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Background: Entresto™ is a new heart failure (HF) therapy that includes the neprilysin (NEP) inhibitor sacubitril. One of the NEP substrates is B-type natriuretic peptide (BNP); its augmentation by NEP inhibition is considered as a possible mechanism for the positive effects of Entresto. We hypothesized that the circulating products of BNP proteolysis by NEP might reflect NEP impact on the metabolism of active BNP. We suggest that NEP-based BNP cleavage at position 17-18 results in BNP ring opening and formation of a novel epitope with C-terminal Arg-17 (BNP-neo17 form). In this study, we use a specific immunoassay to explore BNP-neo17 in a rat model and HF patient plasma., Methods: We injected BNP into rats, with or without NEP inhibition with sacubitril. BNP-neo17 in plasma samples at different time points was measured with a specific immunoassay with neglectable cross-reactivity to intact forms. BNP-neo17 and total BNP were measured in EDTA plasma samples of HF patients., Results: BNP-neo17 generation in rat circulation was prevented by NEP inhibition. The maximum 13.2-fold difference in BNP-neo17 concentrations with and without sacubitril was observed at 2 min after injection. BNP-neo17 concentrations in 32 HF patient EDTA plasma samples ranged from 0 to 37 pg/mL (median, 5.4; interquartile range, 0-9.1). BNP-neo17/total BNP had no correlation with total BNP concentration (with r = -0.175, P = 0.680) and showed variability among individuals., Conclusions: BNP-neo17 formation is NEP dependent. Considering that BNP-neo17 is generated from the active form of BNP by NEP, we speculate that BNP-neo17 may reflect both the NEP activity and natriuretic potential and serve for HF therapy guidance., (© 2019 American Association for Clinical Chemistry.)

Background: In the blood of patients with acute myocardial infarction (AMI), cardiac troponin I (cTnI) presents as an intact molecule with a repertoire of proteolytic fragments. The degradation of cTnI might negatively influence its precise immunodetection. In this study we identified cTnI fragments and calculated their ratio in the blood of patients at different times after AMI to discriminate the most stable part(s) of cTnI., Methods: Serial serum samples were collected from AMI patients within 1 to 36 h after the onset of chest pain both before and after stenting. cTnI and its fragments were immunoextracted from serum samples and analyzed by Western blotting with monoclonal antibodies (mAbs) specific to the different epitopes of cTnI and by 2 in-house immunoassays specific to the central and terminal portions of cTnI., Results: Intact cTnI and its 11 major fragments were detected in blood of AMI patients. The ratio of the fragments in serial samples did not show large changes in the period 1-36 h after AMI. mAbs specific to the epitopes located approximately between amino acid residues (aar) 34 and 126 stained all extracted cTnI. mAbs specific to aar 23-36 and 126-196 recognized approximately 80% to 90% (by abundance) of cTnI., Conclusions: In addition to mAbs specific to the central part of cTnI (approximately aar 34-126), antibodies specific to the adjacent epitopes (approximately aar 23-36 and 126-196) could be used in assays because they recognize ≥80% of cTnI in patients' blood samples within the first 36 h after AMI., (© 2018 American Association for Clinical Chemistry.)

Background: Circulating B-type natriuretic peptide (BNP) is widely accepted as a diagnostic and risk assessment biomarker of cardiac function. Studies suggest that there are significant differences in measured concentrations among different commercial BNP immunoassays. The purpose of our study was to compare BNP-related proteins to determine a form that could be used as a common calibrator to improve the comparability of commercial BNP immunoassay results., Methods: BNP was measured in 40 EDTA-plasma samples from acute and chronic heart failure patients using five commercial BNP assays: Alere Triage, Siemens Centaur XP, Abbott I-STAT, Beckman Access2 and ET Healthcare Pylon. In parallel with internal calibrators from each manufacturer, six preparations containing BNP 1-32 motif a) synthetic BNP, b) recombinant BNP (E. coli), c) recombinant nonglycosylated proBNP (E. coli), d) recombinant His-tagged (N-terminal) nonglycosylated proBNP (E. coli), e) recombinant glycosylated proBNP (HEK cells), and f) recombinant glycosylated proBNP (CHO cells) were also used as external calibrators for each assay., Results: Using the internal standards provided by manufacturers and for five of six external calibrators, up to 3.6-fold differences (mean 1.9-fold) were observed between BNP immunoassays (mean between-assay CV 24.5-47.2%). A marked reduction of the between-assay variability was achieved, when glycosylated proBNP expressed in HEK cells was used as the common calibrator for all assays (mean between-assay CV 14.8%)., Conclusions: Our data suggest that recombinant glycosylated proBNP could serve as a common calibrator for BNP immunoassays to reduce between-assay variability and achieve better comparability of BNP concentrations of commercial BNP immunoassays., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)