, serum salusin- α levels were independently and negatively correlated with the presence of

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Salusin- α and coronary artery disease 341

angiographic CAD (OR 0.113, 95% CI 0.060 – 0.215; p � 0.01).

Correlations between serum salusin- α levels and CAI scores

As demonstrated in Figure 1, serum salusin- α levels negatively correlated with CAI scores ( ρ : � 0.260, p � 0.001). As the CAI scores, NT-proBNP and hs-CRP levels were not normally distributed, loga-rithmic (log) transformed value was performed to meet a normal distribution for multivariate linear

regression analysis. Multivariate linear regression analysis showed that serum salusin- α levels was still the signifi cant and negative determinants of log CAI scores (Table III).

Correlations between serum salusin- α levels and NT-proBNP/hs-CRP levels

We also correlated serum salusin- α levels with other cardiovascular biomarkers such as NT-proBNP and hs-CRP. We demonstrated that serum salusin- α lev-els were neither correlated with NT-proBNP levels

Table II. Logistic regression analysis for the presence of angiographic CAD.

VariablesSimple regression

OR (95% CI) P valueMultiple regression

OR (95% CI) P value

Age (per year) 1.028 (1.004 – 1.052) 0.020 1.027 (0.999 – 1.056) 0.055Male (yes) 0.744 (0.445 – 1.244) 0.260BMI (per kg/mm 2 ) 1.057 (0.958 – 1.167) 0.093SBP (per mm/Hg) 1.011 (0.997 – 1.026) 0.126 1.005 (0.988 – 1.022) 0.588DBP (per mmHg) 1.012 (0.990 – 1.034) 0.282TC (per mmol/L) 1.120 (0.878 – 1.428) 0.363TG (per mmol/L) 1.212 (0.889 – 1.653) 0.224LDL-c (per mmol/L) 1.212 (0.900 – 1.632) 0.205HDL-c (per mmol/L) 0.291 (0.094 – 0.898) 0.032 0.228 (0.058 – 0.895) 0.034NT-proBNP (per ng/L) 1.001 (1.000 – 1.002) 0.173 1.001 (1.000 – 1.002) 0.070Hs-CRP (per mg/L) 1.142 (0.955 – 1.336) 0.145 1.156 (0.951 – 1.405) 0.146Diabetes (yes) 2.196 (1.155 – 4.173) 0.016 1.836 (0.857 – 3.932) 0.118Smoking (yes) 0.623 (0.367 – 1.055) 0.078 0.595 (0.362 – 1.034) 0.103Statins (yes) 0.740 (0.398 – 1.376) 0.341ACEI/ARB (yes) 0.917 (0.538 – 1.563) 0.749Serum salusin- α levels

(per ng/mL)0.118 (0.064 – 0.216) � 0.01 0.113 (0.060 – 0.215) � 0.01

OR, odds ratio; CI, confi dence interval; other abbreviations are as in Table I.

Table I. Baseline clinical characteristics.

VariablesControl ( n � 91)

CAD ( n � 172) P value

Age (years) 61.47 � 10.73 64.91 � 11.43 0.018Male ( n ) 49 105 0.260BMI (kg/mm 2 ) 24.43 � 2.68 24.91 � 2.58 0.158SBP (mm/Hg) 131.21 � 18.41 134.81 � 17.90 0.125DBP (mmHg) 80.70 � 10.79 82.33 � 12.10 0.283TC (mmol/L) 4.54 � 1.10 4.73 � 1.01 0.165TG (mmol/L) 1.84 � 0.84 1.99 � 0.85 0.153LDL-c (mmol/L) 2.66 � 0.79 2.83 � 0.89 0.122HDL-c (mmol/L) 1.04 � 0.19 0.97 � 0.24 0.015NT-proBNP (ng/L) 260 (96 – 322) 285 (210 – 344) 0.077Hs-CRP (mg/L) 0.61 (0.43 – 1.02) 0.87 (0.65 – 1.42) � 0.001Cardiac risk factors (yes)

Diabetes ( n ) 15 52 * 0.013Smoking ( n ) 31 78 0.076

Cardiovascular medication (yes)Statins, ( n ) 18 43 0.336ACEI/ARB ( n ) 31 62 0.749

All values are mean � SD, median with interquartile range or n . CAD, coronary artery disease; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglycerides; LDL-c, low density lipoprotein cholesterol; HDL-c, high density lipoprotein cholesterol; NT-proBNP, N-terminal pro B-type natriuretic peptide; hs-CRP, high-sensitivity C-reactive protein; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker.

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( ρ : � 0.099, p � 0.111) nor correlated with hs-CRP levels ( ρ : � 0.101, p � 0.103).

Discussion

In the current study, we hypothesized that there might be a correlation between serum salusin- α lev-els and the presence and severity of CAD. We have demonstrated that serum salusin- α levels were: (i) Signifi cantly lower in patients with CAD compared with those without CAD, (ii) the independent pre-dictors for the presence of CAD, and (iii) indepen-dently associated the severity of CAD.

Early detection and risk stratifi cation of CAD largely depend on better understanding of the molec-ular mechanisms and identifi cation of reliable bio-markers that could refl ect specifi c biological or pathological processes of the disease. Salusin- α , a peptide consist of 28 amino acids, is expressed in human monocytes/macrophages, vascular endothelial/smooth muscle cells (VSMCs), central nervous system, and the kidney [3,10]. Salusin- α could suppress the expression of acyl-CoA cholesterol

acyltransferase-1 (ACAT-1), which stores cholesterol ester (CE) converted from free cholesterol and spon-taneously increases during monocytic differentiation into macrophages [4,11]. Therefore, salusin- α could suppresses foam cell formation by down-regulation of ACTA-1. As the foam cell formation initiates the development of CAD, salusin- α is considered as a suppressive factor for the development of CAD.

In the present study, we found decreased serum salusin- α levels in patients with CAD compared with controls, which is in accordance with a previous study [4]. It should be recognized that controls enrolled in this study were not really healthy controls because some of them might have had lower stenosis in their coronary arteries, having shown symptoms of CAD. However, a more recent study conducted in Chinese people by Ti et al. demonstrated that serum salusin- α levels detected in healthy controls using the same ELISA kit were even 25% higher than those of ‘ controls ’ in the present study [5]. This study sup-ported our study and further confi rmed the associa-tion between lower serum salusin- α levels and CAD. As there is a signifi cant difference in age between patients with and without CAD, we performed mul-tivariate logistic regression to identify independent predictors for CAD. The results indicated that serum salusin- α levels were independently associated with the presence of CAD. All these results supported the cardiovascular protective role of salusin- α and sug-gested that salusin- α might be a potential biomarker for evaluating future CAD risk. We also demonstrated that serum salusin- α levels were not correlated with other well-established cardiovascular biomarkers such as NT-proBNP and hs-CRP, which provided valuable information on the disease-promoting role of salusin- α signaling pathways in CAD and to truly appreciate salusin- α uniqueness.

CAI is an effective and reliable scoring system to assess the severity of CAD [12]. The present study also revealed a signifi cant negative correlation between serum salusin- α levels and the severity of CAD. The correlation was still signifi cant after adjust-ing for potential confounders. These results indicated that salusin- α might also contribute to delay the pro-gression of CAD and serum salusin- α levels might also be used for risk stratifi cation in CAD patients. However, the precise mechanisms of this biologic action remained to be further investigated. Besides, the correlation between serum salusin- α levels and CAI scores was weak and its clinical signifi cance should be interpreted cautiously.

However, there are still some limitations to be found in this study. First, most of the patients included were referred for coronary angiography because of the occurrence of cardiac symptoms. These might have resulted in a higher CAD preva-lence in this study population group than those in the common population. Second, this study was cross-sectional with a relatively small sample size,

Figure 1. Correlations between serum salusin- α levels and the severity of FCAD evaluated by CAI score. CAI, coronary atherosclerosis index.

Table III. Multivariate linear regression analysis for the determinants of log CAI scores.

Variables β t P value

Age � 0.041 � 0.555 0.579BMI 0.094 1.261 0.209SBP 0.120 1.478 0.141DBP � 0.083 � 1.044 0.298TC 0.028 0.372 0.710TG 0.065 0.866 0.388LDL-c 0.006 0.083 0.934HDL-c 0.104 1.348 0.180log NT-pro BNP levels 0.055 0.731 0.466log hs-CRP levels 0.198 2.537 0.012Serum salusin- α levels � 0.250 � 3.198 0.002

All abbreviations are as in Table I.

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Salusin- α and coronary artery disease 343

which may cause errors in the correlations due to other factors not taken into account in the statisti-cal analysis. Third, all the patients recruited were middle-aged or elderly Chinese population. Future research should therefore make use of a wider range of subjects in terms of age, sex, and race.

To summarize, CAD patients had signifi cantly lower serum salusin- α levels compared to those with-out CAD. Moreover, serum salusin- α levels were negatively correlated with the presence and severity of CAD. These fi ndings indicated that salusin- α might serve as a potential biomarker for predicting the development and progression of CAD and ther-apeutic interventions by promoting salusin- α signal-ing pathways to delay the progressive process of CAD warrants further investigations.

Declaration of interest : The authors report no confl ict of interest. The authors alone are responsible for the content and writing of the article.

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