The Brain Natriuretic Peptide (BNP) Precursor Is the Major Immunoreactive Form of BNP in Patients with Heart Failure

Seferian, Karina R.; Tamm, Natalia N.; Semenov, Alexander G.; Mukharyamova, K.S.; Tolstaya, Anastasya A.; Koshkina, Ekaterina V.; Kara, A. N.; Krasnoselsky, Mihail I.; Apple, Fred S.; Esakova, Tatiana V.; Филатов, Владимир Владимирович; Katrukha, Alexey G.

doi:10.1373/clinchem.2006.076141

Cited by 138 publications

(120 citation statements)

References 9 publications

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“…For proBNP, this observation contradicts an earlier study [15] stating that proBNP is either a trimer or tetramer. Our data lend further support to the conclusion of [9] that the "high-molecularweight component" discussed previously [15,17] is unlikely to be an oligomer of NT-proBNP or proBNP. Interestingly, the four identified O-linked carbohydrate sites in HEK-proBNP have no obvious influence on either the secondary or quaternary structure of proBNP and presumably NT-proBNP as well.…”

Section: Resultssupporting

confidence: 86%

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A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

Crimmins

Kao

2008

Archives of Biochemistry and Biophysics

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The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general.A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays. Immunoassays utilize antibodies that react with specific analyte (usually protein) epitope(s). If these epitopes become unreactive due to say proteolytic processing, or masked by interaction with other molecule(s), or are post-or co-translationally modified, then the immunoassay can generate incorrect results. It becomes important then to identify the circulating forms of the analytes. This analysis can include primary sequence, post-translational modifications, and quaternary structure as homo-and hetero-interaction partners. Knowledge of such can aid in the development of immunoassays that accurately detect the circulating species. Keywords GlycosylatedPrevious characterization of human NT-proBNP whether as a recombinant product or synthetic peptide showed this N-terminal fragment to be a largely unfolded, flexible protein, termed an IUP, which exists as a monomer in solution [7,8]. No comparable data were hitherto available for proBNP and a recently discovered [9] glycosylated form of proBNP. Here we use a combination of CD to assess the global average secondary structure of proBNP and glycosylated-proBNP...

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Section: Resultssupporting

confidence: 86%

“…Several circulating versions of BNP or its processed fragments have been described [17]. These include BNP, proBNP, and NT-proBNP [16], plus a recently isolated glycosylated form of proBNP [9].…”

Section: Resultsmentioning

confidence: 99%

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

Crimmins

Kao

2008

Archives of Biochemistry and Biophysics

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“…Despite the 1:1 stoichiometric ratio, the molar plasma concentration of NT-proBNP is higher than the concentration of BNP likely because NTproBNP has slower clearance from the circulation (17 ). ProBNP in nonprocessed form is found in healthy individuals and patients with HF (18 ). ProBNP is posttranslationally modified in patients with HF by O-glycosylation at several threonine and serine residues within the N-terminal region (amino acid residues 1-76), but not within the BNP-portion of proBNP (amino acid residues 77-108) (19 ).…”

Section: Bnpmentioning

confidence: 99%

“…HyTest designed an assay based on a capture MAb specific for the region 26 -32 of the BNP peptide and a detection antibody specific for the fragment 13-20 of proBNP peptide (18 ). This highly sensitive immunoassay is not affected by the glycosylation of proBNP molecules, since the epitopes are not glycosylation sites.…”

Section: Probnp Immunoassaysmentioning

confidence: 99%

Natriuretic Peptides and Analytical Barriers

Vasile

Jaffe

2017

Clinical Chemistry

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BACKGROUND:The natriuretic peptide system is an endocrine, autocrine and paracrine system that plays an important role in the maintenance of cardiovascular homeostasis. Biomarkers based on these peptides are important diagnostic and prognostic tools for myocardial function.CONTENT: Although natriuretic peptides were discovered more than 2 decades ago, their intricate and complex biology is associated with important questions not yet elucidated. The diversity of circulating forms of natriuretic peptides, the distinct expression of these forms in particular patients, and the heterogeneity of heart failure forms, along with specific assay-related and preanalytic issues, cause assays to be poorly harmonized.

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“…Immunoassays targeting BNP are currently calibrated with recombinant or synthetic BNP and the samples distributed by most of the quality assurance schemes are plasma spiked with BNP. It is, however, now accepted that proBNP, 1-32 BNP and NT-proBNP are not the only forms circulating [9][10][11]. The correlation between a reference method specific for what believed to be the measurand and immunoassays becomes in this case the first mandatory step to evaluate the potential of reference measurement procedures to improve measurement accuracy and precision in clinical laboratories.…”

Section: Introductionmentioning

confidence: 99%

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

Torma¹,

Groves²,

Biesenbruch³

et al. 2017

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: B-type natriuretic peptide (BNP) is a 32 amino acid cardiac hormone routinely measured by immunoassays to diagnose heart failure. While it is reported that immunoassay results can vary up to 45%, no attempt of standardization and/or harmonization through the development of certified reference materials (CRMs) or reference measurement procedures (RMPs) has yet been carried out. Methods: B-type natriuretic peptide primary calibrator was quantified traceably to the International System of Units (SI) by both amino acid analysis and tryptic digestion. A method for the stabilization of BNP in plasma followed by protein precipitation, solid phase extraction (SPE) and liquid chromatography (LC) mass spectrometry (MS) was then developed and validated for the quantification of BNP at clinically relevant concentrations (15-150 fmol/g). Results: The candidate reference method was applied to the quantification of BNP in a number of samples from the UK NEQAS Cardiac Markers Scheme to demonstrate its applicability to generate reference values and to preliminary evaluate the commutability of a potential CRM. The

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The Brain Natriuretic Peptide (BNP) Precursor Is the Major Immunoreactive Form of BNP in Patients with Heart Failure

Cited by 138 publications

References 9 publications

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein

Natriuretic Peptides and Analytical Barriers

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

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