Myeloperoxidase and Paraoxonase Activity in Acute Coronary Syndrome Patients

Background: Myeloperoxidase (MPO), an oxidative stress related enzyme is elevated in Coronary Artery Disease (CAD) and is involved in development of atherosclerotic plaque. Paraoxonase (PON) an enzyme protein associated with HDL serves as an antioxidant and plays an important role in preventing the formation of Oxidized LDL (OxLDL). This suggests a conflicting role of MPO and PON in development of cardiovascular disease and atherosclerosis. Aim: Present study was done to evaluate and compare MPO/PON ratio in Acute Coronary Syndrome (ACS) patients with controls. The study evaluates and compares the pro oxidant and pro inflammatory enzyme, MPO and anti-oxidant and anti-inflammatory enzyme, PON in ACS patients with controls. Oxidative marker, Malondialdehyde (MDA) and anti-oxidant marker, Reduced Glutathione (GSH) was assessed in ACS patients and compared with controls. An attempt was also made to correlate MPO/PON ratio to markers of oxidative stress (MDA and GSH). Methods: Cross-sectional study carried out in Dr. Somervell Memorial CSI Medical College, Trivandrum, Kerala, India.50 ACS patients from Cardiac Care Unit and 50 age and sex matched controls without CAD from Medical College Health Checkup was selected. Original Research Article Varadhan et al.; JPRI, 33(48B): 108-114, 2021; Article no.JPRI.76142 109 Results: MPO and MPO/PON ratio were significantly high and PON was significantly lower in ACS patients compared to controls. Total cholesterol, Triglyceride, LDL, MDA were significantly high in ACS patients. Statistically significant positive correlation was observed between MPO/PON and MDA. Significant negative correlation was observed between MPO/PON and GSH. Conclusion: Myeloperoxidase and MPO/PON ratio was significantly high in ACS patients than controls. This is suggestive of the role of MPO in oxidative damage to lipoproteins in CAD patients. Prooxidant, Paraoxonase ,and antioxidant, GSH is lowered in ACS patients as a result of the increased oxidative stress. This study suggests that MPO/PON1 ratio can be used as a predictive marker of ACS.


INTRODUCTION
Coronary artery disease (CAD) isone of the principal reasons behind death due to Non communicable diseases in developed countries. World Health Organizationhas stated that close to seven million people die of CAD every year all around the world [1]. Among these sudden deaths, 80% are triggered by atherosclerotic CAD [2]. Atherosclerosis occurs as a result of development of fatty lesions,plaque formation andresulting inflammation. This sequence of events may occur as a consequence of continuous oxidative stress building up inside the cell [3].Acute coronary syndrome (ACS) is acomplex form of coronary heart disease (CHD). It includes unstable angina (UA), non-ST segment elevation myocardial infarction (NSTEMI), and ST segment elevation myocardial infarction (STEMI). Rupture of atherosclerotic plaque and the thrombosis resulting from this are the leading cause of ACS [4,5]. One established reason for atherosclerotic plaque formation is the low-density lipoproteins (LDL) undergoing oxidative modifications and formation of oxLDL inside the arterial wall.
Among the enzymes that bring about oxidation of LDL, most important one is Myeloperoxidase (MPO).It is a lysosomal heme containing protein capable of breaking up different types of peroxides. Profusely present in the neutrophil granulocytes, MPO displays potent pro-oxidative and proinflammatory properties. MPO is involved in the reaction of hydrogen peroxide and chloride ion to form hypochlorous acid. This is responsible for the microbicidal action of phagocytes. It is found to be present in large amounts in ruptured plaque [6]. MPO is also involved in the oxidation of lipids associated with LDL cholesterol and for the oxidative modification of lipoproteins in the artery wall [7]. MPO is apparently elevated in ACS patients and is therefore an established marker of inflammation and resulting oxidative stress.
Low Density Lipoprotein has direct correlation with development of CAD [8]. On the contrary, HDL and its major Apo lipoprotein, Apo AI, are inversely related to risk of CAD. HDL becomes an easy target for oxidation by MPO, which converts this atheroprotective lipoprotein to a dysfunctional one. Because of this alteration in vivo, HDL is considered as an objective for therapeutically preventing many vascular diseases [9].Through its antioxidative and antiatherogenic processes, this lipoprotein safeguards against development of atherosclerosis [10,11]. This therapeutic capacity of HDL is attributed to effect of Paraoxonase1 (PON1), an anti atherogenic enzyme associated with this lipoprotein [12,13].
Paraoxonaseis an enzyme protein made up of 354 amino acids. It has a molecular weight of 43 kDa [14,15].Action of PON is to bind reversibly to its substrate like organophosphorouscompounds and it is hydrolyzed.PON has materialized as an essential part of HDL that helps it to metabolize lipid peroxides and prevents them from accumulating on LDL particles. Lipid peroxidation of LDL particles was prevented by purified PON to a great extent [16,17]. PON1 was extensively effective in protecting LDL against oxidation more than Lecithin Cholesterol Acyl Transferase, LCAT or apoA-I.It has been proven that antioxidant effect of HDL is also due to associated Paraoxonase enzyme [18].
Based on these conclusions, PON can be further studied as a causative factor for development of Coronary Artery Disease. Even though many studies have been conducted to test the ability of PON to prevent atheroma, further information is required about the pharmacological and therapeutic effects of this HDL associated enzyme. More light can be thrown into the protective effect of Paraoxonase by studying in detail its interrelationship with other atherogenic inflammatory enzymes like myeloperoxidase.
The specific objective of the present study was to assess and compare the Myeloperoxidase/ Paraoxonase (MPO/PON) ratio in patients with Acute Coronary Syndrome and normal subjects. An attempt was also made to correlate MPO/PON ratio to markers of oxidative stress (MDA and GSH).

MATERIALS AND METHODS
This study was carried out in Department of Biochemistry, Dr. Somervell Memorial CSI Medical College, Trivandrum, Kerala, India.

Statistical Analysis
In case of normally distributed variables, data are presented as mean ± SD. Difference in baseline characteristic between two groups were tested using t test.Correlation Analysis between MPO/PON and MDA and GSH were tested by calculating Karl Pearson Correlation Coefficient. P value of <0.05 was considered to be statistically significant. The statistical analysis was performed using SPSS software.

RESULTS
The study was conducted to assess the contradictory effects of prooxidant enzyme Myeloperoxidase, and antioxidant enzyme Paraoxonase in ACS patients. Pro inflammatory MPO, anti atherogenic PON-1, lipidperoxidation product MDA, an endogenous antioxidant GSH and serum lipid profile were estimated in ACS patients. These values were compared with that of age and sex matched controls.
The test population comprised of 50 subjects that included 34 (68%) males and 16 (32%) females. Control group consisted of 50 subjects that included 33 (65%) males and 17 (35%) females. The age of the test group varied from 35 to 70 years with mean age 52.5 years, whereas that of control subjects varied from 36 to 70 years with a mean age 53 years. Baseline characteristics of ACS patients and controls are shown in Table  1a. Distribution of test and control group according to the conventional risk factors of CAD is given in Table 1b.

DISCUSSION
Many conventional risk factors like diabetes, hypertension, smoking and family history have already been established in the progress of CAD. Hypertension is a principal risk creating factor for Results expressed as mean ± SD Results are expressed in mean ± SD  Results are expressed in mean ± SD cardiovascular disease [19]. Diabetes is also exceedingly prevailing in patients with ACS. Previous studies have demonstrated considerable excess risk in mortality rate and many adverse cardiovascular events were seen [20]. The present study also agrees with the fact that hypertension and Diabetes Mellitus forms a major risk factor for development of Coronary Artery Disease.
Our study clearly indicates an increase in oxidative stress enzyme myeloperoxidase in ACS patients when compared to controls. Study conducted in 2019 [21] suggests that increased Myeloperoxidase is associated with increased risk of mortality. They also suggest that MPO can be used in risk analysis models that can be used to guide therapy in ACS patients.
Also in our study, PON was lowered in ACS patients. It is seen in previous studies that reduced PON1 arylesterase activity can be used as predictive elements for mortality rate after Myocardial infarction [22]. MPO/PON ratio calculated in our study subjects were elevated in patients with ACS compared to controls. In an earlier study by Bacchetti et al, it is clearly mentioned that high MPO/PON1 ratio characterizes patients with atherosclerotic cardiovascular disease ASCVD and can be used as a potential marker of dysfunctional HDL [23]. Another study by Vasylchenko et al shows that myeloperoxidase/ paraoxonase-1 ratio was elevated many folds in patients with cardiovascular complications [24].
In order to assess the oxidant and antioxidant status in the study subjects, Lipid peroxidation product MDA, and endogenous antioxidant GSH levels were estimated in both ACS patients and controls. MDA levels in ACS patients included in this study were significantly higher compared to control subjects. Level of GSH was significantly lowered in control subjects when compared to ACS patients. This finding is well in accordance with study conducted by Nguyen et al. [25] where MDA levels in the ACS group were significantly higher than that present in controls.
Another objective of this study was to correlate MPO/PON ratio with oxidative stress markers, like MDA and GSH in ACS patients. We observed a positive correlation between MPO/PON and MDA, which was statistically significant. Also a negative correlation with statistical significance was observed between MPO/PON and GSH.

CONCLUSION
Oxidative stress inducing MPO, antioxidant and antiatherogenic PON-1 plays a significant conflicting role in the initiation of ACS. The present study shows that Myeloperoxidase and MPO/PON ratio was significantly high in ACS patients than controls. This is suggestive of the role of MPO in oxidative damage to lipoproteins in CAD patients. Paraoxonase, a prooxidant enzyme as well as GSH an antioxidant is lowered in ACS patients as a result of this increased oxidative stress. Conclusions derived from this study is suggesting that MPO/PON1 ratio can be used as a predictive marker of ACS.

LIMITATIONS
Even though we have made much progression in understanding the pathogenesis of atherosclerosis, care should be taken before using these markers in clinical practice. Welldesigned studies with large sample sizes are required to confirm the interaction between these inflammatory markers and to provide a scientific explanation behind this correlation. Genetic studies of well-defined groups would help us to learn more about the precise role of the above markers in development and management of atherosclerosis.

CONSENT
All authors declare that written consent to participate in the study was obtained from the participants before the start of the study.