In-Depth Analysis of Molecular Heterogeneity of Circulating N-Terminal pro-BNP: Does Detailed Characterization of Analyte Structure Really Matter for Its Diagnostic Use?

Begin at the beginning, the King said, very gravely, “and go on till you come to the end: then stop.”—Alice's Adventures in Wonderland B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are the criterion biomarkers for diagnostic evaluation of heart failure (HF); both peptides are embedded in major medical societies’ guidelines and are used globally as useful and cost-effective biomarkers for the diagnosis, risk stratification, and therapy monitoring of acute and chronic HF (1). Immunoassays are used as the standard method of BNP and NT-proBNP detection in a clinical biochemistry laboratory. The first BNP and NT-proBNP immunoassays were developed in the mid-1990s. Since then, several assays utilizing various antibodies and diverse calibrator materials have been introduced into clinical practice. Initially, BNP and NT-proBNP were considered as well-defined analytes not exhibiting high molecular heterogeneity. However, our understanding of the complexity of circulating BNP-related forms came later than the introduction of most assays into clinical practice, thereby resulting in relatively poor comparability between different immunoassays and platforms and complicating the interpretation of results. BNP is a circulating bioactive peptide produced in the heart in response to increased wall stretch and volume overload (2, 3,). Active BNP molecules are released into the circulation along with physiological inert NT-proBNP as a result of endoproteolytic cleavage of a 108 amino acid prohormone, proBNP1-108, which is most likely mediated by furin (4,). Although BNP (32 amino acid residues [aar]) and NT-proBNP (76 aar) are released at a 1:1 ratio, the half-life of NT-proBNP in the circulation is longer than that of BNP, resulting in higher concentrations. During its maturation in cardiomyocytes, proBNP undergoes posttranslational modifications via O-glycosylation in the 1–76 amino acid region of the molecule (5,). Halfinger et al. characterized the exact glycosylation sites of endogenous proBNP and NT-proBNP and showed the coexistence of nonglycosylated forms in each site (6). BNP is present in the circulation as a mixture of multiple proteolytic forms (7,). An additional level of complexity arises from glycosylated proBNP, which is a predominant circulating BNP-immunoreactive form in both HF and healthy individuals (8, 9). However, commercial assays for BNP and NT-proBNP detect all fragments containing the epitope recognized by utilized antibodies. There is currently no commercially available BNP or NT-proBNP assay that does not cross-react with proBNP, owing to the presence of common epitopes. Both proBNP and NT-proBNP are represented by a mixture of glycosylated and nonglycosylated forms. The glycosylation of NT-proBNP/proBNP (designated as a mixture of NT-proBNP and proBNP, as both forms are present in the circulation) has been reported to interfere with immunodetection, as the presence of glycosidic residues at the epitopes recognized by antibodies results in weak binding of the antibodies to the modified regions (10, 11,). The current second-generation NT-proBNP assay from Roche Diagnostics is based on 2 monoclonal antibodies that recognize epitopes within amino acids 27–31 and 42–46 of NT-proBNP. Owing to the specificity of detection antibodies that bind to region 42–46, the assay can detect only a subfraction of endogenous NT-proBNP/proBNP, which is not glycosylated at serine 44 (12). Despite its specificity for a certain fraction of NT-proBNP/proBNP, this assay has a good clinical performance. Notably, glycosylation of circulating NT-proBNP/proBNP in the blood of HF patients is characterized by high interindividual variability. Further investigations are needed to determine whether the higher NT-proBNP/proBNP concentrations measured by immunoassays targeting nonglycosylated regions of NT-proBNP/proBNP are advantageous for distinguishing the initial stages or mild forms of HF (for example, in a population screening). Apart from glycosylation, the diversity of circulating NT-proBNP/proBNP forms may arise from the coexistence of forms generated by the activity of proteolytic enzymes in the circulation. Elucidation of protease-mediated forms of BNP has attracted more attention than NT-proBNP/proBNP owing to the effects of BNP proteolysis on the physiological activity of BNP. The corresponding proteases (i.e., neprilysin, insulin-degrading enzyme, and dipeptidyl-peptidase IV) involved in BNP degradation in the circulation have been investigated, and some attempts are currently ongoing to find a BNP form that provides the most realistic picture of patient dynamics under treatment with certain HF drugs (e.g., EntrestoTM) (13, 14). However, the detailed characterization of truncated NT-proBNP/proBNP forms has been lacking until recently. In this issue of Clinical Chemistry, Amplatz et al. describe important results that add a few more bricks to the wall of our current understanding of the molecular heterogeneity of NT-proBNP/proBNP circulating forms (15). The authors of the study describe 16 previously unknown fragments of NT-proBNP/proNP (9 located in the N-terminal and 7 in the C-terminal regions) present in the circulation, revealing a more profound degradation of NT-proBNP than previously known. Analysis of endogenous proteins that are present at very low concentrations and represented by a mixture of forms is known to be a laborious process requiring complex and sophisticated methodological approaches. The authors utilized immunoprecipitation with antibodies targeting the 1–21 region of NT-proBNP/proBNP, followed by high-resolution nano flow liquid chromatography electrospray mass spectrometric analysis. An elegant approach using 18O isotope labelling during proteolytic in-solution digestion to validate the specificity of the cleavage by the endoproteinase Lys-C should be specially highlighted. Moreover, this is the first report describing NT-proBNP/proBNP forms in healthy subjects, successfully overcoming the challenging conditions for detection due to very low analyte concentrations. The cutting-edge quantitative mass spectrometry-based analysis showed that certain forms of NT-proBNP/proBNP are more abundant than the others. These results provide a rationale for choosing ideal epitopes to be recognized by antibodies for developing next-generation diagnostic assays for clinical use. Considering the quite profound degradation at the N-terminus and the previously reported O-glycosylation sites, the authors suggest that the epitope ideally targeted by next-generation NT-proBNP immunoassays appears to be restricted to the region 16–35 aar, as it is not affected by degradation and O-glycans. Of special interest are 2 identified fragments cleaved at proline 78 and methionine 80. Such extended NT-proBNP fragments have not been previously described. They might indicate alternative processing of proBNP to generate BNP3-32 and BNP5-32, respectively. This may provide a new view on the generation of these truncated BNP forms in the circulation, not as products of BNP proteolysis, but as those of proBNP cleavage. The nature of proteases responsible for the cleavage of proBNP at these sites has yet to be revealed in future studies. There are several limitations of the study by Amplatz et al. that should be considered. For some reason, the authors did not perform separation of proBNP and NT-proBNP prior to mass spectrometric analysis, which could be accomplished by means of affinity matrix with BNP-specific antibodies to bind all molecules containing BNP-related epitopes. Because of this, it is not possible for readers to make any conclusions about any possible differences in N-terminal truncation between NT-proBNP and proBNP. The choice of antibodies specific to region 1–21 for immunoextraction of NT-proBNP/proBNP does not seem to be optimal for the efficient binding of all possible circulating fragments. Moreover, from the clinical perspective, it would be interesting to see if certain peptides might be more disease-specific than others (for instance, the C-terminal peptides ending at proline 78 and methionine 80). However, this lies beyond the scope of the study. What are the consequences of the findings of Amplatz and coworkers for routine diagnostic use of natriuretic peptides in clinical practice? Doubt could be expressed about whether such profound analysis of a well-established and widely used biomarker is actually needed. Considering how much research has already been conducted to elucidate the nature of circulating BNP and NT-proBNP, some may argue that it is already enough for clinical diagnostics needs. The answer to this question requires reference to how science works. Science is known to be a gradual process of never-ending updates that slowly push the boundaries of our knowledge and require our extra focus on the subject. As has been well exemplified by cardiac troponins, detailed characterization of an analyte’s structure and metabolism may take decades of research, ultimately resulting in novel assays with improved precision. Therefore, tiny pieces of data generated in numerous studies are absolutely critical for the development of highly sensitive and accurate assays for application in everyday practice, taking diagnostics to the next level of performance. Although all currently Food and Drug Administration-approved immunoassay platforms for NT-proBNP detection are designed in a similar way utilizing antibodies and calibrators manufactured by Roche Diagnostics, the situation may change with the introduction of novel NT-proBNP immunoassays based on antibodies with diverse specificity. Considering that HF is constantly increasing in prevalence, it can be expected that the number of NT-proBNP assays will increase to meet the growing demand in HF diagnostics. In particular, the introduction of point-of-care-testing assays may contribute to assay variability. Detailed characterization of an analyte’s structure and metabolism, as well as the possible factors that may impact the measurements, will be absolutely crucial to understand the reasons for potential discrepancies in the measurements obtained by different assays and platforms. The authors are to be congratulated for undertaking a thorough investigation of the molecular heterogeneity of NT-proBNP/proBNP circulating forms. The obtained data may help in making steps towards more sophisticated BNP/NT-proBNP-based diagnostics, and assay standardization. This study should encourage further research and may contribute to the development of optimized next-generation NT-proBNP/proBNP immunoassays. BNP, B-type natriuretic peptide; NT-proBNP, N-terminal proBNP; HF, heart failure; proBNP, BNP precursor; aar, amino acid residue. All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 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; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.