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.

Full-Size Cardiac Troponin I and Its Proteolytic Fragments in Blood of Patients with Acute Myocardial Infarction: Antibody Selection for Assay Development.

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.

Human pro-B-type natriuretic peptide is processed in the circulation in a rat model.

BACKGROUND: The appearance of B-type natriuretic peptide (BNP) in the blood is ultimately caused by proteolytic processing of its precursor, proBNP. The mechanisms leading to the high plasma concentration of unprocessed proBNP are still poorly understood. The goals of the present study were to examine whether processing of proBNP takes place in the circulation and to evaluate the clearance rate of proBNP and proBNP-derived peptides. METHODS: We studied the processing of human proBNP in the circulation and the clearance rate of proBNP and proBNP-derived peptides (BNP and N-terminal fragment of proBNP, NT-proBNP) in rats by injecting the corresponding peptides and analyzing immunoreactivity at specific time points. Glycosylated and nonglycosylated proBNP and NT-proBNP were used in the experiments. We applied immunoassays, gel filtration, and mass spectrometry (MS) techniques to analyze the circulation-mediated processing of proBNP. RESULTS: ProBNP was effectively processed in the circulation into BNP (1-32) and various truncated BNP forms as confirmed by gel filtration and MS analysis. Glycosylation of proBNP close to the cleavage-site region suppressed its processing in the circulation. The terminal half-life for human glycosylated proBNP was 9.0 (0.5) min compared with 6.4 (0.5) min for BNP. For NT-proBNP, the terminal half-lives were 15.7 (1.4) min and 15.5 (1.3) min for glycosylated and nonglycosylated forms, respectively. CONCLUSIONS: In rats, processing of human proBNP to active BNP occurs in the circulation. The clearance rate of proBNP is quite similar to that of BNP. These observations suggest that peripheral proBNP processing may be an important regulatory step rather than mere degradation.

Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases.

BACKGROUND: B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. METHODS: We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. RESULTS: Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1-32, whereas corin-mediated cleavage led to the production of BNP 4-32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. CONCLUSIONS: Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.

Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site.

BACKGROUND: Processing of the brain natriuretic peptide (BNP) precursor, proBNP, is a convertase-dependent reaction that produces 2 molecules--the active BNP hormone and the N-terminal part of proBNP (NT-proBNP). Although proBNP was first described more than 15 years ago, very little is known about the cellular mechanism of its processing. The study of proBNP processing mechanisms is important, because processing impairments could be associated with the development of heart failure (HF). METHODS: The biochemical properties of recombinant proBNP and NT-proBNP and the same molecules derived from the blood of HF patients were analyzed by gel-filtration chromatography, site-directed mutagenesis, and different immunochemical methods with a panel of monoclonal antibodies (MAbs). RESULTS: Part of the proBNP molecule (amino acid residues 61-76) located near the cleavage site was inaccessible to specific MAbs because of the presence of O-glycans, whereas the same region in NT-proBNP was completely accessible. We demonstrated that a convertase (furin) could effectively cleave deglycosylated (but not intact) proBNP. Of several mutant proBNP forms produced in a HEK 293 cell line, only the T71A variant was effectively processed in the cell. CONCLUSIONS: Only proBNP that was not glycosylated in the region of the cleavage site could effectively be processed into BNP and NT-proBNP. Site-directed mutagenesis enabled us to ascertain the unique suppressing role of T71-bound O-glycan in proBNP processing.

Novel immunoassay for quantification of brain natriuretic peptide and its precursor in human blood.

BACKGROUND: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis. METHODS: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11-22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5. RESULTS: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the "single-epitope sandwich assay"), which requires only a short BNP fragment (fragment 11-22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays. CONCLUSIONS: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.

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.

Diagnostic utility of a single-epitope sandwich B-type natriuretic peptide assay in stable coronary artery disease: data from the Akershus Cardiac Examination (ACE) 1 Study.

OBJECTIVES: To assess the merit of a novel single-epitope sandwich (SES) assay specific to the stable part of BNP in patients with reversible myocardial ischemia as post-translational modifications of BNP may influence assay performance. DESIGN AND METHODS: We measured BNP concentration by a conventional assay and the SES-BNP assay in 198 patients referred for myocardial perfusion imaging (MPI). BNP concentration was determined before and immediately after exercise stress testing, and 1.5 and 4.5h later. Patients were categorized according to MPI results. RESULTS: BNP concentration was higher with both assays at all time points in patients with reversible myocardial ischemia (n=19) compared to the other patients (n=179). Measuring BNP after stress testing or calculating the changes in BNP concentration did not improve diagnostic accuracy compared to baseline measurements: SES-BNP: AUC 0.71 (95% CI 0.58-0.84) vs. conventional BNP: 0.71 (0.59-0.83), p=0.96. By linear regression analysis, reversible myocardial ischemia was significantly associated with baseline SES-BNP concentration (p=0.043), but not with measurements by the conventional assay (p=0.089). In multivariate logistic regression models, only baseline measurement with the SES-BNP assay was significantly associated with reversible myocardial ischemia: odds ratio [logarithmical transformed BNP] 2.00 (95% CI 1.16-3.47), p=0.013. The SES-BNP assay, but not the conventional BNP assay, reclassified a significant proportion of the patients towards their correct category on top of the best clinical model of our data set: NRI=0.47, p=0.04. CONCLUSIONS: The SES-BNP assay was significantly associated with reversible myocardial ischemia as assessed by several statistical indices, while a conventional BNP assay was not.

Measurement of B-type natriuretic peptide by two assays utilizing antibodies with different epitope specificity.

OBJECTIVES: To compare plasma BNP values determined by a conventional-type and a novel-type of BNP assays. DESIGN AND METHODS: Plasma samples (n=94) from HF patients were analyzed by the novel-type "Single Epitope Sandwich" (SES assay prototype) and the conventional-type Siemens ADVIA Centaur BNP assays. Both assays were calibrated using recombinant proBNP (expressed in E. coli). RESULTS: The SES assay measured 1.2- to 7.2-fold (2.1±0.9; mean, SD) greater BNP concentrations compared to the Siemens assay. A subset of six samples (6.4%) demonstrated the largest (3- to 7.2-fold higher) difference. CONCLUSIONS: The SES prototype assay appears to more accurately measure the absolute concentrations of BNP immunoreactive forms.

Immunodetection of glycosylated NT-proBNP circulating in human blood.

BACKGROUND: Brain natriuretic peptide (BNP) or NT-proBNP (N-terminal fragment of BNP precursor) measurements are recommended as aids in diagnosis and prognosis of patients with heart failure. Recently it has been shown that proBNP is O-glycosylated in human blood. The goal of this study was to map sites on the NT-proBNP molecule that should be recognized by antibodies used in optimal NT-proBNP assays. METHODS: We analyzed endogenous NT-proBNP by several immunochemical methods using a broad panel of monoclonal antibodies specific to different epitopes of the NT-proBNP molecule. RESULTS: Treatment of endogenous NT-proBNP by a mixture of glycosidases resulted in significant improvement of the interaction between deglycosylated NT-proBNP and monoclonal antibodies (MAbs) specific to the mid-fragment of the molecule. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) were able to recognize glycosylated and deglycosylated protein with similar efficiency. CONCLUSIONS: The central part of endogenous NT-proBNP is glycosylated, making it almost "invisible" for the antibodies specific to the mid-fragment of the molecule. Thus sandwich assays using even one antibody (poly- or monoclonal) specific to the central part of the molecule could underestimate the real concentration of endogenous NT-proBNP. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) are the best candidates to be used in an assay for optimal NT-proBNP immunodetection.

The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure.

BACKGROUND: Peptides derived from brain natriuretic peptide (BNP) precursor (proBNP), BNP, and the N-terminal fragment of proBNP (NT-proBNP) are used as biomarkers of heart failure. It remains unclear which forms of these peptides circulate in blood and which forms are measured by assays for these natriuretic peptides. METHODS: To design assays for immunodetection of proBNP, NT-proBNP, and BNP, we used a panel of BNP- and NT-proBNP-specific monoclonal antibodies (MAbs). All MAbs were tested in 2-site combinations in time-resolved fluoroimmunoassays with recombinant or synthetic antigens and plasma from heart failure (HF) patients. ProBNP and related molecules were assayed in HF plasma samples and plasma extracts by means of gel filtration fast protein liquid chromatography (FPLC) before and after protein fractionation on Sep-Pak C18 cartridges. RESULTS: The limits of detection for BNP, proBNP, and NT-proBNP assays were 0.4, 3, and 10 ng/L, respectively. Gel filtration-FPLC studies revealed 1 peak of NT-proBNP (approximately 25 kDa), 1 peak of proBNP (approximately 37 kDa), and 2 peaks of BNP immunoreactivity, a major peak (approximately 37 kDa) for proBNP, and a minor peak (approximately 4 kDa) for BNP. In patient plasma, the molar concentration of NT-proBNP was almost 10 times that of proBNP. The mean proBNP:BNP ratio in patient plasma was 6.3, ranging from 1.8 to 10.8. CONCLUSIONS: ProBNP is the major BNP-immunoreactive form in human blood. The proBNP:BNP ratio in plasma samples is dependent on the methods used for sample handling and for the measurement of the peptides.