Immunoluminometric Assay for Measurement of the C-Terminal Endothelin-1 Precursor Fragment in Human Plasma

Abstract

Endothelin-1 (ET-1), a potent vasoconstrictor, is difficult to measure because of its instability and its binding to receptors and plasma proteins. We report a rapid, robust way to indirectly quantify ET-1 release by measuring the C-terminal ET-1 precursor fragment (CT-proET-1) without an extraction step. In plasma samples from healthy individuals, patients with congestive heart failure (CHF), and patients with sepsis, we measured CT-proET-1 with a sandwich immunoluminometric assay that uses 2 polyclonal antibodies to amino acids 168-212 of pre-proET-1. We also correlated CT-proET-1 concentrations with bigET-1 measurements. The assay yielded results within 3 h and showed linear dilution with an analytical detection limit of 0.4 pmol/L and an interlaboratory CV <10% for values >10 pmol/L. Ex vivo CT-proET-1 was stable (<10% loss of immunoreactivity) in EDTA-, heparin-, and citrate-plasma for at least 4 h at room temperature, 6 h at 4 degrees C, and in EDTA-plasma for at least 6 months at -20 degrees C. CT-proET-1 values followed a gaussian distribution in healthy individuals (mean, 44.3 pmol/L; range, 10.5-77.4 pmol/L) without significant differences between males and females. The correlation coefficient for CT-proET-1 vs age was 0.25 (P <0.0001). CT-proET1 was significantly (P <0.0001) increased in patients with CHF (median, 104 pmol/L; range, 50.8-315 pmol/L) and patients with sepsis (median, 189 pmol/L; range, 34.6-855 pmol/L). The correlation between CT-proET-1 and bigET-1 for 43 samples was 0.80 (P <0.0001). CT-proET-1 measurement is a rapid and easy method for indirectly assessing the release of ET-1 in critically ill patients.