Baseline Glutathione Peroxidase Activity Affects Prognosis after Acute Coronary Syndromes

Abstract

Oxidative stress is associated with atherosclerosis and plaque lesions in experimental in vitro models. Few in vivo studies have examined the association between redox status and the prognosis of acute coronary syndromes.

We undertook a prospective, observational study of 137 patients who had been admitted because of an acute coronary syndrome. We determined glutathione peroxidase activity (a marker of systemic antioxidant status) and recorded clinical and angiographic features and cardiovascular events (cardiovascular death, reinfarction, readmission with a new ischemic event, or need for coronary revascularization).

The mean age of the patients (78% of whom were men) was 61.7 ± 10.9 years; 76% were admitted with non-ST-segment-elevation acute coronary syndrome. Left ventricular ejection fraction was normal in 61%. In the 23.4% who experienced cardiovascular events, glutathione peroxidase activity was higher (mean, 2.38 vs 1.76 mU/mg of protein; P < 0.01). Two-year event-free survival was lower in patients whose glutathione peroxidase activity was higher than the 50th percentile (63% vs 82%; P = 0.01). Multivariate analysis showed a direct independent relationship between glutathione peroxidase activity and cardiovascular events (hazard ratio, 3.72; 95% confidence interval, 1.53–9.02; P < 0.01).

We conclude that patients who experienced acute coronary syndromes and events during follow-up had higher plasma glutathione peroxidase activity, and that glutathione peroxidase activity was an independent predictor of events during follow-up.

Oxidative stress appears to play a vital role in the onset and progression of arteriosclerosis.¹ Oxidation of low-density lipoproteins (LDL) in the vascular wall is one of the pathophysiologic mechanisms by which LDL accumulates in macrophages, but the influence of this process in the evolution of arteriosclerosis is unknown. Great discrepancy exists between the clinical and the experimental evidence concerning the role of antioxidants in the prevention and treatment of arteriosclerotic disease.^2,3

In mammals, glutathione peroxidase (GP) and superoxide dismutase form the main system of antioxidant defense. Glutathione perodixase-1 is the predominant antioxidant system in endothelial cells. This enzyme transforms hydrogen peroxide into water and lipid peroxides into their respective alcohols.⁴ Experimental studies have shown that a deficit of GP-1 results in structural and functional anomalies of the vessels and the heart,⁵ while its overexpression might improve ventricular remodeling after a myocardial infarction.⁶ Nevertheless, only a few translational studies have been undertaken to date, and their results are contradictory.^4,7–12 Most of these studies have focused on examining the influence of the baseline oxidative status on the genesis of systemic or coronary arteriosclerosis, as well as the behavior of antioxidant activity versus situations of myocardial ischemia and postperfusion damage. However, the influence of the systemic oxidative status on prognosis for patients with ischemic heart disease has hardly been evaluated.⁴

The aim of our study was to examine the association between GP activity and the onset of cardiovascular events in a selected sample of patients admitted to the hospital with acute coronary syndrome (ACS) and angiographic evidence of coronary arteriosclerosis.

Patients and Methods

We undertook a prospective study of a consecutive series of 137 patients who were admitted to our center from March 2001 through September 2002 because of ACS. The general characteristics of the patients are shown in Table I; of note was the high percentage of smokers and of patients with dyslipidemia. All patients in the study displayed cardiac-marker elevation (the MB isoenzyme of creatine kinase and troponin), and all underwent diagnostic coronary angiography. The study design and procedures all complied with the Declaration of Helsinki, and the research protocol was approved by our hospital ethics committee. All the patients gave their written, informed consent.

Table I. Characteristics of the Patient Population

Definition of Acute Coronary Syndrome. Acute coronary syndrome was defined according to the guidelines of the Spanish Society of Cardiology that were in effect at the time of the study.¹³

Coronary Arteriography and Ventriculography. Coronary arteriography was performed by the standard Judkins technique. The left ventricular ejection fraction (LVEF) and coronary anatomy were evaluated.

Data Collection. Epidemiologic and clinical data were collected, as were the angiographic characteristics and the LVEF. Left ventricular ejection fraction was considered to be significantly depressed if it was ≤0.45. Multivessel disease was defined as the presence of severe lesions (≥70% stenosis) in at least 2 of the 3 main epicardial coronary arteries. Data were also recorded on coronary revascularization (whether percutaneous or surgical) and on treatment prescribed at hospital discharge.

Blood Samples. Within the first 48 hours after hospital admission—after coronary arteriography, and after a 12-hour fast—arterial blood samples were obtained from a canalized artery. The samples were collected in tubes containing EDTA, placed on ice, and centrifuged within 20 minutes of collection at 3,000 rpm for 15 minutes at 4°C. The plasma was separated and frozen at −80°C for later quantitative analysis.

Measurement of Glutathione Peroxidase Activity. Glutathione peroxidase activity was measured over a fixed time by means of glutathione (GSH) consumption.¹⁴ The spontaneous or enzymatically catalyzed reaction of GSH with hydrogen peroxide was stopped after a fixed time by adding a strong acid, and the GSH was later measured by polarography.

Follow-Up. The patients were monitored by means of a telephone survey, a personal interview, a chart consultation, or some combination of these. The following data were recorded: months of follow-up, death due to cardiovascular cause (to calculate the overall survival rate), and onset of major cardiovascular events (MACEs, defined as cardiovascular death, readmission with ACS, or the need for coronary revascularization)—in order to establish the MACE-free survival rate during follow-up.

Data Processing and Statistical Analysis. The data were analyzed by use of SPSS v. 12.0 (SPSS, Inc.; Chicago, Ill). The quantitative variables were expressed as the mean ± SD and the qualitative variables as percentages. Comparison of differences between the qualitative variables was done with the χ² test or Fisher's exact test when the expected frequencies were less than 5. Student's t test was used to compare differences between the quantitative variables (normal distribution of quantitative variables was verified with the Shapiro-Wilk test). The nonparametric variables were studied using the Mann-Whitney test.

The sample was divided into percentiles in order to study survival in accordance with the plasma's glutathione peroxidase activity (GPA). Survival estimates for each group were analyzed via the Kaplan-Meier method, using the log-rank comparison test.

Study of the variables associated with the prognosis was done by multivariate analysis that used a Cox stepwise regression model of proportional risks. The level of statistical significance was set at P < 0.05 for 2 tails.

Results

Coronary revascularization was performed in almost 75% of the patients. Nearly one quarter of the patients had MACEs during the follow-up of 2 years. The mean GPA was 1.9 ± 1.0 mU/mg of protein (median, 1.80 mU/mg; range, 0.45–7.69 mU/mg).

Table II includes the bivariate analysis according to the GPA. The percentage of patients who suffered MACEs was higher among patients whose GPA was above the median. No other statistically significant differences were seen between the other epidemiologic, clinical, and therapeutic variables.

Table II. Bivariate Analysis for Glutathione Peroxidase Activity That Was Above or Below the Median

However, patients with multivessel involvement were more likely to have MACEs during the follow-up period, and those who underwent revascularization were less likely (Table III). No significant differences in susceptibility to MACEs were seen among patients who took β-blockers, statins, or angiotensin-converting enzyme inhibitors. The mean GPA was higher in patients who had MACEs, and the percentage of patients with GPA above the median was also greater (Table III).

Table III. Bivariate Analysis of Patients' Characteristics in Relation to MACEs during Follow-Up

In patients with GPA above the median, the estimated 1-year, event-free survival was significantly lower, as seen in Figure 1 (66.74% vs 88.50%). The estimated 2-year, event-free survival was also lower in patients whose GPA was higher than the median (63% vs 82%; P = 0.01). Overall, as GPA increased, event-free survival fell (Fig. 2), with 1-year estimates of 90.79%, 80.80%, 67.75%, and 65.52% for each of the GPA quartiles.

Fig. 1 Kaplan-Meier curves show event-free survival according to whether glutathione peroxidase activity (GPA) was below or above the median (50th percentile=1.80 mU/mg of protein).

Fig. 2 Kaplan-Meier curves show event-free survival according to the quartile of glutathione peroxidase activity (GPA). Glutathione peroxidase activity is shown in mU/mg protein.

After adjusting for confounding variables, we found that a GPA above the median was independently associated with the onset of events during follow-up (Table IV). In addition, admission due to ACS with no ST-segment elevation, with involvement of the left main coronary artery, with multivessel disease, with a high GRACE (global registry of acute coronary events) Score,¹⁵ or with important left ventricular dysfunction was each an independent predictor of coronary events during follow-up. Revascularization therapy was inversely associated with the onset of MACEs (Table IV).

Table IV. Cox Multivariate Regression Analysis to Identify Independent Predictors of MACEs

Discussion

This prospective study of patients admitted with ACS and angiographic evidence of important coronary lesions showed a direct association between baseline GPA and the onset of MACEs during the medium-term follow-up.

Although the association between oxidative stress and the development of both endothelial dysfunction and coronary arteriosclerosis has been studied before,^8,16–19 the prognostic role of markers of oxidative damage has received comparatively little attention, and the results so far are not clear. Indeed, in the particular case of GPA, only 2 reports^4,7 have examined its association with the onset of cardiovascular events, and the results (an inverse association between higher GPA and the rate of adverse events during follow-up) were in disagreement with those of our study. Admittedly, those study populations comprised mostly persons with stable ischemic heart disease, whereas in our study all patients were admitted with ACS. Another factor to consider is that the method by which we determined GPA was different from that used in earlier studies, which might also yield different results.

An explanation for our findings could be that our patients with greater baseline oxidative stress had a greater systemic antioxidant response; this would yield an indirect measurement of the worse risk profile of these patients. Measurement of some value directly related to baseline oxidative stress would probably have helped to elucidate this possibility.

Weinbrenner and co-authors⁸ noticed an increase in both oxidizing and antioxidizing activity in patients who had stable coronary disease, as compared with a control group. Although these results are more consistent with ours, the design of the Weinbrenner study was different (case-control), and the characteristics of the patients were markedly different (stable ischemic heart disease).

While GPA is considered protective against oxidative damage, we should nevertheless remember that most studies have been experimental^5,20,21 and that only a few in vivo studies have been undertaken in human beings, mostly in patients with stable coronary disease. Other studies have detected a reduction in antioxidant activity in specific situations, such as in reperfusion damage models. This reduction occurs during the initial phases of ACS and is followed by a rise after the appearance of conditions favoring the genesis of free radicals, such as myocardial damage secondary to reperfusion.²² In our study, the coronary revascularization therapy (mainly percutaneous) in a high percentage of the patients may have influenced the results. Most studies that have been reported to date refer to the role of antioxidant complexes in stable patients. In our study, the situation was different: we examined the role of GPA in unstable patients without considering whether it fell or rose in these situations; rather, we evaluated its prognostic influence. The GPA may increase more in reperfused patients, although in our study the multivariate analysis showed an association between GPA and prognosis that was independent of revascularization.

Most prognostic studies have analyzed the onset of cardiovascular events in relation to systemic oxidative status,^6,23–27 whereas the role of antioxidant status has received little attention, perhaps because its action was assumed to be protective against cardiovascular events during follow-up. Our study shows that, in the event of ACS, higher GPA probably reflects greater antioxidant response due to greater oxidative status, but that this response, at least during the acute phase, is associated not with a better prognosis but with a worse prognosis.

Despite the fact that most clinical studies of interventions with antioxidants have failed to detect an improved prognosis, it should be borne in mind that the inclusion criteria did not take into consideration the baseline oxidative or antioxidant status, which we believe to be crucial to the identification of those patients who might benefit from antioxidant therapy.²⁸

Limitations

This study involved a selected group of patients with ACS who underwent only therapeutic coronary catheterization; the results, therefore, are applicable only to this type of patient.

Our results could also be explained by the higher risk, at baseline, of the considerable segment of our study group that would prove susceptible to MACE. Also, our sample size was small, and this could have influenced the results of the study.

The laboratory methods that we used to measure GPA were different from those applied in other studies; these methods could have influenced the conclusions of our study, which differ from those reported previously.