Radioiodinated tyrosyl-ouabain and measurement of a circulating ouabain-like compound

Background: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by l aboratory-specific plasma values that are measured by different assays. Bec ause of this variability, our study focused on the development of a new I-1 25-labeled ouabain derivative for RIA of high sensitivity. Methods: We generated rabbit antisera against a ouabain-thyroglobulin conju gate. 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 ty rosylating reaction was tyrdsylouabain (molecular mass, 702 Da). This was r adioionated 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 t he corresponding H-3 RIAs and 2-20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C-18-extracted plasma samples displaced the I- 125-labeled tyrosyl-ouabain tracer at the same rate at which authentic;ouab ain was displaced, Plasma immunoreactive ouabain coeluted with authentic ou abain 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 I-125-RIA enables reliable measurements of low end ogenous concentrations of a ouabain-like compound fdr both physiological an d dinical purposes. (C) 2001 American Association for Clinical Chemistry.