Glutathione Peroxidase 1 Activity and Cardiovascular Events in Patients with Coronary Artery Disease
Stefan Blankenberg, M.D., Hans J. Rupprecht, M.D., Christoph Bickel, M.D., Michael Torzewski, M.D., Gerd Hafner, M.D., Laurence Tiret, Ph.D., Marek Smieja, M.D., Ph.D., François Cambien, M.D., Jürgen Meyer, M.D., Karl J. Lackner, M.D., for the AtheroGene Investigators
Background Cellular antioxidant enzymes such as glutathioneperoxidase 1 and superoxide dismutase have a central role inthe control of reactive oxygen species. In vitro data and studiesin animal models suggest that these enzymes may protect againstatherosclerosis, but little is known about their relevance tohuman disease.
Methods We conducted a prospective study among 636 patientswith suspected coronary artery disease, with a median follow-upperiod of 4.7 years (maximum, 5.4) to assess the risk of cardiovascularevents associated with base-line erythrocyte glutathione peroxidase1 and superoxide dismutase activity.
Results Glutathione peroxidase 1 activity was among the strongestunivariate predictors of the risk of cardiovascular events,whereas superoxide dismutase activity had no association withrisk. The risk of cardiovascular events was inversely associatedwith increasing quartiles of glutathione peroxidase 1 activity(P for trend <0.001); patients in the highest quartile ofglutathione peroxidase 1 activity had a hazard ratio of 0.29(95 percent confidence interval, 0.15 to 0.58; P<0.001),as compared with those in the lowest quartile. Glutathione peroxidase1 activity was affected by sex and smoking status but retainedits predictive power in these subgroups. After adjustment forthese and other cardiovascular risk factors, the inverse associationbetween glutathione peroxidase 1 activity and cardiovascularevents remained nearly unchanged.
Conclusions In patients with coronary artery disease, a lowlevel of activity of red-cell glutathione peroxidase 1 is independentlyassociated with an increased risk of cardiovascular events.Glutathione peroxidase 1 activity may have prognostic valuein addition to that of traditional risk factors. Furthermore,increasing glutathione peroxidase 1 activity might lower therisk of cardiovascular events.
Many aspects of the pathogenesis of atherosclerosis have beenunraveled in recent years, and an important potential role ofoxidative mechanisms has been elucidated.1,2 This research hasled to the assumption that oxidation of low-density lipoproteinrepresents a key event in atherogenesis, even though the resultsof trials of antioxidants in the prevention of human atherosclerosishave been mostly negative.3
Oxidative stress may be defined as an imbalance between theproduction and degradation of reactive oxygen species such assuperoxide anion, hydrogen peroxide, lipid peroxides, and peroxynitrite.Enzymatic inactivation of reactive oxygen species is achievedmainly by glutathione peroxidase, superoxide dismutase, andcatalase.4 In mammalian cells, glutathione and the glutathioneperoxidases constitute the principal antioxidant defense system.5,6There are at least four different glutathione peroxidases, allof which contain selenocysteine at their active sites.7
Glutathione peroxidase 1, the ubiquitous intracellular formand key antioxidant enzyme within most cells, including theendothelium, uses glutathione to reduce hydrogen peroxide towater and lipid peroxides to their respective alcohols,8 andit also acts as a peroxynitrite reductase.9 In mice, glutathioneperoxidase 1 deficiency results in abnormal vascular and cardiacfunction and structure.10 Similarly, superoxide dismutase isrepresented by three different ubiquitously expressed enzymesthat convert superoxide anion to hydrogen peroxide: cytosoliccopper- and zinc-containing superoxide dismutase, mitochondrialmanganese-containing superoxide dismutase, and extracellularsuperoxide dismutase. Extracellular superoxide dismutase ismost active in the vessel wall and has been shown to regulatethe availability of nitric oxide by scavenging superoxide anion.11
On the basis of the experimental evidence, we addressed thehypothesis that enhanced activity of cellular glutathione peroxidase1 and superoxide dismutase would be protective against cardiovascularevents in a large prospective cohort of patients with coronaryartery disease.
Methods
Study Population
Between November 1996 and December 1997, 732 patients referredto the Department of Medicine II of the Johannes Gutenberg Universityin Mainz, Germany, with suspected coronary artery disease wereenrolled in the AtheroGene registry. Fourteen patients withacute myocardial infarction and 75 patients in whom glutathioneperoxidase 1 activity could not be determined immediately wereexcluded from the present analysis. Thus, the final study cohortconsisted of 643 patients, 133 with symptoms of unstable anginaand 510 with symptoms of stable angina. Coronary angiographywas performed in all patients. Relevant coronary artery disease,defined by greater than 30 percent stenosis in at least onemajor coronary artery, was detected in 558 patients. The studydesign has been described in detail elsewhere.12 The exclusioncriteria were evidence of hemodynamically significant valvularheart disease, surgery or trauma within the previous month,known cardiomyopathy, known cancer, febrile conditions, or useof oral anticoagulant therapy within the previous four weeks.
We considered patients who were receiving dietary treatmentor medication for diabetes or whose current fasting blood glucoselevel was above 125 mg per deciliter to have diabetes mellitus.Patients who had received antihypertensive treatment or whohad received a diagnosis of hypertension (blood pressure above160/90 mm Hg) were considered to have hypertension. Patientswere classified as currently smoking, as having smoked in thepast (if they had stopped more than 4 weeks and less than 40years earlier), or as never having smoked (if they had neversmoked or had stopped 40 or more years earlier).
Among the 643 patients, 636 (98.9 percent) were followed fora median of 4.7 years (maximum, 5.4). There were 64 deaths fromcardiovascular causes, 21 deaths from other causes, and 19 nonfatalmyocardial infarctions. Information about the causes of deathand clinical events was obtained from hospital and general-practitionercharts.
The study was approved by the ethics committee of the Universityof Mainz. Participation was voluntary, and each patient gavewritten informed consent.
Laboratory Methods
Blood was drawn under standardized conditions before coronaryangiography was performed. Glutathione peroxidase 1 activityand superoxide dismutase activity were determined in washedred cells obtained immediately after sampling from whole bloodanticoagulated with EDTA. Hemolyzed cells were stored frozenfor up to one week; freezing does not lead to changes in enzymeactivity. Glutathione peroxidase 1 was measured as previouslydescribed,13 with minor modifications (Ransel test kit, Randox).The intraassay and interassay coefficients of variation were6.7 percent and 9.9 percent, respectively.
Superoxide dismutase activity was determined by the followingmethod. Superoxide radicals generated by the xanthine oxidasereaction convert 1-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazoliumchloride quantitatively to a formazan dye (Ransod test kit,Randox). Conversion of superoxide radicals to hydrogen peroxideby superoxide dismutase inhibits dye formation and serves asa measure of superoxide dismutase activity. The intraassay andinterassay coefficients of variation were 5.1 percent and 5.5percent, respectively. Serum lipid levels (the levels of totalcholesterol, triglycerides, and high-density lipoprotein cholesterol)were measured immediately by routine methods; low-density lipoproteincholesterol was calculated by the Friedewald formula.
For all other biologic markers measured in the study, plasmaand serum were stored at –80°C until analysis, whichwas performed after a mean of 1.5 years of storage time. C-reactiveprotein was measured with a highly sensitive, latex-particle–enhancedimmunoassay (Roche Diagnostics; range of detection, 0.1 to 20mg per liter; interassay coefficient of variation, 1.0 percentfor values of 15 mg per liter and 6.5 percent for values below4 mg per liter). Interleukin-6 and soluble intercellular adhesionmolecule 1 were measured with commercially available immunoassays(EASIA, Biosource Europe). Homocysteine was measured by high-pressureliquid chromatography (interassay coefficient of variation,7.1 percent) and selenium by carbon-furnace atomic-absorptionspectrometry with Zeeman compensation, as previously described.14
Statistical Analysis
The mean levels and proportions of base-line cardiovascularrisk factors were calculated for study participants in whoma cardiovascular event subsequently occurred and in those withoutsuch an event. The significance of differences between the meansfor the two groups was assessed with Student's t-test, and thesignificance of differences in proportions was tested with thechi-square statistic. Variables with a skewed distribution werepresented as medians, and the Wilcoxon rank-sum test was applied.The cumulative event plots according to quartile of glutathioneperoxidase 1 activity were estimated by the Kaplan–Meiermethod and compared with use of the log-rank test. In all survivalanalyses, the end point was death from cardiovascular causesor nonfatal myocardial infarction. Data from patients who diedfrom other causes were censored at the time of death. Hazardratios for future coronary events according to quartile of glutathioneperoxidase 1 activity were estimated by Cox regression modelsadjusted for potential confounders. Three adjusted models wereconstructed. We adjusted first for age and sex and second forother traditional risk factors. The final model included clinicaland therapeutic variables as well as C-reactive protein, homocysteine,and creatinine.
To evaluate the combined effect of glutathione peroxidase 1activity and smoking status on cardiovascular risk, we dividedthe study participants into six groups according to whetherthe glutathione peroxidase 1 activity was above the median orat or below the median and according to their smoking-statuscategory. In these analyses, Cox regression was used to assesssimultaneously the risk of future cardiovascular events in eachof the six groups, with the group of patients whose glutathioneperoxidase 1 activity was above the median and who had neversmoked as the reference group. The hypothesis that smoking andthe level of glutathione peroxidase 1 activity had an interactiveeffect on the risk of future cardiovascular events was formallytested in a Cox regression model that included a term for themultiplicative interaction of smoking (across the three categoriesof smoking status) and glutathione peroxidase 1 activity. Thehazard ratios and their 95 percent confidence intervals arereported. The P values are two-sided; a P value of less than0.05 was considered to indicate statistical significance. Allcomputations were carried out with SPSS software, version 10.07.
Results
Table 1 gives the base-line characteristics of the 83 studyparticipants who subsequently died from cardiovascular causesor had a nonfatal myocardial infarction and the 553 who didnot have a cardiovascular event, including the results of base-lineblood-chemistry analysis. Glutathione peroxidase 1 activitywas normally distributed among the study participants. It rangedfrom 7.4 to 99.6 units per gram of hemoglobin, with a mean (±SD)of 49.2±11.6, a median of 48.3, and an interquartilerange of 42.0 to 56.3 units per gram of hemoglobin. The base-linelevel of glutathione peroxidase 1 activity was significantlylower among those who died from cardiac causes or had a nonfatalmyocardial infarction than among those who did not. This resultwas stable when data from the subgroups with fatal events (64patients) and nonfatal events (19 patients) were analyzed separately(45.7±13.5 and 44.1±10.4 units per gram of hemoglobin,respectively, vs. 49.8±11.3 units per gram of hemoglobinfor those who did not have cardiovascular events or die fromnoncardiovascular causes; P=0.009 and P=0.03, respectively).The base-line level of glutathione peroxidase 1 activity inpatients who died from noncardiovascular causes (21 patients)was the same as that in event-free patients (49.7±11.1vs. 49.8±11.3 units per gram of hemoglobin).
Table 1. Base-Line Characteristics of the Study Patients.
Figure 1 shows the Kaplan–Meier curves for event-freesurvival according to quartile of glutathione peroxidase 1 activity.The unadjusted rate of cardiovascular events increased in astepwise fashion across decreasing quartiles of base-line glutathioneperoxidase 1 activity. The difference between the lowest andhighest quartiles and the trend across all quartiles were significant(P<0.001 for both comparisons). The event rate for patientsin the lowest quartile of glutathione peroxidase 1 activity(20.8 percent) was approximately three times that for patientsin the highest quartile (7.0 percent). To place the effect inperspective, Table 2 presents the hazard ratios for cardiovascularevents associated with an increase of 1 SD in various risk factors.
Figure 1. Kaplan–Meier Curves Showing Cardiovascular Events According to Quartile of Glutathione Peroxidase 1 Activity.
The numbers of cardiovascular events were 33, 23, 16, and 11 in quartiles 1, 2, 3, and 4, respectively. Glutathione peroxidase 1 activity is shown in units per gram of hemoglobin.
Table 2. Age- and Sex-Adjusted Analysis of the Association between Novel and Traditional Risk Factors and the Risk of a Cardiovascular Event.
The strongest predictors of the level of glutathione peroxidase1 activity were smoking status and sex. Significantly lowerlevels of glutathione peroxidase 1 activity were observed incurrent smokers than in those who had never smoked (45.7 vs.51.6 units per gram of hemoglobin, P<0.001). Former smokersalso had lower levels of enzyme activity than those who hadnever smoked (48.2 vs. 51.6 units per gram of hemoglobin); however,this difference was not statistically significant. Furthermore,the level of glutathione peroxidase 1 activity was lower inmen than in women (48.5 vs. 51.1 units per gram of hemoglobin,P=0.009). Women below 55 years of age had higher levels of glutathioneperoxidase 1 activity than older women (54.1 vs. 50.5 unitsper gram of hemoglobin, P=0.12). No difference in glutathioneperoxidase 1 activity was detected between patients with stableangina and those with unstable angina.
Although statin medication had no significant association withglutathione peroxidase 1 activity in the whole study population,the percentage of patients receiving statins was significantlyhigher among those in the highest quartile of glutathione peroxidase1 activity than among those in the other three quartiles (30.6percent vs. 19.5 percent, P=0.003). With this exception, noassociation was observed between the use of any cardiovascularmedication and glutathione peroxidase 1 or superoxide dismutaseactivity. There was a weak but significant correlation of glutathioneperoxidase 1 activity with homocysteine (r=–0.09, P=0.03)and selenium (r=0.09, P=0.04). Of all the inflammatory markersmeasured in this study, only soluble intercellular adhesionmolecule 1 showed a moderate inverse correlation with glutathioneperoxidase 1 activity (r=–0.11, P=0.02).
To assess the independent predictive value of glutathione peroxidase1 activity, we used a series of Cox predictive models (Table 3).The inverse relation between glutathione peroxidase 1 activityand relative risk remained nearly unchanged after adjustmentfor cardiovascular risk factors and clinical features (model2). Further adjustment for therapeutic variables as well asC-reactive protein (as a cluster representative of the inflammatorymarkers), homocysteine, and creatinine (model 3) also did notattenuate the relative risk associated with glutathione peroxidase1 activity; patients in the highest quartile of glutathioneperoxidase 1 activity had a hazard ratio of 0.29 (95 percentconfidence interval, 0.14 to 0.60; P=0.001) as compared withthose in the lowest quartile. Inclusion of interleukin-6 insteadof C-reactive protein in the Cox predictive model had no effecton the hazard ratios. Similarly, in a subgroup of 566 patientsin whom the ejection fraction had been measured, adjustmentfor this variable did not alter the hazard ratio associatedwith increasing quartiles of glutathione peroxidase 1 activity(data not shown).
Table 3. Hazard Ratios for Future Cardiovascular Events According to Quartile of Base-Line Glutathione Peroxidase 1 Activity.
In previous reports from the entire AtheroGene cohort, the levelsof several inflammatory markers, such as interleukin-18 andsoluble vascular adhesion molecule 1, were found to be elevatedamong those at risk for subsequent coronary events. No statisticallysignificant correlations were observed between glutathione peroxidase1 activity and these two proteins. Inclusion of these variablesin multivariate analyses did not attenuate the risk of futurecoronary events associated with decreased levels of glutathioneperoxidase 1 activity.
Because smoking status was associated with base-line glutathioneperoxidase 1 activity as well as with coronary risk, the interactionbetween smoking status and glutathione peroxidase 1 activitywas analyzed in more detail. The association between smokingand cardiovascular events was observed predominantly in patientswith levels of glutathione peroxidase 1 activity below the median.As illustrated in Figure 2, among those with low levels of glutathioneperoxidase 1 activity (at or below the median value of 48.32units per gram of hemoglobin), former smokers were at significantrisk for cardiovascular events (hazard ratio, 3.0, as comparedwith patients who had never smoked and had high glutathioneperoxidase 1 activity; 95 percent confidence interval, 1.5 to5.9; P=0.001), as were current smokers (hazard ratio, 5.6; 95percent confidence interval, 2.4 to 13.5; P<0.001). To alesser extent, a significant increase in cardiovascular riskwas also observed in former or current smokers in the subgroupof those with levels of glutathione peroxidase 1 activity abovethe median.
Figure 2. Age- and Sex-Adjusted Hazard Ratios for Cardiovascular Events According to the Level of Glutathione Peroxidase 1 Activity and Smoking Status.
The interaction tested is between smoking category and glutathione peroxidase 1 activity (as a continuous variable). A high level of glutathione peroxidase 1 activity was defined as more than the median value of 48.32 units per gram of hemoglobin, and a low level as 48.32 or fewer units per gram of hemoglobin.
Discussion
In this prospective cohort of patients with angiographicallydocumented coronary artery disease, we demonstrated that erythrocyteintracellular glutathione peroxidase 1 activity is inverselyassociated with future fatal and nonfatal cardiovascular events.Erythrocyte glutathione peroxidase 1 is probably a suitablesurrogate marker for cellular glutathione peroxidase 1 activityin general, but this has not been proved by a systematic analysis.The risk of a cardiovascular event among those in the highestquartile of glutathione peroxidase 1 activity was approximately30 percent of that among those in the lowest quartile. Thisdifference did not change appreciably after adjustment for mostpotential confounders, indicating that the relation betweenglutathione peroxidase 1 and future cardiovascular events isindependent of other risk factors and clinical features.
Because glutathione peroxidase 1 appears to have a major rolein the prevention of oxidative stress, it may also be an importantantiatherogenic enzyme.2 In fact, reduced expression of glutathioneperoxidase 1 has been shown to increase cell-mediated oxidationof low-density lipoprotein in mice.15 Furthermore, mice thatare heterozygous for glutathione peroxidase 1 deficiency haveendothelial dysfunction combined with structural vascular abnormalities,such as increased periadventitial inflammation, neointimal formation,and collagen deposition surrounding the coronary arteries.10Glutathione peroxidase 1 deficiency apparently decreases bioavailablenitric oxide in mice,16 an effect that can be aggravated byhyperhomocysteinemia.17 In addition, glutathione peroxidase1 activity is decreased or absent in carotid atheroscleroticplaques, and the lack of glutathione peroxidase 1 activity inatherosclerotic lesions appears to be associated with the developmentof more severe lesions in humans.18 Since catalase activityis absent from human vascular cells19 and superoxide dismutaseis poorly effective against cellular oxidant damage,6 the mostimportant antioxidative shield is reduced in atheroscleroticplaque.
The variability in glutathione peroxidase 1 activity withinan individual patient has been reported to be about half thevariability between patients,20 which is in accordance withour own observations (unpublished data). The causes of variabilitybetween patients are not well established. The few genetic polymorphismsidentified so far do not show an association with glutathioneperoxidase 1 activity.21 Smoking consistently reduces glutathioneperoxidase 1 activity, whereas the effect of commonly used drugsappears to be negligible.20 The level of glutathione peroxidase1 activity is higher in women than in men. In our study thisdifference was most pronounced in premenopausal women, as hasbeen described previously.22
Cigarette smoking is strongly associated with dysfunctionalvasomotor responses, diminished nitric oxide levels, and time-dependentdecreases in the content of endothelial nitric oxide synthasemessenger RNA.23 In accordance with previous studies,24 glutathioneperoxidase 1 activity was decreased in smokers and former smokers.However, the association between low levels of glutathione peroxidase1 activity and high cardiovascular risk was also observed insmokers. Therefore, measurement of glutathione peroxidase 1should identify smokers who are at highest risk for cardiovascularevents.
Glutathione peroxidase 1 was recently shown to inhibit 5-lipoxygenasein monocytic cells.25 Since 5-lipoxygenase is induced in monocytesand macrophages within progressing atherosclerotic lesions26and strongly contributes to atherosclerotic susceptibility inmice,27 the interference of glutathione peroxidase 1 with 5-lipoxygenasemight constitute a protective function of the enzyme, in additionto its antioxidant activity.
The role of selenium, which is known to increase glutathioneperoxidase 1 gene expression and activity,28,29 in cardiovasculardisease is controversial. Epidemiologic studies have been inconclusive,30,31,32and prospective, controlled trials of selenium supplementationare lacking.33 In the present study, a weak but statisticallysignificant association was observed between selenium levelsand glutathione peroxidase 1 activity. The weakness of thisassociation might be due to the fact that the average seleniumlevel was above 70 ng per milliliter, which is probably withinthe range where the effect of selenium on glutathione peroxidase1 activity plateaus.34,35
From a clinical perspective, our data show that low erythrocyteglutathione peroxidase 1 activity identifies patients with coronaryartery disease who are at the highest risk for cardiovascularevents. Adjustment for inflammatory markers such as solubleadhesion molecules or interleukin-18, which have been shownto be predictive of cardiovascular events in this study population,36,37does not affect the association between glutathione peroxidase1 and prognosis. This observation suggests that measurementof glutathione peroxidase 1 activity provides additional informationon risk and might be useful in identifying patients who wouldbenefit from preventive antioxidative treatment.
Supported by grants from the Stiftung Rheinland-Pfalz fürInnovation, Ministry for Science and Education, Mainz, Germany(AZ 15202-386261/545); INSERM, Paris (to Dr. Blankenberg), andthe Schleicher Stiftung, Dresdner Bank, Frankfurt, Germany.
* Members of the AtheroGene study group are listed in the Appendix.
Source Information
From the Departments of Medicine II (S.B., H.J.R., J.M.) and Clinical Chemistry and Laboratory Medicine (M.T., G.H., K.J.L.), Johannes Gutenberg University, Mainz, Germany; INSERM Unité 525, Faculté de Médecine Pitié–Salpêtrière, Paris (S.B., L.T., F.C.); Innere Abteilung, Bundeswehrzentralkrankenhaus, Koblenz, Germany (C.B.); and the Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ont., Canada (M.S.).
Address reprint requests to Dr. Blankenberg at the Department of Medicine II or to Dr. Lackner at the Department of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany, or to stefan.blankenberg{at}uni-mainz.de or lackner{at}zentrallabor.klinik.uni-mainz.de.
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Appendix
The other members of the AtheroGene study group are as follows:Department of Medicine II, Johannes Gutenberg University, Mainz,Germany — C. Espinola-Klein; INSERM Unité 525,Paris — O. Poirier, V. Nicaud, D. Tregouet, and J.-L.Georges. AtheroGene recruitment centers were the Departmentof Medicine II, Johannes Gutenberg University, Mainz, Germany,and Innere Abteilung, Bundeswehrzentralkrankenhaus, Koblenz,Germany.
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