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Isolation and characterization of a digoxin-like immunoreactive substance from human urine by affinity chromatography

DASRS, Aerospace Medicine Department and Chemistry & Technology Department, Aeroporto Pratica di Mare, 00040 Pomezia, Roma, Italy.
¹ I Clinica Medica, Università di Roma "La Sapienza", Viale del Policlinico, 00161 Roma, Italy.

² Department of Internal Medicine, Università dell'Aquila, via S. Sisto n. 22, 67100 Aquila, Italy.
^a Author for correspondence. Fax +39 6 9160 1079;

A series of observations has suggested that one or more digoxin-like immunoreactive substances (DLIS) in biological fluids is able to cross-react with the antidigoxin antibody. Whether this substance is the endogenous inhibitor of Na⁺/K⁺ ATPase has not been well established. The aim of this study was to identify and characterize DLIS from human urine. Treated urine from healthy men was run on an affinity chromatography column at a flow rate of 1 mL/min in which the ligand was an antibody (antiserum) to digoxin. Eluates from affinity chromatography were applied onto analytical reversed-phase HPLC. The active material was eluted 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 mL/L acetonitrile in water. We found a single peak showing cross-reactivity with antidigoxin antibody as measured by RIA. It showed the same retention time as that of a digoxin calibrator. This highly purified substance is able to displace [³H]ouabain from dog kidney-derived Na⁺/K⁺ ATPase, to inhibit Na⁺/K⁺ ATPase activity as measured by the⁸⁶Rb⁺ uptake in red blood cells and by coupled enzyme assay. Our results are consistent with the hypothesis that DLIS, as isolated by this particular digoxin antibody, is a single substance and an inhibitor of Na⁺/K⁺ ATPase.

Key Words: indexing terms: endogenous inhibitor • adenosine triphosphatase • radioimmunoassay

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