ISOLATION AND CHARACTERIZATION OF A DIGOXIN-LIKE IMMUNOREACTIVE SUBSTANCE FROM HUMAN URINE BY AFFINITY-CHROMATOGRAPHY

A series of observations has suggested that one or more digoxin-like i mmunoreactive substances (DLIS) in biological fluids is able to cross- react with the antidigoxin antibody. Whether this substance is the end ogenous inhibitor of Na+/K+ ATPase has not been well established. The aim of this study was to identify and characterize DLIS from human uri ne. Treated urine from healthy men was run on an affinity chromatograp hy column at a flow rate of 1 mL/min in which the ligand was an antibo dy (antiserum) to digoxin. Eluates from affinity chromatography were a pplied onto analytical reversed-phase HPLC. The active material was el uted with a linear gradient of acetonitrile (from 350 to 650 mL/L) and water. A second step in HPLC was carried out isocratically with 280 m L/L acetonitrile in water. We found a single peak showing cross-reacti vity with antidigoxin antibody as measured by RIA. It showed the same retention time as that of a digoxin calibrator. This highly purified s ubstance is able to displace [H-3]ouabain from dog kidney-derived Na+/ K+ ATPase, to inhibit Na+/K+ ATPase activity as measured by the Rb-86( +) uptake in red blood cells and by coupled enzyme assay. Our results are consistent with the hypothesis that DLIS, as isolated by this part icular digoxin antibody, is a single substance and an inhibitor of Na/K+ ATPase.