ABC | Volume 113, Nº4, October 2019

Original Article Abolhasani et al. Serum levels of markers in CAD prediction Arq Bras Cardiol. 2019; 113(4):667-674 a central molecule linked to pathogenesis and progression of thrombotic vascular events, including stroke. In addition, elevated plasma PAI-1 levels are associated with vascular thrombosis. 12 A previous study suggested that high levels of PAI-1 in CAD are associatedwith the risk of endothelial dysfunction and premature atherosclerosis. 13 Sialic acid (SA) is derivative of neuraminic acid, and comprises the terminal sugar part of the oligosaccharide chain in glycolipids and glycoproteins, acting as a cofactor in several cell surface receptors, such as LDL receptor. Its intake of LDL occurs prominently in smooth muscle cells of blood vessels and is increased in several pathological and inflammatory states, such as in atherosclerosis. 5,14,15 Therefore, following an inflammatory reaction or injury, desquamating or secretion from damaged cells can lead to an elevated concentration of SA. 16 Oxidative stress and inflammation also play vital roles in the pathogenesis and progression of CAD. 6 Oxidized low density lipoprotein (OX‑LDL) and correlated composites are also observed in lesion formation at the later stages of atherosclerosis. Hence, OX-LDL could play a major role in both atherogenesis and plaque complications. 17,18 Additionally, malonaldehyde (MDA) results from lipid peroxidation, and its measurement is an undependable marker of oxidative damage, making MDA a suitable indicator and marker for identification and further evaluation of patients with CAD. 17 Among several markers of inflammation, highly sensitive C‑reactive protein (hs‑CRP) has been established as significant in peoplewithCAD. Several studies demonstrated that hs‑CRP is associated with increased CAD risk. 19 Previous findings reported elevated VN, MDA, OX-LDL, PAI-1, hs-CRP and SA levels, which were positively correlated with CAD. Although the pathological aspects of these risk factors have been studied, their role has not been recognized in the early and accurate diagnosis of atherosclerosis in patients with CAD. This study aimed to determine concentrations of the prognostic value of serum levels of hs‑CRP, SA, VN, PAI-1, OX‑LDL andMDA in patients with CAD, all of which can manifest in CAD, in an effort to examine the importance of combining these biomarkers with the diagnosis of CAD. Methods Subjects The sample size was 180 subjects, based on a convenience sample. The subjects were divided into 4 groups according to angiography results. The control group was a no Stenosis group which included 40 subjects with non-significant disease that had no clogged vessels but suffered from chest pain such as angina pectoris; 40 with single occluded vessel disease (1VD); 40with double occluded vessel disease (2VD); and 40 individuals with triple occluded vessel disease (3VD). In addition, the control group was composed of healthy individuals without any presentation of CAD (n = 20). Peripheral blood sampling was obtained after one night of fasting in the Shahid Madani hospital, located in East Azerbaijan, Iran. Ethics Before the beginning of the study, the protocol was presented to the independent ethics committee of the Medical Faculty of the Tabriz University of Medical Sciences (ethics number 91/2-3/5). An informed consent was obtained from all of participants. All patients with the renal disease, lung disorders, liver dysfunction, autoimmune disease, infectious diseases and cancer were excluded from the study. Laboratory methods All blood samples were obtained from a peripheral vein after 12 hours of overnight fasting. Subsequent plasma and serum were separated within 30 minutes, and samples were stored at –70°C until the tests were performed. Measurement of parameters Enzyme-linked immunosorbent assay (ELISA) procedures were used to determine serum levels of OX-LDL (Glory Science co. Ltd, Cat. No: 93614), PAI-1 (Boster Science co. Ltd, Cat. No: EK0859) and VN (Glory Science co. Ltd, Cat. No: 11668). SA was also measured by ELISA, using a commercial kit (Crystal Day, China). Serum MDA was measured based on reaction with Thiobarbituric Acid (TBA); extraction accompanied with normal butanol; absorption measured by spectrophotometer and value calculated according to a standard curve. Serum levels of hs-CRP were estimated by high-sensitivity turbidimetry method using Biosystems kit (Barcelona, Spain, COD 31927); the assay was evaluated on semi-autoanalyser (Alcyon 300, made in USA) in the Biochemistry lab. Analytical methods All statistical analyses were carried out using SPSS software, version 20.0 (SPSS Inc., Illinois, USA). All quantitative variables were expressed as mean ± standard deviation or median and interquartile range. The qualitative variables were expressed in numbers and percentage. The normality of the data was evaluated by the normal curve (skewness and standard deviation of skewness) of Kolmogorov-Smirnov test. Differences for multiple groups were analyzed using independent t-test, and one-way analysis of variance (ANOVA). Also, the chi-square test was used for categorical variables. The Kruskal-Wallis test was used for the quantitative variables if the normality assumption of the residuals was not met. Moreover, ROC curve analysis was used to evaluate the diagnostic effect of VN, MDA, OX-LDL, PAI-1, hs‑CRP and SA by logistic regression model. A statistical analysis was defined when p < 0.05. Results The prognostic indicators were used in the current study. Table 1 lists the general characteristics of the study groups. Differences between patient and control groups based on age and sex distribution were subject to an independent-samples t-test. The results showed that there were no significant differences between the groups (p < 0.3). However, smoking, hypertension and diabetes showed significant difference between the patient and control groups (p value: 0.004, 0.01, and 0.02, respectively). Table 2 represents groupmean parameter values obtained froma one-way ANOVA analysis; the mean serum levels of parameters that have been compared among subgroups were categorized based on number of occluded vessels of study population. Significant differences in all cardiovascular risk factors measured 668