integrated natriuretic peptide system and that BNP Clinical Chemistry 60: (2014)

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1 Clinical Chemistry 60: (2014) Proteomics and Protein Markers Prognostic Value of Midregional Pro A-Type Natriuretic Peptide and N-Terminal Pro B-Type Natriuretic Peptide in Patients with Stable Coronary Heart Disease Followed over 8 Years Mahir Karakas, 1 Andrea Jaensch, 2 Lutz P. Breitling, 3 Hermann Brenner, 3 Wolfgang Koenig, 1* and Dietrich Rothenbacher 2,3 BACKGROUND: Pathophysiological studies suggest that A-type natriuretic peptides (ANPs) might provide valuable information beyond B-type natriuretic peptides (BNPs) about cardiac dysfunction in patients with coronary heart disease (CHD). We aimed to assess the predictive value of midregional pro A-type natriuretic peptide (MR-proANP) for recurrent cardiovascular disease (CVD) events in stable CHD patients for whom information on N-terminal probnp (NTproBNP) was already available. METHODS: Plasma concentrations of MR-proANP and NT-proBNP were measured at baseline in a cohort of 1048 patients aged years with CHD who were participating in an in-hospital rehabilitation program. Main outcome measures were cardiovascular mortality, nonfatal myocardial infarction, and nonfatal stroke. RESULTS: During a median follow-up of 8.1 years, 150 patients (incidence 21.1 per 1000 patient-years) experienced a secondary CVD event. MR-proANP was associated with a hazard ratio (HR) of 1.89 (95% CI, ) when the top quartile was compared to the bottom quartile in the fully adjusted model (P for trend 0.011). For NT-proBNP the respective HR was 2.22 (95% CI, ) with a P for trend Finally, MR-proANP improved various model performance measures, including c-statistics and reclassification metrics, but without being superior to NT-proBNP. CONCLUSIONS: Although we found an independent association of MR-proANP as well as NT-proBNP when used as single markers with recurrent CVD events after adjustment for established risk factors, the results of a simultaneous assessment of both markers indicated that MR-proANP fails to provide additional prognostic information to NT-proBNP in the population studied American Association for Clinical Chemistry Stratification for residual risk of cardiovascular events among high-risk individuals is of considerable interest because of the potential to guide use of primary and secondary preventive therapies (1 4). Such efforts have been undertaken by investigating biomarkers like troponins and natriuretic peptides (5, 6). B-type natriuretic peptide (BNP) 4 and the amino terminal fragment of the prohormone BNP (NTproBNP) appear to provide prognostic information in individuals following an acute coronary syndrome (ACS) (7). A-type natriuretic peptide (ANP) is equivalent to BNP or NT-proBNP in the diagnosis of acute heart failure in patients presenting to the emergency department with shortness of breath and has biological effects similar to those of BNP (8). Although ANP is primarily produced and stored in atrial granules available for ready release, BNP is also produced in the ventricle (9). This suggests that ANP and BNP form a dual, integrated natriuretic peptide system and that BNP 1 Department of Internal Medicine II-Cardiology, University of Ulm Medical Center, Ulm, Germany; 2 Institute of Epidemiology and Medical Biometry, Ulm University, Ulm, Germany; 3 Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Heidelberg, Germany. Current affiliation: Department of General and Interventional Cardiology, University Heart Center Hamburg, Germany. * Address correspondence to this author at: Department of Internal Medicine II - Cardiology, University of Ulm Medical Center, Albert-Einstein-Allee 23, D Ulm, Germany. Fax ; wolfgang.koenig@uniklinikulm.de. Received December 8, 2013; accepted July 28, Previously published online at DOI: /clinchem American Association for Clinical Chemistry 4 Nonstandard abbreviations: BNP, B-type natriuretic peptide; NT-proBNP, amino terminal fragment of the prohormone BNP; ACS, acute coronary syndrome; ANP, A-type natriuretic peptide; MR-proANP, midregional proanp; LA, left atrium; CHD, coronary heart disease; ICD, International Classification of Diseases; CABG, coronary artery bypass grafting; CVD, cardiovascular disease; MI, myocardial infarction; hs-ctnt, high-sensitivity cardiac troponin T; CRP, C-reactive protein; Lp-PLA 2, lipoprotein-associated phospholipase A 2, spla 2, secretory PLA 2 ; HR, hazard ratio; LVEF, left ventricular ejection fraction; BMI, body mass index; PCI, percutaneous coronary intervention; ACE, angiotensinconverting enzyme; NRI, net reclassification improvement; IDI, integrated discrimination improvement; GRACE, Global Registry of Acute Coronary Events; IQR, interquartile range; AUC, area under the curve. 1441

2 may be a backup hormone activated only after prolonged ventricular overload and raises the question of whether ANP might be the more sensitive marker in stable disease (10). The development of an assay for the midregional proanp (MR-proANP) has allowed a better understanding of the peptide (11). MR-proANP is secreted from both the left atrium (LA) and the left ventricle and is stimulated to enter the circulation by atrial stretch (12). In patients without heart failure, both NTproBNP and MR-proANP concentrations are independently associated with LA enlargement, thereby partially explaining the well-recognized value of natriuretic peptides for use in risk stratification (13). In current European guidelines both peptides are regarded as equal for diagnosis of chronic and acute heart failure (14). So far, few data exist in patients with stable coronary heart disease (CHD) and long-term follow-up, especially in relatively unselected patients coming from routine medical care compared to patients included in clinical trials. We therefore assessed the association of plasma MR-proANP concentrations with subsequent cardiovascular events and mortality in a cohort of 1048 patients with stable CHD for whom information on NTproBNP was already available. Methods STUDY POPULATION All patients with CHD [International Classification of Diseases (ICD), 9th Revision codes ] aged years and participating in an in-hospital rehabilitation program between January 1999 and May 2000 in 2 cooperating hospitals (Schwabenland-Klinik, Isny, and Klinik im Südpark, Bad Nauheim, Germany) were enrolled in the study (initial response rate 58%). In Germany, all patients after an ACS or elective coronary artery bypass graft (CABG) are offered a comprehensive in-hospital rehabilitation program after discharge from the acute care hospital. The aim of this 3-week program is the reduction of cardiovascular risk factors, improvement of health-related quality of life, and the preservation of the ability to work (the latter only if an individual was still at work at the onset of disease, otherwise the prevention of nursing care). This in-hospital rehabilitation program usually starts approximately 3 weeks after the acute event or CABG. In the current study, only patients who were admitted within 3 months of the acute event or CABG were included. All patients gave written informed consent. The study was approved by the Ethics Boards of the Universities of Ulm and Heidelberg and of the Physicians Chamber of the States of Baden-Wuerttemberg and Hessen (Germany). DATA COLLECTION At the beginning of the in-hospital rehabilitation program all participants were given a standardized questionnaire containing sociodemographic information and medical history. In addition, information was taken from the patients hospital charts. In all patients, active follow-up was conducted 1, 3, 4.5, 6, and 8 years after discharge from the rehabilitation center. Information was obtained from the patients using a mailed standardized questionnaire. Information regarding adverse cardiovascular disease (CVD) events and treatment since discharge from the in-hospital rehabilitation clinic was obtained from the primary care physicians and also by means of a standardized questionnaire. If an individual had died during follow-up, the death certificate was obtained from the local public health department and the main cause of death was coded according to the ICD (ICD-9 positions and ICD-10 positions I00 I99 and R57.0). Adverse CVD events were defined either as CVD as the main cause of death (as stated in the death certificate), nonfatal myocardial infarction (MI), or nonfatal stroke. All nonfatal adverse events were reported by the primary care physicians. LABORATORY METHODS Blood at baseline was drawn at discharge from the rehabilitation center [on average 43 days after the acute event (first quartile 36 days, third quartile 51 days)] in a fasting state under standardized conditions and stored at 80 C until analysis. MR-proANP concentrations were measured with an automated sandwich chemiluminescence immunoassay on a KRYPTOR system (BRAHMS AG) (11). The interassay CVs were 3.2% and 3.9% at 92.3 and 474 pmol/l. NT-proBNP was measured by electrochemiluminescence on an Elecsys 2010 (15) (Roche Diagnostics) (interassay CVs between 3% and 7%), as was high-sensitivity cardiac troponin T (hs-ctnt). For hs-ctnt, a concentration of 14 ng/l has recently been reported to represent the 99th percentile in a healthy reference population (16). The interassay CVs were 4.1% and 2.8% at concentrations of 27 and ng/l. C-reactive protein (CRP) concentrations were measured by an hs immunonephelometric assay on a Behring Nephelometer II (N Latex CRP mono), for which the interassay CV was 4.1%. Cystatin C was determined on the same device (17) (Dade-Behring) (interassay CV, 3.8%). Lipoproteinassociated phospholipase A 2 (Lp-PLA 2 ) and secretory phospholipase A 2 (spla 2 ) mass and activity were determined as reported previously (18, 19). Creatinine, blood lipids, and leukocyte count were performed by routine methods in both participating hospitals. All biomarkers were measured in a blinded fashion Clinical Chemistry 60:11 (2014)

3 MR-proANP and NT-proBNP Show Similar Prognostic Value STATISTICAL METHODS The study population was described with respect to various sociodemographic and medical characteristics. The associations of sociodemographic characteristics, various cardiovascular risk factors, and medication with MR-proANP concentrations were assessed by means of the nonparametric Kruskal Wallis test. Partial Spearman correlation coefficients, adjusted for age and sex, were calculated to characterize the relationship of MR-proANP concentrations with various established and emerging laboratory risk factors. The relation of MR-proANP concentrations with CVD events during follow-up was assessed by the Kaplan Meier and life table method and tested for statistical significance by means of the log-rank test. The Cox proportional hazards model was employed to assess the independent association of MR-proANP and NT-proBNP concentrations with the risk of adverse CVD events [hazard ratios (HR) and their 95% CI]. A basic model was adjusted for age (years) and sex. In a second step, in addition to the variables age and sex, the following potential confounders were considered in multivariable analyses: left ventricular ejection fraction (LVEF), body mass index (BMI) (kg/m 2 ), smoking status (never, current, ex-smoker), duration of school education ( 10 years, 10 years), hospital site (Isny, Bad Nauheim), family status (married, other), history of MI (yes, no), history of hypertension (yes, no), history of diabetes mellitus (yes, no), severity of CHD (number of affected epicardial coronary vessels at baseline), initial management of CHD [conservative, percutaneous coronary intervention (PCI), CABG], discharge prescription of -blockers (yes, no), discharge prescription of angiotensin-converting enzyme (ACE) inhibitors (yes, no), discharge prescription of diuretics (yes, no), discharge prescription of lipid-lowering drugs (yes, no), HDL cholesterol (mg/dl), LDL cholesterol (mg/dl), and hs-ctnt. To avoid overadjustment, only those variables which were significant predictors of an adverse event at an - level of 0.1 or which changed the parameter estimates for the main variable (MRproANP) by more than 10% were added to the model. In the last step, adjustment for hscrp, cystatin C, and NT-proBNP was done. Measures of model fit, discrimination (20), and reclassification were assessed with Cox proportional hazards regression. We calculated the net reclassification improvement (NRI) by adding MR-proANP or NT-proBNP as well as both markers simultaneously to a basic model, which also included LVEF and hs-ctnt, according to the risk strata of 10%, 10% 20%, and 20% of predicted probability for a cardiovascular event. Furthermore, we assessed the integrated discrimination improvement (IDI), which estimates the extended model s improvement in the difference in Table 1. Sociodemographic, clinical, and laboratory characteristics in 1,048 patients with clinically manifest coronary heart disease. a predicted probabilities (21, 22). To further demonstrate the prognostic value of MR-proANP and NTproBNP, we used a modified Global Registry of Acute Coronary Events (GRACE) score (4), in which the information about ST-segment depression during the acute event was replaced by information regarding ischemic signs during exercise stress tests in the rehabilitation clinic. All statistical procedures were carried out with the SAS statistical software package (release 8.2; SAS Institute Inc. 1999). Results Characteristics at baseline Age, years No. of men 892 (85.1) History of MI 610 (58.2) History of heart failure 129 (12.7) Clinical score, angiographic evaluation 1-vessel disease 256 (24.4) 2-vessel disease 281 (26.8) 3-vessel disease 446 (42.6) Unknown 50 (4.8) School education 10 years 625 (59.6) BMI, kg/m History of diabetes 179 (17.1%) GRACE score 96.0 (20.8) Total cholesterol, mg/dl b (32.8) LDL cholesterol, mg/dl b (29.1) HDL cholesterol, mg/dl b 39.4 (10.5) CRP, mg/l 3.48 (1.25; 8.34) Creatinine clearance, ml/min (78.18; ) Cystatin C, mg/l 1.03 (0.93; 1.19) NT-proBNP, ng/l (277.90; ) hs-ctnt, ng/l 10.9 (5.1; 18.9) MR-proANP, pmol/l (95.2; 185.8) a Data are presented as mean SD, n (%), or median (25th; 75th quantile cut point). b 38.6 mg/dl 1.0 mmol/l. The main sociodemographic and laboratory characteristics in 1048 patients with clinically manifest CHD are shown in Table 1. The mean age of CHD patients was 58.9 years, and 85.1% were men. Main laboratory characteristics are further displayed in this table. The median NT-proBNP concentration was ng/l Clinical Chemistry 60:11 (2014) 1443

4 Table 2. MR-proANP distribution according to various patient characteristics, (sociodemographic, cardiovascular risk factors, and electrocardiogram findings). Characteristic n Median MR-proANP distribution, pmol/l P value a Sex 0.99 Female Male Age, years School education years years Family status 0.56 Married Other BMI, kg/m Smoking status Never Ex Current History of diabetes Yes No History of MI 0.24 Yes No History of hypertension Yes No History of heart failure Yes No Angiographic score, no. affected vessels 0/ Initial management of CHD Conservative PCI CABG LV function, degree of impairment No/only little Modest/severe Unknown Intake of lipid-lowering agent Yes No a Kruskal-Wallis Test. Table 3. Partial Spearman rank correlation coefficients (R) between established and emerging risk factors of CHD (age and sex adjusted). MR-proANP (pmol/l) [interquartile range (IQR), ] and the MR-proANP concentration was pmol/l (IQR, ). The mean LDL cholesterol was fairly low at mg/dl. Table 2 shows the distribution of MR-proANP according to various sociodemographic characteristics and cardiovascular risk factors. Interestingly, there was no sex difference in MR-proANP concentrations (P 0.99). MR-proANP values increased with age, were inversely related to BMI and smoking, and increased in diabetic and hypertensive patients and in those with a history of heart failure. Furthermore, patients had higher serum concentrations of MR-proANP with multivessel CHD, with impaired LV function, and following CABG. Table 3 shows partial Spearman rank correlation coefficients between MR-proANP concentrations and various established and emerging risk factors after adjustment for age and sex. MR-proANP concentrations were not related to lipids, but were positively related to hscrp (r 0.21), interleukin-6 (r 0.20), adiponectin (r 0.19), and cystatin C (r 0.43). In contrast, a negative correlation was seen with creatinine clearance (r 0.32). The strongest correlations however, were seen with hs-ctnt (r 0.51) and with NT-proBNP (r 0.72). R P value HDL cholesterol Total cholesterol LDL cholesterol Leukocytes CRP 0.21 Interleukin Adiponectin 0.19 Creatinine 0.27 Creatinine clearance 0.32 Cystatin C 0.43 NT-proBNP 0.72 Lp-PLA 2 mass 0.15 hs-ctnt 0.51 Hemoglobin A 1c spla 2 mass 0.15 Intercellular adhesion molecule Clinical Chemistry 60:11 (2014)

5 MR-proANP and NT-proBNP Show Similar Prognostic Value Fig. 1. Kaplan Meier estimates of secondary fatal and non-fatal CVD events during follow-up (time days) according to quartiles of MR-proANP. During a mean follow-up of 8.1 years, 150 patients (14.3%) experienced an adverse CVD event (39% cardiovascular death, 33% nonfatal MI, and 27% nonfatal stroke). Baseline concentrations of MR-proANP were higher in patients with an event compared to event-free individuals. Fig. 1 shows Kaplan Meier curves presenting the incidence of adverse CVD events per 1000 patient years according to quartiles of MR-proANP at baseline. Of note, in patients in the top quartile of MR-proANP, the incidence was 42.1 per 1000 patient years compared to only 12.7 per 1000 patient years in the bottom quartile (P ). Table 4 shows the association of MR-proANP with the composite endpoint after adjustment for various confounders. In the basic model, which adjusted for age and sex only, MR-proANP in the top quartile was associated with an HR of 3.00 (95% CI, ) compared to the bottom quartile. Notably, this association was slightly attenuated (HR, 1.89; 95% CI, ) in the multivariate model, which in addition to age and sex included LVEF, hospital site, smoking status, history of diabetes, and initial management of CHD, as well as cystatin C, LDL cholesterol, HDL cholesterol, and treatment with lipid-lowering drugs (of which 90% were statins) (for details of model selection see Methods), and further attenuated when hsctnt was included in the multivariate model (HR, 1.63; 95% CI, ). Analyzing MR-proANP as a continuous variable resulted in similar patterns of risk estimates across all models and remained statistically significant in the final model (HR, 1.78; 95% CI, ). NT-proBNP, on the other hand, was associated with adverse cardiovascular events in the basic model in the top quartile, with an HR of 4.02 (95% CI, ) compared to the bottom quartile and an HR of 2.22 (95% CI, ) in the fully adjusted model. Clinical Chemistry 60:11 (2014) 1445

6 Table 4. Association of MR-proANP with fatal and nonfatal cardiovascular events during follow-up. HR (95% CI), adjusted for age and sex Results of multivariable analysis HR (95% CI), adjusted for multiple covariates a HR (95% CI), adjusted for multiple covariates b MR-proANP, quartile Bottom 1 referent 1 referent 1 referent Second 1.06 ( ) 0.85 ( ) 0.80 ( ) Third 1.29 ( ) 1.21 ( ) 1.11 ( ) Top 3.00 ( ) 1.89 ( ) 1.63 ( ) P for trend P for trend P for trend Per unit increase, ln 2.69 ( ) 1.99 ( ) 1.78 ( ) NT-proBNP, quartile Bottom 1 referent 1 referent 1 referent Second 1.08 ( ) 0.85 ( ) 0.81 ( ) Third 2.29 ( ) 1.66 ( ) 1.53 ( ) Top 4.02 ( ) 2.22 ( ) 1.95 ( ) P for trend P for trend P for trend Per unit increase, ln 1.63 ( ) 1.29 ( ) 1.21 ( ) a Model adjusted for age, sex, rehabilitation clinic, history of diabetes mellitus, smoking status, PCI, CABG, HDL cholesterol, LDL cholesterol, treatment with lipid-lowering drugs, cystatin C (log transformed), LVEF. b Model additionally adjusted for hs-ctnt (log transformed). Adding hs-ctnt to the multivariate model again slightly attenuated the association (HR, 1.95; 95% CI, ). Analysis using NT-proBNP as a continuous variable yielded an HR of 1.29 (MR-proANP, 1.99) per unit increase in the extended model, and 1.21 (95% CI, ) when hs-ctnt was included. Although the correlation coefficient between NTproBNP and MR-proANP was very high (r 0.72, P ), indicating a strong correlation between 2 independent variables and possibly leading to the undesired effect of multicollinearity with an increase of the SEs of the coefficients and inflating CIs, we also included both in the fully adjusted model simultaneously. The resulting HR for quartiles of MR-proANP were 0.72 (95% CI, ), 0.84 (95% CI, ), and 1.09 (95% CI, ) when the second, third, and top quartile were compared to the bottom quartile (P-value for trend 0.52). The respective HRs for NT-proBNP were 0.88 (95% CI, ), 1.60 (95% CI, ), and 1.84 (95% CI, ) (P-value for trend 0.06). We then quantified the incremental value of the 2 markers. The c-statistic [synonymous with the area under the curve (AUC)] for the age- and sex-adjusted model was 0.57 (95% CI, ). The addition of NT-proBNP resulted in an AUC of 0.66 (95% CI, ). Adding MR-proANP to the age- and sexadjusted model increased the AUC to 0.63 (95% CI, ), and if both were included the AUC was 0.67 (95% CI, ). Table 5 shows various measures of model accuracy with and without MRproANP or NT-proBNP, and with both included simultaneously, after full adjustment for covariates. In all models, the addition of MR-proANP or NTproBNP to a basic risk estimation model comprising age, sex, rehabilitation clinic, history of diabetes mellitus, smoking status, PCI, CABG, HDL cholesterol, LDL cholesterol, treatment with lipid-lowering drugs, cystatin C, and LVEF significantly improved the model fit. c-statistics demonstrated a slight but significant increase in the AUC, from 0.69 to 0.71 through addition of MR-proANP, but also for NT-proBNP to the basic model. The NRI was 8.5% (P 0.037) when MRproANP was added to the model, and 14.6% when NTproBNP was added (P ). The IDI was (P 0.039) for the MR-proANP based risk model and (P ) for an NT-proBNP based model. If MR-proANP was added to the model already including NT-proBNP, the c-statistics did not improve (0.71) and NRI improved only little, to 15.1% (P 0.001). Discussion To our knowledge, this is the first study to evaluate the predictive role of MR-proANP for recurrent CVD events in a routine cohort of stable CHD patients for 1446 Clinical Chemistry 60:11 (2014)

7 MR-proANP and NT-proBNP Show Similar Prognostic Value Table 5. Measures of model accuracy with and without MR-proANP or NT-proBNP (both as quartiles). Model accuracy Basic model plus measure Basic model a MR-proANP Model fit Basic model plus NT-proBNP Basic model plus MR-proANP and NT-proBNP LR b 79.3 (df 15, P ) 85.7 (df 18, P ) 88.7 (df 18, P ) 91.2 (df 21, P ) AIC BIC Discrimination c-statistic, AUC (95% CI) 0.69 ( ) 0.71 ( ) 0.71 ( ) 0.71 ( ) Calibration ( 2 ) 7.9 (P 0.54) 2.7 (P 0.97) 5.5 (P 0.79) 7.3 (P 0.60) reclassification IDI (P 0.039) (P ) (P ) NRI 8.5% (P 0.037) 14.6% (P ) 15.1% (P 0.001) Patients with CVD event, 16/9 23/13 21/10 n up /n down Patients without CVD event, n up /n down 83/ / /172 a Adjusted for age, sex, rehabilitation clinic, history of diabetes mellitus, smoking status, PCI, CABG, HDL cholesterol, LDL cholesterol, treatment with lipid-lowering drugs, cystatin C (log transformed), LVEF, hs-ctnt (log transformed). b LR, likehood ratios; AIC, Akaike s information criterion; BIC, Bayesian information criterion. whom information on NT-proBNP was already available during long-term follow-up. In this prospective study we found an independent association between the risk of recurrent CVD events and concentrations of MRproANP and NT-proBNP when each of these natriuretic peptides was used as a single variable in different models after adjustment for various established cardiovascular risk factors. Risk of adverse cardiovascular events was doubled in patients with MR-proANP as well as with NTproBNP concentrations in the top quartile compared to the bottom quartile in the fully adjusted model including markers of inflammation and renal function. However, simultaneous assessment of both markers indicated that MR-proANP failed to provide additional prognostic information in addition to NT-proBNP. PATHOPHYSIOLOGICAL IMPLICATIONS Pathophysiological studies suggest a dual natriuretic peptide system that is upregulated under pathophysiological stress. Three issues need to be considered here. First, the synthesis, secretion, and clearance of BNP differ from those of ANP, suggesting discrete physiological and pathophysiological roles of both peptides (23). ANP secretion occurs from a previously synthesized pool in the secretory granules of atrial cardiomyocytes. Therefore, the regulation of ANP release occurs mainly at the level of hormone secretion, whereas most BNP regulation is done during gene expression, with the majority of BNP being synthesized during bursts of activation from pathophysiological stimuli (24). Second, the natriuretic response may vary depending on the type of pathophysiological stress. For example, in the early phase of ACS, and in human cardiac allograft acute rejection, BNP gene expression increases considerably, whereas ANP concentrations increase only slightly (25). In contrast, in cardiac hypertrophy, plasma ANP concentrations increase as atrial pressure increases, whereas BNP plasma concentrations increase when ventricular hypertrophy develops. Third, hypoxic conditions, which are mainly caused by underlying ischemic disease, directly stimulate the natriuretic system. Accumulated experimental data clearly show that hypoxia leads to an increased synthesis of ANP in both the normal and the hypertrophied myocardium (26). Although hypoxia-response elements have also been found in the promoter sequence of the BNP gene, and it has been shown that hypoxia also stimulates the release of BNP, data from perfused rat ventricular myocardium showed that hypoxia-stimulated ANP release was more pronounced than BNP release (27). Despite plausible pathophysiological explanations, we could not demonstrate an additional value for MRproANP in individuals with stable CHD for whom NTproBNP was already available for the prediction of adverse cardiovascular events. Both natriuretic peptides had strong prognostic implications, but performed equally well if one or the other was used in the final model. MR-proANP AND NT-proBNP IN STABLE CVD Previous studies mainly investigated the role of MRproANP in the setting of heart failure. In the BACH (Biomarkers in Acute Heart Failure) trial, a prospective study of 1641 patients presenting to the emergency de- Clinical Chemistry 60:11 (2014) 1447

8 partment with dyspnea, MR-proANP concentrations above 120 pmol/l proved noninferior to NT-proBNP concentrations above 100 ng/l for the diagnosis of acute heart failure (8). In subgroup analyses, MRproANP measurements added to the utility of NTproBNP in patients with intermediate BNP values and with obesity. Supporting these findings are the results of another study, which evaluated the prognostic value of MR-pro-ANP serum concentrations for all-cause mortality in chronic heart failure patients; at 48 months of follow-up, MR-proANP significantly outperformed NTproBNP (AUC, 0.79 vs 0.75, P 0.03) (28). In apparently healthy individuals, 2 populationbased studies have shown that MR-proANP is associated with cardiovascular events in the general population (29, 30). In a cohort of 5067 participants without prevalent CVD, a 1-SD increase in MR-proANP was associated with an adjusted HR of 1.12 for incident cardiovascular events, whereas this value was 1.22 for a 1-SD increase in NT-proBNP (30). Similarly, in the most recently published prospective observational study, PREVEND (Prevention of Renal and Vascular End-stage Disease), increased plasma concentrations of both natriuretic peptides in patients with clinically important albuminuria were associated with increased risks of all-cause mortality and cardiovascular events after a median follow-up of 10.5 years. However, in contrast to NT-proBNP, MR-proANP was not independently related to cardiovascular mortality (29). This finding might be attributable to the fact that natriuretic peptides were measured in individuals with impaired renal function. Renal extraction ratios of different natriuretic peptides vary, with ANP having about a 4 times as high a renal extraction rate under physiological situations as BNP (31). Even in cardiac patients with normal glomerular filtration rates, the relationship between natriuretic peptides and glomerular filtration rate is continuous and linear (32). However, Franz et al. have shown that there is an increased excretion of ANPs in renal disease and that the increase occurs even before serum creatinine begins to rise (33). So, one may hypothesize that circulating MR-proANP concentrations are much more subject to alterations due to renal response in those with clinically important albuminuria than those of NT-proBNP, and therefore the predictive value of MR-proANP in these individuals may be distorted. Most recently, Sabatine et al. reported on the predictive value of MR-proANP in patients with stable CHD (34). In 3717 patients who were randomized to trandolapril or placebo as part of the Prevention of Events with Angiotensin Converting Enzyme (PEACE) trial, increased concentrations of MR-proANP were independently associated with the risk of cardiovascular death or heart failure (HR per 1 SD, 1.97). As a limitation, these results cannot be transferred to stable CHD cohorts in general, because only patients with preserved LVEF had been included in PEACE. Meanwhile, a second clinical trial reported an independent association between MR-proANP and all-cause mortality, cardiovascular mortality, and cardiovascular events (29). The strongest limitation regarding both trials is that they somehow represent an artificial setting, not reflecting the true patient spectrum in routine daily practice: The PEACE trial included patients with stable CHD and preserved LVEF who were randomized to trandolapril or placebo, whereas the PREVEND trial was designed to assess the effects of the ACE inhibitors, fosinopril and pravastatin, on cardiovascular morbidity and mortality in microalbuminuric individuals without hypertension or hypercholesterolemia. In contrast, our study is the first that evaluates the predictive role of MR-proANP for adverse cardiovascular events in a routine cohort of stable CHD patients during long-term follow-up. STUDY LIMITATIONS Our study has several limitations that must be addressed. In our study population, as in all typical CHD populations, women are clearly underrepresented. Although we had a large sample of patients with CHD (over 50% with a history of MI), fatal CVD events were limited in this study population. This is explained by the fact that mortality of MI is highest during the pre- and early in-hospital phase. As the acute events leading to diagnosis of CHD or MI had occurred at least 3 weeks before inclusion in this study, selection of patients with a better prognosis compared to a patient population within the early phase of a newly diagnosed CHD must be assumed. Furthermore, not all patients were willing or able to participate in an in-hospital rehabilitation program. This may provide a further explanation for the underrepresentation of severely ill patients in our study sample. However, it does not explain the positive association between natriuretic peptides and CVD events, but suggests that their true prognostic value may be even stronger than shown in our study. Conclusion Although we found an independent association of MR-proANP with recurrent CVD events when used as single markers after adjustment for established risk factors, the results of a simultaneous assessment of NTproBNP indicate that MR-proANP fails to provide additional prognostic information to NT-proBNP in the population studied. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting 1448 Clinical Chemistry 60:11 (2014)

9 MR-proANP and NT-proBNP Show Similar Prognostic Value or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest. Role of Sponsor: No sponsor was declared. References Acknowledgments: The contributions of the participating general practitioners, clinicians, documentation officers and assistants are gratefully acknowledged. Drs. Koenig and Rothenbacher had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. 1. Wijeysundera HC, Machado M, Farahati F, Wang X, Wittemann W, van der Velde G, et al. Association of temporal trends in risk factors and treatment uptake with coronary heart disease mortality, JAMA 2010;303: Morrow DA, Antman EM, Parsons L, de Lemos JA, Cannon CP, Giugliano RP, et al. 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