Journal of the American College of Nutrition, Vol. 17, No. 4, 337-341 (1998)
Published by the American College of Nutrition
Plasma Antioxidants and Lipid Peroxidation in Acute Myocardial Infarction and Thrombolysis
Yishai Levy, MD,
Peter Bartha, MD,
Ami Ben-Amotz, PhD,
J. Gerald Brook, MD,
Gertrude Dankner,
Shai Lin, MD, PhD and
Haim Hammerman, MD
Lipid Research Unit (Y.L., P.B., J.G.B., G.D.), Haifa, ISRAEL
National Oceanographic Research Institute (A.B-A.), Haifa, ISRAEL
Department of Clinical Epidemiology (S.L.), Haifa, ISRAEL
Department of Cardiology (H.H.), Rambam Medical Center and Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, ISRAEL
Address reprint requests to: Yishai Levy, MD, Dept. Medicine D, Rambam Medical Center, Haifa 31096 ISRAEL
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ABSTRACT
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Objectives: The aim of this study was to investigate the balance between prooxidative and protective mechanisms in patients with acute myocardial infarction (AMI) throughout streptokinase (STK) therapy.
Methods: Patients who presented to coronary care unit within 3 hours of infarction were followed. Blood was collected before, 2 and 24 hours post STK. Plasma lipid peroxidation was analyzed by a free radical generating system (AAPH) and malondialdehyde equivalents and conjugated dienes quantitated. Plasma vitamins A, E and ß-carotene, were analyzed by HPLC. Patients’ results were compared with those from age-matched, healthy control subjects.
Results: In 38 patients with AMI, baseline plasma antioxidant vitamin concentration was reduced compared with a healthy control group. Upon STK therapy, there was a significant drop in plasma vitamin E concentration. Successful reperfusion was followed by an increased plasma oxidizability. Plasma lipids were not significantly different in the AMI patients except for a lower HDL-cholesterol concentration.
Conclusions: Patients with AMI showed a drop in plasma antioxidant vitamins. Upon thrombolysis, there was an enhanced lipid peroxidation. These alterations indicate the significance of free radical generation processes in reperfusion injury in AMI patients, and suggest the potential involvement of antioxidants in the management of AMI treated by thrombolysis.
Key words: thrombolysis, lipid peroxidation, antioxidants, acute myocardial infarction
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INTRODUCTION
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Free radicals contain one or more unpaired electrons. They play an important role in the pathogenesis of tissue damage in many different clinical disorders [1–4]. Oxygen free radicals are produced continuously. Normally, there is a balance between tissue oxidant and antioxidant activity [3]. The latter is achieved by the antioxidant scavenger system which includes enzymes (superoxide dismutase, catalase, glutathione peroxidase) and antioxidant vitamins (C, A, E and other carotenoids) [5–8].
Early reperfusion by thrombolytic drugs is now accepted as an effective treatment of acute myocardial infarction (AMI), both to restore coronary patency and to limit myocardial damage [9]. However, reperfusion may result in transient or permanent myocardial injury (reperfusion injury), assumed to be oxygen free radical-mediated [9–12]. Oxygen free radicals produce peroxidation of the membrane lipids with structural and functional changes. These mechanisms can explain some manifestations of the reperfusion injury such as myocardial stunning and reperfusion arrhythmias [10–12]. In addition, the accumulation of polymorphonuclear (PMN) cells [11] by their supplementary oxygen free radical production may initiate the reocclusion of damaged vessels, explaining why there is only a partial success of revascularization procedures.
In a limited number of earlier studies, there has been evidence of enhanced production of oxygen free radicals in blood drawn from the coronary sinus of patients who underwent coronary angiography [13]. Evidence of increased oxygen free radical production was demonstrated by exhaled pentane (a direct index of lipid peroxidation) in patients with AMI [14]. In another study, increased oxygen free radical production was measured directly by electron spin resonance and indirectly by enhanced lipid peroxidation products. Malondialdehyde (MDA), and conjugated dienes (CD) were increased in coronary blood of patients who underwent coronary angioplasty [15]. Pharmacological reperfusion by infusion of streptokinase (STK) resulted in an elevated lipid peroxidation in peripheral blood, measured by thiobarbituric acid related substances (TBARS) in patients with coronary angiographically assessed arterial patency [16].
In the present study, we investigated the balance between plasma oxidation and plasma antioxidant vitamins in patients with AMI and control subjects, and the effect of reperfusion with STK on this balance.
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PATIENTS AND METHODS
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Patients
The study consisted of 38 consecutive patients, 27 men and 11 women with a mean age of 61±12 years and the diagnosis of AMI, admitted to the Intensive Coronary Care Unit, Rambam Medical Center, Haifa, who underwent reperfusion by thrombolysis. The diagnosis of AMI was established according to clinical criteria: chest pain which lasted for up to 3 hours, ECG changes (ST elevation of 2 mm or more in at least two leads) and CPK elevation.
Treatment with STK was initiated by intravenous infusion of 1,500,000 U for 1 hour within 3 hours from onset of chest pain. As the opening of a coronary artery occlusion in the early hours of AMI will cause early and higher peaking (at about 8 to 12 hours instead of 24 hours) of CPK, this enzyme was followed hourly after the infusion of STK.
The control group consisted of 20 healthy, age-matched subjects, 12 men and 8 women, recruited from an annual check-up program. All had a normal resting ECG and standard exercise tests.
Laboratory Methods
Blood tests were done at 0 (baseline), 2 and 24 hours after initiation of thrombolysis with streptokinase. Blood was drawn into Na2 EDTA (1 mmol) tubes and centrifuged at 2000 g for 10 minutes. 4 ml of plasma was dialyzed against phosphate buffered saline (PBS) at pH 7.4 overnight at 4°C. 2 ml of dialyzed plasma was incubated for 2 hours at 37°C without (controls) and with 100 mmol of 2,2-azobis,a-amidinopropane hydrochloride (AAPH) (Polysciences, Warington, PA). AAPH is a water soluble azo-compound which thermally decomposes and, thus, generates water soluble peroxy radicals at a constant rate [17]. Plasma lipid peroxidation was determined by the TBARS assay (which measures MDA equivalents by the absorbance at 532 nm) [18] and the formation of CD in the plasma lipids extracted by hexane/isopropanol and precipitated in H2SO4, measuring the absorbance at 234 nm [19].
Plasma concentrations of vitamins A, E and carotenoids were determined by high performance liquid chromatography analysis [6–8]. These lipid soluble vitamins were expressed as both absolute figures and as divided by the sum of the plasma cholesterol and triglycerides.
Plasma lipids (total and HDL-cholesterol, triglycerides), glucose and CPK were measured by enzymatic methods.
Statistical Analysis
The SPSS/PS statistical package was used. Two way analysis of variance (ANOVA) and co-variance (ANCOVA) adjusting for sex, age and smoking habits was done. Paired t-test and Pearson’s correlation coefficients were calculated. All results represent mean±SD.
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RESULTS
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The metabolic characteristics of the two groups are illustrated in Table 1. The patients with AMI had higher blood glucose levels. Blood lipids showed a significant lower concentration of HDL cholesterol. Plasma vitamin levels are shown in Table 2. All the vitamins analyzed were significantly lower in the AMI group, with a 33% decrease in vitamin A, 10% in vitamin E and 37% in carotenoid levels. As lipid soluble vitamins were measured in this study, they were standardized against the sum of plasma cholesterol and triglycerides. However, in the face of similar blood lipids, these equations did not change the difference in plasma antioxidant vitamins between the groups both at baseline and following STK treatment (data not shown).
Table 3 describes the results of plasma oxidation by AAPH. There were no significant differences between the groups. However, oxidation by AAPH resulted in a 165% elevation in MDA and a 95% elevation in CD in AMI, and a 156% elevation in MDA and an 81% elevation in CD in control group. Compared with controls, there were no significant differences in the extent of AAPH-induced oxidation of plasma.
Fig. 1 shows the effect of STK infusion on plasma vitamins. Only vitamin E showed a significant (20%) drop at 24 hours after STK infusion (Fig. 1).
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Fig. 1. Plasma antioxidants vitamin A, E and ß-carotene in patients with AMI at baseline (0), 2 and 24 hours post STK treatment (*p<.005).
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Fig. 2 illustrates the effect of STK on plasma oxidation.
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Fig. 2. Plasma MDA generation at baseline and after 2 hours incubation at 37°C with 100 mM AAPH. Blood was collected from patients with AMI at 0, 2 and 24 hours after treatment by thrombolysis with STK (*p<0.05).
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There was a significant elevation in both basal and AAPH-induced MDA generation at 24 hours (Fig. 2).
The CPK level peaked to 3405±1248 IU/dl after 9.8±4.5 hours from the beginning of STK infusion. Early CPK peak indicated successful reperfusion of a coronary vessel. Thus, the occurrence of peak CPK was related to the extent of lipid peroxidation. There was a significant inverse correlation between peak CPK occurrence and the values of CD at 2 and 24 hours after the start of thrombolysis (p=0.03 and 0.09, respectively). Moreover, there was a strong correlation between the peaking of CPK values before 10 hours from the beginning of thrombolysis and the CD values in the plasma (p=0.045) (Fig. 3). Peak CPK values occurring less than 10 hours after STK initiation are considered successful reperfusion of the myocardium.
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Fig. 3. Baseline conjugated dienes (CD) generation related to STK treatment intervals. A) CD generation separated into 2 groups of patients who showed a peak CPK elevation before and 10 hours post STK treatment (*p<0.05). B) Correlation between peak CPK occurrence in hours and CD generation post STK treatment.
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In summary, patients with AMI had low plasma antioxidant vitamin levels and an increase in basal plasma oxidation. STK treatment resulted in some drop in plasma vitamins. Increased generation of lipid peroxides occurred in patients who apparently underwent a successful thrombolysis.
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DISCUSSION
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The value of thrombolysis in improving short term survival after AMI is established, but in the long term there is evidence of increased rate of failures attributed to reocclusion.
Improvement in left ventricular function and survival after thrombolysis has been shown, but there are also numerous unfavorable events, such as myocardial stunning and arrhythmias during the reperfusion, which result from injury induced by oxidative stress. Demonstration of an enhanced free radicals production which produce cellular damage, including peroxidation or isomerization of lipids, confirms these findings [9–12].
Thrombolysis by streptokinase is widely utilized in treatment of AMI. Some previous studies demonstrated an increase in plasma oxidation in both AMI and angina [14,20]. Our results reveal no significant increase in plasma oxidation in AMI in the basal state compared with controls. The reasons for this discrepancy may relate to our smaller sample size of the control and certain confounding factors which were not adjusted for by our statistical analysis.
The antioxidant vitamin concentrations were found decreased in plasma of patients with angina and AMI [8,20–22]. Our study confirms these findings. In AMI patients we found significantly lower levels of vitamins A, E and carotenoids compared with controls. Many studies suggest that vitamin C represents the first line of defense against free radicals. We have not measured this antioxidant which may have shown a more rapid decline followed by subsequent declines in other antioxidants. A word of caution relates to the lipids and, hence, the lipid soluble antioxidants in AMI. Total cholesterol and LDL are known to be reduced in the setting of AMI which may complicate the interpretation of vitamin levels. Also, increased glucose concentration in AMI may reflect the impact of acute stress and glucose intolerance in our patients. There is, so far, no evidence on a direct influence of STK on plasma levels of antioxidant vitamins.
During reperfusion, a drop in the plasma levels of these vitamins was demonstrated [22]. In our study, there was a significant drop at 24 hours in plasma vitamin E only. Successful reperfusion with enhanced oxygen delivery to the ischemic myocardium apparently resulted in oxidative stress and this washout of oxygen free radicals may have appeared in the peripheral blood system. Previous studies demonstrated such increased oxidation in coronary and peripheral blood after coronary angioplasty and thrombolysis [13,15,16]. In our study, after 24 hours there was a significant elevation in MDA generation upon successful thrombolysis.
The importance of our findings is that it confirms the existence of an abnormal balance between the oxidative and protective mechanisms in AMI patients. With low initial plasma antioxidants, and a continuous drop on reperfusion, supported by other experimental and clinical evidence, it seems appropriate to introduce antioxidant therapy into thrombolysis trials in order to improve treatment [23–25]. However, this study is limited by the fact that we measured the oxidative/antioxidative status in the peripheral blood only — peripheral blood may not be representative of the coronary sinus blood. There were differences between the patients and the control groups, and for correction purposes we used the statistical analysis adjusted for sex and age. The multivariate analysis was specifically aimed at adjusting for smoking patterns. Smokers have lower levels of antioxidants (vitamin C and carotenoids) and generally are under more oxidative stress. Our analysis made sure that smoking, which is an important factor for AMI, will not explain some of the differences between cases and controls in our study. The patients’ stress caused by AMI is a possible source of error and other medications taken by the patients could influence the results. Finally, there is no way to separate the direct effect of STK from other reperfusion effects on oxidation.
In recent studies, Singh et al reported on plasma antioxidants, oxidative stress and the effect of antioxidant vitamins in AMI [26–27]. There was a significant drop in vitamins C, E, A and ß-carotene, whereas lipid peroxides were significantly higher in AMI compared with controls [26]. Provision of vitamins A, C, E and ß-carotene in patients with AMI resulted in a decrease in serum lipid peroxides compared with a placebo group. Also, there were fewer complications in the treatment group [27].
In summary, both reduction in antioxidants and elevation in lipid peroxide generation indicate the significance of free radical generating processes in reperfusion injury in AMI patients, and suggest the future introduction of antioxidants to the management of patients with AMI treated by thrombolysis.
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ACKNOWLEDGMENTS
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The study was supported by an Israeli Ministry of Health Chief Scientists Grant in Aid.
Received May 1, 1997.
Accepted October 1, 1997.
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ABSTRACT
INTRODUCTION
