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).
... 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, proBNP 1-108 , 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).