title: Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides.
abstract: 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.
authors.affiliation: Department of Physiology, Biocenter Oulu, University of Oulu, Finland.
authors.forename: Minna
authors.initials: M
authors.lastname: Ala-Kopsala
authors.forename: Jarkko
authors.initials: J
authors.lastname: Magga
authors.forename: Keijo
authors.initials: K
authors.lastname: Peuhkurinen
authors.forename: Jaana
authors.initials: J
authors.lastname: Leipälä
authors.forename: Heikki
authors.initials: H
authors.lastname: Ruskoaho
authors.forename: Juhani
authors.initials: J
authors.lastname: Leppäluoto
authors.forename: Olli
authors.initials: O
authors.lastname: Vuolteenaho
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D009320:Atrial Natriuretic Factor / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D009203:Myocardial Infarction / Q000097:blood*; D020097:Natriuretic Peptide, Brain / Q000097:blood*; D009419:Nerve Tissue Proteins / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood* / Q000138:chemical synthesis; D011498:Protein Precursors / Q000097:blood / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D018709:Statistics, Nonparametric
chemical_list: D015415:Biomarkers; D000939:Epitopes; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain; D009320:Atrial Natriuretic Factor