Human placenta radioreceptor assay with digoxin and ouabain to detect endogenous digitalis-like factor(s) in human plasma and urine

Abstract

We describe the optimization and validation of a clinically feasible radioreceptor assay to detect endogenous digitalis-like factor(s) (EDLF) in human plasma and urine. The assay is based on the competitive replacement of 125I-labeled digoxin on human placenta membranes by ligands present in sample extracts. Digoxin and ouabain were used as calibrators. We also describe simple and effective methods for extraction and enrichment of EDLF from human plasma and urine. Assay sensitivity and precision were enhanced by using a sequential saturation technique with appropriate concentrations of tracer and receptors. Filtration was used to separate bound from free ligand. A two-step solid-state extraction with acetonitrile allowed the separation of two EDLFs with different polarity (EDLF-1 and EDLF-2) from the same plasma sample. A one-step solid-state extraction with methanol was suitable for urine. EDLF-1 and EDLF-2 in healthy adults were respectively 204 +/- 155 and 207 +/- 423 pmol/L ouabain equivalents, or 312 +/- 241 and 302 +/- 581 pmol/L digoxin equivalents. Plasma concentrations of EDLFs in newborns and pregnant women were higher than in healthy adults, and the concentrations in urine were higher than in plasma. Several cross-reactivity experiments showed that physiological concentrations of endogenous steroids and lipids did not inhibit binding, and supported the hypothesis that EDLFs are endogenous compounds other than the steroids and lipids also investigated.