Depletion of proBNP1-108 in Patients with Heart Failure Prevents Cross-Reactivity with Natriuretic Peptides

Abstract

Background

After synthesis by cardiomyocytes, precursor proBNP1-108 is cleaved into NT-proBNP and BNP. Recently, cross-reactivity between these assays was discussed. The aim of this study was to characterize the cross-reactivities, through a new biochemical innovative approach consisting in the total depletion of the circulating proBNP1-108 in patients with heart failure (HF).

Methods

This prospective study included 180 patients with chronic HF. BNP and NT-proBNP were dosed with commercial kits. ProBNP1-108 was determined using an ELISA research assay specific to the precursor. ProBNP1-108 depletion was performed by immunocapture with a specific antibody targeting exclusively the ProBNP1-108 hinge region. ProBNP1-108, BNP and NT-proBNP levels were determined before and after depletion using this process in HF patients.

Results

Mean age was 74.34 +/-12.5 y, and 69% of patients were males. NYHA classes II and III were the most frequent (32% and 45% respectively). Before depletion, ProBNP1-108, NT-proBNP and BNP levels were 316.8+/-265.9 pg/ml; 6,054.0+/-11,539 pg/ml and 684.3+/-82.1 pg/ml respectively, and were closely correlated with NHYA classes. After immuno-depletion, proBNP1-108 was decreased in mean by 96% (p<0.0001), BNP by 53% (p<0.0001) and NT-proBNP by 5%. The relationship between BNP or NT-proBNP and NHYA classes remained unchanged.

Conclusion

Current BNP and NT-proBNP assays measured as well proBNP molecule. This cross reactivity percentage has been controversial. Thanks to the removal of circulating proBNP1-108 with our immunodepletion process, we are now able to assess the remaining “true” BNP and NT-proBNP molecules and further evaluate their clinical relevance.

About the Authors:

François Roubille

* E-mail: [email protected]

Affiliations Cardiology Department, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France, Research Center, Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada

Delphine Delseny

Affiliation: Cardiology Department, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Jean-Paul Cristol

Affiliation: Department of Biochemistry, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Delphine Merle

Affiliation: CNRS UMR3145 Bio-Rad, SysDiag, Montpellier, France

Nicolas Salvetat

Affiliation: CNRS UMR3145 Bio-Rad, SysDiag, Montpellier, France

Catherine Larue

Affiliation: Bio-Rad Laboratories, Marnes la Coquette, France

Jean-Marc Davy

Affiliation: Cardiology Department, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Florence Leclercq

Affiliation: Cardiology Department, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Jean-Luc Pasquie

Affiliation: Cardiology Department, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Luc Guerrier

Affiliation: Bio-Rad Laboratories, Marnes la Coquette, France

Jeannette Fareh

Contributed equally to this work with: Jeannette Fareh, Anne-Marie Dupuy

Affiliation: CNRS UMR3145 Bio-Rad, SysDiag, Montpellier, France

Anne-Marie Dupuy

Contributed equally to this work with: Jeannette Fareh, Anne-Marie Dupuy

Affiliation: Department of Biochemistry, University Hospital of Montpellier, Université de Montpellier 1, Montpellier, France

Introduction

The human BNP (B-type Natriuretic Peptide) gene encodes for a pre-proBNP molecule constituted of 134 amino acids. The pre-proBNP is cleaved out of the proBNP1-108. ProBNP1-108 was initially considered to be cleaved inside the cardiomyocytes into NT-proBNP (the biologically inactive NH2-terminal peptide fragment proBNP1-76) and the BNP (the biologically active hormone) [1,2]; both are more and more useful for clinical application [1,3,4], including diagnosis [3], treatment [3], prognosis [3], follow-up of patients with cardiac involvement, mainly heart failure (HF). However, intact proBNP1-108 was recently evidenced in plasma in significant amounts in patients with HF [2,5-7] with a lower biological activity than the BNP. In addition, cross-reactivity with the precursor intact proBNP1-108 between the commercial assays for BNP or NT-proBNP is high, with an inter-tests recovery ranging from 5 to 38% or 0-249% respectively [8], challenging the analytical specificities of available commercial kits, although at different levels. Recently, Nishikimi et al. described a direct immunochemiluminescent assay for proBNP and total BNP in 116 healthy subjects and in 32 patients with HF [9].

Little is known about such cross-reactivities in clinical settings, although this point appears critical to better characterize what routinely-used immunoassays measure, to better interpret results, and increase the clinical signification of the natriuretic peptides. In particular, the process we propose could help to better classify patients with atypical profiles and help in medical decision-making for appropriate therapy.

In this study, we aimed at characterizing the cross-reactivities of the natriuretic peptides assays, through a new biochemical approach consisting in the total immunodepletion of the circulating proBNP1-108 in patients at various stages of HF.

Methods

Between May 2010 and February 2011, 180 patients with Heart Failure (HF) were prospectively included in a single University Hospital (CHRU Montpellier, France). The study was performed according to the Declaration of Helsinki (revised version of Somerset West, Republic of South Africa, 1996) and according to the European Guidelines of Good Clinical practice (version 11, July 1990) and French laws. The study protocol was approved by the local Ethics Committee of the University Hospital of Montpellier, written informed consent was obtained from all patients and the biological collection registered by the French government (research Ministery, # DC-2009-1052).

To be eligible to the study, the patients were previously (at least 6 months before the inclusion) diagnosed with acute or chronic HF, as recommended by the European Society of Cardiology [3]. Main inclusion criteria were the ability to give informed consent, age>18 years and confirmed diagnosis of HF, irrespectively of the cause or treatments. Main exclusion criteria were unstable angina or acute coronary syndrome in the past month, cardiac surgery and chemotherapy.

Venous blood was collected in EDTA tubes and was immediately centrifuged (the samples are transported a mean total delay of less than 3 hours (all inclusive until frozen); in the biochemistry lab 95% are treated in less than an hour and a half and 50% in less than one hour) and frozen (-80°C) until tested, three years later.

This was the first thawing.

The BNP levels were determined using an immunochemilumiscent method applied on the Access 2® analyzer (Beckman Coulter, Villepinte, France). Reagents, calibrators and controls were used according to the Biosite Package Insert. The NT-proBNP levels were determined using an inmmuno-electrochemiluminescence assay on the on the Cobas8000/e602® immunochemistry system (Roche Diagnostics, Meylan, France).

ProBNP was determined by an ELISA (Enzyme Linked ImmunoSorbent Assay) method for research use only as previously well described by Giuliani et al. [7] and Waldo et al. [10]. The limits of the detection as provided by the manufacturer are respectively 5 pg/ml for the NT-proBNP and 1 pg/ml for the BNP.

The proposed proBNP ELISA method is mainly characterized by the absence of cross-reactivity with circulating BNP and NT-proBNP, allowing specific determination of the proBNP1-108 form [7]. The proBNP1-108 removal was performed thanks to an immunodepletion system, consisting of NHS-activated beads (Affi-Gel 10, Bio-Rad Laboratories, hercules, CA, USA) coupled to a solution of proBNP-specific antibody (5 mg) according to manufacturer’s instructions. The mice monoclonal antibody binds specifically the proBNP hinge region or anti-irrelevant (myoglobin) antibody. After bead washing, EDTA samples were added to proBNP1-108 coated beads (5:10 ratio) for the depletion step (30 min at 4°C). After a rapid spin column centrifugation (500 rpm), the immunodepleted supernatant was collected for BNP, NTproBNP and proBNP1-108 measurements using the adapted platforms.

Statistical analysis

All statistics and figures were performed with R 2.15.0 (www.R-project.org) or GraphPad Prism (version 4.0, GraphPad Software, San Diego California USA). Results are expressed as means with standard deviation except for study of reduction in peptides, presented as median and interquartile ranges, as more representative of the values’ dispersion. Comparisons were performed through Student t-tests or Wilcoxon rank-sum test depending on the normal distribution (Shapiro Wilks test) and homoscedasticity (F-test) of peptides, especially to analyze statistical significance of change-in-peptides comparisons. All data distributions are illustrated as medians and box plots for each peptide. Correlations were calculated with Spearman tests and Bland-Altman analysis to have the magnitude of difference in BNP and NTproBNP between before and after proBNP depletion. Distributions in different classes were compared using Kruskal-Wallis test.

Results

Mains Main characteristics of the population

Main characteristics of the population are described in the Table 1. The mean age of the 180 subjects was 74.34 +/-12.5y, and a majority was males (69%). Conventional risk factors were first hypertension (61%) and overweight (57%), whereas tabagism (34%) and diabetes (29%) were less frequent. NYHA classes II and III were the most frequent (32% and 45% respectively). Ischemic cardiomyopathy was found in more than half of the patients. Treatments indicated in patients with HF followed ESC guidelines, with 83% of the patients under beta-blockers, and about ¾ under ACE-inhibitors, but only 24% under mineraloreceptor antagonists.

Characteristics Mean+/-SD or n/total (%)

Main characteristics

Age (y) 74.3+/-12.5

Sex (Males) 125/180 (69)

Heart rate (bpm) 80.8+/-23.95

LVEF (%) 37.0+/-13.9

Cardiovascular risk factors

Overweight 97/171 (57)

BMI† 26.8+/-0.4

Hypertension 87/143 (61)

Tabagism 49/143 (34)

Diabetes 52/143 (36)

Dyslipidemia 62/143 (43)

NYHA

I 8/171 (5)

II 55/171 (32)

III 77/171 (45)

IV 31/171 (18)

Etiology of the cardiopathy

Ischemic 103/180 (57)

Dilated cardiomyopathy 31/180 (17)

Valvulopathy 18/180 (10)

Rhythmic disease 16/180 (9)

Hypertrophic 7/180 (4)

Mixed disease 5/180 (3)

Treatments

Beta-blockers 135/163 (83)

ACE-inhibitors/ARB 126/163 (77)

Loop-diuretics 120/164 (73)

Eplerenone-spironolactone 39/163 (24)

Table 1. Baseline characteristics of the study population.

† The body-mass index is the weight in kilograms divided by the square of the height in meters.

SD: standard deviation, LVEF: left ventricular ejection fraction.ACE Inhibitor: angiotensin-converting-enzyme inhibitor.

ARB: angiotensin II receptor inhibitor.

CSV

Download CSV

BNP peptides.

(Table 2) Before depletion, ProBNP1-108, NT-proBNP and BNP levels were 316.8+/-265.9 pg/ml, 6,054.0+/-11,539 pg/ml and 684.3+/-1,092 pg/ml respectively.

The technique of depletion was efficient and led to a total depletion of 96%, p<0.001 (Table 2). Depletion of proBNP1-108 resulted in a deep decrease in BNP (decrease in means was 53% for the 180 samples ; median decrease was 48.3% (interquartile range 44.8%-53.0%) for 178 paired analyses), highly statistically significant (p<0.0001) (Figure 1). It also resulted in a mild decrease in NT-proBNP (decrease in means was 5% for the 180 samples ; median variation was -0.86% (interquartile range -8.0% -+7.1%) for 177 paired analyses), statistically significant (p=0.02) as shown in Figure 2.

Values* Before depletion After depletion Mean reduction (%) P value

BNP (pg/ml) 684.3+/-1,092.0 317.9+/-435.3 53% P<0.0001

NT-proBNP (pg/ml) 6,054.0+/-11,539.0 5,760.0+/-11,510.0 5% P=0.02

proBNP (pg/ml) 316.8+/-265.9 13.3+/-29.1 96% P<0.0001

Table 2. Main biochemical results.

CSV

Download CSV

[Figure omitted. See PDF.]

Figure 1. Circulating BNP levels before and after depletion of proBNP1-108..

BNP decreased significantly by 53% (when means are compared), p<0.0001.

A: Median and interquartile range of reduction for paired values (n=177).

B: Statistical distributions for the 180 patients (median and interquartile range, 16 values are out of the axis limits).

https://doi.org/10.1371/journal.pone.0075174.g001

[Figure omitted. See PDF.]

Figure 2. Circulating NT-proBNP levels before and after depletion of proBNP1-108.

NT-proBNP decreased significantly by 5%, (when means are compared), p=0.02.

A: Median and interquartile range of reduction for paired values (n=178).

B: Statistical distributions for the 180 patients (median and interquartile range, 48 values are out of the axis limits).

https://doi.org/10.1371/journal.pone.0075174.g002

Correlation coefficients were very good between the two assessments (before and after depletion), as presented on Figure 3, respectively for BNP, r=0.97, p<0.0001; and for NT-proBNP r=0.99, p<0.0001. As shown in the Bland-Altman plot (Figure 4), the magnitude of the BNP difference for each patient increased between both conditions: before and after proBNP depletion. Around 95% of patients have a difference in BNP levels between before and after proBNP depletion (bias=-287.7), whereas the NTproBNP presents no magnitude modification between the two conditions (bias=-47.8).

[Figure omitted. See PDF.]

Figure 3. Correlations between BNP and NTproBNP assays before and after depletion of proBNP1-108.

A: BNP. Correlation between the two assays is very strong, in spite of the deep reduction of the values: n=178, r=0.97; p<0.0001.

B: NT-proBNP. Correlation between the two assays is very strong, in spite of the mild reduction of the values N=178, r=0.99; p<0.0001.

https://doi.org/10.1371/journal.pone.0075174.g003

[Figure omitted. See PDF.]

Figure 4. Bland Altman representation of the individual variations before and after depletion, reported to the start level of peptides BNP (A), NT-proBNP (B).

A: BNP. The relationship is clearly linear, whatever the start level of BNP before the depletion procedure.

B: pro-BNP: the relashionship is not strong.

https://doi.org/10.1371/journal.pone.0075174.g004

The three peptides, proBNP, BNP and NT-proBNP, were closely related to NYHA classes. Differential analyses did not reveal significant differences between the three natriuretic peptides: all of them are strictly correlated with the functional classes, (data presented on Figure 5), with regards to discrimination of HF classes, even after depletion. The mean ratio proBNP1-108 to NT-proBNP was 0.105+/-0.0005, irrespective of the HF stage.

[Figure omitted. See PDF.]

Figure 5. Distribution according to NYHA classes of: BNP before depletion (A), after depletion (B) or NTproBNP before depletion (C), after depletion (D), and proBNP1-108 before depletion (E).

All of these Natriuretic peptides are strictly correlated with the functional classes, with p<0.001 (specific values after Kruskal-Wallis analyses are indicated).

https://doi.org/10.1371/journal.pone.0075174.g005

Interestingly, beyond all the three natriuretic peptides that increase significantly following the functional NYHA classes, urea but not creatininemia, and CRP were significantly positively correlated with the NYHA functional classes. They all increase in case of renal impairment and this increase was strictly correlated with the decrease in the renal function (P<0.01 or <0.0001), as represented on Figure 6 (renal function is then estimated by the CKD-EPI, (Chronic Kidney Disease - Epidemiology Collaboration)). CRP not only correlated with NYHA, but also with both BNP and NT-proBNP after depletion, as schematically presented on Figure 7.

[Figure omitted. See PDF.]

Figure 6. Distribution according to renal function (estimated by CKD-EPI) of: BNP before depletion (A), after depletion (B) or NTproBNP before depletion (C), after depletion (D), and proBNP1-108 before depletion (E).

All of these Natriuretic peptides are strictly correlated with the renal function, with p<0.001 (specific values after Kruskal -Wallis analyses are indicated).

https://doi.org/10.1371/journal.pone.0075174.g006

[Figure omitted. See PDF.]

Figure 7. Correlations between CRP, NYHA and BNP or NTproBNP after depletion of proBNP1-108.

A: CRP is correlated with NYHA. CRP levels increase significantly when the NYHA class becomes higher (P<0.0001).

B: BNP after depletion is correlated with CRP levels. BNP levels are considered in quartiles. P=0.0003.

C: NT-proBNP after depletion is correlated with CRP levels. BNP levels are considered in quartiles. P<0.0001.

https://doi.org/10.1371/journal.pone.0075174.g007

Discussion

In this study, for the first time, we demonstrated that effective immuno-depletion of the precursor proBNP1-108 (up to 96% of the circulating proBNP1-108) revealed a clear cross-reactivity of proBNP in BNP assays and in a lesser extent of NTproBNP in routine clinical assays in HF management. The BNP levels are overestimated by more than 50% in absence of depletion of proBNP1-108. Secondly, we confirm that intact proBNP1-108 is correlated to HF stage and increases in case of renal impairment (as well as BNP or NT-proBNP). CRP appears to be correlated both to functional classes and natriuretic peptides after depletion deserves to be mentioned, as this correlation is not always so strong in recent studies [11], and as this could explain -at least partly- the progression of the disease [12].

From a clinical point-of-view, the simultaneous measurement of two peptides representing on the one hand the biologically inactive intact precursor proBNP1-108 and on the other hand the active derived BNP peptide, could allow clinicians to follow better the status of the patient, especially in pathological conditions. For instance, in ambulatory patients with chronic HF, combined assessment of BNP and proBNP provided additional information with regards to risk of adverse clinical outcomes [13]. In these patients, this combined measurement was particularly promising in patients with low levels of BNP, in which the clinicians could be falsely reassured. Clinical evaluation of combinations of highly specific assays for the assessment of BNP peptides are still needed [14] especially in patients with chronic renal failure since the half-life of the 3 peptides and/or their respective renal clearance is poorly understood [15].

Secondly, our findings rise concerns about the currently available methods for assessing natriuretic peptides: either to encourage more specific methods, such as the new process presented here, or to redefine cut-off values (especially as regards the “grey zone” even if this was not specifically the point of interest in the present study), taking into account the lack of specificity of the currently used methods. Dries et al showed indeed that simultaneous assessment of unprocessed proBNP1-108 in addition to processed BNP32 could improve identification of high-risk ambulatory patients with HF [13]. Our method could provide similar information, so that clinical implications deserve to be clarified.

Surprisingly, the clinical signification of BNP overestimation remains unclear as the sample pre-treatment technique did not allow us to better stratify the HF patients and did not contribute to a better patient management care. At least two reasons could be proposed.

First, the two different methods for assessment of peptides before or after depletion seem to provide similar clinical information on this HF cohort of patients, as suggested by the very strong correlations between them (at least for BNP).

Secondly, BNP peptides are already powerful tools to stratify HF patients, so that it could be difficult to improve significantly the clinical meaning in a small population or in a real-life population: only class III patient for instance should have been included to get a more specific population with better predefined endpoints, and a long follow-up. This hypothesis is consistent with the study on a specific not commercially available test in a large cohort [16]. Disappointingly, although proBNP1-108 was a sensitive (78.8%) and specific (86.1%) biomarker for detecting left ventricular systolic dysfunction, this biomarker was comparable but not better than BNP and was even less informative than NT-proBNP1-76 [16], at least in this HF cohort.

In addition, in a recent cohort of 187 Class III-IV HF patients, pro-BNP1-108, standard assay BNP and troponin T (cTnT) were investigated in relation to the primary endpoint of death or cardiac transplantation [17]. Both elevated proBNP1-108 and BNP were associated with increased clinical events. More importantly, elevated levels of proBNP1-108 or BNP identified by serial monitoring similarly predicted events.

The second surprising result of this study is that no significant benefit for immunodepletion could be observed on the NT-proBNP molecule measurement itself (5% decrease for NT-proBNP1-76 versus 50% decrease for BNP). Knowing that an equimolar ratio is expected in the proBNP1-108 cleavage by furin/corin, one could guess to observe a significant decrease in the NT-proBNP1-76 molecule while using the immunodepletion method. To understand this surprising effect, we recognized that O-glycosylated forms of proBNP in heart failure patients were reported, along with uncleaved proBNP [8,18-20]; all reported an overestimation of BNP1-32 and NT-proBNP1-76 due to the cross-reactivity of antibodies towards the proBNP1-108. We therefore hypothesized that the hyperglycosylation of proBNP 1-108at the 1-76 part [18,19,21] might prevent the accessibility of the anti-hinge antibody during the immunodepletion process and therefore decrease its efficiency. We therefore suggest verifying that by using O-glycosidases, neuraminidase and galactosidase enzymes [21], the immunodepletion will stay the same or will benefit from a better access of the anti-hinge antibody. Furthermore, this limited cross-reactivity of the NT-proBNP assay with the proBNP could result from the impact of glycosylation on the detection of NT-proBNP: NT-proBNP could be underdetected because of the glycosylation of the NT-proBNP [22], which could prevent the antibodies from an accurate recognition.

These findings demonstrate that the “BNP” which is supposed to be measured in various clinical settings is not really the true BNP pool Only few publications have already showed the cross-reactivity among natriuretic peptide assays. Luckenbill et al studied pooled samples of healthy donors with established concentrations and demonstrated that BNP assays crossreact with NT-proBNP or proBNP, and calculated the various differences between 5 commercial BNP and 3 commercial NT-proBNP assays [8]. Hawkridge et al, used an immunoaffinity purification assay to isolate endogenous BNP specifically in the plasma of 4 patients ranged NYHA class IV dedicated to subsequent analysis by nano-liquid chromatography, to evidence the absence of circulating BNP in advanced-stage HF patients and they suggested the existence of altered forms of BNP [23]. Here, in a real-life cohort of 180 patients with HF, we established a similar cross-reactivity corroborating that the true BNP pool could represent less than 50% of what is measured by a currently used commercial assay.

Furthermore, it could explain at least partly discrepancies among commercial kits, as specificity could vary from one to another, hence a lack of alignment and difficulties to follow patients when different commercial assays are run in different laboratories.

Limitations

These findings have to be confirmed in a larger population, although this cohort of 180 patients with HF represents a real-life situation. Secondly, it will be of critical importance to test different platforms to demonstrate the general impact of proBNP depletion on BNP levels. Here is provided first technical proof of concept of the impact of proBNP depletion on BNP testing in a pilot study.

In this population, taking into consideration the cross reactivity did not modify the classification of patients. It remains unclear whether considering these true values simply drives all values to 50% lower levels or on the contrary could elicit a profound impact in the classification for specific patients (BNP grey zone, renal impairment, elderly etc). Further larger studies will address this concern.

Finally, it could be suggested that since diagnostic and prognostic performances of BNP and NT-proBNP are roughly similar, if NT-proBNP measurement is not affected by the presence of proBNP, only NT-proBNP and not BNP could be suggested to be used. Nevertheless, as performances are similar, our results could not by themselves support such conclusions. They should on the contrary suggest to develop more specific assays to better understand what is currently assayed (other interactions could obviously be searched as regards NT-proBNP as previously discussed) and propose perhaps more accurate cut-offs.

Conclusion

Routinely used BNP assays are not specific to BNP molecule but also cross react with the proBNP1-108 precursor. By using an immunodepletion method, we were able to really assess the true BNP measurement. Assessing accurately each of the three major natriuretic peptides could be of interest to better characterize patients and to address appropriate therapy especially in case of decompensated heart failure patients. This could open new fields of interest to better understand unclear medical situations and above all to enlighten the grey zone, in order to better stratify patients and to perform a better drug monitoring. Clinical implications of these new methods remain to be investigated.

Acknowledgments

The authors would like to acknowledge Daniele Dietz, Annie Vapaille, Sonia Soltani and Dominique Piquer for their excellent technical assistance and data management

The authors acknowledge Dr Gang He, MD (Montreal Heart Institute) for his advices and improvement of the manuscript and Valérie Macioce (Montpellier University Hospital) the extensive manuscript editing.

Author Contributions

Conceived and designed the experiments: FR DD JPC DM CL JMD LG JF AMD. Performed the experiments: FR DD DM NS CL JF AMD. Analyzed the data: FR DD NS JF. Contributed reagents/materials/analysis tools: FR DD NS JF JPC AMD. Wrote the manuscript: FR DD JPC DM NS CL JMD FL JLP LG JF AMD. Clinical Pertinence: FL JLP.

Citation: Roubille F, Delseny D, Cristol J-P, Merle D, Salvetat N, Larue C, et al. (2013) Depletion of proBNP1-108 in Patients with Heart Failure Prevents Cross-Reactivity with Natriuretic Peptides. PLoS ONE 8(9): e75174. https://doi.org/10.1371/journal.pone.0075174

References

1. Clerico A, Recchia FA, Passino C, Emdin M (2006) Cardiac endocrine function is an essential component of the homeostatic regulation network: physiological and clinical implications. Am J Physiol Heart Circ Physiol 290: H17-H29. PubMed: 16373590.

2. Goetze JP (2010) Biosynthesis of cardiac natriuretic peptides. Results Probl Cell Differ 50: 97-120. PubMed: 19859677.

3. McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Böhm M et al. (2012) ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J 33: 1787-1847. doi:https://doi.org/10.1093/eurheartj/ehs104. PubMed: 22611136.

4. Jessup M, Abraham WT, Casey DE, Feldman AM, Francis GS et al. (2009) 2009 focused update: ACCF/AHA Guidelines for the Diagnosis and Management of Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the International Society for Heart and Lung Transplantation. Circulation 119: 1977-2016. doi:https://doi.org/10.1161/CIRCULATIONAHA.109.192064. PubMed: 19324967.

5. Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA et al. (2007) The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 53: 866-873. doi:https://doi.org/10.1373/clinchem.2006.076141. PubMed: 17384012.

6. Mogelvang R, Goetze JP, Schnohr P, Lange P, Sogaard P et al. (2007) Discriminating between cardiac and pulmonary dysfunction in the general population with dyspnea by plasma pro-B-type natriuretic peptide. J Am Coll Cardiol 50: 1694-1701. doi:https://doi.org/10.1016/j.jacc.2007.07.073. PubMed: 17950153.

7. Giuliani I, Rieunier F, Larue C, Delagneau JF, Granier C et al. (2006) Assay for measurement of intact B-type natriuretic peptide prohormone in blood. Clin Chem 52: 1054-1061. doi:https://doi.org/10.1373/clinchem.2005.061770. PubMed: 16574763.

8. Luckenbill KN, Christenson RH, Jaffe AS, Mair J, Ordonez-Llanos J et al. (2008) Cross-reactivity of BNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC Committee for Standardization of Markers of Cardiac Damage. Clin Chem 54: 619-621. doi:https://doi.org/10.1373/clinchem.2007.097998. PubMed: 18310156.

9. Nishikimi T, Okamoto H, Nakamura M, Ogawa N, Horii K et al. (2013) Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure. PLOS ONE 8: e53233. doi:https://doi.org/10.1371/journal.pone.0053233. PubMed: 23365636.

10. Waldo SW, Beede J, Isakson S, Villard-Saussine S, Fareh J et al. (2008) Pro-B-type natriuretic peptide levels in acute decompensated heart failure. J Am Coll Cardiol 51: 1874-1882. doi:https://doi.org/10.1016/j.jacc.2007.12.051. PubMed: 18466803.

11. Savic-Radojevic A, Radovanovic S, Pekmezovic T, Pljesa-Ercegovac M, Simic D et al. (2013) The Role of Serum VCAM-1 and TNF-alpha as Predictors of Mortality and Morbidity in Patients with Chronic Heart Failure. J Clin Lab Anal.

12. Choi EY, Yan RT, Fernandes VR, Opdahl A, Gomes AS et al. (2012) High-sensitivity C-reactive protein as an independent predictor of progressive myocardial functional deterioration: the multiethnic study of atherosclerosis. Am Heart J 164: 251-258. doi:https://doi.org/10.1016/j.ahj.2012.05.010. PubMed: 22877812.

13. Dries DL, Ky B, Wu AH, Rame JE, Putt ME et al. (2010) Simultaneous assessment of unprocessed ProBNP1-108 in addition to processed BNP32 improves identification of high-risk ambulatory patients with heart failure. Circ Heart Fail 3: 220-227. doi:https://doi.org/10.1161/CIRCHEARTFAILURE.109.903153. PubMed: 20107190.

14. Emdin M, Passino C, Clerico A (2011) Natriuretic peptide assays revisited: do we need pro-B-type natriuretic peptide? J Am Coll Cardiol 57: 1396-1398. doi:https://doi.org/10.1016/j.jacc.2010.09.075. PubMed: 21414537.

15. Bargnoux AS, Klouche K, Fareh J, Barazer I, Villard-Saussine S et al. (2008) Prohormone brain natriuretic peptide (proBNP), BNP and N-terminal-proBNP circulating levels in chronic hemodialysis patients. Correlation with ventricular function, fluid removal and effect of hemodiafiltration. Clin Chem Lab Med 46: 1019-1024. PubMed: 18624622.

16. Macheret F, Boerrigter G, McKie P, Costello-Boerrigter L, Lahr B et al. (2011) Pro-B-type natriuretic peptide(1-108) circulates in the general community: plasma determinants and detection of left ventricular dysfunction. J Am Coll Cardiol 57: 1386-1395. doi:https://doi.org/10.1016/S0735-1097(11)61386-7. PubMed: 21414536.

17. Miller WL, Grill DE, Jaffe AS (2012) Comparison of novel pro-BNP(1-108) and standard BNP assays in heart failure patients. Clin Chim Acta 413: 920-926. doi:https://doi.org/10.1016/j.cca.2012.02.007. PubMed: 22366122.

18. Liang F, O’Rear J, Schellenberger U, Tai L, Lasecki M et al. (2007) Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 49: 1071-1078. doi:https://doi.org/10.1016/j.jacc.2006.10.063. PubMed: 17349887.

19. Lam CS, Burnett JC Jr., Costello-Boerrigter L, Rodeheffer RJ, Redfield MM (2007) Alternate circulating pro-B-type natriuretic peptide and B-type natriuretic peptide forms in the general population. J Am Coll Cardiol 49: 1193-1202. doi:https://doi.org/10.1016/j.jacc.2006.12.024. PubMed: 17367664.

20. Schellenberger U, O’Rear J, Guzzetta A, Jue RA, Protter AA et al. (2006) The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 451: 160-166. doi:https://doi.org/10.1016/j.abb.2006.03.028. PubMed: 16750161.

21. Semenov AG, Postnikov AB, Tamm NN, Seferian KR, Karpova NS et al. (2009) Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 55: 489-498. doi:https://doi.org/10.1373/clinchem.2008.113373. PubMed: 19168558.

22. Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV et al. (2008) Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem 54: 866-873. doi:https://doi.org/10.1373/clinchem.2007.100040. PubMed: 18339697.

23. Hawkridge AM, Heublein DM, Bergen HR 3rd, Cataliotti A, Burnett JC Jr. et al. (2005) Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Natl Acad Sci U S A 102: 17442-17447. doi:https://doi.org/10.1073/pnas.0508782102. PubMed: 16293687.