Scholarly article on topic 'Comparative analysis of biochemical parameters in diabetic and non-diabetic acute myocardial infarction patients'

Comparative analysis of biochemical parameters in diabetic and non-diabetic acute myocardial infarction patients Academic research paper on "Basic medicine"

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{"Type 2 diabetes" / "Acute myocardial infarction" / "Cardiac marker" / "Oxidative stress"}

Abstract of research paper on Basic medicine, author of scientific article — Fatima Ali, Syed Ali Shabaz Naqvi, Mehwish Bismillah, Nadia Wajid

Abstract Background Diabetes is a metabolic disorder characterized by enhanced production of free radicals hence oxidative stress. The aim of this study was to evaluate the activity of cardiac and antioxidant enzymes in diabetic and non-diabetic acute myocardial infarction (AMI) patients. Methods This case–control study was conducted on 450 subjects (70–85 years). Subjects were divided into three groups (Normal, N; Non-diabetic AMI, N-AMI; and Diabetic AMI, D-AMI). Each individual was subjected to a detailed history, clinical examination, and cardiovascular parameters analysis (fasting blood sugar, HbA1c, systolic and diasystolic blood pressure, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), TC/HDL and LDL/HDL ratios). Cardiac markers (Troponin-I, creatine phosphokinase (CPK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), C-reactive protein (CRP) and aspartate aminotransferase (AST)) and oxidative stress markers (superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT)) were also assessed. All these parameters were compared between diabetic and non-diabetic AMI patients. Results D-AMI individuals had high level of TC, TG, LDL, and low level of HDL in comparison to N-AMI individuals. Study suggests that cardiac markers such as Troponin I, CPK, CK-MB, AST, LDH, and CRP levels were significantly increased in patients suffering from myocardial infarction with diabetes mellitus (DM) compared to patients of myocardial infarction without DM. The activity levels of antioxidant SOD and GSH were lower in D-AMI patients than in N-AMI. However, levels of MDA and CAT were higher in D-AMI than in N-AMI controls. Conclusion Study suggests elevated cardiac markers and reduced antioxidants in D-AMI patients compared to N-AMI patients.

Academic research paper on topic "Comparative analysis of biochemical parameters in diabetic and non-diabetic acute myocardial infarction patients"

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Original Article

Comparative analysis of biochemical parameters in diabetic and non-diabetic acute myocardial infarction patients

Fatima Ali*, Syed Ali Shabaz Naqvi, Mehwish Bismillah, Nadia Wajid

Institute of Molecular Biology and Biotechnology (IMBB) & Centre for Research In Molecular Medicine (CRIMM), The University of Lahore, Raiwind Road, Lahore, Pakistan

ARTICLE INFO

ABSTRACT

Article history: Received 7 February 2015 Accepted 28 September 2015 Available online xxx

Keywords: Type 2 diabetes Acute myocardial infarction Cardiac marker Oxidative stress

Background: Diabetes is a metabolic disorder characterized by enhanced production of free radicals hence oxidative stress. The aim of this study was to evaluate the activity of cardiac and antioxidant enzymes in diabetic and non-diabetic acute myocardial infarction (AMI) patients.

Methods: This case-control study was conducted on 450 subjects (70-85 years). Subjects were divided into three groups (Normal, N; Non-diabetic AMI, N-AMI; and Diabetic AMI, D-AMI). Each individual was subjected to a detailed history, clinical examination, and cardiovascular parameters analysis (fasting blood sugar, HbA1c, systolic and diasystolic blood pressure, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), TC/HDL and LDL/HDL ratios). Cardiac markers (Troponin-I, creatine phosphoki-nase (CPK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), C-reactive protein (CRP) and aspartate aminotransferase (AST)) and oxidative stress markers (superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT)) were also assessed. All these parameters were compared between diabetic and non-diabetic AMI patients.

Results: D-AMI individuals had high level of TC, TG, LDL, and low level of HDL in comparison to N-AMI individuals. Study suggests that cardiac markers such as Troponin I, CPK, CK-MB, AST, LDH, and CRP levels were significantly increased in patients suffering from myocardial infarction with diabetes mellitus (DM) compared to patients of myocardial infarction without DM. The activity levels of antioxidant SOD and GSH were lower in D-AMI patients than in N-AMI. However, levels of MDA and CAT were higher in D-AMI than in N-AMI controls.

Conclusion: Study suggests elevated cardiac markers and reduced antioxidants in D-AMI patients compared to N-AMI patients.

© 2015 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.

* Corresponding author. E-mail address: Fatemei.ali@gmail.com (F. Ali). http://dx.doi.org/10.1016/j.ihj.2015.09.026

0019-4832/© 2015 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.

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1. Introduction

Diabetes mellitus (DM) increases the incidence of cardiovascular diseases (CVDs) and increases the risk of CVD-induced mortality in diabetic subjects compared to non-diabetic subjects.1,2 Coronary artery disease (CAD) contributed to myocardial infarction (MI) and heart failure, attributed to most of the mortalities around the globe.3-5 Acute myocardial infarction (AMI) is associated with obstruction of coronary artery, myocardial ischemia leading to myocardial necrosis and generation of reactive oxygen species (ROS).6 Previous studies show that hyperglycemia promotes ROS-induced complications of heart by reacting with lipids, protein, and DNA7; this oxidative damage is rescued by myocardial antioxidants.8 Several studies depicted that antioxidants functioning is diminished in diabetic subjects,9 which may further augment the oxidative stress-induced pathogenesis of AMI.10 Diabetes, dyslipidemia, hypertension, family history, obesity, and smoking are well documented risk factors for the development of AMI.11

The purpose of the study was to assess the oxidative stress-induced damage to heart in diabetic and non-diabetic AMI patients. This study emphasizes that antioxidants imbalance may be a key indicator of diabetes-induced myocardial damage as other indicators such as ECG and cardiac biomarkers. The data showed significant increase in lipid parameters (total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cardiac markers, i.e. troponin-I (TnI), creatine phosphokinase (CPK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), C-reactive protein (CRP) within 12 h after the onset of chest pain in D-AMI patients compared to N-AMI patients. Oxidative stress markers such as malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) also increased in D-AMI patients compared to N-AMI patients. Study results suggest that antioxidants based interventions in D-AMI patients might assist to reduce oxidative stress-induced damage in D-AMI patients.

2. Methods

2.1. Subjects and study design

This case-control study included 450 subjects; out of which, 150 subjects (90 males and 60 females) were with normal blood glucose level and with normal ECG (Normal, N), 150 subjects (85 males and 65 females) were with normal blood glucose level and AMI (non-diabetic and AMI, N-AMI), and 150 subjects (98 males and 52 females) were with diabetes and AMI (Diabetic and AMI, D-AMI), visiting the outpatient clinic at Department of Cardiology at Services Hospital Lahore, Punjab Institute of Cardiology Lahore and Ittefaq Hospital Lahore, Pakistan from September 2013 to May 2014.

Diabetes was diagnosed by analyzing the level of glycated hemoglobin level (HbA1c > 6.5%).12 Diagnosed cases of diabetic and non-diabetic AMI patients were included after obtaining a written consent from their caretakers to take part in the

study. Questionnaires were duly filled in with bio-data of the patients, detailed medical history, blood pressure, electrocar-diography (ECG), complete blood count (CBC) along with available additional information. This study was approved by the local ethical committee at The University of Lahore, Pakistan.

2.2. Inclusion criteria and exclusion criteria

Subjects of all ages and both genders with the history of AMI were included. AMI diagnosis was based on a history of chest pain, ECG changes, and elevated cardiac enzymes.6,13 Diabetic and non-diabetic AMI patients were included in this study. The control subjects were selected on basis of being normotensive and with normal ECG. Subjects who have the history of smoking, obesity, or any other disease were excluded from this study.

2.3. Collection of blood and isolation of serum

Blood samples were collected from Department of Cardiology at Services Hospital Lahore, Punjab Institute of Cardiology Lahore and Ittefaq Hospital Lahore. Preprandial venous blood were drawn from cubital vein from all subjects and instantly transferred from hospital to CRIMM laboratory in an icebox. Blood samples were centrifuged at 2000 x g for 10 min at 4 °C. Serum was aspirated, aliquoted, and stored at -20 °C for analysis.

2.4. Evaluation of cardiovascular parameters

Serum levels of TC, TG, and HDL were measured spectropho-tometrically using commercial assay kits (Randox laboratories Ltd, United Kingdom). LDL was calculated by using Friedewald formula.14

2.5. Analysis of cardiac markers

Levels of various cardiac enzymes including troponin-I (TnI), CPK, CK-MB, LDH, AST, and CRP were assessed using commercial kits (Randox laboratories Ltd, United Kingdom).

2.6. Estimation of oxidative stress

Oxidative stress was measured by analyzing the serum level of MDA, CAT, SOD, and GSH at Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore.

2.7. Determination of SOD activity

SOD activity was determined by the method of Kakkar et al.15 Homogenate was prepared by mixing serum and trichloroa-cetic acid (50%) in 1:1 ratio and centrifuged at 13,000 rpm for 10 min at 25 °C. 15 mL supernatant was added to 120 mL sodium pyrophosphate buffer (52 mM, pH 8.3), 12 mL phenazine methosulphate, 36 mL nitroblue tetrazolium. Reaction was started by addition of 24 mL nicotinamide adenine dinucleo-tide. After incubation at 37 °C for 90 s, reaction was stopped by addition of 12 mL of glacial acetic acid. The reaction mixture was stirred vigorously with 400 mL of n-butanol. The mixture

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was incubated for 10 min and then centrifuged at 2000 rpm for 5 min at 25 °C and butanol layer was separated. The color intensity of chromogen in butanol layer was measured at 560 nm against n-butanol using a spectrophotometer.

2.8. Estimation of GSH

Reduced GSH was assayed according to method of Beutler et al.16 Homogenate was prepared by mixing serum and trichloroacetic acid (10%) in 1:1 ratio and centrifuged at 1000 rpm for 10 min at 25 °C. 40 mL supernatant was mixed with 150 mL of 0.3 M disodium phosphate buffer. Then 25 mL of 0.001 M freshly prepared DTNB [5,5'-dithiobis (2-nitrobenzoic acid) dissolved in 1% sodium citrate] was added. Reduction of DTNB with GSH produces a yellow compound, whose absor-bance was noted spectrophotometrically at 412 nm. The reduced chromogen is directly proportional to GSH concentration.

2.9. Estimation of CAT activity

Activity of CAT was monitored by using method described by Sinha.17 40 mL serum was mixed with 360 mL phosphate buffer (10 mM, pH 7.0) and centrifuged at 13,000 rpm for 10 min at 25 °C. 21 mL of the supernatant and 180 mL phosphate buffer (10 mM, pH 7.4) were added in an eppendorf. Reaction was started by addition of freshly prepared 75 mL H2O2 (0.2 M). 360 mL potassium dichromate acetic acid reagent (5%) was added to reaction mixture and incubated for 10 min in boiling water, cooled, and absorbance was measured at 530 nm.

2.10. Estimation of MDA level

Level of MDA, a free radical species, was evaluated by measuring thiobarbituric acid (TBA) reactive substances via method of Ohkawa et al.18 For this, 40 mL of serum was taken and a homogenate was prepared in 360 ml phosphate buffers (10 mM, pH 7.4) and centrifuged at 13,000 rpm for 10 min at 25 °C. 15 mL supernatant was mixed with 15 mL SDS (8.1%), 96 mL TBA (0.8%), 96 mL acetic acid (20%) and 18 mL distilled water and incubated at 90 °C for 60 min. Afterwards, 60 mL distilled water and 300 mL n-butanol-pyridine mixture (15:1) was added and the mixture was shaken vigorously and

centrifuged at 4000 rpm at 25 °C for 10 min. The upper butanol layer was separated and its absorbance was taken at 532 nm.

2.11. Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). At first one-way ANOVA was used to compare quantitative variables between three groups and then Bon-ferroni post hoc test was used to confirm where the differences occurred between groups. All data were presented as mean ± standard deviation (SD).p < 0.05 was considered statistically significant.

3. Results

3.1. Demographic characteristics of subjects

Study subjects were divided on the basis of health conditions into N group, normal (150 subjects); N-AMI group, non-diabetic with AMI (150 subjects); and D-AMI group, diabetic with AMI (150 subjects). N-AMI patients had mean age of 80.2 ± 19.0 years, whereas D-AMI patients had mean age 70.2 ± 11.4 years. Systolic blood pressure (SBP) and diasystolic pressure (DBP) were high in D-AMI and N-AMI compared with normal group. Fasting blood glucose (FBG) and HbA1c levels were significantly high in D-AMI group (p < 0.001) compared with N-AMI and normal group. Results of basic demographic characteristics are illustrated in Table 1.

3.2. Evaluation of cardiovascular parameters

Alterations in levels of all the lipid constituents among all groups are presented in Fig. 1. D-AMI group showed significant increase in TC (270.3 ± 12.4 mg/dL), TG (301.3 ± 18.5 mg/dL), LDL (291.7 ±22.4 mg/dL) levels compared to that of N-AMI group for TC (238.2 ± 10.4 mg/dL), TG (229.2 ± 23.3 mg/dL), LDL (247.7 ± 24.2 mg/dL) and N group for TC (181.2 ± 14.2 mg/dL), TG (151.0 ± 29.0 mg/dL), LDL (139.0 ± 13.0 mg/dL), respectively (Fig. 1A-C). Whereas, D-AMI group showed significantly lower level of HDL (26.3 ± 7.0 mg/dL) in comparison to N-AMI group (31.5 ± 1.9 mg/dL) and N group (40.3 ± 5.2 mg/dL), respectively

Table 1 - General demographic characteristics.

Characteristics N group (150 subjects) N-AMI group (150 subjects) D-AMI group (150 subjects) p-Value

Male/Female (n) 90/60 85/65 98/52 -

Age (years) 80.0 ± 10.1 80.2 ± 18.9 70.2 ± 11.4 <0.001"'*

FBG (mm/L) 4-3 ± 0.6 5.2 ± 0.8 8.7 ± 1.6 <0.001"'#'a

HbAlc (%) 3.5 ± 1.2 5.8 ±2.7 9.6 ± 1.9 <0.001"'*'a

SBP (mm Hg) 122 ± 10 133 ± 24 160 ± 22 <0.001"'#'a

DBP (mm Hg) 80 ±9 84 ± 14 93 ± 20 <0.001"'#'a

All values are mean ± SD.

* p value for D-AMI versus N-AMI.

* p for D-AMI versus N group. a p for N-AMI versus N group.

D-AMI, diabetic and myocardial infarction; DBP, diasystolic pressure; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; N, normal; N-AMI, non-diabetic and myocardial infarction; SBP, systolic pressure.

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Fig. 1 - Evaluation of cardiovascular parameters. (A) Total cholesterol (TC), (B) triglycerides (TG), (C) low-density lipoproteins (LDL), (D) high-density lipoproteins (HDL), (E) TC/HDL ratio, and (F) LDL/HDL ratio. All value were expressed as mean ± SD. D-AMI versus N-AMI, *p < 0.05; D-AMI versus N group, #p < 0.05; N-AMI versus N group, and ap < 0.05.

(Fig. 1D). Also, D-AMI subjects showed high value of TC/HDL and LDL/HDL ratios compared to N-AMI (Fig. 1E and F).

3.3. Evaluation of cardiac markers

D-AMI patients had significantly higher level of CRP (7.8 ± 0.3 mg/L) as compared to N-AMI (4.4 ± 0.6 mg/L) patients (Fig. 2A).

TnI level was also found significantly high in D-AMI patient (3.3 ± 0.2 ng/mL) than N-AMI group (1.8 ± 0.2 ng/mL). The data demonstrated significant elevations of CPK (1075.0 ± 28.4 IU/L) and CK-MB (235.3 ± 16.2 IU/L) in D-AMI patients compared to N-AMI for CPK and CK-MB (324.2 ± 18.3 IU/L) (104.7 ± 5.0 IU/L) (Fig. 2B-D).

Level of LDH was found to be elevated in serum of D-AMI (1004.0 ± 30.1 IU/L) compared to N-AMI (615.0 ± 105.1 IU/L). Similar to LDH, D-AMI patients showed significant elevations of AST (102.0 ± 3.6 IU/L) compared to those of N-AMI patients (62.3 ± 14.6 IU/L) (Fig. 2E and F).

3.4. Assessment of oxidative stress markers

Oxidative stress induced in AMI was measured by evaluating levels of MDA, SOD, GSH, and CAT. There was an increase in MDA level (0.09 ± 0.0) and CAT activity (0.60 ± 0.1) in D-AMI group compared to N-AMI group for MDA (0.05 ± 0.0) and CAT (0.90 ± 0.0) (Fig. 3A and B). Compared with N-AMI group, SOD activity (0.07 ± 0.0), and GSH level (0.07 ± 0.0) were decreased

in D-AMI group for SOD (0.05 ±0.0) and GSH (0.05 ±0.0), respectively (Fig. 3C and D).

4. Discussion

AMI is initiated by myocardial ischemia due to enhanced production of ROS,10 activation of proinflammatory reac-tions,19 impaired functioning of antioxidants,20 and increased lipid peroxidation.21 All these events elicit the activation of plaque, coronary blockage and ultimately heart attack. The large segment of Pakistani population suffer from AMI.22,23 There are numerous risk factors associated with the development of AMI, such as diabetes, dyslipidemia, hypertension, smoking, obesity, advancing age, etc.6 Bartels et al.24 reported that diabetes increases the risk of CVD in diabetic subjects compared with non-diabetic subjects. The present study, presented the effect of hypertension, diabetes, and dyslipide-mia in D-AMI patients. Type 2 diabetes was found to alter lipids and lipoproteins utilization and induce atherogenic dyslipidemia.25,26 Our results show significantly higher levels of TC, TG, and LDL however; low level of HDL in D-AMI patients, and this suggests an important role of atherogenic dyslipidemia in the development of AMI in diabetic subjects.

Atherogenic dyslipidemia favors the oxidative modification of proteins along with lipids specially LDL and thus induces a local and systemic inflammatory responses.27,28 These inflammatory responses trigger myocardial tissue injury which

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Fig. 2 - Evaluation of cardiac markers. (A) C-reactive protein (CRP), (B) troponin-I (TnI), (C) creatine phosphokinase (CPK), (D) creatine kinase-MB (CK-MB), (E) lactate dehydrogenase (LDH), and (F) aspartate aminotransferase (AST). All values were expressed as mean ± SD. D-AMI versus N-AMI, *p < 0.05; D-AMI versus N group, #p < 0.05; N-AMI versus N group, ap < 0.05.

Ш N group E3 N-AMI group П D-AMI group

N group N-AMI group D-AMI group N group N-AMI group D-AMI group

Fig. 3 - Assessment of oxidative stress markers. (A) Lipid peroxidation was assessed by measuring Malondialdehyde (MDA) generation in groups, (B) catalase (CAT), (C) superoxide dismutase (SOD), and (D) reduced glutathione (GSH). All values were expressed as mean ± SD. D-AMI versus N-AMI, *p < 0.05; D-AMI versus N group, #p < 0.05; N-AMI versus N group, ap < 0.05.

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is detected by measuring the CRP level. Indeed, CRP is systemic inflammation marker and gives prognostic information of cardiovascular events such as atherosclerosis and CAD.29,30 In this study, increased CRP was found in D-AMI patients compared to N-AMI.

Heart contractility is evaluated by measuring the myocar-dial tissue specific protein Trop I, involved in cardiac contractility. Previous studies indicated that Trop I is highly sensitive and specific marker of myocardial damage and therefore used as a diagnostic marker for AMI.31 In this study, significantly raised level of Trop I was found in D-AMI patients compared to N-AMI patients indicating that cardiac muscle cell death increases in diabetic subjects.

CPK and CK-MB are two important indicators of myocardial necrosis32 and a significant elevation of CPK and CK-MB was documented in D-AMI group in this study. We also found statistically significant difference in LDH and AST values between D-AMI and N-AMI groups, the two markers being advocated for diagnosis of infarct previously.33

Hyperlipidemia and hyperglycemia-induced oxidative stress has been regarded as contributors to progression of AMI.2 The oxidative stress results in disturbance between free radicals and antioxidant defense mechanism. SOD, one of the important defense enzymes catalyzes the dismutation of superoxide radicals into either oxygen (O2) or hydrogen peroxide (H2O2).31 Glutathione peroxidase (GPX) or CAT catalyzes the reduction of H2O2 into H2O, CAT catalyzes this reduction independently without any cofactor, whereas GPX relies on GSH,34 GSH also inhibits lipid peroxidation.35 Previous reports showed that lipid peroxidation increased in AMI patients,36 and this increased lipid peroxidation is a consequence of hyperglycemia-induced oxidative stress. This study revealed significant decrease in antioxidants including SOD, GSH, and CAT, and an increase in MDA, which is lipid peroxidation product in D-AMI patients compared to N-AMI patients.

Our study indicates the significance of atherosclerosis and its associated complications such as dyslipidemia and inflammation in D-AMI patients. In conclusion, our study demonstrates a significant increase in traditional cardiac markers (CPK, CK-MB, LDH, and AST) and non-traditional cardiac markers such as CRP in D-AMI patients compared to N-AMI. The study shows a significant increase of oxidative stress parameter MDA while levels of antioxidants CAT, SOD, and GSH are reduced in D-AMI patients. These results will be helpful for clinicians in therapy of MI patients with DM.

4.1. Limitation of study

Following limitation should have to be considered for interpretation of the study: The data were collected in a short time period and from a few local hospitals. We also have limited resources for this project, so we relied on present data. Although some similar reports have been published before, but to the best of our knowledge it is the first organized data of diabetic and acute myocardial infarcted patients of local population.

Conflicts of interest

The authors have none to declare.

Acknowledgments

This work was supported by research grants from The University of Lahore, Lahore, Pakistan.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ihj.2015.09.026.

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