Radioiodinated Tyrosyl-Ouabain and Measurement of a Circulating Ouabain-like Compound

Abstract

Abstract Background: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by laboratory-specific plasma values that are measured by different assays. Because of this variability, our study focused on the development of a new 125I-labeled ouabain derivative for RIA of high sensitivity. Methods: We generated rabbit antisera against a ouabain-thyroglobulin conjugate. A tyrosylated ouabain derivative for radioiodination was synthesized using periodate and sodium cyanoborohydride reagents. Results: Mass spectrometric analyses showed that the main product of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702 Da). This was radioiodinated with Chloramine-T and used as a tracer in a RIA, which gave an assay detection limit of 5 pmol/L (4 ng/L), 2–100 times lower than that in the corresponding 3H-RIAs and 2–20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C18-extracted plasma samples displaced the 125I-labeled tyrosyl-ouabain tracer at the same rate at which authentic ouabain was displaced. Plasma immunoreactive ouabain coeluted with authentic ouabain in two different HPLC conditions. Using the new RIA, we found plasma ouabain concentrations, assayed as immunoreactive equivalents, of 10.0 ± 1.3 pmol/L in healthy women and 12.0 ± 0.9 pmol/L in healthy men (mean ± SE; n = 10), as well as 41.2 ± 9.6 pmol/L in rats. The concentrations were 2–90 times lower than those previously reported using different assay methods. Conclusions: Our ouabain 125I-RIA enables reliable measurements of low endogenous concentrations of a ouabain-like compound for both physiological and clinical purposes.