DIFFERENT METHODS FOR MEASURING ENDOGENOUS DIGITALIS-LIKE FACTOR(S)

It is well recognized that one of the difficulties in the search for e ndogenous ligand(s) with digitalis-like properties (endogenous digital is-like factor(s), EDLF) in mammals has been the lack of a unique, spe cific method for the accurate measurement of EDLF. Using C-18 solid-ph ase extracts of plasma from normal adults and various patient groups, and purified extracts from umbilical cord plasma by affinity resin chr omatography and HPLC, different methods to measure EDLF were evaluated . These were: (a) a human placenta radioreceptor assay (RRA) developed on the premise that competition for cardiac glycoside receptors was a n absolute requirement for EDLF; (b) the inhibition of Rb-86 uptake in human erythrocytes to estimate the potassium transport by the sodium pump; (c) an enzyme immunoassay specific for ouabain recently introduc ed in the market (DuPont Ouabain EIA Reagent Pack). The human placenta RRA was found to have the same ease of application as immunoassay, bu t could have major advantages in detecting active molecules, being ''b iologically more meaningful''. Ouabain immunoreactivity correlated wit h EDLF values obtained by RRA, but in some instances the two assays we re completely unrelated. Moreover, the high specificity of the DuPont antibody for ouabain (< 3% cross reactivity with digoxin) could be dis advantageous to detect EDLF not strictly resembling ouabain. The Rb-86 uptake inhibition method correlated with RRA for EDLF purified by HPL C. It tested the complete enzymatic cycle and could therefore better r eflect the in-vivo inhibitory activity of EDLF. However, it appeared n ot suitable for the routine EDLF evaluation in clinical studies since it was susceptible to sample osmolarity and required daily isolation o f human erythrocytes possibly from the same donor. Results of the pres ent study demonstrate that every assay has its limitations, and would suggest the use of multiple assays for EDLF detection.