Evaluations of Antioxidant Enzyme Activities, Total Sialic Acid and Trace Element Levels in Coronary Artery Bypass Grafting Patients

Introduction In this study, patients before and after cardiac surgery with cardiopulmonary bypass (CPB) and control subjects were evaluated for erythrocyte glutathione peroxidase, catalase and superoxide dismutase enzyme activities, in addition to glutathione, malondialdehyde, serum total sialic acid, lipid-bound sialic acid, total antioxidant status, trace elements and mineral levels. The correlation of these variables with coronary artery disease (CAD) was also assessed. Methods A total of 30 CAD patients and 30 control subjects were included in the study. CAD patients were divided into three groups: before surgery (BS), first day after surgery (1st day AS) and seventh day after surgery (7th day AS). Results Malondialdehyde (MDA) and total sialic acid (TSA) levels were significantly higher in CAD (BS) than in the control group (P<0.05, P<0.05). In addition, GSH and TAS levels were significantly lower in the 1st day AS group than in the control group (P<0.001, P<0.01). Moreover, Co, Cu, Mg, Se, V and Zn levels were significantly lower in CAD (BS) group than in the control group (P<0.01, P<0.01, P<0.01, P<0.01, P<0.05, P<0.001). Conclusions It was concluded that the levels of LDL-C, total cholesterol, triglycerides and CRP significantly associated with parameters, as well as Cu, Ca and SOD activity, should be measured together to monitor CAD. It is also considered that measuring TSA and MDA might be an appropriate choice for biomarkers of CAD.


INTRODUCTION
Coronary artery disease (CAD), which is mediated by multiple interactions of environmental and genetic factors, is the main cause of disability and death worldwide [1] . Oxidative stress and chronic inflammation may play a role in the pathogenesis of CAD and atherosclerosis [2] . The risk factors due to excess free radicals are smoking, diet, pollution and metabolic abnormalities that lead to increased oxidative stress to the heart [3] . A series of inflammatory and immunological alterations, which trigger oxidative stress, may be involved during cardiac surgery with cardiopulmonary bypass (CPB) [4] .
Reactive oxygen species (ROS) upregulate atherosclerotic events such as cell infiltration, platelet activation, adhesion and migration. These ROS oxidize cellular biomolecules, including lipids, nucleic acids and proteins, causing endothelial impairments [5] . The unbalanced production of ROS could trigger protein and lipid oxidation in the vascular wall [2,6] . One of the main risk factors for CAD is hyperlipidaemia. The incidence of CAD is higher in the group of patients with low-density lipoprotein cholesterol values [7] . Destruction of endothelium, disruption of the routine vasomotor system and increase in thrombosis thought to be caused by ROS are the mechanisms of atherogenesis [8] .
In energy transfer and physiological heart function, micronutrients, which are enough in trace amount, are fundamental cofactors. Micronutrients are compounds that inhibit oxidation and play a role in signal transduction pathways [9] . Trace elements (TE) play an important role in the balance of normal structure and physiological functions of cells. The cardiovascular system may be directly affected by TE levels and may also be affected indirectly in the vascular system through the role of trace elements in lipid metabolism [10] . Mg is essential, hence, it participates in many biochemical, physiological and cellular processes for an appropriate cardiovascular function [11] .
The purpose of this study was to evaluate the alterations in erythrocytes glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) enzyme activities, the glutathione (GSH) and malondialdehyde (MDA) levels, in the serum total sialic acid (TSA), lipid bound sialic acid (LSA), total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), trace element (As, Be, Cu, Cr, Co, Pb, Li, Sr, Se, Ti, Zn, V), mineral (Mg, Ca, Na, Cl, K), lipid profile parameters (total cholesterol, LDL-C, triglycerides) and some biochemical parameters (albumin, ALT, AST, BUN, CRP, glucose, uric acid, Ca, K, Na, Cl) in relation to indices of CAD in patients before (BS) and in the first and seventh day after surgery (1 st day AS and 7 th day AS) with the use of CPB to compare the control subjects. In addition, the correlations between the parameters were used to evaluate the relationship with CAD.

Study Population
Before the beginning of the study, the necessary approval was received from the ethics committee of the Measurements of biochemical parameters: albumin, aspartate transaminase (AST), blood urea nitrogen (BUN), alanine transaminase (ALT), C-reactive protein (CRP), glucose, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), uric acid (UA), sodium (Na), calcium (Ca), potassium (K) and chloride (Cl) were carried out using standard methods with the biochemical analyzer Architect CI16200 (Abbott Diagnostics, Abbott Park, IL) at the laboratory of the Faculty of Medicine of Van YYU. The level of CRP was measured by a nephelometer (Siemens BN II, Siemens Healthcare GmbH).
Erythrocyte GSH was analysed according to the method described by Rizzi et al. [13] . Erythrocyte MDA was evaluated using the method defined by Jain et al. [14] Erythrocyte activities of antioxidant enzymes (GSH-Px, CAT and SOD) were evaluated by a spectrophotometer (Shimadzu UV-1800 UV-VIS, Kyoto). The results are expressed as enzyme units per gram of haemoglobin (IU/g Hb). Erythrocyte GSH-Px (E.C.1.11.1.9) activity was evaluated at a wavelength of 340 nm according to the method described by Paglia et al. [15] . SOD (EC 1.15.1.1) activity was measured at a wavelength of 505 nm based on the method described by Sun et al. [16] . CAT (EC 1.11.1.6) activity was measured at a wavelength of 240 nm by the decomposition of H 2 O 2 , using the method described by Aebi [17] . Serum TSA level was determined according to the method described by Sydow et al. [18] . Serum LSA level was determined using the method specified by Katopodis et al. [19] .
The total antioxidant status (TAS) is often used to evaluate the overall antioxidant state of the body and to monitor antioxidant therapy. The serum TAS was determined with 2,2-azinobis-3ethylbenzothiazoline-6-sulfonate (ABTS) and was measured at a wavelength of 660 nm. The results are expressed as mmol Trolox equivalent/L [20] . The total oxidant status (TOS) was used to determine the patient's overall oxidative status. Serum total oxidation status (TOS) was measured at 530 nm using method, developed by Erel. The results are expressed in terms of millimoles of hydrogen peroxide (H 2 O 2 ) equivalent per liter (mmol H 2 O 2 equivalent/L) [21] .
The oxidative stress index (OSI) is defined as the ratio between the TOS and TAS levels. The OSI is an indicator of the degree of oxidative stress. The OSI was calculated using the ratio between the TOS and TAS levels.

Statistical Analysis
The results are expressed as mean±standard error of the mean ( X ±SEM). The analysis of variance (ANOVA) or the Kruskal-Wallis test was used for statistical analysis, and then Tukey test was carried out for post-hoc comparison of mean values. Pearson's correlation analysis was used to assess the levels of biochemical parameters for correlation studies in the coronary artery disease (CAD) in patients of the BS group. The statistical analysis was performed using the statistical software SPSS® v23 (SPSS Inc., Chicago, Il, USA).

RESULTS
The comparison of clinical and biochemical characteristics in CAD patients and control subjects is shown in Table 1.
As shown in Table 1, the age of healthy controls and CAD patients was 62.97±1.84 and 63.23±1.79 years and the body mass index (BMI) of the healthy group and CAD patients was 25.48±0.72 kg/m 2 and 25.95±0.60 kg/m 2 . No significant difference was observed between CAD and control groups in terms of age and BMI (P>0.05).
It was observed that a significant reduction in GSH-Px and CAT enzyme activities, GSH, TAS (P<0.01, P<0.01, P<0.001, P<0.01), and MDA, TSA and OSI concentrations (P<0.05, P<0.05, P<0.01) were significantly higher in the CAD (BS) group than in the healthy control group. However, the after surgery with CPB (1 st day AS) group was also significantly lower than the control group regards to CAT, TAS and GSH (P<0.05, P<0.01, P<0.001, respectively), and a significant increase in MDA, TSA, LSA, TOS, OSI concentrations (P<0.001, P<0.01, P<0.001, P<0.01, P<0.001). CAT enzyme activity, GSH and TAS (P<0.05, P<0.05, P<0.01) were significantly lower than the control group in the after surgery with CPB (7 th day AS) group, while the GSH level was lower in the CAD (BS) group than after surgery with CPB (7 th day AS) (P<0.001) group. Similarly, the GSH level was significantly lower and the LSA level was importantly higher in the after surgery with CPB (1 st day AS) group than in the after surgery with CPB (7 th day AS) group (P<0.01, P<0.01). However, the comparison of the SOD enzyme activity revealed no statistical significance between groups of CAD patients and healthy controls (P>0.05) (Figures 1 to 3).
The mean trace element (As, Be, Cu, Cr, Co, Pb, Li, Sr, Se, Ti, Zn, V), mineral (Mg) and trace element ratio (Cu/V, Se/Co, V/Zn) levels of CAD patients before, after undergoing CABG with CPB and control subjects are shown in Table 3.
To assess the correlation analysis in CAD (BS) cases, a positive linear correlation was found between the levels of Se, Co and Mg in subjects with CAD (BS) (r=0.621; P=0.001, r=0.412; P=0.033), between K and GSH, age levels (r=0.421; P=0.020, r=0.424;

DISCUSSION
In this study, in CAD patients before and after CABG with CPB and in healthy control groups, erythrocytes antioxidant enzyme activities (GSH-Px, CAT and SOD), GSH, MDA in serum TAS, TOS, TSA, LSA, TE (As, Be, Cu, Cr, Co, Pb, Li, Sr, Se, Ti, Zn, V), mineral (Mg) and biochemical parameters (total cholesterol, LDL-C, TG albumin, ALT, AST, BUN, CRP, glucose, uric acid, Ca, K, Na, Cl) levels were assessed, in addition, correlations between the parameters were to evaluate the relationship with CAD patients (before surgery).
CABG is a recognized therapeutic approach in people with symptomatic multivessel CAD. Cardiac surgery with CPB leads to ischemia reperfusion-mediated oxidative stress. Inflammation and oxidative stress are associated with the pathogenesis of atherosclerosis and increase the risk of postoperative atrial fibrillation and graft failure [22] Trace elements are necessary for a wide range of primary cellular functions and are especially crucial for various enzymes involved in the generation and neutralization of ROS that are normally produced by the cell [23] . Trace elements such as Cu and Co may contribute to myocardial dysfunction [4] .
To our knowledge, there is no published information on some parameters such as TSA, LSA, As, Be, Co, Be, As, Cr, Li, Ti, V, V/Zn levels in patients with CAD before and after surgery with CPB and correlations between biochemical parameters in CAD patients before surgery. In the current study, statistical analysis clearly demonstrates that comparison of CAD patients and controls indicated a significant elevation of TC (P<0.05), LDLC (P<0.01) and triglycerides (P<0.05) concentrations. In this study, significant increases in triglycerides, total cholesterol and LDL-C values were consistent with the literature [8] . Via oxidation of LDL-C and free radical formation, the oxidative stress contributed to CAD pathogenesis [8] . We also observed increased lipid peroxidation and oxidation of LDL among our CAD patients.
In a study by Lee et al. [7] , conducted in erythrocyte samples collected from CAD patients, activities of erythrocyte SOD X X X X  reported by other authors [22,24] . However, the decrease in the TAS level agree with the data of Tani et al. [25] . In a different study by Bayir, serum values of Zn, Se and Cu in male and female patients in acute coronary syndrome and control groups were reported to be significantly (P<0.001, P<0.05, P<0.001) decreased compared to healthy control groups [10] . The levels of Cu, Se and Zn in the CAD (BS) group were importantly lower than in the healthy group (P<0.01, P<0.01, P<0.001). However, a comparison of the levels of As, Be, Cr, Li, Pb, Sr, Ti, Cu/V and Se/Co revealed no statistical significance between CAD patients and controls (P>0.05). Our findings (the significant decrease in Zn, Se, Cu values) were in parallel with the results of other studies [8,10] . These results show that low levels of Cu, Zn and Se can present a risk for CAD. It has been reported that the majority of cardiac surgery patients presented a significant Se deficiency before surgery, which was increased by the intraoperative procedure [22] . There was no study determining the levels of trace elements As, Be, Co, Be, As, Cr, Li, Ti and V.
It was thought that Cu could contribute to myocardial dysfunction in heart failure, and that Cu deficiency decreases the activity of cardiac cytochrome c oxidase [10] . Two of the most significant micronutrients are Zn and Cu, which play a major role in the oxidant/antioxidant mechanism [9] . Cu deficiency leads to an increase in total cholesterol and in LDL-cholesterol, increasing its atherogenicity. Low level of Cu elevates the risk of CAD [8] .
In the study, it was observed that the levels of Mg in the CAD (BS) group were significantly lower than in the healthy group (P<0.01). Moreover, the after surgery with CPB (1 st day AS) and control groups differed significantly in the Mg concentration (P<0.05). Mg is essential to the etiopathology of several cardiovascular diseases, including atherosclerosis, CAD, hypertension, cardiac arrhythmias and congestive heart failure [11] .
The findings of the present study provide evidence that the inflammation marker (CRP) is intimately linked with CAD. Our results suggested that high V/Zn ratio, MDA, TSA, LSA, TOS, OSI and low Co, Cu, Se, V, Zn, Mg, CAT enzyme activity, TAS and GSH can be associated risk factors of CAD and should be considered together with CRP, LDL-C, total cholesterol and triglycerides monitoring for development of CAD. In this study, the results clearly indicate that CAD can be caused by oxidative stress and chronic inflammation.
Coronary artery risk factors (total cholesterol, CRP, triglycerides) were correlated with serum Ca, Cu and albumin levels. The results suggest that Cu, albumin and Ca levels may have a predictive value for healthy subjects. In the current study, the serum Zn level was found to be considered significantly lower in patients with CAD compared with the control subjects; moreover, it was found to be correlated with the SOD enzyme activity SOD-Zn (r=0.399; P=0.029). However, no significant correlations were found between GSH-Px, CAT, MDA, TOS, OSI, TSA, As, Be, Cd, Cr, Li, Pb, Sb, Sr, Ti, V, Na, BUN and UA parameters in CAD patients (BS).

CONCLUSION
As a result of the analysis, it was observed that, in terms of changes in V/Zn ratio, MDA, TSA, LSA, TOS, OSI, Co, Cu, Se, V, Zn, (P=0.034), CAT (P=0.033), GSH-Px, (P=0.042) were reported to have significantly decreased activity compared to the healthy control groups. In a study by Palazhy et al. [24] , on the samples taken from patients with CAD, erythrocyte GSH-Px and GSH were reported to be significantly (P=0.001) and (P=0.001) decreased when compared to healthy control groups. However, a significant increase in MDA level (P=0.001) was found. Almzaiel et al. [8] observed that, in serum samples taken from patients with ischemic heart disease, the levels of Zn, Cu, SOD and CAT were importantly decreased (P<0.05, P<0.05, P<0.05 and P<0.05) when compared with healthy control groups. Conversely, LDL-C and triglycerides levels increase significantly (P<0.01, P<0.01). In another study, Tani et al. reported that the serum MDA level increased significantly in CAD compared with control group (P<0.001), whereas the TAS value was significantly lower in patient group (P<0.001) [25] .
In the study, the results of reduced CAT, GSH-Px activities and GSH levels in CAD were consistent with the results of other studies [7,8,24] , while findings of elevated SOD activity in CAD patients were not supported by other researchers [7,24] . The significant increase in MDA value was consistent with those Mg, CAT enzyme activity, TAS and GSH levels, the relationship between oxidative stress and inflammation was found in different groups of cardiac surgery patients who underwent cardiopulmonary bypass.
In the correlation analyses, we found a significant association between LDL-C-total cholesterol, triglycerides-Ca, SOD-Zn, CRP-albumin and total cholesterol-Cu. Our results showed that increased levels of LDL-C, CRP and triglycerides have also been found to be directly related to increased risk of CAD. It was concluded that the levels of LDL-C, total cholesterol, triglycerides and inflammatory markers such as CRP, significantly associated with parameters, as well as Cu, Ca and SOD enzyme activity, should be measured together for monitoring CAD. Our study also suggests that measurement of TSA and MDA might be an appropriate choice for biomarkers of CAD.