Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction

Sumayya Shahzad; Asif Hasan; Abul Faiz Faizy; Somaiya Mateen; Naureen Fatima; Shagufta Moin

doi:10.1177/1076029617725602

. 2017 Sep 25;24(5):780–789. doi: 10.1177/1076029617725602

Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction

Sumayya Shahzad ¹, Asif Hasan ², Abul Faiz Faizy ¹, Somaiya Mateen ¹, Naureen Fatima ¹, Shagufta Moin ^1,^✉

PMCID: PMC6714874 PMID: 28946755

Abstract

Background:

Ischemic tissue damage in myocardial infarction (MI) is allied with the exaggerated production of reactive oxygen species (ROS) beyond the countering capability of chain-breaking radical scavengers, fallouts in the form of oxidatively burdened myocardial tissue.

Methods:

One hundred and twenty five patients with MI were included in the study to evaluate the dynamics of redox status of patients by monitoring the antioxidant potential, biomarkers of oxidative stress, lipid indices, RBC membrane damage when compared to healthy individuals in patients with MI congregated on the basis of Global Registry of Acute Coronary Events (GRACE) score, risk factors, and age.

Results:

Higher levels of malondialdehyde, 8-hydroxy-2-deoxyguanosine, lipid indices, ROS content, and membrane deterioration in erythrocytes were seen in patients with MI. Furthermore, reduced activities of erythrocyte antioxidant enzymes and lower concentrations of antioxidant molecules, plus reduced total antioxidant capacity, were observed in plasma of all patients with MI with respect to control. However, elevation in oxidative stress was found to be significantly marked in patients having GRACE score >100, risk factors, and MI >45 years when compared to patients with GRACE score ≤100, without risk factors, and MI ≤45 years, respectively.

Conclusion:

These findings indicate the existence of increased oxidative damage and reduced antioxidant potential in patients with MI have a potent relationship with their GRACE risk score, risk factors, and age.

Keywords: myocardial infarction, oxidative stress, reactive oxygen species, antioxidant enzymes, 8-hydroxy-2-deoxyguanosine

Introduction

Reactive oxygen species (ROS) serve as important mediators of many physiological cellular signaling pathways in relation to cell growth, adhesion, differentiation, senescence, and apoptosis¹ in many types of cells within the cardiovascular system and elsewhere, at low-to-moderate concentrations. But in larger concentrations, they cause oxidative damage to cells, either directly or as intermediates in diverse signaling pathways, this ensues in cell dysfunction, apoptosis, and necrosis. Many evidences have shown that ROS act by adverse modification of cell components, such as proteins, lipids, and DNA.^2–4 Briefly, oxidative stress befalls when dynamic balance between ROS and antioxidant defense of the cell is disrupted, in the direction of former.

Many evidences suggest that ROS play an important role in the pathophysiology of myocardial infarction (MI).⁵ In MI two diverse types of damage occur to the heart: ischemic and reperfusion injury, both of which significantly contribute to ROS production.⁶ The ROS then react with lipid bilayer of cardiomyocytes, thereby opening the self-perpetuating chain reactions of peroxidation of lipid molecules in the membranes. The ROS also causes mitochondrial dysfunction in heart cells and may also lead to the development of serious complications like remodeling of left ventricle and infarct expansion after MI.⁷ Moreover, redox state of arterial blood can be a predicting factor for heart functioning following MI. The disease is also associated with many metabolic and hormonal disturbances, like changes in the serum lipid profile particularly the low levels of high-density lipoprotein-cholesterol (HDLc).⁸ Thus, MI is an acute inflammatory reaction supplemented with the change in lipid oxidation.

This study intends to find out the extent of oxidative stress by measuring the levels of biomarkers of lipid, protein and DNA oxidation, and various antioxidants and lipid indices in patients with MI, so as to elucidate the expanse of various oxidative stress markers in these patients. We also aimed to examine the correlation between total antioxidant status and various other parameters in patients with MI grouped according to Global Registry of Acute Coronary Events (GRACE) risk score into two groups: one with GRACE score ≤ 100 (GS ≤ 100), while other group consists of patients with GRACE score >100 (GS > 100) because many clinical practice guides recommend risk stratification in these patients using GRACE score.^9,10 Same patients were also alienated on the basis of risk factors (with [MI+RF] and without risk factors [MI]) and age (less than or equal to 45 years [MI ≤ 45 years] and >45 years [MI > 45 years]) because in recent studies, Indians were found to have increased MI incidence at young age.¹¹ Furthermore, control individuals were also divided into two age-groups (control ≤ 45 years and control >45 years). The risk factors included were hypertension, diabetes mellitus, hyperlipidemia, cigarette smoking, and family history of coronary artery disease (CAD). Calculation of GRACE score was done for each patient at the time of admission using GRACE 2.0 calculator¹² by allocating appropriate number of points for each of the specific variables: age, heart rate, systolic blood pressure, creatinine, Killip class, cardiac arrest at admission, elevated cardiac markers, and ST-segment deviation in electrocardiogram.

Materials and Methods

One hundred twenty-five consecutive patients diagnosed with MI were selected for the study. Diagnosis was made according to the criteria of MI.¹³ Normal individuals (57) were selected, who belong to the same age-group, upon their visit to the hospital for their routine checkups. Written informed consent was obtained from relative of the patients and healthy volunteers, after full explanation of the study. The current study was approved by Institutional Ethics and Research Advisory Committee of Faculty of Medicine, J. N. Medical College & Hospital, A.M.U., Aligarh, India. Patients older than 75 years and those presenting with clinically significant valvular heart disease or arrhythmia, serious conduction disturbances, and heart failure were excluded.

Collection and Processing of Blood Samples

Venous blood sample was withdrawn from the patients at the time of admission, under all aseptic conditions in two different vacutainers. One with coagulant for serum separation for lipid profile and 8-hydroxy-2-deoxyguanosine (8OHdG) analysis. Another vacutainer was containing EDTA for separation of plasma and RBCs. From RBCs, 10% hematocrit and hemolysate was prepared. Parameters involving RBC lysate and hematocrit were assessed on the same day, whereas those involving plasma were determined the next day.

Estimation of Lipid Indices

Serum total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDLc), low density lipoprotein-cholesterol (LDLc) concentrations were determined using enzymatic spectrophotometric kits (Siemens[Siemens Healthcare Diagnostics Inc, Newyork, U.S.A]) in the Aeroset automatic analyzer in central facility (J.N. Medical College & Hospital).

Antioxidant Status Assays

Total antioxidant status was assessed using ferric reducing antioxidant power (FRAP) assay in plasma¹⁴ and percentage quenching of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in hemolysate.¹⁵

Evaluation of Nitric Oxide Level

Plasma nitric oxide (NO) concentration present in the reaction mixture was assessed using Griess reagent.¹⁶

Dichlorodihydrofluorescein Assay

To determine intracellular production of ROS in hematocrit, dichlorodihydrofluorescein-diacetate ester fluorescent dye was used according to the method devised by Keller et al.¹⁷

Determination of Hemoglobin and Plasma Protein

Hemoglobin (Hb) present in hemolysate was measured using Drabkins reagent. Protocol of Lowry et al¹⁸ was used for calculation of protein concentration in plasma.

Assay for Lipid Peroxidation, Carbonylation of Plasma Proteins, Total Thiol Groups, and Oxidative DNA Damage

Estimation of lipid peroxidation was done by measuring plasma malondialdehyde (MDA) level by following Beuge and Aust method.¹⁹ Plasma protein carbonylation was determined by method of Reznick²⁰ and Sedlak and Lindsay²¹ method was used to assess total thiol (TSH) groups in plasma. The 8OHdG was assessed as a biomarker of DNA damage using highly sensitive 8-OHdG enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay) kit (Qayee Biotechnology Co, Ltd; QY-E05181).

Estimation of Reduced Glutathione and Plasma Vitamin C Level

Reduced intracellular glutathione (GSH) was measured using the Jollow et al method²² with slight modifications, and Kyaw²³ method was used to estimate plasma vitamin C level.

Determination of Antioxidant Enzymes in Hemolysate

Superoxide dismutase (SOD) activity was assayed by the method of Marklund and Marklund²⁴ whereas that of catalase was determined by the method of Claiborne.²⁵ Glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR) was estimated by the methods of Flohe and Gunzler²⁶ Habig²⁷ and Carlberg and Mannerick²⁸ respectively.

Scanning Electron Microscopy

Hematocrit (10%) of erythrocytes (RBCs) was incubated with glutraldehyde, followed by centrifugation at 1500 rpm. Pellet obtained was washed twice with phosphate-buffered saline and then suspended in same buffer. This sample was mounted on glass slide, and after incubation was washed with ethanol and distilled water. After coating, samples were examined using a scanning electron microscope (SEM).

Statistical Analysis

Quantitative values of the groups are given as the mean (standard deviation [SD]). In order to find the significance of results, values of patients and controls were compared using Mann-Whitney U test, as data values does not show normal distribution upon analysis using Shapiro-Wilk normality test. Also, different variables were correlated by utilizing Spearman correlation coefficient test by Graph Pad Prism software (Graph Pad Software Inc, San Diego, CA, USA), version 7.0, with P values of ≤ .05 considering to be statistically significant.

Results

The baseline characteristics of the study population are shown in Table 1. Among risk factors, higher prevalence was observed in hyperlipidemia (36%), followed by hypertension (23%), smoking (22%), diabetes (17%), and family history of CAD (14%).

Table 1.

Demographic Characteristics of the Study Groups.

Variables	Control, n = 57	MI, n = 125
Age	50.28 (9.71)^a	51.70 (12.09)^a
Sex (M/F)	40/17	96/29
BMI (kg/m²)	23.28 (2.48)^a	24.17 (3.19)^a
Family history of CAD	4 (7%)	18 (14%)
HTN	–	29 (23%)
DM	–	21 (17%)
Hyperlipidemia	–	45 (36%)
Smoking	–	27 (22%)

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Abbreviations: MI, myocardial infarction; BMI, body mass index; CAD, coronary artery disease; HTN, hypertension; DM, diabetes mellitus.

^aValues are expressed as mean (SD).

We observed that patients with MI had significantly higher mean level of TC, TG, and LDLc compared tocontrol (TC = 158.41 ± 18.26, mean [SD]; TG = 121.35 ± 19.11; LDLc = 89.05 ± 23.15 mg/dL), as anticipated. But HDLc levels in healthy control = 45.08 ± 10.22 mg/dL was substantially elevated than other MI groups. These parameters were also significantly different among MI (TC = 172.50 ± 14.01; TG = 136.14 ± 26.56; HDLc = 40.37 ± 9.26; LDLc = 104.90 ± 19.67 mg/dL) and MI+RF groups (TC = 203.40 ± 28.36; TG = 151.08 ± 23.42; HDLc = 35.47 ± 7.92; LDLc = 137.71 ± 28.79 mg/dL). However, lipid profile of groups GS > 100 (TC = 191.08 ± 23.56; TG = 148.58 ± 23.13; HDLc = 36.38 ± 7.75; LDLc = 124.97 ± 25.90 mg/dL) and MI >45 years (TC = 190.74 ± 24.57; TG = 146.74 ± 21.93; HDLc = 36.22 ± 8.98; LDLc = 125.16 ± 27.44 mg/dL) were found to be altered from the groups GS ≤ 100 (TC = 188.20 ± 31.02; TG = 140.63 ± 27.73; HDLc = 38.81 ± 9.66; LDLc = 121.25 ± 33.19 mg/dL) and MI ≤ 45 years (TC = 187.38 ± 32.84; TG = 140.04 ± 31.70; HDLc = 40.32 ± 8.05; LDLc = 119.04 ± 33.96 mg/dL), respectively, although this alteration was not statistically noteworthy (Table 2).

Table 2.

Lipid Profile of Different Study Groups.

Parameters, mean (SD)	Control, n = 57	GS ≤ 100, n = 66	GS > 100, n = 59	MI, n = 56	MI+ RF, n = 69	MI ≤ 45, n = 44	MI > 45, n = 81
Total cholesterol, mg/dL	158.41 (18.26)	188.20 (31.02)^a	191.08 (23.56)^a	172.50 (14.01)^a	203.40 (28.36)^a,b	187.38 (32.84)^a	190.74 (25.57)^a
Triglycerides, mg/dL	121.35 (19.11)	140.63 (27.73)^a	148.58 (23.13)^a	136.14 (26.56)^a	151.08 (23.42)^a,b	140.04 (31.70)^a	146.74 (21.93)^a
HDLc, mg/dL	45.08 (10.22)	38.81 (9.66)^a	36.38 (7.75)^a	40.37 (9.26)^a	35.47 (7.92)^a,b	40.32 (8.05)^a	36.22 (8.98)^a
LDLc, mg/dL	89.05 (23.15)	121.25 (33.19)^a	124.97 (25.90)^a	104.90 (19.67)^a	137.71 (28.79)^a,b	119.04 (33.96)^a	125.16 (27.44)^a

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Abbreviations: GS, GRACE score; MI, myocardial infarction; RF, risk factors; HDLc, high-density lipoprotein-cholesterol; LDLc, low-density lipoprotein-cholesterol.

^aP ≤ .05 vs control group.

^bP ≤ .05 vs MI.

Our findings showed antioxidant status of plasma in the form of FRAP values and antioxidant power of hemolysate as represented by their DPPH radical scavenging ability (Figure 1A and B). It can be noted that FRAP value and percentage quenching of DPPH decreased significantly in MI groups with respect to control (FRAP = 775.67 ± 100.41 μmol/L; % quenching of DPPH radical = 52.88 ± 8.96). This decrement was pronounced in diseased group with GS > 100 (FRAP = 549.67 ± 97.53 μmol/L; percentage quenching of DPPH radical = 40.98 ± 9.85) as compared to GS ≤ 100 (FRAP = 642.21 ± 99.58 μmol/L; % quenching of DPPH radical = 45.88 ± 9.85). Antioxidant capacity of plasma and hemolysate was also found to fall in MI+RF group (FRAP = 575.71 ± 116.10 μmol/L; % quenching of DPPH radical = 40.67 ± 9.66) when compared to MI alone (FRAP = 626.58 ± 92.10 μmol/L; % quenching of DPPH radical = 47.15 ± 9.57), and in higher age-group MI (FRAP = 553.46 ± 99.94 μmol/L; % quenching of DPPH radical = 41.57 ± 9.72) than lower age-group patients with MI (FRAP = 681.50 ± 67.61 μmol/L; percentage quenching of DPPH radical = 47.26 ± 9.89 P ≤ .05, Table 3).

Figure 1. — A, FRAP values in control and patients with MI grouped on the basis of GRACE score (GS ≤ 100 and GS > 100); risk factors (MI and MI+RF). B, DPPH radical scavenging activity of hemolysate in various study groups. C, Plasma NO concentration in healthy and diseased individuals. D, Variations in oxidative intermediates production in different study groups, determined by DCFH assay. Results are expressed as mean (SD); * p ≤ .05 vs control; # P ≤ .05 vs GS ≤ 100; † p ≤ .05 vs MI; ¶ p ≤ .05 vs MI ≤ 45 yrs. FRAP indicates ferric reducing antioxidant power; MI, myocardial infarction; GRACE, Global Registry of Acute Coronary Events; RF, risk factors; DPPH, 2,2-diphenyl-1-picrylhydrazyl; NO, nitric oxide; DCFH, dichlorodihydrofluorescein; GS, GRACE score.

Table 3.

Biochemical Parameters of Healthy Control Individuals and Patients with MI of Lower (≤ 45 years) and Higher (>45 years) Age Groups.

Biochemical parameters	Control ≤ 45 years (n = 18)	MI ≤ 45 years (n = 44)	Control >45 years (n = 39)	MI >45 years (n = 81)
FRAP, μmol/L	788.37 ± 78.16	681.50 ± 67.61^a	769.81 ± 109.60	553.46 ± 99.94^b,c
% quenching of DPPH	55.00 ± 11.02	47.26 ± 9.89^a	51.90 ± 7.80	41.57 ± 9.72^b,c
NO, μmol/mL	2.71 ± 1.67	7.19 ± 3.29^a	3.28 ± 2.02	9.07 ± 3.70^b,c
MDA, nmol/mL	1.66 ± 1.27	3.57 ± 2.35^a	2.81 ± 1.42^a	5.00 ± 2.39^b,c
Protein carbonyl, nmol/mg protein	1.63 ± 0.98	2.29 ± 1.01^a	1.97 ± 0.87	2.94 ± 0.91^b,c
TSH groups, μmol/L	380.14 ± 67.47	342.20 ± 47.34^a	361.49 ± 50.20	291.28 ± 53.23^b,c
8OHdG, ng/mL	3.63 ± 1.56	5.35 ± 0.82^a	4.21 ± 1.40	8.18 ± 1.48^b,c
GSH, nmol/mg protein	6.52 ± 1.18	5.15 ± 1.75^a	6.34 ± 2.00	3.37 ± 1.66^b,c
Vitamin C, mg/dL	1.41 ± 0.38	0.58 ± 0.24^a	1.28 ± 0.36	0.50 ± 0.23^b
SOD, U/mg Hb	17.20 ± 3.41	14.48 ± 4.53^a	16.43 ± 2.81	12.66 ± 4.10^b,c
Catalase, U/mg Hb	8.32 ± 0.99	6.47 ± 1.15^a	8.07 ± 1.16	5.03 ± 0.96^b,c
GPx, U/mg Hb	14.03 ± 2.01	10.89 ± 3.04^a	12.06 ± 2.23^a	9.60 ± 2.66^b,c
GST, U/mg Hb	6.29 ± 0.83	4.20 ± 1.13^a	6.23 ± 1.82	3.90 ± 1.20^b
GR, U/mg Hb	9.67 ± 1.49	6.17 ± 2.11^a	9.20 ± 1.52	5.64 ± 1.67^b

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Abbreviations: MI, myocardial infarction; FRAP, ferric reducing antioxidant power; DPPH, 2,2-diphenyl-1-picrylhydrazyl; NO, nitric oxide; MDA, malondialdehyde; TSH, total thiol hormone; 8OHdG, 8-hydroxy-2-deoxyguanosine; GSH, glutathione; SOD, superoxide dismutase; Hb, hemoglobin; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase.

^aP ≤ .05 vs control ≤ 45 years,

^bP ≤ .05 vs control >45 years,

^cP ≤ .05 vs MI ≤ 45 years.

The NO levels estimated in patients with MI displayed noteworthy escalation, in relation to control individuals (3.10 ± 1.92 μmol/mL). Statistical increment in NO level was exhibited in GS > 100 (9.66 ± 3.15 μmol/mL), MI+RF (9.17 ± 3.48 μmol/mL), and MI > 45 years (9.07 ± 3.70 μmol/mL) groups, as compared to GS ≤ 100 (7.29 ± 3.75 μmol/mL), MI (7.48 ± 3.69 μmol/mL), and MI ≤ 45 years (7.19 ± 3.29 μmol/mL) groups, respectively (Figure 1C).

In Figure 1D, mean concentration of ROS in all healthy patients was found to be lesser to a much greater extent than all MI groups. Significant change was found amid MI groups when patients were segregated according to GS (GS ≤ 100 = 147.55 + 26.71; GS > 100 = 162.08 + 26.53 % of control), risk factors (MI = 143.93 + 24.37; MI+RF = 162.91 + 27.12 % of control), and age (MI ≤ 45 years = 141.58 + 29.81; MI > 45 years = 161.38 + 23.56 % of control; Table 3).

All patients with MI exhibited higher MDA level than control group (2.45 ± 1.47 nmol/mL). Patients with GS ≤ 100 (4.01 ± 2.47 nmol/mL) was having MDA level lower than patients with MI having GS > 100 (5.09 ± 2.34 nmol/mL). Of all patients, younger ones (3.57+2.35 nmol/mL) and those without any risk factor (3.75 + 2.22 nmol/mL) had decreased lipid peroxidation level than MI > 45 years (5.00 + 2.39 nmol/mL) and MI + RF (5.11 + 2.50 nmol/mg) groups, respectively (P ≤ .05; Figure 2A, Table 3).

Figure 2. — A, Plasma MDA level in control and patients with MI. B, Carbonylation of plasma protein and total thiol groups (C) in plasma of patients with respect to control group. D, Extent of 8OHdG in serum of healthy and disease individuals. Results are expressed as mean (SD); * P ≤ .05 vs control; # P ≤ .05 vs GS ≤ 100; † P ≤ .05 vs MI. MDA indicates malondialdehyde; MI, myocardial infarction; 8OHdG, 8-hydroxy-2-deoxyguanosine; GS, GRACE score.

The overall mean protein carbonyl concentration in nondisease group (1.86 ± 0.91 nmol/mg protein) was prominently diminished in comparison to diseased groups. Moreover, protein carbonyl level among various disease groups was differing statistically significantly (GS ≤ 100 = 2.51 + 1.00; GS > 100 = 2.94 + 0.95; MI = 2.39 + 0.96; MI+RF = 2.97 + 0.95; MI ≤ 45 years = 2.29 +1.01; MI > 45 years = 2.94 + 0.91 nmol/mg protein; Figure 2B). Plasma TSH level was also markedly elevated in healthy individuals serving as control (367.38 ± 56.29 μmol/L) with respect to MI groups (GS ≤ 100 = 319.78 ± 51.79; GS > 100 = 297.37 ± 59.73; MI = 327.35 ± 49.95; MI+RF = 294.48 ± 57.69; MI ≤ 45 years = 342.20 ± 47.34; MI > 45 years = 291.28 ± 53.23 μmol/L; Figure 2C). Also, there exists difference in amount of TSH amid MI groups (P ≤ .05).

There was significant difference observed in DNA damage between the patients and the control groups, as assessed by serum 8OHdG level. It was increased in patients with MI when compared to the healthy volunteers (4.03 ± 1.47 ng/mL; Figure 2D, Table 3). The results also showed an upsurge in 8OHdG concentration in GS > 100 (7.94 ± 1.75 ng/mL) with respect to GS ≤ 100 group (6.51 ± 1.71 ng/mL); in MI+RF (7.79 ± 1.81 ng/mL) group when compared to MI group (6.44 ± 1.67 ng/mL); in MI > 45 years (8.18 ± 1.48 ng/mL) than MI ≤ 45 years group (5.35 ± 0.82 ng/mL) at P ≤ .05.

We also got significant decrease in GSH, a nonenzymatic antioxidant in all the groups of MI when compared to control (6.40 ± 1.77 nmol/mg Hb). Moreover, GSH level was higher in GS ≤ 100 (4.70 ± 1.74 nmol/mg Hb), MI (4.51 ± 1.73 nmol/mg Hb), MI ≤ 45 years (5.15 ± 1.73 nmol/mg Hb) groups in comparison with GS > 100 (3.17 ± 1.72 nmol/mg Hb), MI+RF (3.55 ± 1.91 nmol/mg Hb), MI > 45 years (3.37 ± 1.66 nmol/mg Hb) groups, respectively. Plasma vitamin C content was shrinked in all the MI groups (MI = 0.55 ± 0.21 mg/dL; MI+RF = 0.51 ± 0.26; MI ≤ 45 years = 0.58 ± 0.24 mg/dL; MI > 45 years = 0.50 ± 0.23 mg/dL) when compared to the healthy group (1.32 ± 0.37 mg/dL). Remarkable diminution in ascorbic acid concentration was found in GS > 100 group (0.44 ± 0.21 mg/dL) when compared to GS ≤ 100 (0.60 ± 0.24 mg/dL) at P ≤ .05, as shown in Figure 3A and B and Table 3.

Figure 3. — (A) Plasma GSH and vitamin C (B) in healthy control and patients with MI. Results are expressed as mean (SD); * P ≤ .05 vs control; # P ≤ .05 vs GS ≤ 100; † P ≤ .05 vs MI. GSH indicates glutathione; MI, myocardial infarction.

The mean SOD activity dropped down in the patient groups (GS ≤ 100 = 14.21 ± 4.16; GS > 100 = 12.28 ± 4.32; MI = 14.49 ± 4.20; MI+RF = 12.39 ± 4.21; MI ≤ 45 years = 14.48 ± 4.53; MI > 45 years = 12.66 ± 4.10 U/mg Hb) than control group (16.67 ± 3.00 U/mg Hb; P ≤ .05). Also, the activity of catalase was found to be repressed as compared to control (8.15 ± 1.11 U/mg Hb) in patient groups (GS ≤ 100 = 5.73 ± 1.22; GS > 100 = 5.33 ± 1.24; MI = 6.01 ± 1.12; MI+RF = 5.16 ± 1.20; MI ≤ 45 years = 6.47 ± 1.15; MI > 45 years = 5.03 ± 0.96 U/mg Hb; Figure 4A and B). Similarly, the activities of GPx (GS ≤ 100 = 10.78 ± 2.58; GS > 100 = 9.24 ± 2.95; MI = 10.87 ± 2.76; MI+RF = 9.39 ± 2.77; MI ≤ 45 years = 10.89 ± 3.04; MI > 45 years = 9.60 ± 2.66 U/mg Hb); GST (GS ≤ 100 = 4.09 ± 1.21; GS > 100 = 3.90 ± 1.41; MI = 4.19 ± 1.26; MI+RF = 3.85 ± 1.09; MI ≤ 45 years = 4.20 ± 1.13; MI > 45 years = 3.90 ± 1.20 U/mg Hb); and GR (GS ≤ 100 = 5.95 ± 1.72; GS > 100 = 5.69 ± 1.99; MI = 5.95 ± 1.90; MI+RF = 5.73 ± 1.81; MI ≤ 45 years = 6.17 ± 2.11; MI > 45 years = 5.64 ± 1.67 U/mg Hb) showed remarkable decrease from healthy control (GPx = 12.68 ± 2.34; GST = 6.25 ± 1.57; GR = 9.35 ± 1.51 U/mg Hb; Figure 4C to E). The GPx activity in higher age-group controls was found to be statistically significant than their lower age-group counterparts (Table 3). Furthermore, variance in antioxidant enzyme activities was statistically significant at P value ≤.05 in GS ≤ 100, MI, and MI ≤ 45 years groups when compared to GS > 100, MI+RF, and MI > 45 years groups, respectively, except for catalase in GS ≤ 100 and GS > 100 group; and GST and GR in different groups of patients with MI.

Figure 4. — Shift in the activities of several antioxidant enzymes: (A) SOD, (B) catalase, (C) GPx, (D) GST, (E) GR in healthy individuals and patients. Results are expressed as mean (SD); * P ≤ .05 vs control; # P ≤ .05 vs GS ≤ 100; † P ≤ .05 vs MI. SOD indicates superoxide dismutase; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; MI, myocardial infarction; GS, GRACE score.

Figure 5A and B shows typical biconcave shape of RBCs, but RBCs of patients with MI exhibited visible morphological changes. Extended projections and alteration in size and shape were found due to oxidative deformation of membrane of these cells.

Percentage quenching of DPPH in patients with MI was found to had positive correlation with FRAP, TSH, GSH, vitamin C, SOD, catalase, GPx, and GS. We also discovered significant inverse correlations between DPPH percentage quenching and NO, ROS, MDA, protein carbonyl, and 8OHdG level. Correlation between percentage quenching of DPPH and GR, GST was not found to have any significant P value.

Ferric reducing antioxidant power of plasma in patients with MI showed positive correlations with DPPH, TSH, GSH, vitamin C, SOD, catalase, GPx, GST, GR and GS. We also observed FRAP values to be negatively correlated with NO, ROS, MDA, protein carbonyl, and 8OHdG level (supplementary material).

Discussion

Altered lipid profile is a conventional risk factor for MI, and the results of the present work indicated exceedingly high levels of TC, TG, and LDLc in the patients with MI. Higher fat and carbohydrate in diet could be a reason for raised TC, TG, and lower HDLc in the patients. Moreover, decreased HDLc denoted weakened reverse cholesterol transport in the patients. Increased LDLc content further indicates its prospective deposition in arterial intima of the patients which had culminated in the form of infarction.

Myocardial ischemia leads to ultrastructural damage, contractility dysfunction, increase in ROS production, and change in myocardial metabolism, which culminates in necrosis of cells. We found elevated ROS level in patients with MI. Thus, implying such a grave increment in ROS generation, which antioxidant defense, is unable to tackle. The ROS content was also found to be increased in heart of rats that suffered myocardial ischemic/reperfusion injury.²⁹

Nitric oxide is produced constitutively by eNOS at low level, but we detected exaggeration in NO content of patients with MI. This is consistent with similar findings in earlier studies.³⁰ Higher NO level can be accredited to increased activity of iNOS in activated macrophages, although NO is a vasodilator but such exceedingly high NO level may combine with superoxide radical producing more potent radical peroxynitrite that particularly oxidizes zinc fingers, iron/sulfur centers, and thiol groups in proteins, thereby driving toward more oxidant-induced stress in patients with MI.

The total antioxidant capacity of plasma and that of RBCs was found to be significantly lessened in diseased patients, inferring the weakened defense mechanism against oxidative damage. In the same line of thought, Eva Sedláková et al found an immediate and significant reduction in plasma 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).³¹

Lipid peroxidation is a part of normal metabolism, but increase in lipid peroxidation is an outcome of oxidative stress and increased concentrations of MDA found in plasma of patients with MI indicates elevated amount of TBARS in circulation of these patients. Also, higher MDA level was found in older control group than younger one, indicating that lipid peroxidation advances with age, and ischemia of myocardium further afflicts it. This increase in peroxidation of lipid molecules is attributed to a deficiency of antioxidant defense mechanism as well as larger ROS production. All this climax into intrinsic cell membrane properties alteration, and polymerization of membrane components due to physicochemical changes in oxidized lipids. Our results are in accordance with previous report of Chamblee et al.³²

One of the markers of protein oxidation, protein carbonyl, was raised prominently in patients with MI than control. Protein carbonyl is formed upon reaction of proteins with products of lipid peroxidation or due to oxidation of certain amino acid residues. This points the level of ROS-driven oxidative protein damage as a consequence of MI. The major part of plasma TSH group is derived from proteins, particularly albumin. Thiol groups are highly susceptible to oxidation, and we found significant low amount of TSH in plasma of patients with MI. This indicates enlarged consumption of TSH in neutralizing increased ROS level. Reduction in TSH availability may also affect other functions of protein inside cell such as their catalyzing ability.

In addition, oxidative DNA damage is gravely deleterious and can result in apoptosis through induction of various proapoptotic proteins. Moreover, modified DNA bases appear to play a crucial part in mutagenesis.³³ Higher extent of 8-OHdG in serum of patients with MI reflects greater polynucleotide oxidative damage in them. In this context, previous study has also shown increased level of the modified guanosine in plasma of patients with MI.³⁴

The GSH is the chief endogenous antioxidant and directly scavenges oxidized molecules and not the GSH is the chief endogenous antioxidant and directly scavenges oxidized molecules. It serves as reductant of GPx which further reduces other substrates. Our study showed significantly lessened erythrocyte GSH content in diseased individuals. This can be explained as increased utilization of GSH to counter exaggerated level of free radicals and protection of thiol (SH) group possessing proteins from oxidation. Drop in GSH content in MI model of rat had also been reported earlier.³⁵ We also detected diminution in vitamin C, another main antioxidant molecule, this could be due to its increased consumption in scavenging the oxyradicals or could be due to declined GSH level because it is involved in vitamin C recycling.

Upon excessive free radical generation, scavenging enzymes such as SOD, catalase, and GPx comes into play. The SOD dismutates superoxide anion radical and catalase decomposes hydrogenperoxide (H₂O₂) and not the SOD dismutates superoxide anion radical and catalase decomposes hydrogenperoxide (H₂O₂). Declined activity of SOD and catalase may be a result of their exhaustion, upon increased utilization in combating free radicals. Reduction in SOD activity could also be due to irreversible inhibition by its reaction product H₂O₂, which is capable of inducing changes in charges or denaturation of the enzyme. But in contrast to our study, Lubrano et al did not found any significant difference in SOD levels among patients of single and multivessel CAD.³⁶ This might be probably due to the absence of ischemic injury in these patients. Another study of Gupta et al showed upregulation in SOD and catalase level in patients with CAD, and these enzymes decreased significantly in the patients with late stage of the disease.³⁷

The GPx eliminates hydrogen and lipid peroxides, thereby shielding cellular and subcellular membranes from the peroxidative damage. We found decrease in the GPx activity in RBCs of patients with MI, which might be due to increased exploitation of the enzyme in countering aldehydic by-products of lipid peroxidation, and this also explains significant decrement in GPx activity in older controls with respect to younger ones, as former group was having much elevated level of MDA. Another contributing factor for decreased GPx activity may be reduced GSH concentration. Since GST carry out detoxification by conjugating biomolecular electrophilic centers to GSH through thiol group for the purpose of making them more water soluble, whereas GR is involved in reduction of oxidized GSH. Diminution in activities of GST and GR in RBCs of patients with MI may be owing to their fastened consumption in GSH-mediated oxidant neutralization. The GR inactivation terminates into accumulation of oxidized GSH, which is known to inhibit protein synthesis and enzymes containing SH-group. Low concentration of GST and GR had been reported in MI-induced rats.³⁸

Intact shape and structure of RBCs in case of control as seen in SEM images indicate no substantial oxidative damage to membrane, while distorted biconcave-shaped membrane projections were observed in RBCs of patients which reflects the consequence of peroxidative membrane damage.

On inspection of the study results, we observed that patients of MI with greater severity in terms of GRACE score were found to be associated with upsurge in oxidative stress, DNA damage, and alteration in lipid indices than patients with less severity. This signifies the disturbance of oxidant–antioxidant balance with stiffness of the disease. However, we found greater decrease in activity of antioxidant enzymes, SOD, and GPx in higher risk patient (GS > 100) than with lower risk (GS ≤ 100). This can be explained as grave shrinkage of these antioxidant enzymes in patients with higher risk, owing to their utilization in countering free radicals produced in greater amount in them, than former group. In addition, the oxidative stress seen in patients with MI was not only a result of residual oxidative stress but also due to the pathology itself. Moreover, this stress became exaggerated in old patients (MI ≤ 45 years) and those possessing risk factors in comparison with younger (MI ≤ 45 years) and without risk factor patients, respectively. This outcome is probably due to the fact that injury triggered by risk factors in MI was greater than the damage caused by infarction alone, and risk factors plus increasing age hold the ability to alter oxidative indices and add to disease burden. Thus, our findings demonstrate the details of oxidative damage during infarction of myocardial tissue and may prove beneficial in further understanding of disease’s pathophysiology as well as management.

Conclusion

Oxidative stress and DNA damage get widen critically with disease severity, in addition to risk factors and age of patients with MI. Thus, grave production of free radicals with concomitant reduced amounts of antioxidants may be causative factor for severity of disease. Taken together, these results suggest that GRACE risk score may serve as direct indicator of oxidative stress status in patients with MI and supplementation of antioxidants to such patients might be beneficial.

Supplemental Material

Supplementary_material - Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction

Click here for additional data file.^{(101.6KB, pdf)}

Supplementary_material for Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction by Sumayya Shahzad, Asif Hasan, Abul Faiz Faizy, Somaiya Mateen, Naureen Fatima, and Shagufta Moin in Clinical and Applied Thrombosis/Hemostasis

Acknowledgments

We are highly thankful for technical assistance to Chairperson, Department of Biochemistry, Faculty of Medicine, J.N Medical College, A.M.U, Aligarh.

Authors’ Note: S Moin and S Shahzad conceived and designed the study; S Shahzad and A Hasan contributed in data collection; S Shahzad performed the experiments and wrote the article; S Moin, S Shahzad, and S Mateen contributed with data analysis and statistical insight.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental Material: Supplementary material for this article is available online.

References

1. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82(1):47–95. [DOI] [PubMed] [Google Scholar]
2. Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160(1):1–40. [DOI] [PubMed] [Google Scholar]
3. Mateen S, Moin S, Khan AQ, Zafar A, Fatima N. Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis. Plos One. 2016;11(4):e0152925. [DOI] [PMC free article] [PubMed] [Google Scholar]
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5. Loeper J, Goy J, Rozensztajn L, Bedu O, Moisson P. Lipid peroxidation and protective enzymes during myocardial infarction. Clin Chim Acta. 1991;196(2-3):119–125. [DOI] [PubMed] [Google Scholar]
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7. Ohsawa M, Tsuru R, Hojo Y, et al. Relationship between redox state of whole arterial blood glutathione and left ventricular function after acute myocardial infarction. J Cardiol. 2004;44(4):141–146. [PubMed] [Google Scholar]
8. González-Pacheco Héctor, Vargas-Barrón Jesús, Vallejo Maite, et al. Prevalence of conventional risk factors and lipid profiles in patients with acute coronary syndrome and significant coronary disease. Ther Clin Risk Manag. 2014;10:815–823. [DOI] [PMC free article] [PubMed] [Google Scholar]
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12. GRACE ACS Risk Score. http://www.Outcomes%20umassmed.org/ grace/acs_risk/acs_risk, (accessed January 9–14, 2017).
13. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. J Am Coll Cardiol. 2007;50(22):2173–2195. [DOI] [PubMed] [Google Scholar]
14. Benzie IF, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem. 1996;239(1):70–76. [DOI] [PubMed] [Google Scholar]
15. Janaszewska A, Bartosz G. Assay of total antioxidant capacity: comparison of four methods as applied to human blood plasma. Scand J Clin Lab Invest. 2002;62(3):231–236. [DOI] [PubMed] [Google Scholar]
16. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide. 2001;5(1):62–71. [DOI] [PubMed] [Google Scholar]
17. Keller A, Mohamed A, Dröse S, Brandt U, Fleming I, Brandes RP. Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. Free Radic Res. 2004;38(12):1257–1267. [DOI] [PubMed] [Google Scholar]
18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275. [PubMed] [Google Scholar]
19. Buege JA, Aust SD. Microsomal lipid peroxidation. Meth Enzymol. 1978;52:302–310. [DOI] [PubMed] [Google Scholar]
20. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Meth Enzymol. 1994;233:357–363. [DOI] [PubMed] [Google Scholar]
21. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem. 1968;25(1):192–205. [DOI] [PubMed] [Google Scholar]
22. Jollow D, Mitchell J, Zampaglione NA, Gillette J. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 1974;11(3):151–169. [DOI] [PubMed] [Google Scholar]
23. Kyaw A. A simple colorimetric method for ascorbic acid determination in blood plasma. Clin Chim Acta. 1978;86(2):153–157. [DOI] [PubMed] [Google Scholar]
24. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469–474. [DOI] [PubMed] [Google Scholar]
25. Claiborne A. Catalase activity In: Greenwald RA. (Ed.) CRC Handbook of Methods for Oxygen Radical Research. Boca Raton, FL: Chemical Rubber Company Press; 1985;283–284. [Google Scholar]
26. Flohé L, Günzler WA. Assays of glutathione peroxidase. Meth Enzymol. 1984;105:114–120. [DOI] [PubMed] [Google Scholar]
27. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130–7139. [PubMed] [Google Scholar]
28. Mannervik B, Carlberg I. Glutathione reductase. Methods Enzymol. 1985;113:484–490. [DOI] [PubMed] [Google Scholar]
29. Yu P, Zhang J, Yu S, et al. Protective effect of Sevoflurane postconditioning against cardiac ischemia/reperfusion injury via ameliorating mitochondrial impairment, oxidative stress and rescuing autophagic clearance. PLoS One. 2015;10(8):e0134666. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. LoPresti R, Catania A, D’Amico T, Montana M, Caruso M, Caimi G. Oxidative stress in young subjects with acute myocardial infarction: evaluation at the initial stage and after 12 months. Clin Appl Thromb Hemost. 2007;14(4):421–427. [DOI] [PubMed] [Google Scholar]
31. Sedláková E, Rácz O, Lovásová E, et al. Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary intervention. Cent Eur J Med. 2009;4(1):26–31. [Google Scholar]
32. Chamblee BB, Timm TC, Hunsaker LA, Vander Jagt DL. Relationship of oxidative stress indices to decreased LDL-cholesterol after acute myocardial infarction. Clin Biochem. 2000;33(5):423–426. [DOI] [PubMed] [Google Scholar]
33. Kuchino Y, Mori F, Kasai H, et al. Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature. 1987;327(6117):77–79. [DOI] [PubMed] [Google Scholar]
34. Himmetoglu S, Dincer Y, Bozcali E, Vural VA, Akcay T. Oxidative DNA damage and antioxidant defense after reperfusion in acute myocardial infarction. J Investig Med. 2009;57(4):595–599. [DOI] [PubMed] [Google Scholar]
35. Karthikeyan K, Bai BS, Devaraj SN. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. Int J Cardiol. 2007;115(3):326–333. [DOI] [PubMed] [Google Scholar]
36. Lubrano V, Di Cecco P, Zucchelli GC. Role of superoxide dismutase in vascular inflammation and in coronary artery disease. Clin Exp Med. 2006;6(2):84–88. [DOI] [PubMed] [Google Scholar]
37. Gupta S, Sodhi S, Mahajan V. Correlation of antioxidants with lipid peroxidation and lipid profile in patients suffering from coronary artery disease. Expert Opin Ther Targets. 2009;13(8):889–894. [DOI] [PubMed] [Google Scholar]
38. Li H, Xie YH, Yang Q, et al. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PLoS One. 2012;7(11):e48872. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary_material - Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction

Click here for additional data file.^{(101.6KB, pdf)}

[bibr1-1076029617725602] 1. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82(1):47–95. [DOI] [PubMed] [Google Scholar]

[bibr2-1076029617725602] 2. Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact. 2006;160(1):1–40. [DOI] [PubMed] [Google Scholar]

[bibr3-1076029617725602] 3. Mateen S, Moin S, Khan AQ, Zafar A, Fatima N. Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis. Plos One. 2016;11(4):e0152925. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bibr4-1076029617725602] 4. Weidinger A, Kozlov AV. Biological activities of reactive oxygen and nitrogen species: oxidative stress versus signal transduction. Biomolecules. 2015;5(2):472–484. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bibr5-1076029617725602] 5. Loeper J, Goy J, Rozensztajn L, Bedu O, Moisson P. Lipid peroxidation and protective enzymes during myocardial infarction. Clin Chim Acta. 1991;196(2-3):119–125. [DOI] [PubMed] [Google Scholar]

[bibr6-1076029617725602] 6. Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A. 1987;84(5):1404–1407. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bibr7-1076029617725602] 7. Ohsawa M, Tsuru R, Hojo Y, et al. Relationship between redox state of whole arterial blood glutathione and left ventricular function after acute myocardial infarction. J Cardiol. 2004;44(4):141–146. [PubMed] [Google Scholar]

[bibr8-1076029617725602] 8. González-Pacheco Héctor, Vargas-Barrón Jesús, Vallejo Maite, et al. Prevalence of conventional risk factors and lipid profiles in patients with acute coronary syndrome and significant coronary disease. Ther Clin Risk Manag. 2014;10:815–823. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bibr9-1076029617725602] 9. Anderson JL, Adams CD, Antman EM, et al. ACC/AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients with Unstable Angina/Non-ST-Elevation Myocardial Infarction). J Am Coll Cardiol. 2007;50(7):e1–e157. [DOI] [PubMed] [Google Scholar]

[bibr10-1076029617725602] 10. Granger CB, Goldberg RJ, Dabbous O, et al. Predictors of hospital mortality in the global registry of acute coronary events. Arch Intern Med. 2003;163(19):2345–2353. [DOI] [PubMed] [Google Scholar]

[bibr11-1076029617725602] 11. Yusuf S, Hawken S, Ôunpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case–control study. Lancet. 2004;364(9438):937–952. [DOI] [PubMed] [Google Scholar]

[bibr12-1076029617725602] 12. GRACE ACS Risk Score. http://www.Outcomes%20umassmed.org/ grace/acs_risk/acs_risk, (accessed January 9–14, 2017).

[bibr13-1076029617725602] 13. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. J Am Coll Cardiol. 2007;50(22):2173–2195. [DOI] [PubMed] [Google Scholar]

[bibr14-1076029617725602] 14. Benzie IF, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem. 1996;239(1):70–76. [DOI] [PubMed] [Google Scholar]

[bibr15-1076029617725602] 15. Janaszewska A, Bartosz G. Assay of total antioxidant capacity: comparison of four methods as applied to human blood plasma. Scand J Clin Lab Invest. 2002;62(3):231–236. [DOI] [PubMed] [Google Scholar]

[bibr16-1076029617725602] 16. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide. 2001;5(1):62–71. [DOI] [PubMed] [Google Scholar]

[bibr17-1076029617725602] 17. Keller A, Mohamed A, Dröse S, Brandt U, Fleming I, Brandes RP. Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. Free Radic Res. 2004;38(12):1257–1267. [DOI] [PubMed] [Google Scholar]

[bibr18-1076029617725602] 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275. [PubMed] [Google Scholar]

[bibr19-1076029617725602] 19. Buege JA, Aust SD. Microsomal lipid peroxidation. Meth Enzymol. 1978;52:302–310. [DOI] [PubMed] [Google Scholar]

[bibr20-1076029617725602] 20. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Meth Enzymol. 1994;233:357–363. [DOI] [PubMed] [Google Scholar]

[bibr21-1076029617725602] 21. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem. 1968;25(1):192–205. [DOI] [PubMed] [Google Scholar]

[bibr22-1076029617725602] 22. Jollow D, Mitchell J, Zampaglione NA, Gillette J. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 1974;11(3):151–169. [DOI] [PubMed] [Google Scholar]

[bibr23-1076029617725602] 23. Kyaw A. A simple colorimetric method for ascorbic acid determination in blood plasma. Clin Chim Acta. 1978;86(2):153–157. [DOI] [PubMed] [Google Scholar]

[bibr24-1076029617725602] 24. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469–474. [DOI] [PubMed] [Google Scholar]

[bibr25-1076029617725602] 25. Claiborne A. Catalase activity In: Greenwald RA. (Ed.) CRC Handbook of Methods for Oxygen Radical Research. Boca Raton, FL: Chemical Rubber Company Press; 1985;283–284. [Google Scholar]

[bibr26-1076029617725602] 26. Flohé L, Günzler WA. Assays of glutathione peroxidase. Meth Enzymol. 1984;105:114–120. [DOI] [PubMed] [Google Scholar]

[bibr27-1076029617725602] 27. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130–7139. [PubMed] [Google Scholar]

[bibr28-1076029617725602] 28. Mannervik B, Carlberg I. Glutathione reductase. Methods Enzymol. 1985;113:484–490. [DOI] [PubMed] [Google Scholar]

[bibr29-1076029617725602] 29. Yu P, Zhang J, Yu S, et al. Protective effect of Sevoflurane postconditioning against cardiac ischemia/reperfusion injury via ameliorating mitochondrial impairment, oxidative stress and rescuing autophagic clearance. PLoS One. 2015;10(8):e0134666. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bibr30-1076029617725602] 30. LoPresti R, Catania A, D’Amico T, Montana M, Caruso M, Caimi G. Oxidative stress in young subjects with acute myocardial infarction: evaluation at the initial stage and after 12 months. Clin Appl Thromb Hemost. 2007;14(4):421–427. [DOI] [PubMed] [Google Scholar]

[bibr31-1076029617725602] 31. Sedláková E, Rácz O, Lovásová E, et al. Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary intervention. Cent Eur J Med. 2009;4(1):26–31. [Google Scholar]

[bibr32-1076029617725602] 32. Chamblee BB, Timm TC, Hunsaker LA, Vander Jagt DL. Relationship of oxidative stress indices to decreased LDL-cholesterol after acute myocardial infarction. Clin Biochem. 2000;33(5):423–426. [DOI] [PubMed] [Google Scholar]

[bibr33-1076029617725602] 33. Kuchino Y, Mori F, Kasai H, et al. Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature. 1987;327(6117):77–79. [DOI] [PubMed] [Google Scholar]

[bibr34-1076029617725602] 34. Himmetoglu S, Dincer Y, Bozcali E, Vural VA, Akcay T. Oxidative DNA damage and antioxidant defense after reperfusion in acute myocardial infarction. J Investig Med. 2009;57(4):595–599. [DOI] [PubMed] [Google Scholar]

[bibr35-1076029617725602] 35. Karthikeyan K, Bai BS, Devaraj SN. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. Int J Cardiol. 2007;115(3):326–333. [DOI] [PubMed] [Google Scholar]

[bibr36-1076029617725602] 36. Lubrano V, Di Cecco P, Zucchelli GC. Role of superoxide dismutase in vascular inflammation and in coronary artery disease. Clin Exp Med. 2006;6(2):84–88. [DOI] [PubMed] [Google Scholar]

[bibr37-1076029617725602] 37. Gupta S, Sodhi S, Mahajan V. Correlation of antioxidants with lipid peroxidation and lipid profile in patients suffering from coronary artery disease. Expert Opin Ther Targets. 2009;13(8):889–894. [DOI] [PubMed] [Google Scholar]

[bibr38-1076029617725602] 38. Li H, Xie YH, Yang Q, et al. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PLoS One. 2012;7(11):e48872. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction

Sumayya Shahzad, PhD

Asif Hasan, MD

Abul Faiz Faizy, MD

Somaiya Mateen, PhD

Naureen Fatima, PhD

Shagufta Moin, MD

Abstract

Background:

Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Collection and Processing of Blood Samples

Estimation of Lipid Indices

Antioxidant Status Assays

Evaluation of Nitric Oxide Level

Dichlorodihydrofluorescein Assay

Determination of Hemoglobin and Plasma Protein

Assay for Lipid Peroxidation, Carbonylation of Plasma Proteins, Total Thiol Groups, and Oxidative DNA Damage

Estimation of Reduced Glutathione and Plasma Vitamin C Level

Determination of Antioxidant Enzymes in Hemolysate

Scanning Electron Microscopy

Statistical Analysis

Results

Table 1.

Table 2.

Figure 1.

Table 3.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Discussion

Conclusion

Supplemental Material

Acknowledgments

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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