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Prognostic value of plasma myeloperoxidase concentration in patients with stable coronary artery disease
Prognostic value of plasma myeloperoxidase concentration in patients with stable coronary artery disease Ada Stefanescua Siegmund Braun, MD,b Gjin Ndrepepa, MD,b Tobias Koppara, MD,b Herribert Pavaci, MD,c Julinda Mehilli, MD,b Albert Scho ¨ mig, MD,b,c and Adnan Kastrati, MDb Montreal, Quebec, Canada; Munich, Germany; and Rome, Italy
Background There are no studies yet on the usefulness of myeloperoxidase (MPO) as a prognostic tool in patients with stable coronary artery disease (CAD). Methods The study included 382 patients with clinical and angiographic confirmation of stable CAD. Blood samples for MPO measurement were taken before angiography. Myeloperoxidase was determined using an enzyme immunoassay. The primary end point of the study was all-cause mortality. Results Patients were categorized into 2 groups: the high-MPO group included patients in the third tertile of MPO levels (N75.0 μg/L; 127 patients), and the low-MPO group included patients in the first (b52.6 μg/L) and second tertiles (52.6-75.0 μg/L) of MPO levels (255 patients). The median follow-up was 3.5 [3.3-4.8] years. There were 35 deaths (9.2%) during the follow-up. The MPO concentration was 60.1 [47.0; 83.8] μg/L in survivors and 72.7 [54.8; 105.1] μg/L in nonsurvivors (P = .06). There were 17 deaths in the high-MPO level and 18 deaths in the low-MPO group: Kaplan-Meier estimates of mortality were 18.3% and 10.5% with an odds ratio of 1.96 (95% confidence interval [1.02-3.76], P = .04). The Cox proportional hazards model adjusting for correlates of mortality showed that plasma MPO was not an independent correlate of mortality (hazard ratio 1.06, 95% confidence interval [0.71-1.59], P = .77 for 1 SD increase in the log variable). Conclusion
Although elevated plasma MPO concentration is associated with a more advanced cardiovascular risk profile, plasma MPO does not predict mortality independent of other cardiovascular risk factors in patients with stable CAD. (Am Heart J 2008;155:356-60.)
Myeloperoxidase (MPO) is an enzyme of the mammalian peroxidase family secreted by activated leukocytes, mainly polymorphonuclear neutrophils.1 Myeloperoxidase is part of the innate immune system defenses against bacteria and parasites and has been shown to play a significant role in promoting inflammation. In particular, MPO is secreted by polymorphonuclear neutrophils undergoing site-specific activation in the coronary artery endothelium2,3 and is present in increased levels in the plasma of patients with coronary artery disease (CAD),4 acute coronary syndrome
From the aFaculty of Medicine, McGill University, Montreal, Quebec, Canada; bKlinik für Herz- und Kreislauferkrankungen and Institut für Laboratoriumsmedizin, Deutsches Herzzentrum München, Munich, Germany; 1. Medizinische Klinik Klinikum rechts der Isar, Technische Universität München, Munich, Germany; and cDepartment of Heart and Great Vessels bAttilio RealeQ, University of Rome, Rome, Italy. Submitted August 1, 2007; accepted October 12, 2007. Reprint requests: Adnan Kastrati, MD, Deutsches Herzzentrum, Lazarettstr. 36, 80636 Munich, Germany. E-mail: [email protected] 0002-8703/$ - see front matter © 2008, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2007.10.017
(ACS),5,6 and acute myocardial infarction.7 Suggested mechanisms through which MPO promotes inflammation and plaque formation include the production of hydrochlorous acid, which disrupts intracellular signaling8; the oxidation of lipids such as high- and lowdensity lipoproteins and other endothelial cell-surface lipoproteins,9,10 which promotes macrophage activation and foam cell formation; and the depletion of nitric oxide in vascular endothelium, which reduces endogenous regulation of vessel dilatation.11 Myeloperoxidase has been shown to be a strong predictor of adverse events in patients with ACS,5,6 chronic heart failure,12 acute myocardial infarction,7 and of future CAD in a healthy population.13 Stable CAD is one of the earliest and most common presentation of CAD14,15 but is often poorly diagnosed and managed.15,16 In particular, this is due to variability in clinical presentations and degrees of severity, especially in the elderly and in diabetic patients—two high-risk groups.16 Biomarkers such as N-terminal probrain natriuretic peptide17,18 and C-reactive protein (CRP)19 have been shown to have prognostic value for adverse events in patients with stable CAD; as of yet, however, there have been no
American Heart Journal Volume 155, Number 2
studies on the usefulness of MPO as such a prognostic tool in these patients. The purpose of this study is to assess the prognostic value of plasma MPO concentration in patients with stable CAD.
Methods Patients The study included 382 patients with stable CAD undergoing coronary angiography at the Deutsches Herzzentrum, Munich, Germany. Patients were recruited in a prospective cohort study to investigate the prognostic value of biochemical markers in patients with angiographically proven CAD. The diagnosis of stable CAD was based on the presence of chest pain that did not change its pattern during the preceding 2 months. We did not include patients who presented with ACS, diagnosed on the basis of ST-segment changes on electrocardiogram, or abnormal creatine kinase (CK) or cardiac troponin tests. We did not include patients with unstable angina, defined as a history of crescendo angina, angina at rest or with minimal exertion, or angina of new onset (within 1 month) in the absence of clearcut electrocardiographic and cardiac enzyme changes diagnostic of an acute myocardial infarction. Furthermore, patients with end-stage renal disease, inflammatory diseases, or malignancies were excluded. All patients gave informed consent for the participation in the study. The study protocol was approved by the institutional ethics committee. Percutaneous coronary interventions (stent implantation in most cases) and periprocedural care were performed according to standard criteria. Bare metal stents were used. Antiplatelet therapy consisted of clopidogrel (600 mg as a loading dose followed by 75 mg/d for ≥4 weeks) and aspirin (200 mg/d administered orally and continued indefinitely).
Definitions Angiographic CAD was diagnosed in the presence of coronary stenosis with ≥50% lumen obstruction in at least 1 of the 3 major coronary arteries. Arterial hypertension was defined in patients under active treatment with antihypertensive agents or with a systolic blood pressure of N140 mm Hg and/or diastolic blood pressure of N90 mm Hg on at least 2 separate occasions. Hypercholesterolemia was defined as a documented total cholesterol value ≥240 mg/dL. The patients who admitted to ongoing nicotine consummation were considered current smokers. Diabetes mellitus was defined in patients under an active treatment with insulin or oral hypoglycemic medication. For patients on dietary treatment alone, documentation of abnormal fasting blood glucose or a previous abnormal glucose tolerance test, according to the World Health Organization criteria, were required for the diagnosis of diabetes.20
Biochemical measurements Blood samples for MPO measurement were taken from a peripheral vein before coronary angiography and heparin administration in tubes containing EDTA (Sarstedt, Nümbrecht, Germany) and promptly centrifuged at 1550g for 10 min. After separation, plasma aliquots were stored frozen at −80°C until assayed within batches. Blood count, serum lipids, and other
Stefanescu et al 357
metabolites were determined immediately after collection using standard methods. The concentration of MPO in EDTA plasma was determined with an enzyme immunoassay (Mercodia, Uppsala, Sweden). The immunoassay is based on the sandwich technique, in which 2 monoclonal antibodies are directed against separate antigenic determinants on the MPO molecule. Samples were measured in duplicate after a 1 + 10 dilution with sample buffer. The measuring range of this assay is between 3 and 300 μg/L. Intraassay imprecision ranges between 1.24% (median MPO = 62.5 μg/L) and 7.38% coefficient of variation (median MPO = 8.7 mg/L). Interassay imprecision is 8.96% (median MPO = 53.3 μg/L) and 15.5% (median MPO = 9.5 μg/L). The plasma concentrations of CRP were measured with a fully automated latex-enhanced immunoturbidometric assay on a Cobas Integra (Roche Diagnostics, Mannheim, Germany). The CRP protein assay has an analytical sensitivity of 0.085 mg/L and a measuring range up to 160 mg/L. The upper limit of the reference range of CRP in healthy adults is 5 mg/L. Laboratory personnel were unaware of the results of clinical diagnosis or coronary artery angiography.
Angiographic evaluation Digital angiograms were analyzed offline with an automated edge detection system (CMS; Medis Medical Imaging Systems, Nuenen, The Netherlands). The complexity of lesions was defined according to the modified American College of Cardiology/American Heart Association grading system. Complex lesions were defined as American College of Cardiology/American Heart Association class B2 and C lesions. Left ventricular ejection fraction was calculated using left ventricular angiograms.
Study end points and follow-up The primary end point of the study was all-cause mortality. Other adverse events assessed were myocardial infarction and target-vessel revascularization. The diagnosis of myocardial infarction was based on the presence of typical chest pain accompanied by either the appearance of pathologic Q waves on electrocardiogram or an increase in the levels of cardiac markers (CK-MB isoenzyme N3 times the upper limit of normal). Coronary bypass surgery or repeat percutaneous transluminal coronary angioplasty in the presence of angiographic restenosis of the target lesion, performed during the follow-up period because of symptoms or signs of myocardial ischemia, are considered target-lesion revascularization. Clinical follow-up consisted of telephone interviews at 1 month, at 6 months, and yearly afterwards. All patients were advised to contact our outpatient clinic or their referring physicians whenever they experienced cardiac symptoms. In case of symptoms, at least 1 clinical and electrocardiographic checkup was performed. Collection of baseline characteristics of the patients, follow-up information, as well as adjudication of adverse events, was performed by medical staff unaware of MPO levels.
Statistical analysis Data are presented as median (with 25th-75th percentiles) or counts and proportions (percentages). The normality of distribution of the data was assessed by 1-sample Kolmogorov-
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358 Stefanescu et al
Table I. Baseline clinical and angiographic characteristics Characteristic Age (y) Women (n [%]) Diabetes mellitus (n [%]) Arterial hypertension (n [%]) Current smoking (n [%]) Hypercholesterolemia (n [%]) Previous myocardial infarction Prior coronary artery bypass grafting (n [%]) Myeloperoxidase (μg/L) High-sensitivity CRP (mg/L) Creatinine (mg/dL) LV ejection fraction (%) No. of narrowed coronary arteries 1 2 3 Complex lesions (n [%]) Coronary vessel treated Left main coronary artery (n [%]) Left anterior descending artery (n [%]) Left circumflex artery (n [%]) Right coronary artery (n [%]) Venous graft (n [%]) Lesion length (mm) Vessel size (mm) MLD before intervention (mm) MLD after intervention (mm) Therapy at discharge Statins (n [%]) β-Blockers (n [%]) ACE inhibitors (n [%])
High-MPO group (n = 127)
Low-MPO group (n = 255)
P
68.4 [62.7; 74.9] 36 (28.3) 38 (29.9) 82 (65.0) 21 (16.5) 89 (70.1) 59 (46.0) 24 (18.9) 102.2 [85.8; 161.8] 4.17 [2.08; 9.48] 1.2 [1.1; 1.3] 55.0 [44.0; 65.0]
64.7 [59.0; 72.7] 54 (21.2) 72 (28.2) 150 (59.0) 37 (14.5) 171 (67.1) 101 (40.0) 41 (16.1) 52.6 [42.5; 61.2] 1.93 [0.93; 3.98] 1.1 [1.0; 1.3] 59.0 [49.0; 66.0]
.002 .12 .73 .28 .60 .55 .20 .49 b.001 b.001 .021 .038 .72
31 32 64 90
(24.4) (25.2) (50.4) (70.9)
53 (20.8) 67 (26.3) 135 (52.9) 172 (67.5)
.50 .73
4 (3.1) 50 (39.4) 33 (26.0) 32 (25.2) 8 (6.3) 11.11 [7.77; 15.86] 2.83 [2.46; 3.24] 1.13 [0.74; 1.40] 2.66 [2.19; 3.09]
8 (3.1) 93 (36.5) 58 (22.7) 82 (32.2) 14 (5.5) 11.03 [7.86;15.18] 2.71 [2.39; 3.21] 1.05 [0.77; 1.40] 2.75 [2.30; 3.19]
.90 .28 .96 .37
70 (55.0) 93 (73.2) 77 (61.0)
147 (58.0) 194 (76.1) 163 (64.0)
.64 .54 .53
Data are median [interquartile range] or number of patients (%). LV, Left ventricle; MLD, minimal lumen diameter; ACE, Angiotensin-converting enzyme.
Smirnov test. Categorical data were compared with χ2 test. Continuous data were compared with Kruskal-Wallis rank sum test. Survival analysis was performed by applying the KaplanMeier method. Differences in survival were assessed with the log-rank test. The Cox proportional hazards model was used to assess the association between MPO and mortality while adjusting for other potential confounding variables. All analyses were performed using S-plus statistical package (S-PLUS; Insightful Corp, Seattle, WA). P b .05 was considered to indicate statistical significance.
Results Myeloperoxidase values did not follow a normal distribution. Patients were categorized into 2 groups based on the tertile values of MPO levels. The high-MPO group included patients in the third tertile of MPO levels (N75.0 μg/L; 127 patients). The low-MPO group levels included patients in the first (b52.6 μg/L) and second tertiles (52.6 to b75.0 μg/L) of MPO levels (255 patients).
Baseline characteristics Baseline clinical and angiographic characteristics are shown in Table I. Patients in the high-MPO group were older, had higher levels of CRP and creatinine, and
lower left ventricular ejection fractions than patients in the low-MPO group. The remaining clinical and angiographic characteristics did not differ significantly between the groups.
Clinical outcome The median follow-up of patients was 3.5 [3.3-4.8] years without differences in both groups. During the follow-up period, there were 35 deaths (9.2%). There were 17 deaths in the high-MPO group and 18 deaths in the low-MPO group (Kaplan-Meier estimates of mortality 10.5% and 18.3%, respectively, odds ratio 1.96, 95% confidence interval [1.02-3.76], P = .04) (Figure 1). The MPO concentration was 60.1 [47.0; 83.8] μg/L in survivors and 72.7 [54.8; 105.1] μg/L in nonsurvivors (P = .06). Myocardial infarction occurred in 5 patients (3.9%) in the high-MPO group and 13 patients (13%) in the lowMPO group (P = .61). Target-lesion revascularization was required in 34 patients (26.8%) in the high-MPO group and 70 patients (27.5%) in the low-MPO group (P = .88). Results of multivariable analysis The Cox proportional hazards model was used to test the observed association between MPO level and
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Figure 1
Kaplan-Meier curves of mortality in groups with high- and lowMPO level.
mortality (in the univariate analysis). The following variables were entered into the model: age, sex, diabetes, arterial hypertension, hypercholesterolemia, smoking, previous myocardial infarction, multivessel disease, baseline creatinine level, baseline CRP level, left ventricular ejection fraction, and MPO level. Because of skewed distribution, log values of MPO were entered into the multivariable model. The model showed that MPO level was not an independent correlate of mortality (hazard ratio 1.06, 95% confidence interval [0.71-1.59], P = .77 for 1 SD increase in the log variable).
Discussion In this study, we assessed the prognostic value of plasma MPO concentration in patients with stable CAD. We used a cohort of well-characterized patients with stable CAD, in whom the presence of disease was identified by using coronary angiography. The main findings of the study can be summarized as follows: (1) elevated MPO plasma concentration is associated with an increased risk of death in patients with stable CAD, and (2) elevated MPO concentration does not predict the increased risk of death independently of other known cardiovascular risk factors. Inflammation has been involved in all stages of atherosclerotic process from initial endothelial dysfunction to plaque rupture with ensuing clinical complications.21 Myeloperoxidase is a known marker of inflammatory status, and elevated plasma MPO levels reflect a heightened inflammatory state. Several studies have demonstrated that patients with CAD have higher levels of MPO than normal subjects4 and that elevated MPO is a prognostic marker in patients with ACS.5-7
Stefanescu et al 359
Several mechanisms support a role of MPO in the genesis of instable atherosclerotic plaques that precipitate ACS. For instance, fissured thrombosed plaques of patients with fatal ACS have been found to have marked neutrophil infiltration and high MPO levels.22,23 Furthermore, intracoronary endothelial desquamation by MPOgenerated hypochlorous acid24 and increased catabolism of nitric oxide by MPO11 have been demonstrated. All of these studies seem to directly implicate MPO in the genesis of ACS and may explain the observed association between elevated MPO concentration and short-5 and long-term25 mortality in these patients. The present study in patients with stable CAD showed that 5-year risk of death is significantly higher among patients in the upper tertile of plasma MPO concentration than among patients in lower tertiles (mortality estimates 18.3% and 10.5%, respectively). Furthermore, patients dying within the 5-year follow-up period tended to have higher MPO plasma concentrations than patients who survived the 5-year follow-up period. However, patients in the upper tertiles of MPO concentration had a more adverse cardiovascular risk profile as indicated by their significantly greater age, lower left ventricular ejection fraction, and more impaired renal function. Adjustment in the multivariable analysis for these and other cardiovascular risk factors abolished the association between MPO and mortality as observed in the univariate analysis. Thus, our data show that although elevated MPO concentration is an index of increased cardiovascular risk, MPO does not predict increased risk of death independent of other cardiovascular risk factors. In fact, multiple studies have demonstrated that the presence of several cardiovascular risk factors influences MPO plasma levels. A previous study by our own group has shown that in patients with CAD, plasma MPO concentration was independently predicted by several cardiovascular risk factors such as ACS, inflammatory status (assessed by CRP), impaired renal function, reduced left ventricular function, and smoking.26 Some of these factors are well known for promoting leukocyte activation and an inflammatory state. In addition, other studies as well have demonstrated that MPO correlated positively with factors such as age and CRP levels both in healthy individuals13,27 and in patients with cardiovascular disease.3,5 Although our study does not evidence an independent role of MPO in predicting increased risk of death in patients with stable CAD, a role of MPO as a bridging factor through which various cardiovascular risk factor participate in destabilization of atherosclerotic plaques cannot be refuted. The limited number of patients, in particular of those who completed the 5-year follow-up, is a weakness of this study; additional studies with larger number of patients are needed to further assess the prognostic role of MPO in predicting mortality in patients with stable CAD. In conclusion, in patients with stable CAD, plasma concentration of MPO is not an independent predictor of
360 Stefanescu et al
mortality. However, because elevated plasma MPO is associated with a more adverse cardiovascular risk profile, it can be seen as an index of increased cardiovascular risk.
References 1. Shishehbor MH, Aviles RJ, Brennan ML, et al. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. JAMA 2003;289:1675-80. 2. Daugherty A, Dunn JL, Rateri DL, et al. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994;94:437-44. 3. Buffon A, Biasucci LM, Liuzzo G, et al. Widespread coronary inflammation in unstable angina. N Engl J Med 2002;347:5–12. 4. Zhang R, Brennan ML, Fu X, et al. Association between myeloperoxidase levels and risk of coronary artery disease. JAMA 2001;286: 2136-42. 5. Brennan ML, Penn MS, Van Lente F, et al. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med 2003; 349:1595-604. 6. Baldus S, Heeschen C, Meinertz T, et al. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation 2003;108:1440-5. 7. Mocatta TJ, Pilbrow AP, Cameron VA, et al. Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction. J Am Coll Cardiol 2007;49:1993-2000. 8. Lau D, Baldus S. Myeloperoxidase and its contributory role in inflammatory vascular disease. Pharmacol Ther 2006;111:16-26. 9. Malle E, Marsche G, Panzenboeck U, et al. Myeloperoxidasemediated oxidation of high-density lipoproteins: fingerprints of newly recognized potential proatherogenic lipoproteins. Arch Biochem Biophys 2006;445:245-55. 10. Zhang R, Brennan M-L, Shen Z, et al. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 2002;277:46116-22. 11. Baldus S, Heitzer T, Eiserich JP, et al. Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion. Free Radic Biol Med 2004;37:902-11. 12. Tang WWH, Tong W, Troughton RW, et al. Prognostic value and echocardiographic determinants of plasma myeloperoxidase levels in chronic heart failure. J Am Coll Cardiol 2007;49:2364-70. 13. Meuwese MC, Stroes ES, Hazen SL, et al. Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals: the EPIC-Norfolk prospective population study. J Am Coll Cardiol 2007;50:159-65.
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14. Ben-Dor I, Battler A. Treatment of stable angina. Heart 2007;93: 868-74. 15. Daly CA, Clemens F, Sendon JLL, et al. The clinical characteristics and investigations planned in patients with stable angina presenting to cardiologists in Europe: from the Euro Heart Survey of stable angina. Eur Heart J 2005;26:996–1010. 16. Abrams J. Clinical practice. Chronic stable angina. N Engl J Med 2005;352:2524-33. 17. Kragelund C, Gronning B, Kober L, et al. N-terminal pro-B-type natriuretic peptide and long-term mortality in stable coronary heart disease. N Engl J Med 2005;352:666-75. 18. Ndrepepa G, Braun S, Niemöller K, et al. Prognostic value of N-terminal pro-brain natriuretic peptide in patients with chronic stable angina. Circulation 2005;112:2102-7. 19. Dibra A, Mehilli J, Braun S, et al. Association between C-reactive protein levels and subsequent cardiac events among patients with stable angina treated with coronary artery stenting. Am J Med 2003; 114:715-22. 20. World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications: Report of a WHO Consultation. Part 1. Diagnosis and classification of diabetes mellitus. Geneva: World Health Organization; 1999. 21. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340:115-26. 22. Naruko T, Ueda M, Haze K, et al. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation 2002;106: 2894-900. 23. Sugiyama S, Okada Y, Sukhova GK, et al. Macrophage myeloperoxidase regulation by granulocyte macrophage colony-stimulating factor in human atherosclerosis and implications in acute coronary syndromes. Am J Pathol 2001;158:879-91. 24. Sugiyama S, Kugiyama K, Aikawa M, et al. Hypochlorous acid, a macrophage product, induces endothelial apoptosis and tissue factor expression: involvement of myeloperoxidase-mediated oxidant in plaque erosion and thrombogenesis. Arterioscler Thromb Vasc Biol 2004;24:1309-14. 25. Cavusoglu E, Ruwende C, Eng C, et al. Usefulness of baseline plasma myeloperoxidase levels as an independent predictor of myocardial infarction at two years in patients presenting with acute coronary syndrome. Am J Cardiol 2007;99:1364-8. 26. Ndrepepa G, Braun S, Mehilli J, et al. Myeloperoxidase level in patients with stable coronary artery disease and acute coronary syndromes. Eur J Clin Invest 2007 [in press]. 27. Hoy A, Trégouët D, Leininger-Muller B, et al. Serum myeloperoxidase concentration in a healthy population: biological variations, familial resemblance and new genetic polymorphisms. Eur J Hum Genet 2001;9:780-6.
Background There are no studies yet on the usefulness of myeloperoxidase (MPO) as a prognostic tool in patients with stable coronary artery disease (CAD). Methods The study included 382 patients with clinical and angiographic confirmation of stable CAD. Blood samples for MPO measurement were taken before angiography. Myeloperoxidase was determined using an enzyme immunoassay. The primary end point of the study was all-cause mortality. Results Patients were categorized into 2 groups: the high-MPO group included patients in the third tertile of MPO levels (N75.0 μg/L; 127 patients), and the low-MPO group included patients in the first (b52.6 μg/L) and second tertiles (52.6-75.0 μg/L) of MPO levels (255 patients). The median follow-up was 3.5 [3.3-4.8] years. There were 35 deaths (9.2%) during the follow-up. The MPO concentration was 60.1 [47.0; 83.8] μg/L in survivors and 72.7 [54.8; 105.1] μg/L in nonsurvivors (P = .06). There were 17 deaths in the high-MPO level and 18 deaths in the low-MPO group: Kaplan-Meier estimates of mortality were 18.3% and 10.5% with an odds ratio of 1.96 (95% confidence interval [1.02-3.76], P = .04). The Cox proportional hazards model adjusting for correlates of mortality showed that plasma MPO was not an independent correlate of mortality (hazard ratio 1.06, 95% confidence interval [0.71-1.59], P = .77 for 1 SD increase in the log variable). Conclusion
Although elevated plasma MPO concentration is associated with a more advanced cardiovascular risk profile, plasma MPO does not predict mortality independent of other cardiovascular risk factors in patients with stable CAD. (Am Heart J 2008;155:356-60.)
Myeloperoxidase (MPO) is an enzyme of the mammalian peroxidase family secreted by activated leukocytes, mainly polymorphonuclear neutrophils.1 Myeloperoxidase is part of the innate immune system defenses against bacteria and parasites and has been shown to play a significant role in promoting inflammation. In particular, MPO is secreted by polymorphonuclear neutrophils undergoing site-specific activation in the coronary artery endothelium2,3 and is present in increased levels in the plasma of patients with coronary artery disease (CAD),4 acute coronary syndrome
From the aFaculty of Medicine, McGill University, Montreal, Quebec, Canada; bKlinik für Herz- und Kreislauferkrankungen and Institut für Laboratoriumsmedizin, Deutsches Herzzentrum München, Munich, Germany; 1. Medizinische Klinik Klinikum rechts der Isar, Technische Universität München, Munich, Germany; and cDepartment of Heart and Great Vessels bAttilio RealeQ, University of Rome, Rome, Italy. Submitted August 1, 2007; accepted October 12, 2007. Reprint requests: Adnan Kastrati, MD, Deutsches Herzzentrum, Lazarettstr. 36, 80636 Munich, Germany. E-mail: [email protected] 0002-8703/$ - see front matter © 2008, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2007.10.017
(ACS),5,6 and acute myocardial infarction.7 Suggested mechanisms through which MPO promotes inflammation and plaque formation include the production of hydrochlorous acid, which disrupts intracellular signaling8; the oxidation of lipids such as high- and lowdensity lipoproteins and other endothelial cell-surface lipoproteins,9,10 which promotes macrophage activation and foam cell formation; and the depletion of nitric oxide in vascular endothelium, which reduces endogenous regulation of vessel dilatation.11 Myeloperoxidase has been shown to be a strong predictor of adverse events in patients with ACS,5,6 chronic heart failure,12 acute myocardial infarction,7 and of future CAD in a healthy population.13 Stable CAD is one of the earliest and most common presentation of CAD14,15 but is often poorly diagnosed and managed.15,16 In particular, this is due to variability in clinical presentations and degrees of severity, especially in the elderly and in diabetic patients—two high-risk groups.16 Biomarkers such as N-terminal probrain natriuretic peptide17,18 and C-reactive protein (CRP)19 have been shown to have prognostic value for adverse events in patients with stable CAD; as of yet, however, there have been no
American Heart Journal Volume 155, Number 2
studies on the usefulness of MPO as such a prognostic tool in these patients. The purpose of this study is to assess the prognostic value of plasma MPO concentration in patients with stable CAD.
Methods Patients The study included 382 patients with stable CAD undergoing coronary angiography at the Deutsches Herzzentrum, Munich, Germany. Patients were recruited in a prospective cohort study to investigate the prognostic value of biochemical markers in patients with angiographically proven CAD. The diagnosis of stable CAD was based on the presence of chest pain that did not change its pattern during the preceding 2 months. We did not include patients who presented with ACS, diagnosed on the basis of ST-segment changes on electrocardiogram, or abnormal creatine kinase (CK) or cardiac troponin tests. We did not include patients with unstable angina, defined as a history of crescendo angina, angina at rest or with minimal exertion, or angina of new onset (within 1 month) in the absence of clearcut electrocardiographic and cardiac enzyme changes diagnostic of an acute myocardial infarction. Furthermore, patients with end-stage renal disease, inflammatory diseases, or malignancies were excluded. All patients gave informed consent for the participation in the study. The study protocol was approved by the institutional ethics committee. Percutaneous coronary interventions (stent implantation in most cases) and periprocedural care were performed according to standard criteria. Bare metal stents were used. Antiplatelet therapy consisted of clopidogrel (600 mg as a loading dose followed by 75 mg/d for ≥4 weeks) and aspirin (200 mg/d administered orally and continued indefinitely).
Definitions Angiographic CAD was diagnosed in the presence of coronary stenosis with ≥50% lumen obstruction in at least 1 of the 3 major coronary arteries. Arterial hypertension was defined in patients under active treatment with antihypertensive agents or with a systolic blood pressure of N140 mm Hg and/or diastolic blood pressure of N90 mm Hg on at least 2 separate occasions. Hypercholesterolemia was defined as a documented total cholesterol value ≥240 mg/dL. The patients who admitted to ongoing nicotine consummation were considered current smokers. Diabetes mellitus was defined in patients under an active treatment with insulin or oral hypoglycemic medication. For patients on dietary treatment alone, documentation of abnormal fasting blood glucose or a previous abnormal glucose tolerance test, according to the World Health Organization criteria, were required for the diagnosis of diabetes.20
Biochemical measurements Blood samples for MPO measurement were taken from a peripheral vein before coronary angiography and heparin administration in tubes containing EDTA (Sarstedt, Nümbrecht, Germany) and promptly centrifuged at 1550g for 10 min. After separation, plasma aliquots were stored frozen at −80°C until assayed within batches. Blood count, serum lipids, and other
Stefanescu et al 357
metabolites were determined immediately after collection using standard methods. The concentration of MPO in EDTA plasma was determined with an enzyme immunoassay (Mercodia, Uppsala, Sweden). The immunoassay is based on the sandwich technique, in which 2 monoclonal antibodies are directed against separate antigenic determinants on the MPO molecule. Samples were measured in duplicate after a 1 + 10 dilution with sample buffer. The measuring range of this assay is between 3 and 300 μg/L. Intraassay imprecision ranges between 1.24% (median MPO = 62.5 μg/L) and 7.38% coefficient of variation (median MPO = 8.7 mg/L). Interassay imprecision is 8.96% (median MPO = 53.3 μg/L) and 15.5% (median MPO = 9.5 μg/L). The plasma concentrations of CRP were measured with a fully automated latex-enhanced immunoturbidometric assay on a Cobas Integra (Roche Diagnostics, Mannheim, Germany). The CRP protein assay has an analytical sensitivity of 0.085 mg/L and a measuring range up to 160 mg/L. The upper limit of the reference range of CRP in healthy adults is 5 mg/L. Laboratory personnel were unaware of the results of clinical diagnosis or coronary artery angiography.
Angiographic evaluation Digital angiograms were analyzed offline with an automated edge detection system (CMS; Medis Medical Imaging Systems, Nuenen, The Netherlands). The complexity of lesions was defined according to the modified American College of Cardiology/American Heart Association grading system. Complex lesions were defined as American College of Cardiology/American Heart Association class B2 and C lesions. Left ventricular ejection fraction was calculated using left ventricular angiograms.
Study end points and follow-up The primary end point of the study was all-cause mortality. Other adverse events assessed were myocardial infarction and target-vessel revascularization. The diagnosis of myocardial infarction was based on the presence of typical chest pain accompanied by either the appearance of pathologic Q waves on electrocardiogram or an increase in the levels of cardiac markers (CK-MB isoenzyme N3 times the upper limit of normal). Coronary bypass surgery or repeat percutaneous transluminal coronary angioplasty in the presence of angiographic restenosis of the target lesion, performed during the follow-up period because of symptoms or signs of myocardial ischemia, are considered target-lesion revascularization. Clinical follow-up consisted of telephone interviews at 1 month, at 6 months, and yearly afterwards. All patients were advised to contact our outpatient clinic or their referring physicians whenever they experienced cardiac symptoms. In case of symptoms, at least 1 clinical and electrocardiographic checkup was performed. Collection of baseline characteristics of the patients, follow-up information, as well as adjudication of adverse events, was performed by medical staff unaware of MPO levels.
Statistical analysis Data are presented as median (with 25th-75th percentiles) or counts and proportions (percentages). The normality of distribution of the data was assessed by 1-sample Kolmogorov-
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Table I. Baseline clinical and angiographic characteristics Characteristic Age (y) Women (n [%]) Diabetes mellitus (n [%]) Arterial hypertension (n [%]) Current smoking (n [%]) Hypercholesterolemia (n [%]) Previous myocardial infarction Prior coronary artery bypass grafting (n [%]) Myeloperoxidase (μg/L) High-sensitivity CRP (mg/L) Creatinine (mg/dL) LV ejection fraction (%) No. of narrowed coronary arteries 1 2 3 Complex lesions (n [%]) Coronary vessel treated Left main coronary artery (n [%]) Left anterior descending artery (n [%]) Left circumflex artery (n [%]) Right coronary artery (n [%]) Venous graft (n [%]) Lesion length (mm) Vessel size (mm) MLD before intervention (mm) MLD after intervention (mm) Therapy at discharge Statins (n [%]) β-Blockers (n [%]) ACE inhibitors (n [%])
High-MPO group (n = 127)
Low-MPO group (n = 255)
P
68.4 [62.7; 74.9] 36 (28.3) 38 (29.9) 82 (65.0) 21 (16.5) 89 (70.1) 59 (46.0) 24 (18.9) 102.2 [85.8; 161.8] 4.17 [2.08; 9.48] 1.2 [1.1; 1.3] 55.0 [44.0; 65.0]
64.7 [59.0; 72.7] 54 (21.2) 72 (28.2) 150 (59.0) 37 (14.5) 171 (67.1) 101 (40.0) 41 (16.1) 52.6 [42.5; 61.2] 1.93 [0.93; 3.98] 1.1 [1.0; 1.3] 59.0 [49.0; 66.0]
.002 .12 .73 .28 .60 .55 .20 .49 b.001 b.001 .021 .038 .72
31 32 64 90
(24.4) (25.2) (50.4) (70.9)
53 (20.8) 67 (26.3) 135 (52.9) 172 (67.5)
.50 .73
4 (3.1) 50 (39.4) 33 (26.0) 32 (25.2) 8 (6.3) 11.11 [7.77; 15.86] 2.83 [2.46; 3.24] 1.13 [0.74; 1.40] 2.66 [2.19; 3.09]
8 (3.1) 93 (36.5) 58 (22.7) 82 (32.2) 14 (5.5) 11.03 [7.86;15.18] 2.71 [2.39; 3.21] 1.05 [0.77; 1.40] 2.75 [2.30; 3.19]
.90 .28 .96 .37
70 (55.0) 93 (73.2) 77 (61.0)
147 (58.0) 194 (76.1) 163 (64.0)
.64 .54 .53
Data are median [interquartile range] or number of patients (%). LV, Left ventricle; MLD, minimal lumen diameter; ACE, Angiotensin-converting enzyme.
Smirnov test. Categorical data were compared with χ2 test. Continuous data were compared with Kruskal-Wallis rank sum test. Survival analysis was performed by applying the KaplanMeier method. Differences in survival were assessed with the log-rank test. The Cox proportional hazards model was used to assess the association between MPO and mortality while adjusting for other potential confounding variables. All analyses were performed using S-plus statistical package (S-PLUS; Insightful Corp, Seattle, WA). P b .05 was considered to indicate statistical significance.
Results Myeloperoxidase values did not follow a normal distribution. Patients were categorized into 2 groups based on the tertile values of MPO levels. The high-MPO group included patients in the third tertile of MPO levels (N75.0 μg/L; 127 patients). The low-MPO group levels included patients in the first (b52.6 μg/L) and second tertiles (52.6 to b75.0 μg/L) of MPO levels (255 patients).
Baseline characteristics Baseline clinical and angiographic characteristics are shown in Table I. Patients in the high-MPO group were older, had higher levels of CRP and creatinine, and
lower left ventricular ejection fractions than patients in the low-MPO group. The remaining clinical and angiographic characteristics did not differ significantly between the groups.
Clinical outcome The median follow-up of patients was 3.5 [3.3-4.8] years without differences in both groups. During the follow-up period, there were 35 deaths (9.2%). There were 17 deaths in the high-MPO group and 18 deaths in the low-MPO group (Kaplan-Meier estimates of mortality 10.5% and 18.3%, respectively, odds ratio 1.96, 95% confidence interval [1.02-3.76], P = .04) (Figure 1). The MPO concentration was 60.1 [47.0; 83.8] μg/L in survivors and 72.7 [54.8; 105.1] μg/L in nonsurvivors (P = .06). Myocardial infarction occurred in 5 patients (3.9%) in the high-MPO group and 13 patients (13%) in the lowMPO group (P = .61). Target-lesion revascularization was required in 34 patients (26.8%) in the high-MPO group and 70 patients (27.5%) in the low-MPO group (P = .88). Results of multivariable analysis The Cox proportional hazards model was used to test the observed association between MPO level and
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Figure 1
Kaplan-Meier curves of mortality in groups with high- and lowMPO level.
mortality (in the univariate analysis). The following variables were entered into the model: age, sex, diabetes, arterial hypertension, hypercholesterolemia, smoking, previous myocardial infarction, multivessel disease, baseline creatinine level, baseline CRP level, left ventricular ejection fraction, and MPO level. Because of skewed distribution, log values of MPO were entered into the multivariable model. The model showed that MPO level was not an independent correlate of mortality (hazard ratio 1.06, 95% confidence interval [0.71-1.59], P = .77 for 1 SD increase in the log variable).
Discussion In this study, we assessed the prognostic value of plasma MPO concentration in patients with stable CAD. We used a cohort of well-characterized patients with stable CAD, in whom the presence of disease was identified by using coronary angiography. The main findings of the study can be summarized as follows: (1) elevated MPO plasma concentration is associated with an increased risk of death in patients with stable CAD, and (2) elevated MPO concentration does not predict the increased risk of death independently of other known cardiovascular risk factors. Inflammation has been involved in all stages of atherosclerotic process from initial endothelial dysfunction to plaque rupture with ensuing clinical complications.21 Myeloperoxidase is a known marker of inflammatory status, and elevated plasma MPO levels reflect a heightened inflammatory state. Several studies have demonstrated that patients with CAD have higher levels of MPO than normal subjects4 and that elevated MPO is a prognostic marker in patients with ACS.5-7
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Several mechanisms support a role of MPO in the genesis of instable atherosclerotic plaques that precipitate ACS. For instance, fissured thrombosed plaques of patients with fatal ACS have been found to have marked neutrophil infiltration and high MPO levels.22,23 Furthermore, intracoronary endothelial desquamation by MPOgenerated hypochlorous acid24 and increased catabolism of nitric oxide by MPO11 have been demonstrated. All of these studies seem to directly implicate MPO in the genesis of ACS and may explain the observed association between elevated MPO concentration and short-5 and long-term25 mortality in these patients. The present study in patients with stable CAD showed that 5-year risk of death is significantly higher among patients in the upper tertile of plasma MPO concentration than among patients in lower tertiles (mortality estimates 18.3% and 10.5%, respectively). Furthermore, patients dying within the 5-year follow-up period tended to have higher MPO plasma concentrations than patients who survived the 5-year follow-up period. However, patients in the upper tertiles of MPO concentration had a more adverse cardiovascular risk profile as indicated by their significantly greater age, lower left ventricular ejection fraction, and more impaired renal function. Adjustment in the multivariable analysis for these and other cardiovascular risk factors abolished the association between MPO and mortality as observed in the univariate analysis. Thus, our data show that although elevated MPO concentration is an index of increased cardiovascular risk, MPO does not predict increased risk of death independent of other cardiovascular risk factors. In fact, multiple studies have demonstrated that the presence of several cardiovascular risk factors influences MPO plasma levels. A previous study by our own group has shown that in patients with CAD, plasma MPO concentration was independently predicted by several cardiovascular risk factors such as ACS, inflammatory status (assessed by CRP), impaired renal function, reduced left ventricular function, and smoking.26 Some of these factors are well known for promoting leukocyte activation and an inflammatory state. In addition, other studies as well have demonstrated that MPO correlated positively with factors such as age and CRP levels both in healthy individuals13,27 and in patients with cardiovascular disease.3,5 Although our study does not evidence an independent role of MPO in predicting increased risk of death in patients with stable CAD, a role of MPO as a bridging factor through which various cardiovascular risk factor participate in destabilization of atherosclerotic plaques cannot be refuted. The limited number of patients, in particular of those who completed the 5-year follow-up, is a weakness of this study; additional studies with larger number of patients are needed to further assess the prognostic role of MPO in predicting mortality in patients with stable CAD. In conclusion, in patients with stable CAD, plasma concentration of MPO is not an independent predictor of
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mortality. However, because elevated plasma MPO is associated with a more adverse cardiovascular risk profile, it can be seen as an index of increased cardiovascular risk.
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