A Mixture of Glycosylated proBNP and BNP-32 Is a Suitable Calibrator for BNP Immunoassays

Keiichi Masuta,Yasuaki Nakagawa,Masayo Igarashi,Toshio Nishikimi,Norio Ota,Kenzo Funatsuki,Kazukiyo Horii,Izuru Masuda,Hiroshi Nishi,Youko Inoue

doi:10.4236/ojcd.2019.92004

Abstract

Aim: Measurement of B-type natriuretic peptide (BNP) is widely used as a diagnostic and risk assessment tool for cardiovascular disease. Recent studies have demonstrated that BNP-32 and its precursor proBNP circulate in the blood stream, and that most commercial BNP immunoassays measure both forms. However, recombinant or synthetic BNP-32 is used as the standard for those BNP immunoassays. This gap between clinical samples and the standard might be a potential source of variation in BNP measurements among assays. The purpose of this study is to validate a more suitable calibrator for BNP immunoassays. Methods: External BNP calibrators containing both BNP-32 and glycosylated proBNP were prepared at two concentration levels. Target BNP concentrations of the low and high levels were 40 and 160 pg/mL, respectively. And to reflect clinical samples, the molar ratios of BNP-32 to glycosylated proBNP in these concentration levels were adjusted to 50:50 and 25:75, respectively. BNP concentrations of plasma samples along with the external BNP calibrators were measured at two commercial labs and using an automated analyzer MI02 Shionogi® BNP (MI02). These samples and the calibrators were also measured using an immunoradiometric assay (IRMA) as a standard assay procedure. Concentrations of the plasma samples measured at the labs or using the MI02 were adjusted according to a comparison of the measured concentrations of the external BNP calibrators with the IRMA. Results: After measured concentrations of the plasma samples were adjusted using the external BNP calibrators, the correlation between each measurement and the IRMA was improved. The range of the slopes according to Passing-Bablok regression analysis narrowed from 0.628 - 0.955 to 0.911 - 1.005. Conclusions: Our data suggests that a mixture of BNP-32 and glycosylated proBNP at different ratios by concentration level is suitable for calibration to minimize variations in BNP measurements among immunoassays.

Highlights

B-type natriuretic peptide (BNP) is a 32 amino acid peptide hormone produced by myocardium [1] [2]
Concentrations of the plasma samples measured at the labs or using the MI02 were adjusted according to a comparison of the measured concentrations of the external BNP calibrators with the immunoradiometric assay (IRMA)
All glycosylation sites are located on the NT-proBNP part of proBNP, and the glycosylation status of one amino acid, T71, is crucial for processing proBNP into NT-proBNP and BNP-32 due to the distance between the O-glycosylation and cleavage site [17] [18] [19]. These findings suggest that proBNP in circulation is O-glycosylated, including at amino acid T71, which is nearest to the cleavage site

Summary

Introduction

B-type natriuretic peptide (BNP) is a 32 amino acid peptide hormone produced by myocardium [1] [2]. In the last few decades, BNP has been successfully developed as a biomarker for various life sciences. In cardiovascular and related fields, BNP has become an indispensable biomarker in clinical practice along with troponins. Screening BNP concentrations can establish risk for patients bearing symptoms that imply HF. Various trials have established the use of BNP measurements for other diagnoses and treatments including dyspnea, cardiogenic brain embolism, and general medical wellness [4] [5]. Clinical guidelines recommend measuring BNP to aid in the diagnosis of HF and other diseases and to monitor disease progression [6] [7]

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