CLB-08814; No. of pages: 6; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
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Serum cystatin C levels are associated with coronary artery disease and its severity Gan-nan Wang a, Kai Sun a, De-liang Hu a, Hong-hao Wu a, Xiao-zhi Wang b, Jin-song Zhang a,⁎ a b
Department of Emergency, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, PR China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, PR China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 29 April 2014 Accepted 18 July 2014 Available online xxxx Keywords: Cystatin C Coronary artery disease Gensini score Glomerular filtration rate
a b s t r a c t Objectives: Serum cystatin C has been established as a predictor of cardiovascular events. The aim of this study was to evaluate the role of cystatin C in determining the presence and the severity of patients with coronary artery disease (CAD). Design and methods: A total of 936 subjects without overt renal disease were included in this cross-sectional study. Among them were 714 patients with CAD and 222 without based on coronary angiography. Subjects were further divided into four groups according to cystatin C quartile. Serum cystatin C was measured using particleenhanced immunoassay method. The study analyzed the relationship of cystatin C levels with the presence and severity of CAD, including the number of stenotic vessels involved and Gensini score. Results: Serum cystatin C levels were significantly higher in patients with CAD than those without (P b 0.001), and significantly increased as the involvement of coronary vessels increased (P b 0.001). The prevalence of CAD and its severity assessed by Gensini score were also significantly greater in the highest quartile of cystatin C (P b 0.001). Moreover, cystatin C levels were independently correlated with the presence of CAD in a multivariate logistic regression model (P = 0.023) and were positively correlated with Gensini score by linear regression analysis (standardized β = 0.083, P = 0.010). Conclusions: Elevated serum cystatin C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. It is suggested that cystatin C might play a role in CAD diagnosis and serve as a marker of CAD severity. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Coronary artery disease (CAD) singly is the most frequent cause of death. Over seven million people every year die from CAD, accounting for 11.2% of all deaths [1]. Early reperfusion and adequate drug treatment are the cornerstones of CAD management, limiting cardiac dysfunction and subsequent morbidity and mortality. Early diagnosis and treatment are the most important predictors of long-term outcome [2]. Although the application of high-sensitive troponin has improved the accuracy of the diagnosis, it still needs other markers to evaluate the early diagnosis and severity of CAD [3]. Therefore, the new, early measurable biomarkers might play crucial roles in the initial assessment of CAD. Abbreviations: CAD, coronary artery disease; Cys C, cystatin C; SCr, serum creatinine; eGFR, estimated glomerular filtration rate; CAG, coronary angiography; SBP, systolic blood pressure; DBP, diastolic BP; HR, heart rate; FPG, fasting plasma glucose; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Fg, fibrinogen; ROC, receiveroperating characteristic; AUC, area under the curve. ⁎ Corresponding author. E-mail address: [email protected] (J. Zhang).
Cystatin C (Cys C) is a low molecular mass protein produced by all nucleated cells at a constant rate regardless of variations of the intracellular environment and extracellular environment, and acts as a cysteine protease inhibitor. Cys C dose does not form a complex with other serum proteins in blood, and is filtered by the renal glomeruli to be reabsorbed and degraded in the proximal tubules [4]. Over the past few years, serum levels of Cys C had been proposed as a more reliable marker of kidney function than serum creatinine (SCr), and as an alternative parameter for estimating glomerular filtration rate (GFR) [5]. But Cys C was also associated with CAD independently of renal disease [6]. Serum Cys C has been established as a predictor of cardiovascular events and mortality in patients with non-ST-elevation acute coronary syndrome [7], ST-elevation myocardial infarction [8], and suspected CAD [6], but also in the general population [9] and elderly persons [10]. Although the predictive potential in CAD seems to be high, the diagnostic value of Cys C for acute myocardial ischemia remains unexplored so far. There is limited information on the significance of Cys C as a predictor of fatal events in patients with CAD recruited irrespective of renal function [11]. The present study was designed to evaluate whether there was a relationship between serum Cys C levels and the presence of CAD, its severity,
http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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G. Wang et al. / Clinical Biochemistry xxx (2014) xxx–xxx
and the number of vessels involved in CAD patients documented with coronary angiography (CAG) but without overt kidney disease.
Materials and methods Study subjects The subjects were consecutive 936 patients (62.26 ± 10.74 years old, 65.9% males) who underwent CAG under the suspected diagnosis of CAD between December 2010 and December 2012 in the Department of Cardiology at the First Affiliated Hospital of Nanjing Medical University (Nanjing, China). CAD has been evaluated by CAG based on maximal luminal narrowing of visual stenosis and defined as the presence of at least one stenosis ≥50% in at least one of 15 coronary segments of the three major coronary arteries [12]. Furthermore, the left anterior descending artery, left circumflex artery, and right coronary artery were examined to evaluate the number of stenotic coronary arteries as 0 to 3-vessel disease. If the left main trunk was involved, this was evaluated as a 2-vessel disease by itself [4]. The results of CAG were evaluated by two expert investigators. The severity of coronary stenosis was assessed in the direction that showed the most severe stenosis according to Gensini scoring [13]. Patients with renal dysfunction (creatinine-based estimated GFR b 60 mL/min/1.73 m2, SCr N 133 μmol/L), valvular heart disease, life-threatening arrhythmias, malignant, infectious and inflammatory disease, and thyroid disease were excluded from the study. The study protocol was reviewed and approved by the Institutional Review Boards for Human Studies of Nanjing Medical University. Written informed consent was obtained from all study participants.
Statistical analysis For the present study, all the subjects were divided into 4 quartiles on the basis of baseline serum Cys C levels. Clinical characteristics as well as biochemical and angiographic outcomes were compared in each Cys C quartile. Continuous variables with normal distribution were expressed as mean ± SD, while percentages were used to express categorical variables. The conformity of data with a normal distribution was analyzed using a Kolmogorov–Smirnov test. In multiple-group comparisons, analysis of variance (ANOVA) was used for parameters conforming to a normal distribution. The student's t test was carried out for comparison of two groups. However, the Kruskal–Wallis H test was used for parameters not conforming to a normal distribution in multi-group comparisons while the Mann–Whitney u test in twogroup comparisons. Categorical variables of subjects with and without CAD as well as stratified by baseline Cys C levels were analyzed by the chi-square test. In addition, the relationships between data were tested using Pearson's or Spearman's correlation analysis. A linear regression analysis was applied to determine the correlation between serum Cys C and severity of CAD evaluated by Gensini score. Moreover, factors considered to affect CAD were evaluated by the multivariate logistic regression model. A receiver-operating characteristic (ROC) curve analysis was drawn to identify the optimal cut-off points of Cys C levels (to determine maximal sensitivity and specificity) for predicting CAD. The area under the curve (AUC) value was calculated to determine accuracy of the test. All the statistical tests were performed in SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). A two-tailed P value of less than 0.05 was considered statistically significant. Results
Clinical assessment and laboratory measurements
Comparisons of patients with and without CAD
The detailed histories of patients, including demographics characteristics and cardiovascular risk factors, were recorded. The cardiovascular risk was assessed in terms of smoking (≥1 cigarette per day for one year or more), hypertension (blood pressure ≥140/90 mmHg or the use of antihypertensive drugs), and diabetes (fasting plasma glucose ≥7.0 mmol/L or random plasma glucose ≥11.1 mmol/L with classic symptoms of hyperglycemia or patients was on anti-diabetic medications) [14]. Systolic and diastolic blood pressures (BP) were measured using standard methods. The venous blood samples were collected after overnight fasting before CAG. Cys C, SCr, fasting plasma glucose (FPG), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by an automatic biochemical analyzer (AU5400, Olympus, Tokyo, Japan). The serum concentration of Cys C was determined by particle-enhanced turbidimetric immunoassay (PETIA) method. According to the manufacturer's specification, the assay was linear from 0.20 to 8.00 mg/L (r2 ≥ 0.996). Within-run coefficient of variation (CV) was 4.2 and 2.5% at cystatin C concentrations of 0.20 and 2.00 mg/L, respectively. Between-run CV was 8.5 and 6.6% at the same concentrations. The average analytical recovery was 99%. Bilirubin ≤ 400 μmol/L, hemoglobin ≤ 5 g/L, chyle ≤ 0.50%, Vitamin C ≤ 0.5 g/L and heparin sodium ≤ 100 IU/mL did not interfere with the assay. Moreover, the intraindividual biological variations ranged from 2% to 10%. The measurement was conducted in the laboratory recognized by the China National Accreditation Service for Conformity Assessment (CNAS). Fibrinogen (Fg) was measured using Von Clauss method on an automatic coagulometer (CA-7000, Sysmex, Kobe, Japan). Estimated glomerular filtration rate (eGFR) was calculated according to the simplified Modification of Diet in Renal Disease (MDRD) equation based on the level of SCr (eGFRMDRD = 186.3 ∗ SCr−1.154 ∗ age−0.203 ∗ 0.742 (if female) where eGRF is expressed as mL/min/1.73 m2 of body surface area and SCr is expressed as mg/dL.) [15].
The characteristics of study subjects are presented in Table 1. There were 714 patients with CAD and 222 without CAD according to coronary angiograph results. CAD patients were significantly older, more frequently male and smoking, and also had significantly higher DBP, FPG, AST, SCr, eGFR, Fg, and Gensini score. However, there was no significant difference in histories of hypertension and diabetes, SBP, HR, and lipid profiles between the groups. Moreover, serum Cys C levels were significantly higher in patients with CAD than those without (1.08 ± 0.27 vs. 1.00 ± 0.24 mg/L, P b 0.001; Fig. 1A). All the subjects were further divided into four groups according to the number of stenotic vessels with coronary involvement. The levels of Cys C were 1.00 ± 0.24, 1.05 ± 0.24, 1.08 ± 0.26, and 1.14 ± 0.30 mg/L in groups of patients Table 1 Clinical and biochemical baseline characteristics of study subjects.
Age (years) Males (%) Smoking (%) Hypertension (%) Diabetes (%) SBP (mm Hg) DBP (mm Hg) HR (bpm) FPG (mmol/L) AST (U/L) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) SCr (μmol/L) eGFR (mL/min/1.73 m2) Cys C (mg/L) Fg (g/L) Gensini score
Non-CAD (N = 222)
CAD (N = 714)
P
59.88 ± 10.39 53.6 28.4 66.2 18.0 133.10 ± 16.53 80.89 ± 10.16 71.88 ± 12.90 5.59 ± 1.52 22.35 (18.10–29.50) 4.30 ± 1.01 1.60 ± 1.04 1.14 ± 0.28 2.63 ± 0.75 71.70 ± 15.35 94.6 ± 19.1 1.00 ± 0.24 2.77 ± 0.64 0 (0–4)
63.00 ± 10.75 69.7 36.8 65.5 24.1 132.97 ± 18.08 79.20 ± 10.73 71.71 ± 11.54 6.07 ± 1.92 25.80 (19.98–54.43) 4.31 ± 1.15 1.58 ± 1.05 1.10 ± 0.28 2.70 ± 0.87 76.24 ± 15.78 91.6 ± 19.9 1.08 ± 0.27 3.10 ± 0.88 40 (20–66)
b0.001 b0.001 0.021 0.854 0.059 0.921 0.038 0.849 b0.001 b0.001 0.838 0.768 0.063 0.308 b0.001 0.048 b0.001 b0.001 b0.001
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
G. Wang et al. / Clinical Biochemistry xxx (2014) xxx–xxx
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with 0, 1, 2, and 3-vessel disease respectively, and the Cys C levels significantly ascended as the involvement of coronary vessels increased (P b 0.001) (Fig. 1B). Age, systolic blood pressure (SBP), diastolic BP (DBP), heart rate (HR), fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum creatinine (SCr), estimated glomerular filtration rate (eGFR), cystatin C (Cys C) and fibrinogen (Fg) conformed to normal distributions and were presented as mean ± SD, while aspartate aminotransferase (AST) and Gensini score, which were not normally distributed, were presented as median (interquartile range). Patient characteristics according to cystatin C quartiles The present study further compared the variables of patients according to Cys C quartiles. As shown in Table 2, the ranges of serum Cys C levels for each quartile were 0.21 to 0.88, 0.89 to 1.02, 1.03 to 1.20, and 1.21 to 2.90 mg/L, respectively. As expected, patients in higher Cys C quartiles had higher SCr and lower eGFR, were older, and were more likely to have histories of hypertension and diabetes. Furthermore, SBP and Fg were also higher in the fourth quartile than in the lower ones. The prevalence of CAD and its severity assessed by Gensini score were significantly greater in the highest quartile of Cys C. Association of Cys C levels with the presence of CAD and with CAD severity
Fig. 1. Comparisons of serum cystatin C levels in terms of the presence of coronary artery disease (A) and according to the number of stenotic vessels with coronary involvement (n = 222, 302, 208, and 204, respectively in 0, 1, 2, and 3-vessel groups) (B).
The variables considered to affect CAD were evaluated according to the multivariate logistic regression model, which included age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, LDL-C, HDL-C, SCr, eGFR, Cys C, and Fg. It was found that sex, AST, Cys C, and Fg were correlated independently with the presence of CAD (Table 3). In addition, the ability of Cys C levels to distinguish patients with CAD from those without was assessed using ROC curve analysis. The ROC curve for CAD diagnosis had an AUC of 0.588 (95% CI 0.544–0.631, P b 0.001). A 0.865 mg/L cut-off value of Cys C predicted the presence of CAD with a sensitivity and specificity of 81.8% and 30.6%, respectively (Fig. 2). Spearman's correlation analysis showed that serum Cys C levels was positively correlated with Gensini score (r = 0.133, P b 0.001). A
Table 2 Baseline characteristics and laboratory variables according to cystatin C concentration quartiles. Variables
Age (years) Males (%) Smoking (%) Hypertension (%) Diabetes (%) SBP (mm Hg) DBP (mm Hg) HR (bpm) FPG (mmol/L) AST (U/L) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) SCr (μmol/L) eGFR (mL/min/1.73 m2) Cys C (mg/L) Fg (g/L) CAD (%) Gensini score
Serum cystatin C levels (mg/L)
P
Quartile 1 (b0.89) n = 224
Quartile 2 (0.89–1.02) n = 255
Quartile 3 (1.03–1.20) n = 229
Quartile 4 (N1.20) n = 228
56.77 ± 9.95 62.1 36.2 61.2 24.1 130.87 ± 16.33 80.13 ± 10.69 72.72 ± 10.50 6.12 ± 2.05 25.60 (18.85–58.83) 4.31 ± 1.10 1.50 ± 0.92 1.12 ± 0.27 2.69 ± 0.84 66.46 ± 11.77 105.6 ± 17.8 0.78 ± 0.10 3.00 ± 0.87 68.3 20 (3.25–53.75)
60.60 ± 9.30 65.9 37.3 60.4 20.8 131.69 ± 18.14 79.19 ± 10.44 70.69 ± 11.18 5.87 ± 1.77 24.80 (19.40–44.50) 4.30 ± 1.01 1.60 ± 0.99 1.10 ± 0.27 2.68 ± 0.76 71.78 ± 12.11 96.0 ± 15.8 0.95 ± 0.04 2.92 ± 0.76 75.7 24 (5–53)
63.34 ± 10.46 69.4 36.7 65.1 17.5 132.80 ± 18.01 79.43 ± 11.10 71.78 ± 11.67 5.74 ± 1.66 24.30 (19.70–41.10) 4.30 ± 1.17 1.72 ± 1.39 1.08 ± 0.27 2.66 ± 0.87 76.03 ± 12.55 90.1 ± 15.6 1.11 ± 0.05 3.04 ± 0.87 79.0 26 (6–55)
68.42 ± 9.94 66.2 28.9 76.8 28.5 136.77 ± 17.78 79.70 ± 10.31 71.96 ± 13.89 6.09 ± 1.89 25.15 (19.25–40.70) 4.33 ± 1.19 1.50 ± 0.77 1.14 ± 0.30 2.70 ± 0.91 86.62 ± 18.66 77.5 ± 18.9 1.42 ± 0.23 3.16 ± 0.84 82.0 34 (11.25–64)
b0.001 0.430 0.199 0.001 0.033 0.002 0.796 0.307 0.086 0.763 0.987 0.081 0.089 0.972 b0.001 b0.001 – 0.018 0.005 0.005
Values are given as mean ± SD or median (interquartile range) for continuous variables, and as percentage for categorical data. Group differences (P value) were calculated by ANOVA or Kruskal–Wallis test for continuous and χ2 test for categorical variables.
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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Table 3 Analyses of variables affecting coronary artery disease according to the multivariate logistic regression model. Variables
Odds ratio
95% CI
P
Sex AST Cystatin C Fibrinogen
0.325 1.010 2.673 1.619
0.129–0.819 1.006–1.015 1.142–6.259 1.272–2.061
0.017 b0.001 0.023 b0.001
CI, confidence interval; AST, aspartate aminotransferase. Variables included in the model are age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, HDL-C, LDL-C, SCr, eGFR, cystatin C and fibrinogen.
significant association between Cys C and Gensini score was also found by linear regression analysis adjusted for age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, LDL-C, HDL-C, SCr, eGFR, and Fg (standardized β = 0.083, P = 0.010). Furthermore, the Pearson's correlation analysis demonstrated that serum Cys C levels was positively correlated with age, SBP, SCr, and Fg while negatively with eGFR (P b 0.01). Discussion CAD is the leading cause of morbidity and mortality worldwide. Deaths are mainly caused by acute coronary syndrome (ACS) induced by destabilization or rupture of an atherosclerotic plaque and formation of an occlusive thrombus on the coronary arteries [16]. Serum extracellular vesicle protein (such as Cys C) concentrations provide information regarding the presence of ACS and therefore have a potential role in the evaluation of patients suspected of CAD [17]. The present study was aimed to identify Cys C as an independent risk factor for the development and severity of CAD patients with normal creatinine-based eGFR, and it was found that serum Cys C levels were independently and positively related with CAD and its severity after adjusting for other cardiovascular risk factors. The presence and severity of CAD increased with the increasing quartiles of Cys C. However, previous studies focused on the postulated role of Cys C as a marker in predicting CAD have been controversial. In 2007, the European Cardiology Foundation recommended the prognostic use of Cys C in myocardial infarction and in the detection of long-term risks of death of patients with non-ST-elevation ACS [18]. Likewise, some studies have reported a positive and graded relationship between
Fig. 2. Receiver-operating characteristic (ROC) curve analyses for cystatin C in predicting the presence of coronary artery disease.
higher Cys C levels and increased cardiovascular disease prevalence [19–21]. Furthermore, a meta-analysis has indicated higher Cys C levels were strongly and independently associated with specific endpoints like myocardial infarction [22]. However, other studies have demonstrated that the content of Cys C decreased in the tissues of animals and people with atheromatous plaques [23,24] and an association was detected between a genetically determined decrease in Cys C expression and increased severity of CAD [25]. A recent study has suggested that lower Cys C levels may be associated with increased severity of CAD in clinically stable patients, whereas higher levels may indicate the presence of any vulnerable plaque [26]. It is well recognized that patients with impaired renal function are at significantly higher risk for cardiovascular diseases in contrast to patients without kidney disease [27]. Serum Cys C is a novel endogenous marker for renal dysfunction, whose production is constant and is less influenced by age, sex, race, muscle mass, and drug administration. Several studies have demonstrated the superiority of Cys C compared with SCr or eGFR in predicting cardiovascular events and all-cause mortality [28]. Moreover, studies have also shown that cardiovascular risk associated with higher levels of Cys C was independent of eGFR [29]. The possibility that Cys C might directly influence the occurrence of cardiovascular disease has been raised. The present study evaluated the relationship between Cys C and CAD in a consecutive series of patients with normal renal function in order to avoid the well-known effect of overt kidney disease, and suggested that serum levels of Cys C rather than SCr or eGFR were independently associated with the development of CAD. Similar results were also reported by Wang et al. [30], who demonstrated that elevated Cys C levels were associated with the presence of CAD in subjects with mild renal insufficiency, while SCr and eGFR were not able to predict CAD occurrence. Mild renal insufficiency is associated with inflammatory and procoagulant biomarkers that may affect plaque stability and thrombotic potential after plaque rupture, leading to increased risk of CAD. However, renal dysfunction is also associated with traditional cardiovascular risk factors and may enhance atheroma progression. The mechanism of the association between high serum Cys C levels and cardiovascular events is largely unknown and hypothetical. High serum Cys C levels are assumed to represent early kidney disease. On the other hand, the adverse effect of higher Cys C in patients with cardiovascular diseases is not yet completely explained by the renal dysfunction [28]. Degradation of the extracellular matrix in atherosclerotic lesions predominantly takes place in the pathogenesis of ischemic coronary events. As Cys C is a cysteine protease inhibitor, it was suggested to be involved in the atherosclerotic process. Niccoli et al. [31] found that Cys C concentration was directly proportional to the number of stenotic lesions in ACS patients with normal GFR, and Cys C was thought to play some role in plaque formation. However, Kiyosue et al. [4] demonstrated no clear relationship between Cys C and the number of stenotic coronary arteries in patients with normal renal function, and provided no evidence of a direct relationship between Cys C and atherosclerosis. The present study, in accordance with the findings of Niccoli et al., suggested that Cys C levels were significantly higher in CAD patients with eGFR ≥ 60 mL/min/1.73 m2 and significantly increased as the involvement of stenotic coronary vessels increased. Moreover, a positive association was also found between Cys C levels and Gensini score independent of eGFR, though only 8% (standardized β = 0.083 by linear regression analysis) of the variance in Gensini score can be explained by variation in Cys C, which means correlation analysis between Gensini score and Cys C is not practically meaningful. Those conflicts among all the data lead to the discussion that Cys C plays an active causal role in or is a systemic acute-phase response against the atherosclerotic process. It may be claimed that lower levels of Cys C are associated with increased proteolytic activity. The existence of a balance between cysteine proteases and their inhibitor Cys C at the level of coronary arteries and in circulation provided protection against the development of atherosclerosis [26]. In addition, Cys C is associated
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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with cardiovascular risk factors as well as inflammation, which may promote atherosclerosis. Some studies have suggested that inflammatory cytokines were related to atherosclerosis stimulate lysosomal cathepsins, which may be associated with increased Cys C levels to counterbalance the elastolytic activity of cathepsins [32]. Another study indicated that there were significant associations between Cys C and proinflammatory factors like fibrinogen and C-reactive protein in CAD patients [33]. Consistent with this study, the present study found a significant correlation between Cys C levels and fibrinogen, whereas the relationships between fibrinogen and other renal markers such as SCr and eGFR were weak and nonsignificant (P N 0.05). It has been suggested that Cys C would be a marker of both inflammation and renal function. Thus, atherogenic changes associated with inflammation could be one mechanism related to Cys C and cardiovascular disease. Although Cys C was independently associated with the presence of CAD, in the ROC analysis, the AUC for Cys C falls into the range 0.5– 0.7, which indicates that Cys C as a solo marker was of relatively low predictive value, and the cut-off value of 0.865 mg/L cannot be categorized as an ideal value to identify CAD effectively (lower specificity). Nevertheless, the addition of Cys C may compensate for the inadequacy of other classical parameters such as myocardial enzymes for CAD. Serum levels of Cys C and their clinical and prognostic significance might be determined at the end of meta-analyses to guide diagnostic and therapeutic options as well as the utility of D-dimer, pro-brain natriuretic peptide, etc. in clinical practice. This study also presents several limitations. First, the present study failed to rely on a gold-standard GFR measurement by collecting 24hour urine, and no data on urinary protein excretion were available. SCr-based eGFR showed a significantly reduced renal function in the fourth Cys C quartile, which indicated that elevated Cys C concentrations possibly comprised conditions of preclinical kidney disease that may be prevalent in this study. Additional analyses should be performed to eliminate any effect of this confounder. Second, the recruitment of subjects in the current study was consecutive and angiography-based while this study was cross-sectional. Since CAG is done if there is a clinical suspicion of CAD, there has been an enrichment of patients with CAD compared to non-CAD and it is a matter of fact that the amount of male patients is higher than the number of females. Third, as with any observational study, there might be residual confounding of the association between Cys C and CAD, despite adjusting for well-known cardiovascular confounders.
Conclusion The present study reported that elevated serum Cys C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. Cys C is of a versatile utility in clinical practice especially in CAD diagnosis and is more than a marker of GFR. Classification of serum levels of Cys C and their clinical significance may guide the diagnostic and evaluable options in either emergency rooms or coronary care units. Further larger, population-based prospective studies are required to validate the findings suggested by the current study.
Conflict of interest statement The authors have no conflicts of interest to declare.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Serum cystatin C levels are associated with coronary artery disease and its severity Gan-nan Wang a, Kai Sun a, De-liang Hu a, Hong-hao Wu a, Xiao-zhi Wang b, Jin-song Zhang a,⁎ a b
Department of Emergency, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, PR China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, PR China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 29 April 2014 Accepted 18 July 2014 Available online xxxx Keywords: Cystatin C Coronary artery disease Gensini score Glomerular filtration rate
a b s t r a c t Objectives: Serum cystatin C has been established as a predictor of cardiovascular events. The aim of this study was to evaluate the role of cystatin C in determining the presence and the severity of patients with coronary artery disease (CAD). Design and methods: A total of 936 subjects without overt renal disease were included in this cross-sectional study. Among them were 714 patients with CAD and 222 without based on coronary angiography. Subjects were further divided into four groups according to cystatin C quartile. Serum cystatin C was measured using particleenhanced immunoassay method. The study analyzed the relationship of cystatin C levels with the presence and severity of CAD, including the number of stenotic vessels involved and Gensini score. Results: Serum cystatin C levels were significantly higher in patients with CAD than those without (P b 0.001), and significantly increased as the involvement of coronary vessels increased (P b 0.001). The prevalence of CAD and its severity assessed by Gensini score were also significantly greater in the highest quartile of cystatin C (P b 0.001). Moreover, cystatin C levels were independently correlated with the presence of CAD in a multivariate logistic regression model (P = 0.023) and were positively correlated with Gensini score by linear regression analysis (standardized β = 0.083, P = 0.010). Conclusions: Elevated serum cystatin C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. It is suggested that cystatin C might play a role in CAD diagnosis and serve as a marker of CAD severity. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Coronary artery disease (CAD) singly is the most frequent cause of death. Over seven million people every year die from CAD, accounting for 11.2% of all deaths [1]. Early reperfusion and adequate drug treatment are the cornerstones of CAD management, limiting cardiac dysfunction and subsequent morbidity and mortality. Early diagnosis and treatment are the most important predictors of long-term outcome [2]. Although the application of high-sensitive troponin has improved the accuracy of the diagnosis, it still needs other markers to evaluate the early diagnosis and severity of CAD [3]. Therefore, the new, early measurable biomarkers might play crucial roles in the initial assessment of CAD. Abbreviations: CAD, coronary artery disease; Cys C, cystatin C; SCr, serum creatinine; eGFR, estimated glomerular filtration rate; CAG, coronary angiography; SBP, systolic blood pressure; DBP, diastolic BP; HR, heart rate; FPG, fasting plasma glucose; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; Fg, fibrinogen; ROC, receiveroperating characteristic; AUC, area under the curve. ⁎ Corresponding author. E-mail address: [email protected] (J. Zhang).
Cystatin C (Cys C) is a low molecular mass protein produced by all nucleated cells at a constant rate regardless of variations of the intracellular environment and extracellular environment, and acts as a cysteine protease inhibitor. Cys C dose does not form a complex with other serum proteins in blood, and is filtered by the renal glomeruli to be reabsorbed and degraded in the proximal tubules [4]. Over the past few years, serum levels of Cys C had been proposed as a more reliable marker of kidney function than serum creatinine (SCr), and as an alternative parameter for estimating glomerular filtration rate (GFR) [5]. But Cys C was also associated with CAD independently of renal disease [6]. Serum Cys C has been established as a predictor of cardiovascular events and mortality in patients with non-ST-elevation acute coronary syndrome [7], ST-elevation myocardial infarction [8], and suspected CAD [6], but also in the general population [9] and elderly persons [10]. Although the predictive potential in CAD seems to be high, the diagnostic value of Cys C for acute myocardial ischemia remains unexplored so far. There is limited information on the significance of Cys C as a predictor of fatal events in patients with CAD recruited irrespective of renal function [11]. The present study was designed to evaluate whether there was a relationship between serum Cys C levels and the presence of CAD, its severity,
http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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and the number of vessels involved in CAD patients documented with coronary angiography (CAG) but without overt kidney disease.
Materials and methods Study subjects The subjects were consecutive 936 patients (62.26 ± 10.74 years old, 65.9% males) who underwent CAG under the suspected diagnosis of CAD between December 2010 and December 2012 in the Department of Cardiology at the First Affiliated Hospital of Nanjing Medical University (Nanjing, China). CAD has been evaluated by CAG based on maximal luminal narrowing of visual stenosis and defined as the presence of at least one stenosis ≥50% in at least one of 15 coronary segments of the three major coronary arteries [12]. Furthermore, the left anterior descending artery, left circumflex artery, and right coronary artery were examined to evaluate the number of stenotic coronary arteries as 0 to 3-vessel disease. If the left main trunk was involved, this was evaluated as a 2-vessel disease by itself [4]. The results of CAG were evaluated by two expert investigators. The severity of coronary stenosis was assessed in the direction that showed the most severe stenosis according to Gensini scoring [13]. Patients with renal dysfunction (creatinine-based estimated GFR b 60 mL/min/1.73 m2, SCr N 133 μmol/L), valvular heart disease, life-threatening arrhythmias, malignant, infectious and inflammatory disease, and thyroid disease were excluded from the study. The study protocol was reviewed and approved by the Institutional Review Boards for Human Studies of Nanjing Medical University. Written informed consent was obtained from all study participants.
Statistical analysis For the present study, all the subjects were divided into 4 quartiles on the basis of baseline serum Cys C levels. Clinical characteristics as well as biochemical and angiographic outcomes were compared in each Cys C quartile. Continuous variables with normal distribution were expressed as mean ± SD, while percentages were used to express categorical variables. The conformity of data with a normal distribution was analyzed using a Kolmogorov–Smirnov test. In multiple-group comparisons, analysis of variance (ANOVA) was used for parameters conforming to a normal distribution. The student's t test was carried out for comparison of two groups. However, the Kruskal–Wallis H test was used for parameters not conforming to a normal distribution in multi-group comparisons while the Mann–Whitney u test in twogroup comparisons. Categorical variables of subjects with and without CAD as well as stratified by baseline Cys C levels were analyzed by the chi-square test. In addition, the relationships between data were tested using Pearson's or Spearman's correlation analysis. A linear regression analysis was applied to determine the correlation between serum Cys C and severity of CAD evaluated by Gensini score. Moreover, factors considered to affect CAD were evaluated by the multivariate logistic regression model. A receiver-operating characteristic (ROC) curve analysis was drawn to identify the optimal cut-off points of Cys C levels (to determine maximal sensitivity and specificity) for predicting CAD. The area under the curve (AUC) value was calculated to determine accuracy of the test. All the statistical tests were performed in SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). A two-tailed P value of less than 0.05 was considered statistically significant. Results
Clinical assessment and laboratory measurements
Comparisons of patients with and without CAD
The detailed histories of patients, including demographics characteristics and cardiovascular risk factors, were recorded. The cardiovascular risk was assessed in terms of smoking (≥1 cigarette per day for one year or more), hypertension (blood pressure ≥140/90 mmHg or the use of antihypertensive drugs), and diabetes (fasting plasma glucose ≥7.0 mmol/L or random plasma glucose ≥11.1 mmol/L with classic symptoms of hyperglycemia or patients was on anti-diabetic medications) [14]. Systolic and diastolic blood pressures (BP) were measured using standard methods. The venous blood samples were collected after overnight fasting before CAG. Cys C, SCr, fasting plasma glucose (FPG), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by an automatic biochemical analyzer (AU5400, Olympus, Tokyo, Japan). The serum concentration of Cys C was determined by particle-enhanced turbidimetric immunoassay (PETIA) method. According to the manufacturer's specification, the assay was linear from 0.20 to 8.00 mg/L (r2 ≥ 0.996). Within-run coefficient of variation (CV) was 4.2 and 2.5% at cystatin C concentrations of 0.20 and 2.00 mg/L, respectively. Between-run CV was 8.5 and 6.6% at the same concentrations. The average analytical recovery was 99%. Bilirubin ≤ 400 μmol/L, hemoglobin ≤ 5 g/L, chyle ≤ 0.50%, Vitamin C ≤ 0.5 g/L and heparin sodium ≤ 100 IU/mL did not interfere with the assay. Moreover, the intraindividual biological variations ranged from 2% to 10%. The measurement was conducted in the laboratory recognized by the China National Accreditation Service for Conformity Assessment (CNAS). Fibrinogen (Fg) was measured using Von Clauss method on an automatic coagulometer (CA-7000, Sysmex, Kobe, Japan). Estimated glomerular filtration rate (eGFR) was calculated according to the simplified Modification of Diet in Renal Disease (MDRD) equation based on the level of SCr (eGFRMDRD = 186.3 ∗ SCr−1.154 ∗ age−0.203 ∗ 0.742 (if female) where eGRF is expressed as mL/min/1.73 m2 of body surface area and SCr is expressed as mg/dL.) [15].
The characteristics of study subjects are presented in Table 1. There were 714 patients with CAD and 222 without CAD according to coronary angiograph results. CAD patients were significantly older, more frequently male and smoking, and also had significantly higher DBP, FPG, AST, SCr, eGFR, Fg, and Gensini score. However, there was no significant difference in histories of hypertension and diabetes, SBP, HR, and lipid profiles between the groups. Moreover, serum Cys C levels were significantly higher in patients with CAD than those without (1.08 ± 0.27 vs. 1.00 ± 0.24 mg/L, P b 0.001; Fig. 1A). All the subjects were further divided into four groups according to the number of stenotic vessels with coronary involvement. The levels of Cys C were 1.00 ± 0.24, 1.05 ± 0.24, 1.08 ± 0.26, and 1.14 ± 0.30 mg/L in groups of patients Table 1 Clinical and biochemical baseline characteristics of study subjects.
Age (years) Males (%) Smoking (%) Hypertension (%) Diabetes (%) SBP (mm Hg) DBP (mm Hg) HR (bpm) FPG (mmol/L) AST (U/L) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) SCr (μmol/L) eGFR (mL/min/1.73 m2) Cys C (mg/L) Fg (g/L) Gensini score
Non-CAD (N = 222)
CAD (N = 714)
P
59.88 ± 10.39 53.6 28.4 66.2 18.0 133.10 ± 16.53 80.89 ± 10.16 71.88 ± 12.90 5.59 ± 1.52 22.35 (18.10–29.50) 4.30 ± 1.01 1.60 ± 1.04 1.14 ± 0.28 2.63 ± 0.75 71.70 ± 15.35 94.6 ± 19.1 1.00 ± 0.24 2.77 ± 0.64 0 (0–4)
63.00 ± 10.75 69.7 36.8 65.5 24.1 132.97 ± 18.08 79.20 ± 10.73 71.71 ± 11.54 6.07 ± 1.92 25.80 (19.98–54.43) 4.31 ± 1.15 1.58 ± 1.05 1.10 ± 0.28 2.70 ± 0.87 76.24 ± 15.78 91.6 ± 19.9 1.08 ± 0.27 3.10 ± 0.88 40 (20–66)
b0.001 b0.001 0.021 0.854 0.059 0.921 0.038 0.849 b0.001 b0.001 0.838 0.768 0.063 0.308 b0.001 0.048 b0.001 b0.001 b0.001
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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with 0, 1, 2, and 3-vessel disease respectively, and the Cys C levels significantly ascended as the involvement of coronary vessels increased (P b 0.001) (Fig. 1B). Age, systolic blood pressure (SBP), diastolic BP (DBP), heart rate (HR), fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum creatinine (SCr), estimated glomerular filtration rate (eGFR), cystatin C (Cys C) and fibrinogen (Fg) conformed to normal distributions and were presented as mean ± SD, while aspartate aminotransferase (AST) and Gensini score, which were not normally distributed, were presented as median (interquartile range). Patient characteristics according to cystatin C quartiles The present study further compared the variables of patients according to Cys C quartiles. As shown in Table 2, the ranges of serum Cys C levels for each quartile were 0.21 to 0.88, 0.89 to 1.02, 1.03 to 1.20, and 1.21 to 2.90 mg/L, respectively. As expected, patients in higher Cys C quartiles had higher SCr and lower eGFR, were older, and were more likely to have histories of hypertension and diabetes. Furthermore, SBP and Fg were also higher in the fourth quartile than in the lower ones. The prevalence of CAD and its severity assessed by Gensini score were significantly greater in the highest quartile of Cys C. Association of Cys C levels with the presence of CAD and with CAD severity
Fig. 1. Comparisons of serum cystatin C levels in terms of the presence of coronary artery disease (A) and according to the number of stenotic vessels with coronary involvement (n = 222, 302, 208, and 204, respectively in 0, 1, 2, and 3-vessel groups) (B).
The variables considered to affect CAD were evaluated according to the multivariate logistic regression model, which included age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, LDL-C, HDL-C, SCr, eGFR, Cys C, and Fg. It was found that sex, AST, Cys C, and Fg were correlated independently with the presence of CAD (Table 3). In addition, the ability of Cys C levels to distinguish patients with CAD from those without was assessed using ROC curve analysis. The ROC curve for CAD diagnosis had an AUC of 0.588 (95% CI 0.544–0.631, P b 0.001). A 0.865 mg/L cut-off value of Cys C predicted the presence of CAD with a sensitivity and specificity of 81.8% and 30.6%, respectively (Fig. 2). Spearman's correlation analysis showed that serum Cys C levels was positively correlated with Gensini score (r = 0.133, P b 0.001). A
Table 2 Baseline characteristics and laboratory variables according to cystatin C concentration quartiles. Variables
Age (years) Males (%) Smoking (%) Hypertension (%) Diabetes (%) SBP (mm Hg) DBP (mm Hg) HR (bpm) FPG (mmol/L) AST (U/L) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) SCr (μmol/L) eGFR (mL/min/1.73 m2) Cys C (mg/L) Fg (g/L) CAD (%) Gensini score
Serum cystatin C levels (mg/L)
P
Quartile 1 (b0.89) n = 224
Quartile 2 (0.89–1.02) n = 255
Quartile 3 (1.03–1.20) n = 229
Quartile 4 (N1.20) n = 228
56.77 ± 9.95 62.1 36.2 61.2 24.1 130.87 ± 16.33 80.13 ± 10.69 72.72 ± 10.50 6.12 ± 2.05 25.60 (18.85–58.83) 4.31 ± 1.10 1.50 ± 0.92 1.12 ± 0.27 2.69 ± 0.84 66.46 ± 11.77 105.6 ± 17.8 0.78 ± 0.10 3.00 ± 0.87 68.3 20 (3.25–53.75)
60.60 ± 9.30 65.9 37.3 60.4 20.8 131.69 ± 18.14 79.19 ± 10.44 70.69 ± 11.18 5.87 ± 1.77 24.80 (19.40–44.50) 4.30 ± 1.01 1.60 ± 0.99 1.10 ± 0.27 2.68 ± 0.76 71.78 ± 12.11 96.0 ± 15.8 0.95 ± 0.04 2.92 ± 0.76 75.7 24 (5–53)
63.34 ± 10.46 69.4 36.7 65.1 17.5 132.80 ± 18.01 79.43 ± 11.10 71.78 ± 11.67 5.74 ± 1.66 24.30 (19.70–41.10) 4.30 ± 1.17 1.72 ± 1.39 1.08 ± 0.27 2.66 ± 0.87 76.03 ± 12.55 90.1 ± 15.6 1.11 ± 0.05 3.04 ± 0.87 79.0 26 (6–55)
68.42 ± 9.94 66.2 28.9 76.8 28.5 136.77 ± 17.78 79.70 ± 10.31 71.96 ± 13.89 6.09 ± 1.89 25.15 (19.25–40.70) 4.33 ± 1.19 1.50 ± 0.77 1.14 ± 0.30 2.70 ± 0.91 86.62 ± 18.66 77.5 ± 18.9 1.42 ± 0.23 3.16 ± 0.84 82.0 34 (11.25–64)
b0.001 0.430 0.199 0.001 0.033 0.002 0.796 0.307 0.086 0.763 0.987 0.081 0.089 0.972 b0.001 b0.001 – 0.018 0.005 0.005
Values are given as mean ± SD or median (interquartile range) for continuous variables, and as percentage for categorical data. Group differences (P value) were calculated by ANOVA or Kruskal–Wallis test for continuous and χ2 test for categorical variables.
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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Table 3 Analyses of variables affecting coronary artery disease according to the multivariate logistic regression model. Variables
Odds ratio
95% CI
P
Sex AST Cystatin C Fibrinogen
0.325 1.010 2.673 1.619
0.129–0.819 1.006–1.015 1.142–6.259 1.272–2.061
0.017 b0.001 0.023 b0.001
CI, confidence interval; AST, aspartate aminotransferase. Variables included in the model are age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, HDL-C, LDL-C, SCr, eGFR, cystatin C and fibrinogen.
significant association between Cys C and Gensini score was also found by linear regression analysis adjusted for age, sex, smoking, hypertension, diabetes, SBP, DBP, FPG, AST, TC, TG, LDL-C, HDL-C, SCr, eGFR, and Fg (standardized β = 0.083, P = 0.010). Furthermore, the Pearson's correlation analysis demonstrated that serum Cys C levels was positively correlated with age, SBP, SCr, and Fg while negatively with eGFR (P b 0.01). Discussion CAD is the leading cause of morbidity and mortality worldwide. Deaths are mainly caused by acute coronary syndrome (ACS) induced by destabilization or rupture of an atherosclerotic plaque and formation of an occlusive thrombus on the coronary arteries [16]. Serum extracellular vesicle protein (such as Cys C) concentrations provide information regarding the presence of ACS and therefore have a potential role in the evaluation of patients suspected of CAD [17]. The present study was aimed to identify Cys C as an independent risk factor for the development and severity of CAD patients with normal creatinine-based eGFR, and it was found that serum Cys C levels were independently and positively related with CAD and its severity after adjusting for other cardiovascular risk factors. The presence and severity of CAD increased with the increasing quartiles of Cys C. However, previous studies focused on the postulated role of Cys C as a marker in predicting CAD have been controversial. In 2007, the European Cardiology Foundation recommended the prognostic use of Cys C in myocardial infarction and in the detection of long-term risks of death of patients with non-ST-elevation ACS [18]. Likewise, some studies have reported a positive and graded relationship between
Fig. 2. Receiver-operating characteristic (ROC) curve analyses for cystatin C in predicting the presence of coronary artery disease.
higher Cys C levels and increased cardiovascular disease prevalence [19–21]. Furthermore, a meta-analysis has indicated higher Cys C levels were strongly and independently associated with specific endpoints like myocardial infarction [22]. However, other studies have demonstrated that the content of Cys C decreased in the tissues of animals and people with atheromatous plaques [23,24] and an association was detected between a genetically determined decrease in Cys C expression and increased severity of CAD [25]. A recent study has suggested that lower Cys C levels may be associated with increased severity of CAD in clinically stable patients, whereas higher levels may indicate the presence of any vulnerable plaque [26]. It is well recognized that patients with impaired renal function are at significantly higher risk for cardiovascular diseases in contrast to patients without kidney disease [27]. Serum Cys C is a novel endogenous marker for renal dysfunction, whose production is constant and is less influenced by age, sex, race, muscle mass, and drug administration. Several studies have demonstrated the superiority of Cys C compared with SCr or eGFR in predicting cardiovascular events and all-cause mortality [28]. Moreover, studies have also shown that cardiovascular risk associated with higher levels of Cys C was independent of eGFR [29]. The possibility that Cys C might directly influence the occurrence of cardiovascular disease has been raised. The present study evaluated the relationship between Cys C and CAD in a consecutive series of patients with normal renal function in order to avoid the well-known effect of overt kidney disease, and suggested that serum levels of Cys C rather than SCr or eGFR were independently associated with the development of CAD. Similar results were also reported by Wang et al. [30], who demonstrated that elevated Cys C levels were associated with the presence of CAD in subjects with mild renal insufficiency, while SCr and eGFR were not able to predict CAD occurrence. Mild renal insufficiency is associated with inflammatory and procoagulant biomarkers that may affect plaque stability and thrombotic potential after plaque rupture, leading to increased risk of CAD. However, renal dysfunction is also associated with traditional cardiovascular risk factors and may enhance atheroma progression. The mechanism of the association between high serum Cys C levels and cardiovascular events is largely unknown and hypothetical. High serum Cys C levels are assumed to represent early kidney disease. On the other hand, the adverse effect of higher Cys C in patients with cardiovascular diseases is not yet completely explained by the renal dysfunction [28]. Degradation of the extracellular matrix in atherosclerotic lesions predominantly takes place in the pathogenesis of ischemic coronary events. As Cys C is a cysteine protease inhibitor, it was suggested to be involved in the atherosclerotic process. Niccoli et al. [31] found that Cys C concentration was directly proportional to the number of stenotic lesions in ACS patients with normal GFR, and Cys C was thought to play some role in plaque formation. However, Kiyosue et al. [4] demonstrated no clear relationship between Cys C and the number of stenotic coronary arteries in patients with normal renal function, and provided no evidence of a direct relationship between Cys C and atherosclerosis. The present study, in accordance with the findings of Niccoli et al., suggested that Cys C levels were significantly higher in CAD patients with eGFR ≥ 60 mL/min/1.73 m2 and significantly increased as the involvement of stenotic coronary vessels increased. Moreover, a positive association was also found between Cys C levels and Gensini score independent of eGFR, though only 8% (standardized β = 0.083 by linear regression analysis) of the variance in Gensini score can be explained by variation in Cys C, which means correlation analysis between Gensini score and Cys C is not practically meaningful. Those conflicts among all the data lead to the discussion that Cys C plays an active causal role in or is a systemic acute-phase response against the atherosclerotic process. It may be claimed that lower levels of Cys C are associated with increased proteolytic activity. The existence of a balance between cysteine proteases and their inhibitor Cys C at the level of coronary arteries and in circulation provided protection against the development of atherosclerosis [26]. In addition, Cys C is associated
Please cite this article as: Wang G, et al, Serum cystatin C levels are associated with coronary artery disease and its severity, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.013
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with cardiovascular risk factors as well as inflammation, which may promote atherosclerosis. Some studies have suggested that inflammatory cytokines were related to atherosclerosis stimulate lysosomal cathepsins, which may be associated with increased Cys C levels to counterbalance the elastolytic activity of cathepsins [32]. Another study indicated that there were significant associations between Cys C and proinflammatory factors like fibrinogen and C-reactive protein in CAD patients [33]. Consistent with this study, the present study found a significant correlation between Cys C levels and fibrinogen, whereas the relationships between fibrinogen and other renal markers such as SCr and eGFR were weak and nonsignificant (P N 0.05). It has been suggested that Cys C would be a marker of both inflammation and renal function. Thus, atherogenic changes associated with inflammation could be one mechanism related to Cys C and cardiovascular disease. Although Cys C was independently associated with the presence of CAD, in the ROC analysis, the AUC for Cys C falls into the range 0.5– 0.7, which indicates that Cys C as a solo marker was of relatively low predictive value, and the cut-off value of 0.865 mg/L cannot be categorized as an ideal value to identify CAD effectively (lower specificity). Nevertheless, the addition of Cys C may compensate for the inadequacy of other classical parameters such as myocardial enzymes for CAD. Serum levels of Cys C and their clinical and prognostic significance might be determined at the end of meta-analyses to guide diagnostic and therapeutic options as well as the utility of D-dimer, pro-brain natriuretic peptide, etc. in clinical practice. This study also presents several limitations. First, the present study failed to rely on a gold-standard GFR measurement by collecting 24hour urine, and no data on urinary protein excretion were available. SCr-based eGFR showed a significantly reduced renal function in the fourth Cys C quartile, which indicated that elevated Cys C concentrations possibly comprised conditions of preclinical kidney disease that may be prevalent in this study. Additional analyses should be performed to eliminate any effect of this confounder. Second, the recruitment of subjects in the current study was consecutive and angiography-based while this study was cross-sectional. Since CAG is done if there is a clinical suspicion of CAD, there has been an enrichment of patients with CAD compared to non-CAD and it is a matter of fact that the amount of male patients is higher than the number of females. Third, as with any observational study, there might be residual confounding of the association between Cys C and CAD, despite adjusting for well-known cardiovascular confounders.
Conclusion The present study reported that elevated serum Cys C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. Cys C is of a versatile utility in clinical practice especially in CAD diagnosis and is more than a marker of GFR. Classification of serum levels of Cys C and their clinical significance may guide the diagnostic and evaluable options in either emergency rooms or coronary care units. Further larger, population-based prospective studies are required to validate the findings suggested by the current study.
Conflict of interest statement The authors have no conflicts of interest to declare.
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