Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Nishikimi, Toshio; Okamoto, Hiroyuki; Nakamura, Masahiro; Ogawa, Naoko; Horii, Kazukiyo; Nagata, Kiyoshi; Nakagawa, Yasuaki; Kinoshita, Hideyuki; Yamada, Chinatsu; Nakao, Kazuhiro; Minami, Tetsuto; Kuwabara, Yoshihiro; Kuwahara, Koichiro; Masuda, Izuru; Kangawa, Kenji; Minamino, Naoto; Nakao, Kazuwa

doi:10.1371/journal.pone.0053233

Cited by 56 publications

(62 citation statements)

References 18 publications

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“…The following materials were used in this study: synthetic BNP 1-32 from Bachem (5 ); BNP-specific monoclonal antibody (mAb) KY-BNP-II (epitope 14 -21) from Shionogi (13 ); BNP-specific mAb 24C5 (epitope [11][12][13][14][15][16][17], 50E1 (epitope 26 -32), Ab-BNP2 (specific for immune complex of BNP/proBNP with mAb 24C5), human recombinant proBNP (expressed in Escherichia coli), and human recombinant glycosylated proBNP (expressed in mammalian cells) from HyTest (5,14 ); and recombinant human neprilysin [expressed in CHO (Chinese hamster ovary) cells] from R&D systems (15 ).…”

Section: Methodsmentioning

confidence: 99%

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter

Semenov

Katrukha

2016

Clinical Chemistry

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BACKGROUND:Protease neprilysin is known to be responsible for the degradation of natriuretic peptides. A recent heart failure (HF) drug, LCZ696 (Entresto TM ), that combines a neprilysin inhibitor and an angiotensin II receptor inhibitor was suggested to augment circulating B-type natriuretic peptide (BNP) concentrations, making the results of BNP measurements diagnostically ambiguous. Because the main form of measured BNP in HF patients is represented by its uncleaved precursor, proBNP, it is important to know the susceptibility of proBNP to cleavage by neprilysin.

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Section: Methodsmentioning

confidence: 99%

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter

Semenov

Katrukha

2016

Clinical Chemistry

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“…A blood sample was immediately transferred to chilled disposable tubes containing aprotinin (500 kallikrein inactivator units/ ml) and EDTA (1.5 mg/ml) and centrifuged at 4°C, and the plasma was frozen and stored at Ϫ80°C until required for assay. ProBNP and total BNP (BNP ϩ proBNP) in plasma from control and heart failure patients and in culture medium conditioned by proBNP-transfected myocytes were measured using our recently developed direct immunochemiluminescent assay for proBNP and total BNP (16). This is a two-step immunochemiluminescent assay for total BNP and proBNP using monoclonal antibodies and glycosylated proBNP as a standard.…”

Section: Methodsmentioning

confidence: 99%

Pro-B-type natriuretic peptide is cleaved intracellularly: impact of distance between O-glycosylation and cleavage sites

Nishikimi

Nakagawa

Minamino

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

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-We investigated the molecular mechanism underlying the processing of pro-B-type natriuretic peptide (proBNP). Rat neonatal atrial and ventricular myocytes were cultured separately. We examined the molecular forms of secreted and intracellular BNP in atrial and ventricular myocytes; levels of corin and furin mRNA in atrial and ventricular myocytes; the effect their knockdown on proBNP processing; plasma molecular forms of BNP from rats and humans with and without heart failure; and the impact of the distance between the glycosylation and cleavage sites in wild-type and mutant human proBNP, expressed in rat myocytes transfected with lentiviral vectors. BNP was the major molecular form secreted by atrial and ventricular myocytes. Transfection of furin siRNA reduced proBNP processing in both atrial and ventricular myocytes; however, transfection of corin siRNA did not reduce it. BNP was the major molecular form in rat plasma, whereas proBNP was the major form in human plasma. The relative fraction of human BNP in rat myocytes expressing human proBNP was about 60%, but increasing the distance between the glycosylation and cleavage sites through mutation, increased the processed fraction correspondingly. These results suggest that proBNP is processed into BNP intracellularly by furin. The level of proBNP processing is lower in humans than rats, most likely due to the smaller distance between the O-glycosylation and cleavage sites in humans.B-type natriuretic peptide; atrial natriuretic peptide; furin; processing; corin B-TYPE (ALSO KNOWN AS BRAIN) NATRIURETIC PEPTIDE (BNP) is a cardiac hormone mainly expressed in the ventricles (14). Ventricular expression of BNP increases in response to pressure overload, volume overload, and ischemic injury, and plasma BNP levels increase in proportion to disease severity in patients with heart failure, myocardial infarction, pulmonary hypertension, or cardiac hypertrophy (13). Whereas tissue concentrations of BNP are much higher in the atria than the ventricles (1), and BNP secreted from the atria is also important in cases of atrial fibrillation and atrial overload (6). The precursor proBNP is believed to be cleaved to BNP and NH 2 -terminal proBNP, as it is secreted from myocytes (13), and both molecules have been used for more than a decade as biochemical markers of heart failure. However, several recent studies have shown that in addition to BNP and NH 2 -terminal proBNP, levels of uncleaved proBNP are also greatly increased in heart failure (15,23,28).The precise mechanism by which proBNP is processed is not fully understood at present. Specifically, the molecular form of BNP within cells, the mechanism of proBNP processing, the enzymes catalyzing proBNP processing, whether the patterns of BNP secretion differ between the atria and ventricles, and whether the pattern of BNP secretion differs from that of atrial natriuretic peptide (ANP) all remain poorly understood. Interestingly, recombinant proBNP derived from mammalian cells has seven sites of O-linked oligosaccharide atta...

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“…However, in contrast to the previous model of all proBNP being converted to BNP and NT-proBNP in a 1:1 ratio before release, the revised model suggests that glycosylated proBNP is also released into the circulation (5,6 ). Still, previous studies have found several-fold higher circulating NT-proBNP concentrations compared with proBNP concentrations in the same individuals (6,7 ), so immunoassays that bind to the N-terminal part of proBNP will mainly reflect NTproBNP concentrations. The stability of intracellular proBNP has been attributed to glycosylation.…”

confidence: 70%

“…Hence, early data indicated that proBNP 1-108 is also released into plasma. This has since been validated (6,22 ), although data from other groups have revealed several-fold higher circulating NT-proBNP concentrations compared with proBNP concentrations in the same individuals (6,7 ). Accordingly, assays that bind to the N-terminal part of proBNP 1-108 will mainly reflect NT-proBNP concentrations.…”

Section: Discussionmentioning

confidence: 94%

“…Still, we believe our data are valuable, as alternative approaches to detect glycosylated NTproBNP can be developed in the future. To this end, we and other groups have published data that indicate that novel assays can be designed to circumvent the influence by posttranslational modifications to proBNP peptide measurements (7,27 ). Nevertheless, given the extensive data already available for the diagnostic and prognostic value of the established NT-proBNP assays and the easy use of the established assays for B-type natriuretic peptide measurements in large automatic systems, additional data providing a clear diagnostic and prognostic advantage of deglycosylated NT-proBNP are needed before any change of current practice can be recommended.…”

Section: Discussionmentioning

confidence: 99%

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Influence of Glycosylation on Diagnostic and Prognostic Accuracy of N-Terminal Pro–B-Type Natriuretic Peptide in Acute Dyspnea: Data from the Akershus Cardiac Examination 2 Study

et al. 2015

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BACKGROUND The N-terminal part of pro–B-type natriuretic peptide (NT-proBNP) is glycosylated, but whether glycosylation influences the diagnostic and prognostic accuracy of NT-proBNP measurements is not known. METHODS We measured NT-proBNP concentrations of 309 patients with acute dyspnea by use of standard EDTA tubes and EDTA tubes pretreated with deglycosylation enzymes. The primary cause of dyspnea was classified as heart failure (HF) or non-HF, and the diagnosis was adjudicated by 2 independent physicians. We collected information on all-cause mortality during follow-up. RESULTS In all, 142 patients (46%) were diagnosed with HF. NT-proBNP concentrations in nondeglycosylated samples distinguished HF patients from patients with non-HF related dyspnea [median 3588 (quartiles 1–3 1578–8404) vs 360 (126–1139) ng/L, P < 0.001], but concentrations were markedly higher in samples pretreated with deglycosylation enzymes (total NT-proBNP) [7497 (3374–14 915) vs 798 (332–2296) ng/L, P < 0.001]. The AUC to separate HF patients from patients with non-HF related dyspnea was 0.871 (95% CI 0.829–0.907) for total NT-proBNP compared with 0.852 (0.807–0.890) for NT-proBNP measurements in standard EDTA plasma. During a median follow-up of 816 days, 112 patients (36%) died. Both NT-proBNP and total NT-proBNP concentrations were associated with mortality in separate multivariate models, but only total NT-proBNP concentrations provided added value to the basic risk model of our dataset as assessed by the net reclassification index: 0.24 (95% CI 0.003–0.384). There was a graded increase in risk across total NT-proBNP quartiles, in contrast with the results for NT-proBNP measurements. CONCLUSIONS NT-proBNP concentrations were higher, and diagnostic and prognostic accuracy was improved, by pretreating tubes with deglycosylation enzymes.

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Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Cited by 56 publications

References 18 publications

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter

Pro-B-type natriuretic peptide is cleaved intracellularly: impact of distance between O-glycosylation and cleavage sites

Influence of Glycosylation on Diagnostic and Prognostic Accuracy of N-Terminal Pro–B-Type Natriuretic Peptide in Acute Dyspnea: Data from the Akershus Cardiac Examination 2 Study

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