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

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.