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

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 43; no. 8; pp. 1416 - 1420
• Main Authors: De Angelis, Claudio, Riscazzi, Massimiliano, Salvini, Riccardo, Piccoli, Alfonso, Ferri, Claudio, Santucci, Anna
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.08.1997; American Association for Clinical Chemistry
• Subjects: Analysis; Animals; Antibodies - immunology; Binding, Competitive; Biological and medical sciences; Cardenolides; Cats; Chromatography, Affinity; Chromatography, High Pressure Liquid; Digoxin - immunology; Dogs; Enzyme Inhibitors - isolation & purification; Enzyme Inhibitors - pharmacology; Enzyme Inhibitors - urine; Erythrocytes - metabolism; General pharmacology; Humans; Kidney - enzymology; Male; Medical sciences; Ouabain - metabolism; Pharmacology. Drug treatments; Radioimmunoassay; Rubidium Radioisotopes; Saponins - immunology; Saponins - isolation & purification; Saponins - urine; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - metabolism; Space life sciences; Human; Urine; Biological fluid; Cardiotonic agent; Digoxin; Enzyme; HPLC chromatography; Molecular interaction; K; Immunological method; Na; Radioimmunoassay; Hydrolases; exchanging ATPase; Glycoside; Quantitative analysis
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/43.8.1416

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 the 86Rb+ 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.