Clinical chemistry · 2004
Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides.
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Ala-Kopsala Minna, Magga Jarkko, Peuhkurinen Keijo, Leipälä Jaana, Ruskoaho Heikki, Leppäluoto Juhani, Vuolteenaho Olli
Аннотация
BACKGROUND: The N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) are powerful markers of cardiac function. The current assays require refinement with regard to standardization with native calibrators and the ability to detect the actual circulating forms.
METHODS: The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1-108, and Tyr0-proBNP77-108. Fifteen peptides of 13-22 amino acids, spanning the sequences of NT-proANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC.
RESULTS: According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1-76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30-40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r2 = 0.77-0.85). In patients with acute myocardial infarction, NT-proANP and NT-proBNP concentrations were 1.8-2.3 and 4.2-4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations.
CONCLUSIONS: Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NT-proANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP.
METHODS: The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1-108, and Tyr0-proBNP77-108. Fifteen peptides of 13-22 amino acids, spanning the sequences of NT-proANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC.
RESULTS: According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1-76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30-40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r2 = 0.77-0.85). In patients with acute myocardial infarction, NT-proANP and NT-proBNP concentrations were 1.8-2.3 and 4.2-4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations.
CONCLUSIONS: Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NT-proANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP.
Предметные рубрики MeSH
AdultAgedAged, 80 and overAtrial Natriuretic FactorBiomarkersChromatography, High Pressure LiquidEpitopesFemaleHumansInfant, NewbornMaleMiddle AgedMyocardial InfarctionNatriuretic Peptide, BrainNerve Tissue ProteinsPeptide FragmentsProtein PrecursorsRadioimmunoassayRecombinant ProteinsReproducibility of ResultsStatistics, Nonparametric