Scholarly article on topic 'Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary intervention'

Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary intervention Academic research paper on "Basic medicine"

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Academic research paper on topic "Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary intervention"


Cent. Eur J. Med. • 4(1) • 2009 • 26-31 DOI: 10.2478/s11536-009-0015-8

Central European Journal of Medicine

Markers of oxidative stress in acute myocardial infarction treated by percutaneous coronary


Research Article

Eva Sedlakova1", Oliver Racz1, Eva Lovasova1, Roman Benacka1, Martin Kurpas1, Anna Chmelarova2, Jan Sedlak3, Martin Studencan3

1 Department of Pathophysiology, Faculty of Medicine, Safarik University,

04066 Kosice, Slovakia

2 Department of Experimental Medicine, Faculty of Medicine, Safarik University, 04066 Kosice, Slovakia

3 Eastern Slovak Cardiovascular Institute, 04001 Kosice, Slovakia Received 2 October 2008; Accepted 11 January 2009

Abstract: In the current study, we evaluated the dynamics of oxidative stress markers in patients with acute myocardial infarction (AMI) treated by primary percutaneous coronary intervention (PCI). Thirty consecutive patients with AMI with ST elevation were included. Plasma lipid peroxidation end-product malondialdehyde (MDA) and total antioxidant capacity (TAC) in blood plasma were evaluated. Peripheral venous blood samples were obtained prior to reperfusion and at five time points after reperfusion. The control group consisted of 20 ischemic patients without acute coronary syndrome. TAC in the AMI group at admission was lower than in control patients (1.26 ± 0.32 vs. 1.52 ± 0.24 mmol/l). Within 1 h after reperfusion, in most cases, values significantly declined (1 min, 1.10 ± 0.33 mmol/l;

1 h, 1.06 ± 0.21 mmol/l [p= 0.03]). After 3 h, values began to increase (1.14 ± 0.29 mmol/l) and returned to basal values after 3 d (1.29 ± 0.24 mmol/l). MDA levels in AMI patients at admission were higher than in control patients (1.66 ± 0.55 vs. 1.44 ± 0.55 mmol/l) but showed a sustained decrease over the 3 h after reperfusion of the occluded artery (1 min, 1.57 ± 0.37 mmol/l; 1 h, 1.50

± 0.35 mmol/l; 3 h, 1.35 ± 0.59 mmol/l [p = 0.03]). Reperfusion of the occluded coronary artery by PCI in AMI lead to an immediate decrease in TAC, suggesting formation of reactive oxygen species. However, the MDA level significantly decreased after reperfusion. This may suggests less reperfusion injury after PCI. Keywords: Acute myocardial infarction • Reperfusion • Oxidative stress • Total antioxidant capacity • Malondialdehyde

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

1. Introduction

The main goal of therapy for acute myocardial infarction (AMI) is the early restoration of coronary flow to limit myocardial necrosis. Reperfusion of previously ischemic myocardium, however, may injure myocardial and endothelial cells in coronary arteries. Although the pathogenesis of reperfusion injury is complex, formation of reactive oxygen species (ROS) may play a critical role. In animal models, ROS are important mediators of reperfusion injury to ischemic but viable myocardium [1,2]. Oxidative stress during reperfusion leads to lipid peroxidation. Because direct measurement of liberated

ROS is difficult due to their instability, malondialdehyde (MDA), a stable lipid peroxidation end-product, is frequently used as a marker of ROS production.

Antioxidant scavenging systems defend organism against ROS and thereby inhibiting oxidative damage [3]. The total antioxidant capacity (TAC) of plasma is used as a measure of an organism's ability to defend against ROS. It is determined as a sum of the overall effect of water-soluble (uric acid, ascorbic acid, protein thiols, and bilirubin) and lipid-soluble antioxidants (a-tocopherol, p-carotene, and ubiquinol).

Thrombolytic therapy is used to restore perfusion of infarct-related arteries. Previous studies have found that, after successful reperfusion, there is an increase

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in plasma MDA levels and an decrease in various antioxidants such as as a-tocopherol, p-carotene, vitamin C, superoxide dismutase, and glutathione peroxidase [3,4]. The most effective therapy for reperfusion of infarct-related arteries is primary percutaneous coronary intervention (PCI) [5,6]. In the current study, we examined the dynamics of MDA production and TAC in patients with AMI undergoing successful recanalization of an infarct-related artery by PCI. Our goal was to to evaluate the time-course of oxidative stress to determine the correlation between parameters of oxidative stress and the extent of myocardial damage and duration of ischemia.

2. Statistical methods and Experimental Procedures

2.1. Study conduct

The study was approved by the local ethical committee of the Eastern Slovak Cardiovascular Institute and complied with the Declaration of Helsinki. Written consent was obtained from each patient. Consecutive patients diagnosed with AMI were enrolled. Inclusion criteria were as follows: electrocardiographic evidence of ST elevation of > 1 mV in two or more standard limb leads or 2 mV in two or more precordial leads; typical chest pain lasting more than 20 min; evidence of totally occluded an infarct-related artery on initial coronary angiography. Control patients had ischemic heart disease without acute coronary syndrome.

PCI was confined to the infarct-related artery and was performed using standard techniques and equipment appropriate for the coronary anatomy. One balloon inflation (60 s) was usually enough to restore coronary artery patency, although further inflation or application of stent was performed to optimize the angiographic appearance of the underlying stenosis. The procedure was considered successful if the thrombolysis in myocardial infarction flow was III and the luminal diameter of the residual stenosis was <20%. Only patients with successful reperfusion were included in this study. Patients with cardiogenic shock were excluded. Patients received 10,000 IU of unfractionated heparin during the coronary angiography. Glycoprotein IIb/IIIa inhibitors were not administered to patients in the study group patients.

Peripheral venous blood samples were obtained from each patient immediately before the beginning of PCI (t0) and then after 1 min (t1), 5 min (t5), 1 h (t60), 3 h (t180), and 3 d (t3d). Samples were drawn into plastic tubes with heparin. Blood was centrifuged within 1 h

at 1500 x g for 15 min, and the collected plasma was stored at -30° C until analysis (within 1 month).

2.2. Measurement of MDA

MDA was estimated by a modified thiobarbituric acid reaction as described by Yagi [7]. In the first step, lipoproteins are precipitated. After removal of soluble components, the precipitate was resolved in boiling sulphuric acid. The MDA present in the sample reacts with thiobarbituric acid, producing a fluorescent product that is extracted with n-butanol. The fluorescence in the extract was measured at an excitation wavelength of 515 nm and emission wavelength of 535 nm using the 204 Fluorescence Spectrofluorometer (Perkin Elmer, Norwalk, USA).

2.3. Measurement of plasma TAC

Measurement of plasma TAC is based on the quenching of the ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) radical cation by antioxidants [8,9]. The ABTS radical cation is generated by the interaction of ABTS with highly reactive ferrylmyoglobin radical species, produced by the activation of methmyoglobin with H2O2. The absorbance was measured at 600 nm and 37°C using a Cobas Mira S spectrophotometer (Roche, Switzerland). The concentration was determined by comparing the absorbance with a standard curve generated using the water-soluble vitamin E analog Trolox (Randox Laboratoires, Great Britain) as a standard.

2.4. Assessment of myocardial injury

The extent of myocardial injury was evaluated indirectly by following the time course of changes in activities of total creatine kinase using standard assays.

2.5. Statistical analysis

The data are presented as means ± standard deviation. Statistical significance of the differences during the time course was determined by paired Student's f-test. A value of p<0.05 was considered as indicating a statistically significant difference. The Shapiro-Wilkov W test was used to test for a normal distribution. Correlation analysis was performed using Pearson's correlation coefficient.

3. Results

The study included 30 consecutive patients (17 males and 13 females, mean age 68.2 ± 12.8 years). The control group consisted of twenty patients with ischemic heart disease but without acute coronary syndrome.

Figure 1.

MDA levels In the control group and In patients with AMI before PCI (t). *p = 0.01 for control group vs. AMI group t .

Figure 2. MDA levels In patients with AMI before PCI (t0) and after PCI (t1, 1 min after PCI; t5, 5 min after PCI; t60, 1 h after PCI; t^0, 3 h after PCI). *p = 0.04 for AMI group to vs. group t1. **p = 0.03 for AMI group to vs. groups t5, t , and t

Figure 3. TAC levels In the control group and In patients with AMI before PCI (t0). *p = 0.01 for control group vs. AMI group

Figure 4. ETAC levels in patients with AMI before PCI (group t0) and after PCI (t1, 1 min after PCI; t5, 5 min after PCI; t60, 1 h after PCI; t180, 3 h after PCI). *p = 0.03 for AMI group t0 vs. groups t1, t5, and t60.

For the study group, the mean time from the onset of symptoms of AMI to recanalization was 6.8 ± 5.5 hours. Electrocardiographic infarct sites were as follows: anterior (14 patients), lateral (4 patients), and inferior (13 patients). In all anterior infarctions, the occluded artery was the left anterior descending artery. In all lateral infarctions, the occlusion was on the circumflex coronary artery. The right coronary artery was occluded in 8 out of 10 patients with inferior infarction. In 2 cases, the occlusion was on the circumflex coronary artery. Therapy after PCI was aspirin (93% of patients), low molecular weight heparin (100%), clopidogrel (100%), beta blockers (83%), ACE inhibitors (73%), statins (60%), and intravenous nitrates (10%).

The mean activity of creatine kinase at admission was 6.2 ± 5.5 mkat/l, and the activity was 50.4 ± 35.4, 43.1 ± 32.2, and 35.6 ± 30.9 mkat/l at 6, 12, and 24 h, respectively. Peak creatine kinase levels were detected at 6 h.

The overall mean plasma MDA level before reperfusion (t0) was 1.66 ± 0.55 mmol/l (Figure 1). The mean plasma MDA level of the control group was 1.44 ± 0.61 mmol/l (p = 0.01). A significant decrease of plasma MDA level occurred after 1 min (t1, 1.57 ± 0.37 mmol/l [p = 0.04]). There was a further decrease over the next 3 h.

The mean MDA level at 5 min (t5), 1 h (t60), and 3 h (t180) was 1.56 ± 0.54, 1.50 ± 0.35, and 1.35 ± 0.59 mmol/l, respectively (p = 0.03; Figure 2).

The total antioxidant capacity (TAC) of the plasma before PCI (t0) was 1.26 ± 0.32 mmol/l (Figure 3). For the control group, the mean TAC was 1.52 ± 0.24 mmol/l (p = 0.01). From the first minute after reperfusion, the PCI levels of TAC showed a regular and continuous decrease (t1, 1.10 ± 0.33 mmol/l and t5, 1.08 ± 0.18 mmol/l [p = 0.03]) with minimum values reached usually within the first hour (t60, 1.06 ± 0.21 mmol/l [p= 0.03]). In contrast, there was a variable increase after 3 h (t180, 1.14 ± 0.29 mmol/l) and 3 d (t3d, 1.29 ± 0.24 mmol/l) (Figure 4).

There was no correlation between MDA and TAC levels and serum activity of creatine kinase. We also did not find a significant correlation between plasma MDA or TAC and the duration of coronary occlusion as measured by the time from the occurrence of the symptoms of AMI to the opening of infarct-related artery.

4. Discussion

Early restoration of blood flow to the ischemic myocardium is the only way to prevent progression to myocardial necrosis and thus limit the infarct size. However, a sudden restoration of oxygen supply to previously ischemic myocardium can lead to oxidative stress, with consequent oxidative injury to cell function and structure due to lipid peroxidation.

Several studies have evaluated the level of MDA in patients with AMI after thrombolytic therapy [3,10-14]. In all cases, there was an increase of MDA after thrombolysis, although in some studies, the increase did not reach the level of statistical significance. Also, there were differences in the time course of changes in the MDA level. Beard et al. [11] and Young et al. [13] reported that, after an initial increase, there is a decrease in MDA 2 to 6 h after thrombolysis, and Iqbal reported [12] a decrease after 12 h. Pucheu et al. [14], however, found an increasse in MDA for up to day 10.

In the current study, we examined the global plasma oxidant and antioxidant status as measured by alterations in TAC and MDA levels following the successful reopening of occluded infarct-related arteries by primary PCI. MDA levels in AMI patients were higher than in the control group, and TAC levels were lower. This indicates that oxidative stress is already elevated during ischemia, which is in agreement with previous studies [3,15].

We did not find any correlation between oxidative stress parameters (MDA and TAC) and serum creatine kinase activity, indicating that these aspects of oxidative stress do not depend on the extent of damage to the myocardium. Similarly, Berg et al. [15] did not find a direct relationship between 8-iso-PGF2a, a marker of oxidative stress, and troponin T, a marker of myocardial injury.

The duration of coronary occlusion, evaluated as the time from the occurrence of the symptoms of AMI to opening of infarct related artery, also did not correlate with the plasma MDA and TAC levels. An increase in MDA levels was reported during prolongation of ischemia in rat hearts [16]; however, the longest duration of experimental ischemia in this study was only 30 min. In our clinical cohort, the mean duration of ischemia as evaluated by the history of precordial pain was much longer. It isn't known whether prolonged ischemia (i.e., lasting a few hours) leads to a continuous rise in MDA level or whether the level reaches a plateau.

We found an immediate and significant reduction in TAC after reperfusion. From the first minute and until the first hour after successful reperfusion, there was

a regular and continuous decline in the TAC level. In contrast, starting at the third hour, there was an increase in the TAC level up to the third day. These findings are similar to those reported by Beard et al. [11] and Berg et al. [15]. Beard reported a decrease in plasma glutathione peroxidase lasting 2 h after reperfusion, followed by a continuous increase for up to 2 d. The decrease in TAC after reperfusion may indicate a depletion of antioxidants as a consequence of overproduction of ROS in the damaged area.

All previous studies reported an increase in the MDA level following thrombolytic opening of infarct-related arteries, and Berg et al. reported an increase in oxidative stress as measured by 8-iso-PGF2a levels following PCI [15]. Although we expected a rise in the plasma MDA level, it fell significantly after PCI in this study. Similar findings were reported by Olsson et al. [17]. A decrease in TAC after PCI suggests increased ROS production, but the decease in MDA failed to document increased lipid peroxidation in our patients.

It is unlikely that our results were influenced by additional medical treatments. None of the patients received glycoprotein IIb/IIIa inhibitors. Patients receiving intravenous nitrates had lower MDA levels. Similarly, Ohlin et al. [18] found reduced MDA levels in patients treated with intravenous nitroglycerin. However, the limited number of patients in receiving nitrates in our study precluded any formal subgroup analysis. How thrombolysis may have affected the TAC level is not clear because this has not been previously studied, and it was not possible to compare the reduction in TAC levels between the various reperfusion therapies.

Previous studies also supporting the idea of ROS production after reperfusion by PCI. Grech et al. [20] reported an elevated free radical signal with a peak 2 to 3 h after PCI using electron paramagnetic resonance spectroscopy. Although they did not find significant changes in MDA levels after successful PCI [19], the found a marked increase in conjugated dienes, which, like MDA, are lipid peroxidation products.

In this study, lipid peroxidation was evaluated by the TBARS fluorescence method. This approach has potential limitations with respect to specificity. Another method of evaluating MDA is high-performance liquid chromatography (HPLC). However, MDA levels measured by TBARS fluorescence and HPLC do not agree [17,21,22]. Olsson et al. [17] reported nonsignificant changes in plasma MDA after PCI using TBARS but a significant decrease using HPLC. Although TBARS can detect not only MDA but also non-lipid related, MDA-like, TBA-reactive substances [21], our results suggest that oxidant-mediated injury may actually be modest in our patients. The finding of consistently

decreasing TBARS levels after reperfusion supports this conclusion. Only when defense mechanisms are overwhelmed oxidant attack progresses to cell injury. Likewise, Jansen et al. [23] reported that human hearts subjected to cardioplegia and subsequent reperfusion show oxidative stress without an increase in lipid peroxidation, and Milei et al. [24] found that in patients receiving elective bypass surgery, oxidative stress upon reperfusion after cardioplegic arrest doesn't lead to reperfusion injury.

In contrast to an increase in MDA levels after thrombolytic therapy, the levels fall after reopening of coronary arteries by PCI. This may suggest less severe reperfusion injury after PCI. The discrepancy in MDA dynamics between these methods may be due to the different rate and extent of reperfusion. Gradual


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