Molecular Heterogeneity Has a Major Impact on the Measurement of Circulating N-Terminal Fragments of A- and B-Type Natriuretic Peptides

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Abstract
The N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) are powerful markers of cardiac function. The current assays require refinement with regard to standardization with native calibrators and the ability to detect the actual circulating forms. The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1-108, and Tyr0-proBNP77-108. Fifteen peptides of 13-22 amino acids, spanning the sequences of NT-proANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC. According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1-76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30-40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r2 = 0.77-0.85). In patients with acute myocardial infarction, NT-proANP and NT-proBNP concentrations were 1.8-2.3 and 4.2-4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations. Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NT-proANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP.