Diagnostic utility of total NT-proBNP testing by immunoassay based on antibodies targeting glycosylation-free regions of NT-proBNP.

OBJECTIVES: The N-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) is a widely used heart failure (HF) biomarker. Commercial NT-proBNP immunoassays detect only a subfraction of endogenous NT-proBNP, as the antibodies target a region of NT-proBNP that could be glycosylated at Ser44. The diagnostic utility of immunoassays measuring total NT-proBNP remains unclear. METHODS: NT-proBNP was measured in 183 HF and 200 non-HF patients diagnosed by two independent cardiologists blinded to NT-proBNP results. Plasma samples either non-treated or treated with a mixture of glycosidases were analyzed by the Elecsys proBNP II assay (Roche Diagnostics, based on antibodies targeting a glycosylated region of NT-proBNP) and the SuperFlex NT-proBNP assay (PerkinElmer, based on antibodies targeting regions of NT-proBNP that are free of O-glycans). The diagnostic accuracy of the two assays was analyzed by comparison of ROC curves. RESULTS: The ROC-AUC for the proBNP II assay was 0.943 (95% CI 0.922-0.964) for NT-proBNP measured in untreated samples and 0.935 (0.913-0.958) for NT-proBNP measured in glycosidase-treated samples. The SuperFlex NT-proBNP assay in untreated samples gave a ROC-AUC of 0.930 (95% CI 0.907-0.954). The median percentage of non-glycosylated NT-proBNP to total NT-proBNP was 1.5-1.6-fold lower in the non-HF group compared to that in the HF group. CONCLUSIONS: The clinical value of total NT-proBNP for HF diagnosis was similar to the subfraction of NT-proBNP that was non-glycosylated at Ser44. The lower percentage of non-glycosylated NT-proBNP to total NT-proBNP in non-HF patients suggests that total NT-proBNP might be more sensitive in individuals without current or prior symptoms of HF.

Detection of Neprilysin-Derived BNP Fragments in the Circulation: Possible Insights for Targeted Neprilysin Inhibition Therapy for Heart Failure.

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.

Standardization of BNP and NT-proBNP Immunoassays in Light of the Diverse and Complex Nature of Circulating BNP-Related Peptides.

Brain natriuretic peptide (BNP) and the N-terminal fragment of the BNP precursor (NT-proBNP) are widely used as heart failure (HF) biomarkers. Since the discovery of BNP in 1988, much effort has been allocated to the precise detection of BNP and NT-proBNP levels for reliable HF diagnostics. As a result, measurements of these biomarkers are globally accepted and used in clinical practice for the diagnosis of acute and chronic HF, risk stratification, and monitoring response to therapy. Several immunoassays specific for BNP and NT-proBNP are currently commercially available. Recent comparative studies show that there are marked differences between different BNP and NT-proBNP assays and platforms, and the results of measurements are not comparable enough. The lack of equivalence between the assays complicates the interpretation of the results and renders the cut-off points for diagnostic decisions to be method dependent. Presently, there is no agreement on what kind of BNP or NT-proBNP standard should be used for calibration, and a certified reference material as well as reference measurement procedures are lacking. The aim of this chapter is to summarize the available data on the complex nature of BNP-related peptides, specificity for existing BNP and NT-proBNP immunoassays, and to discuss potential approaches for standardization of BNP and NT-proBNP measurements.

Searching for a BNP standard: Glycosylated proBNP as a common calibrator enables improved comparability of commercial BNP immunoassays.

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.

Characterization of endogenously circulating IGFBP-4 fragments-Novel biomarkers for cardiac risk assessment.

BACKGROUND: Recent findings show that circulating N- and C-terminal fragments of IGF-binding protein-4 (NT-IGFBP-4 and CT-IGFBP-4) can be utilized as biomarkers for cardiac risk assessment in acute coronary syndrome (ACS) patients. The fragments are thought to be the products of pregnancy-associated plasma protein A (PAPP-A)-dependent proteolysis. Two immunoassays for the measurement of IGFBP-4 fragments have been proposed. However, properties of the endogenous IGFBP-4 fragments that could influence the performance of the immunoassays were still not investigated. METHODS: NT- and CT-IGFBP-4 were extracted from pooled ACS plasma using affinity purification, and their concentrations were measured using sandwich immunoassays utilizing antibodies specific to their proteolytic neo-epitopes or internal epitopes. The extracted fragments were characterized by Western blots (WB) and mass-spectrometry. ACS plasma samples were analyzed by size exclusion chromatography (SEC). RESULTS: Immunoassays utilizing the neo-epitope-specific and the internal epitope-specific antibodies measured equal concentrations of the analyte in the endogenous IGFBP-4 fragments preparations. Only the 18 kDa NT-IGFBP-4 and 14 kDa CT-IGFBP-4 were detected in the WB analysis. Using mass-spectrometry, peaks corresponding to intact non-truncated and non-modified NT-IGFBP-4 (14626 Da) and CT-IGFBP-4 (11346 Da) were observed. The absence of complexed forms of IGFBP-4 in patients' plasma was demonstrated using SEC. CONCLUSIONS: Endogenous NT- and CT-IGFBP-4 from ACS patients' plasma correspond to the PAPP-A-derived IGFBP-4 fragments and do not undergo any truncation, modification, or complex formation in the patients' blood. Because of the demonstrated intact state of the circulating IGFBP-4 fragments, the neo-epitope-specific immunoassays perform reliably, allowing further clinical validation of these novel biomarkers.