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Clinical Chemistry 54: 1780-1787, 2008. First published September 11, 2008; 10.1373/clinchem.2008.105635
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(Clinical Chemistry. 2008;54:1780-1787.)
© 2008 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Impact of Epitope Specificity and Precursor Maturation in Pro-B–Type Natriuretic Peptide Measurement

Jens Peter Goetze1,a, Ulf Dahlström2, Jens F. Rehfeld1 and Urban Alehagen2

1 Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark; 2 Department of Cardiology, Linkoping University Hospital, Sweden.

aAddress correspondence to this author at: Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Fax: +45–3545-4640; e-mail JPG{at}dadlnet.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cardiac-derived natriuretic peptides are sensitive plasma markers of cardiac dysfunction. Recent reports have disclosed a more complex molecular heterogeneity of B-type natriuretic peptide precursor (proBNP)-derived peptides than previously suggested. In this study, we examined the impact of epitope specificity and precursor maturation on plasma measurement of proBNP-derived peptides.

Methods: We compared 2 assays, N-terminal proBNP and proBNP 1–76, in a randomly collected set of human plasma specimens (n = 370). Additionally, we evaluated the clinical performance of 4 assays with different epitope specificities in a cohort of elderly patients presenting with symptoms associated with heart failure (n = 415).

Results: Comparison of N-terminal proBNP with proBNP 1–76 measurement in plasma revealed a high correlation on regression analysis (r2 = 0.91, P < 0.0001). Nevertheless, the proBNP 1–76 assay measured lower concentrations in the high range than the N-terminal proBNP assay. Correlations between assay measurements in a clinical setting were comparable for all the assays (r2 approximately 0.57–0.83), and ROC analyses revealed area-under-the-curve values ranging between 0.77 and 0.81 for identifying reduced left ventricular ejection fraction. In parallel, all assays displayed comparable abilities in predicting long-term mortality.

Conclusions: Our results reveal marked assay differences in analytical assay comparison, contrasting the overall comparable clinical performance in cardiovascular diagnostics or prognosis in the elderly.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Plasma concentrations of B-type natriuretic peptide (BNP)1 and its precursor-derived N-terminal fragment (proBNP 1–76 or NT-proBNP) are sensitive markers of cardiac dysfunction (1). In addition, plasma measurements provide valuable information in discriminating between cardiac-related and non–cardiac-related dyspnea in the emergency setting and the general population (2)(3). The present use of the markers is primarily recommended in the diagnostic phase of heart failure, where the clinical presentation can be difficult to differentiate from other medical conditions (4)(5). BNP and proBNP 1–76 concentrations in plasma also offer prognostic information on both morbidity and mortality independent of the underlying medical condition (6). Moreover, recent data indicate that individualized heart failure treatment by use of natriuretic peptide concentrations as effect monitors might reduce morbidity and possibly even mortality (7)(8)(9).

Cardiac BNP gene expression leads to synthesis of a preprostructure, which is N-terminally cleaved to proBNP 1–108 during ribosomal translation into the endoplasmic reticulum. The posttranslational phase of proBNP maturation has long been simplified to a singular endoproteolytic cleavage, which harbors an N-terminal fragment, i.e., proBNP 1–76 or NT-proBNP, and the C-terminal, bioactive proBNP 77–108 or BNP-32 (10). However, recent reports have disclosed that cardiac proBNP processing is considerably more complex than initially suggested. First, proBNP circulates as an intact precursor, which challenges the suggestion of a stoichiometric 1:1 secretion ratio of the N-terminal fragment and the C-terminal hormone (11)(12). Second, the complex profile of proBNP immunoreactivity on gel chromatography seems to be due to O-linked glycosylation in the proBNP midregion (13)(14). This posttranslational modification provides an expedient explanation for the high-molecular-weight proBNP forms observed by several groups (1)(15)(16)(17). Finally, proBNP and BNP-32 seem to be trimmed in the N-termini, leading to truncated forms (18)(19). The new insights into proBNP processing have recently been reviewed (20)(21).

The aim of the present study was to compare 3 NT-proBNP assays and 1 BNP assay to evaluate differences related to molecular heterogeneity in the N-terminal region. Moreover, we wanted to compare the clinical performance of the 4 assays in a clinically relevant cohort of elderly patients presenting with symptoms associated with heart failure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
head-to-head comparison of 2 n-terminal PRObnp assays
To examine possible differences between assays with epitope recognition in the N-terminus or the midregion of proBNP, we collected leftover plasma samples sent for routine NT-proBNP analysis in our department. Samples (n = 370) were collected without knowledge of patient history or current status and were analyzed for NT-proBNP (Roche Modular E) and N-terminal proBNP concentrations using an in-house immunoassay (22). The epitope specificities of the 2 assays are outlined in Fig. 1A . The use of blood samples for proBNP measurement was approved by the local Ethics Committee.


Figure 1
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  		Figure 1. (A), The intact BNP precursor and the epitope sites for 2 immunoassays (PIA and Modular). Note the glycosylated midregion indicated by a box in proBNP.

(B), Bland-Altman plot of the Modular proBNP 1–76 and PIA N-terminal proBNP measurements. Each point represents individual samples.

clinical performance of PRObnp-derived peptide assays in elderly patients
Elderly patients presenting with symptoms associated with heart failure (tiredness, dyspnea, and/or peripheral edema) were included in the study. The design of the study has been described in detail (23). In brief, we reviewed 1168 medical records from patients with symptoms associated with heart failure. In those cases where a possible diagnosis of heart failure could not be excluded, the patients were invited to participate in the study. Of these 548 patients, 510 chose to participate. The initial median age was 73 years (range 65–82 years). An experienced cardiologist clinically evaluated all patients, recording patient history and drug treatment, performing a standard clinical examination, and assessing New York Heart Association (NYHA) functional class. A 12-lead electrocardiography was also performed. Of the 510 patients, 474 agreed to donate a blood sample for plasma analyses. The patients were then followed for a median time period of 5.5 years (range 242–2222 days). During this period, 102 patients died. None of the patients were lost during follow-up. The Ethics Committee of the University Hospital of Linköping, Sweden, approved the study protocol.

We used Doppler echocardiography (Accuson XP-128C) to evaluate systolic and diastolic function. All patients were examined in the supine left position. The systolic function expressed as left ventricular ejection fraction (LVEF) was determined using a semiquantitative method (24)(25)(26), where moderately impaired systolic function was defined as LVEF <40% and normal systolic function was defined as LVEF ≥50%. Diastolic dysfunction was defined as an impaired relaxation or pseudonormal/restrictive filling pattern by abnormal mitral flow E/A ratio and/or abnormal pulmonary venous flow, respectively, using age-adjusted parameters. All patients were fasting and seated and had a 30-min rest before venipuncture. The samples were collected in prechilled plastic tubes containing EDTA (Terumo EDTA K-3), placed on ice, and centrifuged at 3000g for 10 min at 4 °C. Plasma was immediately removed and stored at –70 °C until analysis.

assays
We quantified BNP-32 using a nonextraction immunoradiometric assay (Shionogi). Total interassay CV was 9.3% at 12 pmol/L (n = 10) and 5.4% at 45 pmol/L (n = 10). We measured proBNP 1–76 using the Elecsys 2010 (Roche Diagnostics). This assay uses 2 polyclonal antibodies directed against amino acid sequences 1–21 and 39–50 (Fig. 1AUp ). Total CV was 4.8% at 26 pmol/L and 2.1% at 503 pmol/L (n = 70). We quantified N-terminal proBNP using a nonextraction RIA with an antiserum raised against synthetic proBNP 1–21 conjugated to bovine albumin (27). Synthetic proBNP 1–21 (Peninsula) served as the calibrator. Total interassay CVs were 14.6% at 26 pmol/L (n = 10). We also measured N-terminal proBNP using a processing-independent assay (PIA) that recognizes the N-terminus of human proBNP 1–10 (22). Before incubation, plasma was treated with trypsin, which cleaved both proBNP 1–76 and proBNP 1–108 into a uniform proBNP 1–21 fragment (10). We then measured this fragment using an antiserum specific for the N-terminus of proBNP; i.e., removal of the first amino acid residue abolishes antibody binding. Interassay CVs were 20% at 16 pmol/L and 8% at 70 pmol/L (22). Cross-reactivity of the different assays is only partially known, whereas the Elecsys assay has been shown to fully detect proBNP 1–76 and to a lesser extent the intact precursor (28). This assay does not detect glycosylated proBNP. For the PIA proBNP assay, N-terminal trimming abolishes immunodetection (22). For the detection of glycosylated proBNP forms, no in vitro data are available, but both assays use antibodies raised against sequences where glycosylation does not occur. None of the N-terminal proBNP assays cross-react with the C-terminal BNP forms (28).

statistics
The results are expressed as percentage or mean (SD). Because none of the assays yielded plasma concentrations that were normally distributed, we performed a transformation of values (10log) for all assays. We analyzed survival using Kaplan-Meier survival curves, performed ROC analysis to illustrate the diagnostic sensitivity and specificity, and calculated area under curve (AUC). We performed a Cox proportional hazard regression analysis was identify the weight of the individual risk variables for cardiovascular mortality, as well as a Cox proportional hazard regression analysis including a multivariate evaluation of the different assays. We treated each set of assay data as a continuous variable to compare assays in spite of differences in the ranges of plasma concentrations obtained. The β-coefficient of the assay variable of the Cox regression was divided by the SD of the specific assay. In the evaluation of agreement between the different assays, we used Bland-Altman plots. For the results from the peptide measurements (PIA assay and Elecsys assay), results <5 pmol/L were replaced by 5 pmol/L, because the uncertainty in this lower region is high. A P value <0.05 was considered statistically significant. All data analysis was performed using a commercially available statistical analysis software package (Statistica v. 8, Statsoft Inc.; Analyse-It v. 2.05, Analyse-It Software Ltd.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PRObnp 1–76 vs n-terminal PRObnp assay
Comparison of proBNP 1–76 with N-terminal proBNP measurements revealed a high correlation in plasma concentrations (r2 = 0.91, P < 0.0001). Although overall bias was modest, the proBNP 1–76 assay reported lower concentrations in the high range than the N-terminal proBNP assay (Fig. 1BUp ), reflected as a slope deviating from the identity line on regression analysis. The differences in assay measurement may therefore not be due to simple differences in calibration, but suggest a molecular change in the measured endogenous proBNP forms related to the absolute concentration.

impact of PRObnp-derived peptide assays in a clinical setting
Basal patient characteristics are listed in Table 1 . The mean age of the population was 72.3 years. No patient was categorized to NYHA functional class IV. Mean body mass index was 27.0 (4.2) kg/m2. Of those that accepted Doppler echocardiography and where an acceptable quality of the examination was obtained (n = 415), 48% demonstrated impaired systolic or diastolic function by means of Doppler echocardiography.


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  		Table 1. Clinical characteristics of the study population.

Correlations between the 4 assays ranged from r2 = 0.57 to 0.81 (all P < 0.0001). Bias plots are shown in Fig. 2 . As expected, the median plasma concentration differed between assays: proBNP 1–76, 23 pmol/L (range 0–2208); RIA N-terminal proBNP, 59 pmol/L (range 10–1007); PIA N-terminal proBNP, 18 pmol/L (range 0–570); BNP-32, 13 pmol/L (range 1–335). In addition, we evaluated the diagnostic performance for identifying patients with reduced LVEF (<40%). Using ROC, the assays performed very similarly, with AUC values ranging between 0.77 and 0.81 (Table 2 ). In patients with severely impaired systolic function (LVEF <30%), AUC values were higher (0.84–0.94), with no obvious differences between the assays.


Figure 2
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  		Figure 2. Bland-Altman plots from the N-terminal proBNP and the BNP assays (n = 415).


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  		Table 2. Ability of the 4 assays to identify clinical end points.

In elderly patients presenting with symptoms associated with heart failure, a substantial number display normal systolic but impaired diastolic function. We therefore evaluated patients with relaxation abnormalities by ROC analysis; the resulting AUC values ranged between 0.63 and 0.68, suggesting that none of the assays can effectively identify patients with relaxation abnormalities. However, patients with a pseudonormal or restrictive filling pattern represent a much different population among those with diastolic dysfunction. The ability to identify a pseudonormal or restrictive filling pattern was marginally higher, with AUC values between 0.64 and 0.70, and again no obvious differences between the assays.

PRObnp-derived peptides as prognostic indicators for cardiovascular mortality
Increased risk for cardiovascular mortality is important to identify in heart failure patients. Although the present assays did not display any significant differences in the ability to predict those at increased risk for cardiovascular death, AUC values were still relatively high. Analyzing the prognostic capability to predict cardiovascular mortality, we included 10 years of follow-up and analyzed the population using a Cox proportional hazard regression analysis. In most of the assays, the highest quartile had significant prognostic power, with an increased risk ranging between approximately 3 and 6 times (Table 3 ).


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  		Table 3. Cox proportional hazard regression analysis of clinical variables and assay results.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We examined the impact of assay epitope specificity in proBNP-derived peptide measurement focusing on the N-terminal region. Our results reveal a striking difference between standard assay comparison vs the overall comparable clinical performance in cardiovascular diagnostics or prognosis. Although head-to-head laboratory comparisons suggest a shift in the molecular heterogeneity in plasma as a function of the absolute concentration, the clinical impact is not apparent in a cohort of elderly patients presenting with symptoms associated with heart failure.

ProBNP-derived peptides in plasma have long been thought to consist of 2 forms: BNP-32 (the bioactive hormone) and a complementary N-terminal fragment (proBNP 1–76, referred to as NT-proBNP) (1). However, even the earliest molecular characterization in cardiac tissue extracts as well as plasma suggested the existence of a larger form. Moreover, the presence of the intact precursor would provide an expedient explanation for the paradoxical lack of hormonal effects in heart failure patients with high circulating concentrations (29), which has been referred to as "junk" BNP. One group even suggested that N-terminal proBNP fragments could oligomerize (15). Nevertheless, decisive data on the intact precursor in circulation was only recently provided by development of a specific immunoassay targeted against the uncleaved precursor (11). The troublesome elution profiles from chromatography studies have also been elucidated and seem to be due to glycosylated proBNP forms (13). Moreover, both BNP-32 and proBNP can be trimmed in the N-termini (18)(19).

Epitope recognition is the defining feature of an immunoassay. Paratope/epitope binding usually involves only a short sequence of 4–6 amino acid residues in the ligand. Strictly speaking, a concentration measured by an immunoassay therefore reports on this specific sequence and not necessarily on the remaining molecule. A relevant example in this context is the term NT-proBNP, which initially covered only the proBNP 1–76 form, against which the commercial assay is calibrated. However, the method also measures intact proBNP, with a reduced affinity (28). A second aspect of this widely used assay is the epitope in position 39–50 (Fig. 1AUp ). Intact proBNP and proBNP 1–76 can be glycosylated in this region, and a recent report has revealed abolished immunodetection of a recombinant glycosylated proBNP peptide (28). This suggests that this modification also may have an impact on binding affinity in vivo and could introduce variable bias depending on the molecular heterogeneity in plasma. In extension of our present findings, an early report on N-terminal proBNP vs NT-proBNP measurement in fact indicated such methodological differences in absolute concentrations as a function of the endogenous concentration (30). Whether these findings relate to changes in glycosylation or endoproteolytic cleavage (or both) remains to be established.

Measuring proBNP-derived peptides in plasma has proven to be much more complex than initially perceived. The earlier discrepancies in absolute concentrations by different assays now seem due to both calibration design and the variable endogenous forms, which again may depend on disease status. In severe heart failure, proBNP 1–108 circulates as a glycoprotein (31)(32), but little is still known about proBNP glycosylation in healthy individuals and in patients with mild cardiac dysfunction. To make matters more complicated, the metabolism of the different forms is not known, but it seems reasonable to suspect, for instance, that renal clearance of the glycosylated peptides vs nonglycosylated peptide may differ. To overcome these differences, an assay designed to measure an epitope without variable modifications or degradation would be optimal, and a recent report has suggested that amino acids 13–24 and 63–76 may be the best epitope candidates (32). Our in-house assay (PIA) was initially designed to overcome some of the potential differences in molecular heterogeneity with a focus on proBNP 1–76 vs proBNP 1–108 (22). However, even the free N-terminus has been shown to be subject to enzymatic cleavage, thus introducing another variable (19). The analytical work on measurement of proBNP-derived peptides in plasma thus needs further characterization on the molecular heterogeneity in both health and disease, focusing on what specific forms are affected by different conditions. Notably, we suspect that the heterogeneity might differ most in patients with severe forms of heart failure, i.e., cardiogenic chock or chronic heart failure patients in NYHA class IV. In comparison, the present patient cohort does not reflect the most severe forms of cardiac dysfunction. On the other hand, the everyday need for a heart failure biomarker in elderly patients presenting with symptoms of heart failure in primary care is considerable.

The differences in epitope recognition did not reveal clear differences in clinical performance as evaluated by ROC analyses. The combined measurement of diagnostic specificity and sensitivity for detecting reduced left ventricular systolic function was similar (Table 2Up ). Moreover, the ability to predict increased long-term risk of cardiovascular death was comparable, with a tendency toward better performance by the commercial assays (Table 3Up ). As cutoff values are population-dependent, we also performed the analyses with the peptide data as continuous variables. Again, no clear differences were observed between the 4 assays. We therefore speculate that the relatively small cohort might in itself be a limitation, in that only larger differences would be detected by ROC analysis. On the other hand, we note that the AUC values in this cohort of elderly patients are similar to the clinical performance data in much larger population-based cohorts (33)(34). Thus, differences in the molecular heterogeneity in plasma should be further pursued with a clear assay design targeted at specific forms, for instance the intact precursor (11). Studies addressing medical conditions associated with increased concentrations besides heart failure could also provide important information on the "gray zone" patient, where the current ability to discriminate between cardiac vs noncardiac disease could be improved though measurement of different proBNP-derived peptide forms.

study limitations
The present report primarily addresses the potential impact of molecular heterogeneity in the N-terminal proBNP region. Our findings cannot be extrapolated to assays directed at other regions, including the N-terminal region of proBNP 1–76. Moreover, the results from ROC analyses are population-dependent, and the cutoff values may not apply to other patient cohorts. Finally, the present patient cohort consists of elderly patients presenting with symptoms associated with heart failure. The peptide findings thus refer to this particular clinical setting; we suspect larger differences in molecular heterogeneity may exist in, for instance, patients presenting in the emergency ward with acute, decompensated heart failure or severe renal dysfunction. However, our population reflects a clinical setting, where the need for a heart failure biomarker is considerable.


   Acknowledgments
 
Author Contributions: Each author confirmed he or she has contributed to the intellectual content of this paper and has met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: The study was supported by grants from The County Council of Östergötland, The Swedish Heart and Lung Foundation, and The Linköping University Research Foundation CIRC.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Kerstin Gustavsson, heart failure nurse, for her invaluable help. Laboratory technician Lone Olsen is gratefully acknowledged for her expert assistance with the PIA proBNP measurements.


   Footnotes
 
1 Nonstandard abbreviations: BNP, B-type natriuretic peptide; proBNP, pro-B–type natriuretic peptide; NYHA, New York Heart Association; LVEF, left ventricular ejection fraction; PIA, processing-independent assay; AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M Schou, U Alehagen, J P Goetze, F Gustafsson, and U Dahlstrom
Effect of estimated glomerular filtration rate on plasma concentrations of B-type natriuretic peptides measured with multiple immunoassays in elderly individuals
Heart, September 15, 2009; 95(18): 1514 - 1519.
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