Characterization of endogenously circulating IGFBP-4 fragments-Novel biomarkers for cardiac risk assessment

Clin Biochem. 2015 Aug;48(12):774-80. doi: 10.1016/j.clinbiochem.2015.05.010. Epub 2015 May 27.

Abstract

Background: Recent findings show that circulating N- and C-terminal fragments of IGF-binding protein-4 (NT-IGFBP-4 and CT-IGFBP-4) can be utilized as biomarkers for cardiac risk assessment in acute coronary syndrome (ACS) patients. The fragments are thought to be the products of pregnancy-associated plasma protein A (PAPP-A)-dependent proteolysis. Two immunoassays for the measurement of IGFBP-4 fragments have been proposed. However, properties of the endogenous IGFBP-4 fragments that could influence the performance of the immunoassays were still not investigated.

Methods: NT- and CT-IGFBP-4 were extracted from pooled ACS plasma using affinity purification, and their concentrations were measured using sandwich immunoassays utilizing antibodies specific to their proteolytic neo-epitopes or internal epitopes. The extracted fragments were characterized by Western blots (WB) and mass-spectrometry. ACS plasma samples were analyzed by size exclusion chromatography (SEC).

Results: Immunoassays utilizing the neo-epitope-specific and the internal epitope-specific antibodies measured equal concentrations of the analyte in the endogenous IGFBP-4 fragments preparations. Only the 18 kDa NT-IGFBP-4 and 14 kDa CT-IGFBP-4 were detected in the WB analysis. Using mass-spectrometry, peaks corresponding to intact non-truncated and non-modified NT-IGFBP-4 (14626 Da) and CT-IGFBP-4 (11346 Da) were observed. The absence of complexed forms of IGFBP-4 in patients' plasma was demonstrated using SEC.

Conclusions: Endogenous NT- and CT-IGFBP-4 from ACS patients' plasma correspond to the PAPP-A-derived IGFBP-4 fragments and do not undergo any truncation, modification, or complex formation in the patients' blood. Because of the demonstrated intact state of the circulating IGFBP-4 fragments, the neo-epitope-specific immunoassays perform reliably, allowing further clinical validation of these novel biomarkers.

Keywords: Acute coronary syndrome (ACS); Biomarkers; Cardiovascular risk; IGF-binding protein-4 (IGFBP-4) fragments; Immunoassay; Ischemia; Major adverse cardiac events (MACE); Myocardial infarction (MI); Pregnancy-associated plasma protein A (PAPP-A).

MeSH terms

  • Aged
  • Aged, 80 and over
  • Biomarkers / blood
  • Cardiovascular Diseases / blood*
  • Female
  • Humans
  • Immunoassay / methods
  • Insulin-Like Growth Factor Binding Protein 4 / blood*
  • Male
  • Middle Aged
  • Peptide Fragments / blood
  • Pregnancy-Associated Plasma Protein-A / metabolism
  • Risk Assessment
  • Risk Factors

Substances

  • Biomarkers
  • Insulin-Like Growth Factor Binding Protein 4
  • Peptide Fragments
  • Pregnancy-Associated Plasma Protein-A
  • PAPPA protein, human