Prime Risk Factors to Act as Biomarkers for the Diagnosis of Myocardial Infarction

Aims: This study was done to find out retrospective case-control with respect to myocardial infarction diagnosis on the basis of biochemical markers and lipid profile characteristics. Design and Setting: This study was conducted at the Department of Biochemistry and sample collection at LPS Institute of Cardiology & Cardiac Surgery Department, Kanpur. Methods: The total number of subjects participated in this study (n=178), of either sex (with age>65years) were included in this study from the case collected from Outpatient Department (OPD) and Indoor Patient Department (IPD) and control from patients attendant, which consisted of two subject groups: The group I: myocardial infarction (cases) n= 89 and Group II: Healthy Subjects Original Research Article Verma et al.; JPRI, 33(24B): 1-12, 2021; Article no.JPRI.67562 2 (controls) n= 89. Laboratory methodology was performed to determine the prime important risk factors such as body mass index (BMI), blood pressure and lipid profile characteristics associated with myocardial infarction. Risk factors were analyzed by Chi-square and Pearson correlation, receiver operating characteristic (ROC) curve method considered significant when the critical, P <0.01level was set up, for a 95% confidence interval. Results: Total number of (n=178) participants (males=65; mean age of 65 years, females=24, mean age of 58 years) at 95% CI were considered in this study, out of these, 89 individuals were expected to have risk of myocardial infarction (especially observed in case groups), and remaining 89 individuals were considered as control groups, all were included as, study subjects. Conclusion: The current study concludes the importance of myocardial infarction in presence of prognostic inflammatory markers: higher IL-6 and plasma fibrinogen level, instead of highsensitivity C-reactive protein, incase and control groups.


INTRODUCTION
Myocardial infarction known as acute myocardial infarction (AMI), which is commonly used for an event of heart attack. MI occurs when blood supply is obstructed, due to which oxygen supply is not available to the heart muscles [1] and heart muscles get injured. The basic reason of myocardial infarction or heart attack is narrowing of coronary arterioles, blockage with plaques, cholesterol and fat deposits, resulting in blood clots, which stops the supply of blood into the heart. This is known as hardening of arterial walls; medically termed as atherosclerosis disease [2]. Atherosclerosis is now regarded as an inflammatory disease but the process of atherosclerosis is complex because of the involvement of inflammatory regulatory mechanism. Actually, some pro-inflammatory cytokines like interleukin-6 (IL-6) [3] are thought to be involved in blooming events such as occlusion injury, repair process and scar tissue formation marking the pre-conditional phase of heart. [4] Several parameters of systemic inflammatory markers are also associated with Myocardial Infarction (MI) disease. However, the level of high-sensitivity C-reactive protein (hsCRP) circulating in body fluids have special attention and correlated to IL-6 and Plasma fibrinogen as risk factors, responsible for provoking the myocardial infarction events. [5] Moreover, it has been established that IL-6 induces the hs-CRP level. In addition, many associated risk factors (such as hypercholesterolemia and blood sugar fasting [BSF], dyslipidemia, diabetes, hypertension [HT], alcohol consumption), are linked to, but had significant effect of tobacco intake, cigarette smoking on MI disease progression and complications in a clinical and population-based studies. [6,7] As discussed in prior studies, the age dependent death rate per 100,000 population from cardiovascular disease (CVD)is estimated to be 277.9 (at 95% confidence interval, 16.2% to 12.5%) from 2006 [8]. In a systemic review of coronary artery disease (CAD) incidence from India, Krishnan et al., [9]commented that alone Kerala had a highest prevalence cases of CAD but no recent studies was reported in this state. The MI prevalence varies from 1%-2% in rural populations and 2%-4% in urban populations. [9] Chan et al., [6] reported higher prevalence of MI among Asian group subtypes, Indians, Malaya and Chinese but the risk profile of each racial groups is differed due to genetically differed composition, dietary and lifestyle behavior [6,10]. In the 2016 year, approx. 17.6million (at 95% CI, 17.3-18.1 million) death cases were reported from CVD at the global level, [11] accounting to an increment of 14.5% (at 95% CI, 12.1%-17.1%) from 2006 [8].Thus, the prior studies confirmed difference in mortality risk factors among each Asian group [12]. The aim of the study was to explore Interleukin-6 and Plasma Fibrinogen and association of myocardial infarction (hsCRP, Lipid profile and blood sugar fasting among Myocardial Infarction and healthy controls.

Study Sample
Case groups were confirmed as a study group, and distinguished from Control groups, by following the basic selection criteria prescribed by Physicians and expert's cardiologist. Prescribed selection criteria were as follows:

Measurement of hsCRP, IL-6 and plasma fibrinogen concentration
Admitted patients suffering from myocardial infarction were taken from diagnosed cases of myocardial infarction immediately collected venous blood samples and centrifuged (Thermo Scientific™ Sorvall™ Legend™ Micro 21 Microcentrifuge manufacture by Waltham city, United States) 31 at 4000 x g for 5 minutes. Serum and plasma were separated into Eppendorf tubes for analysis. The case and control subjects for the concentrations of human IL-6 serum and plasma fibrinogen were determined using a commercially available immunoenzymatic assay (ELISA) kit (purchased from Elabscience Biotechnology Co., Ltd. Houston, United States) [17]. Absorbance was read at 450 nm using automated Microplate Reader [18] and washer Thermo [19] Scientific (Multiskan™ FC analyzer manufacture by Waltham city, United States). Specimens were stored at -80 o C in a deep freezer (Thermo Scientific™ Forma™ 89000 Series Ultra-Low Freezers) manufacture by Waltham city, United States.

Body Mass Index (BMI) assessment
Anthropometric, lifestyle, and dietary data were derived from the questionnaire administered to female and male group, with missing information substituted from previous questionnaires.

Blood pressure measurement
Systolic and diastolic blood pressure measured in a sitting position, after a five-minute rest, using a mercurial sphygmomanometer instrument.

Statistical Analysis
Statistical data were compared using brand IBM SPSS Statistics software, statistical package for the social sciences (SPSS) version 21 developed by country of United States. Mean, Standard deviation, testing of hypothesis can be performed by using an un-paired Student t-test, chi square test. While the drawn ROC curves through the SPSS version 21software; the entire stats test were also performed by using this software. Statistical comparative analysis of data was performed in columns, followed by the unpaired t-test, based on data distribution; Graphical plot with diagnostic ability were analyzed using receiver-operating characteristic method. The tested parameters were considered significant when critical, P >0.01level was set up, for a 95% CI.

RESULTS
Current study is case-control, discussing the lipid and biochemical marker characteristics. Total number of (n=178) individuals (males=65; mean age of 65years, females=24, mean age of 58 years at 95% CI)) participated in this study, out of these, 89 individuals were expected with risk of myocardial infarction (cases), and remaining 89 individuals were of healthy persons (controls), study subjects. Case-control population study is summarized in Table 1. discussing the coronary heart disease (CHD) incidence in studied population of participants, involving both males' and females' individuals, all the participated subjects were found to be almost similar age group during the past 5-year study duration.  Table 1 Prevalence of, hypertension, diabetes, dyslipidemia, Tobacco, Sweating, Cough and palpitation characteristics were appeared more significantly in males, at (P <0.01) compared to females, and rest of the factors showed no significance, at P >0.01 level. However, hypertension, diabetes and dyslipidemia conditions, are the most common causes of development of plaque formation and rupture of the capillary artery, which leads to announcement between the lipid content of the plaque and the blood flowing through the arterial lumen, lastly occlusion of the coronary artery by the thrombus reduces the blood supply to the myocardial tissues leading to ischemia and necrosis, eventually causing myocardial infarction. Some symptoms for example: sweating, cough and palpitation were found more significantly expressed in males than females, as in myocardial infarction patients. It means, male individuals were more prone to high risk of myocardial infarction than female individuals. Table 2. Case-control comparison study is represented, depicting, case with myocardial infarction and healthy persons treat as control group, having same age and sex were affected in both the groups. Risk factors which are taken under consideration in this study are blood sugar fasting (BSF), Total cholesterol (TC), Triglyceride (TG), Low density lipoprotein (LDL), Very lowdensity lipoprotein (VLDL), Triglyceride/High density lipoprotein (TG/HDL-c), Total cholesterol/High density lipoprotein (TC/HDL-c), Low density lipoprotein/High density lipoprotein (LDL/HDL-c); high sensitivity c-reactive protein (hsCRP), Interleukin-6 (IL-6) and Plasma Fibrinogen. Current studied risk factors were found significantly higher in level, at (P<0.01), in case groups, except High density lipoprotein (HDL). In Levene's test analyses, proves the key controlling factors are: hsCRP, IL-6, and plasma fibrinogen, and level of these found significantly higher, at (P <0.01),>4mg/l of hsCRP, 36.37 of IL-6 pg/ml and 25.85ng/ml was observed in males (of case group) than control groups.
Highlighted key controlling factors are: hsCRP, IL-6, and plasma fibrinogen, directly influence the lipid and non-lipid profile factors. Out of the considered risk factors, only body-mass index (BMI), total cholesterol (TC), LDL cholesterol, and triglycerides (TG) were found significant with positive correlated in case group, while negatively correlated with HDL cholesterol. Furthermore, the prevalence of smoking, hypertension, and diabetes risk factors had found significant higher in case group, as depicted in Table 3. In univariate analysis was taken under consideration for, hsCRP, IL-6, and plasma fibrinogen risk factors, an observed factor were significantly higher in case groups compare to control group. However, after adjustment with other risk factors, a significant association of hsCRP and hypertension were lost but the significance was observed between the diabetes and dyslipidemia. Fig. 1 described the dyslipidemia association with alcohol intake at P =0.161; diabetes at P =0.674 and least with hypertension at P =0.746 while Fig. 2 discussed the diabetes association, majorly with IL-6 at P =0.172, and hsCRP at P =0.435, least association with plasma fibrinogen, at P =0.795.
Etiological study of myocardial infarction is complex and dependent on several risk factors such as alcohol consumption, hypertension, diabetes, tobacco intake and physical activity significantly and moderately provoked the risk of dyslipidemia, except one, insignificant risk factors, that is, cigarette smoking. This can be demonstrated by ROC curve for smoking, indicated by yellow line, which is fallen below the reference line/baseline, as indicated by sky-blue color. In addition, the most influential risk factors affecting the dyslipidemia condition followed the  2&3. Receiver-operating characteristic (ROC) and Scatter diagram curves depict the Dyslipidemia associated risk factors, interleukin(IL-6)(pg/ml) and Plasma Fibrinogen (ng/ml), for the identification of myocardial infarction. IL-6(pg/ml) had strong positive correlation with Plasma fibrinogen (ng/ml), that is, predicted from r 2 =0.767, and showed the significance, at P=0.000 level, as depicted in a Fig. 3 compare to case group.

Fig. 1. Receiver-operating characteristic (ROC) curves with respective areas under the ROC curve showing the dyslipidemia association with hypertension, diabetes, alcohol, tobacco, smoking and physical activity, for myocardial infarction risk identification in a subject group
Considered risk factors such as IL-6 and plasma fibrinogen, were not perfectly correlated with dyslipidemia, as depicted in a Fig. 2. ROC curve depicted ROC curve, for test variables was found in significant, as predicted in Table 4 representing the area under curve (AUC) values, which lies in between the 0.497-0.527 of IL-6 and Plasma fibrinogen. Since, the (AUC) Table 4 predicts insignificant correlation between the risk factors: IL-6, plasma fibrinogen, with dyslipidemia, at 95% confidence interval (CI). In terms of effectiveness, IL-6 had found more significant effect than plasma fibrinogen on dyslipidemia condition. However, the best cutoff scores value for IL-6 and plasma fibrinogen were 66.6 (AUC 0.497), and 66.6 (0.527). It was noted from these studied cutoff values for IL-6 and plasma fibrinogen factors against dyslipidemia, representing their test sensitivity, which are equally decreased while diagnosing the dyslipidemia condition among case subjects. Observed study demonstrate the dyslipidemia and diabetes condition were appeared in case subject groups because, it follows the descending trends of IL-6 (sensitivity =0.381) and (sensitivity =0.313), plasma fibrinogen (sensitivity =0.409) and (sensitivity =0.374) and hsCRP (sensitivity =0.0) and (sensitivity =0.345) compare to control group.

Fig. 3. Scatter diagram Dyslipidemia with interleukin -6 (pg/ml) and Plasma Fibrinogen (ng/ml) correlated of myocardial infarction
Considered risk factors such as hsCRP, IL-6 and plasma fibrinogen were not perfectly correlated with diabetes as depicted in Fig. 4. ROC curve. Both the risk factors, IL-6 and hsCRP, were equally correlated with diabetes but no significant correlation of plasma fibrinogen was observed in this study. The best cutoff scores of hsCRP, IL-6 and plasma fibrinogen for diagnosing the diabetes were 0.076(AUC 0.423), 0.076(AUC 0.485) and 0.0(AUC 0.456), as depicted in area under curve. Table 5. By following the Youden index (YJ) method. It was noted that the cutoff point value was found low for IL-6 and hsCRP risk factors, but the test for sensitivity, which is used to predict the IL-6 and plasma fibrinogen level was found good but no significant effect of plasma fibrinogen was observed in the study.

DISCUSSION
Myocardial infarction (MI), commonly known as heart attack, is caused by the coronary artery lesions due to insufficient supply of oxygen (also known as ischemia); generally observed in old aged patients of more than 65 years [20]. Limited research studies have demonstrated an association of inflammatory markers with MI [21]. An elevated level of inflammatory marker signals to restrict the supply of blood to the heart muscles, which leads to muscle necrosis and ischemia events, eventually causing myocardial infarction [22,23]. Present study is a case-control comparative study, diagnosing MI risk in hundreds of seventy-eight participants through the marker system characterizing lipid profile factors by demonstrating the level of non-lipid profile risk factors. Earlier reported studies extensively discuss about the hsCRP biomarker [24]. Mean hsCRP level was higher in present study than those earlier reported studies, [25] it was noted from this study, that higher hsCRP level depicts the high risk of patients for the development of cardiovascular disease (CVD) [26,27].
The vast majority of examined case groups were affected and had (>4mg/l of hsCRP) level in >90% case groups were detectable in this study whereas other studies reported above 3mg/l level of hsCRP in 50% patients of CVD [24,28]. Mean of IL-6 and plasma fibrinogen is elevated significantly in this study but lesser than the other reported studies [29]. An elevated level of these markers can be explained by differences in age, body mass index (BMI) ,excessive consumption of alcohol, and other inherent intake of tobacco.
Smoking had a slight activity among the studied case groups. Mean of Lipid profile factors such as total cholesterol (TC), triglycerides (TG) and LDL cholesterol level is enhanced drastically, whereas HDL level is lowered especially in male case groups compared to female case groups.Similarl findings reported by Pokharelet al. [30] Interleukin-6 (IL-6) is the key promoter of hsCRP production and secreted out from hepatocytes. It has also been reported that the IL-6 also predicts the future risk of myocardial infarction in a middle-aged individual [31] similarly reported in this study. The studied mean data of IL-6 represent the development of dyslipidemia and diabetes with myocardial infarction (MI) as previously reported in Pakistan, suggesting that Coronary arterial disease (CAD) were found in patients with diabetes [32]. It was noted that lowered blood serum of IL-6 and higher plasma fibrinogen factors, found in case subjects but it developed diabetes and dyslipidemia condition, compare to control subjects. Same can be found in study done by Ali et al., [33] in a more elaborate way on the basis of hypertension, alcohol consumption but no significant risk was observed by the intake of tobacco, cigarette smoking and physical activity. Mahalleet al., [34] reported that the age, sex, BMI and IL-6 had no significant correlation with the hypertension (HTN), and this study reported the same [34]. Fibrinogen is an acute phase protein and well-known coagulation factor in the blood [35]. It is associated with those patients who had Carotid atherosclerosis and Myocardial infarction disease [36]. A positive relation was found in between the fibrinogen and risk of cell death of myocardial muscles similarly reported in this current study. Constant cardiac output occurred, if the fibrinogen level is exceeded from 400mg/dl plasma concentration [37], as noted in this study, that fibrinogen level (>400mg/dl) is much lesser than reported value, thus, no significant effect of fibrinogen on myocardial infarction was found, in this study. Further studies are warranted to investigate the underlying mechanism of depression emphasized on dyslipidemia and diabetic condition, causing the increased risk of myocardial infarction and CHD death.

CONCLUSION
This study concludes the importance of risk factors involving higher Interleukin-6 and plasma fibrinogen level instead of high sensitivity Creactive protein in coronary arteries blockage or cardiac heart disease. The lipid profiles and markers for lipids are used to distinguish the two studied groups: Case and control groups. These markers play a vital role in diagnosing an association between the lipid and non-lipids profile markers which includes the age, smoking, alcohol consumption, tobacco intake, body mass index and cholesterol, triglyceride level provokes dyslipidemia, diabetes, and arterial hypertension conditions, especially observed in case groups, and no significant effect of physical activity was observed. Body mass index (BMI) was significantly covariate at P =0.01 level with hsCRP of the case group while it was not significantly correlated with hsCRP in the control group. However, study results showed that the cholesterol, LDL cholesterol, and triglycerides were significantly higher in the case group, whereas HDL cholesterol was lower compare to the control group. The current study identifies the male individuals of case groups were found at higher risk than female case groups.

STUDY LIMITATIONS
This study will be considering a large population size of each etiology, the study population comprised of newly diagnosed Myocardial Infarction. We should be added biochemical parameters of Lipoprotein-associated phospholipase A2 (Lp-PLA2) & Fatty acidbinding proteins it is a newly (FABP 3) that will be estimated. FABP 3 is more cardio-specific. The early elevation of FABP 3 in blood detection is approx. 30 mins, time to peak 6-12 hrs, and return to normal in 24 hrs in comparison to myoglobin initially shows elevation in blood approx. 1-3 hrs, time to peak 5-8 hrs and return to normal 16-24 hrs and leads to an earlier diagnosis of myocardial injury whereas current 'gold standard' troponin T initial elevation in blood approx 3-6 hrs, time to peak 10-48 hrs, and return to normal 10-15 days. The positive correlation between IL-6, FABP 3, Plasma Fibrinogen, and Lp-PLA2 new risk marker needs to be biologically plaque formation, measurable, repeatable, and show a strong and graded relationship to Myocardial Infarction. The results of the study may be helping the clinician to develop more novel therapeutic strategies for the management of MI patients.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The study was approved by the Institute Ethics Committee, Santosh University, Ghaziabad, India letter number-F.No. Su/2017/1226(16) and date of 18-December-2017 Questionnaire and informed consent were obtained from all the patients.