Characterization of Molecular Forms of N-Terminal B-Type Natriuretic Peptide In Vitro

Abstract

BACKGROUNDThe heterogeneity of circulating peptides may influence the interpretation of results from N-terminal profragment of BNP (NT-proBNP) assays. Our objective was to characterize the heterogeneity for better usability of the assays.METHODSEndogenous proBNP was purified from patient samples and treated with trifluoromethanesulfonic acid (chemical deglycosylation). The human proBNP gene was introduced into rat hearts by adenoviral transfer. Cell lysates and plasma samples containing proBNP-derived peptides were analyzed by chromatography. The fate of exogenous recombinant NT-proBNP added to fresh whole blood samples was followed by immunoassays and chromatography. The main NT-proBNP components were isolated and identified by mass spectrometry.RESULTSImmunoreactive NT-proBNP in human plasma comprised several molecular forms, as did circulating immunoreactive human NT-proBNP after adenoviral transfer of human proBNP cDNA into rat ventricular myocardium. Incubation of recombinant NT-proBNP1–76 in human plasma or serum resulted in multiple components with the 2 major components identified as NT-proBNP1–36 and NT-proBNP1–62/64. Profiling by different antisera and chromatography indicated masking of the non–mid-region epitopes likely due to formation of oligomers. More than 75% of the original immunoreactivity in the mid-region epitope was retained after 3-week storage of plasma samples at room temperature.CONCLUSIONSThere is marked heterogeneity in immunoreactive NT-proBNP in plasma not related to glycosylation. The mid-region epitope of NT-proBNP is stable even in harsh storage conditions. Careful choice of antibody epitopes can yield extraordinarily robust assays.