The distribution of 3H-labelled cardenolides between isolated guinea-pig atrial tissue and circulating, oxygenated whole blood.

1. An experimental method was developed that allowed the incubation of isolated organs in circulating whole blood. The circulating blood was oxygenated with a specially designed disc-oxygenator and drawn through the system by means of a roller pump.2. The method proved suitable for guinea-pig isolated atria, rabbit duodenum and to a lesser extent for chronically denervated rat diaphragm. Isolated atria could be kept for several hours. Various parameters of the circulating blood (haemolysis, pH, O(2) saturation, concentration of electrolytes) remained satisfactory for at least 5 hr. The method proved convenient for pharmacological and kinetic studies on isolated organs, suspended in whole blood of the corresponding species. The organs showed normal spontaneous mechanical activity and also responded to electrical stimuli and various drugs.3. The uptake of (3)H-labelled ouabain, digoxin, digitoxin and digitoxigenin was studied in guinea-pig isolated atria, suspended in circulating blood. The uptake reached equilibrium after 60-90 min. With respect to the total serum radio-activity the "apparent" tissue/medium (T/M) ratios obtained for the four drugs were within the range 0.4-1.1. If, however, the amount of free, non protein bound drug was taken as a base for the calculations, the following "true" T/M ratios were obtained: (3)H-ouabain 0.45; (3)H-digoxin 1.6; digitoxin 8.8; digitoxigenin 8.4. These values are virtually the same as those obtained with atria, suspended in an aqueous medium. Obviously, the uptake of (3)H-cardenolides from whole blood is determined by the amount of free non-protein-bound drug.4. (3)H-digitoxin and (3)H-digitoxigenin were taken up by guinea-pig erythrocytes to a small extent. No measurable amounts of (3)H-ouabain and (3)H-digoxin were taken up by erythrocytes.

Binding of digitoxin and some related cardenolides to human plasma proteins.

Tritium-labeled digitoxin, digitoxigenin, digoxin, and digoxigenin of established purity and chemcal authenticity were used to study the binding of these compounds to human plasma proteins. 97% of digitoxin in plasma was nondialyzable. Continuous flow paper electrophoresis of plasma containing digitoxin and dialysis experiments in which human serum albumin competed for the glycoside with plasma or plasma protein fractions demonstrated that digitoxin was almost exclusively bound by albumin. Equilibrium dialyses revealed that the interaction was characterized by a single binding site on the albumin molecule and an association constant of 9.62 x 10(4) liter/mole at 37 degrees C. At 1 degrees C the association constant was 4.64 x 10(4) liter/mole. The interaction therefore was endothermic; the gain in enthalpy of 3.5 kcal/mole and the free energy change of - 7.06 kcal/mole was derived from a large change in entropy of 33.8 cal/mole per degrees K. The direction of these thermodynamic changes suggested the formation of a hydrophobic bond between digitoxin and albumin. Quenching of the fluorescence of albumin by digitoxin indicated that the conformation of albumin was altered by the binding process.Digitoxigenin, its mono- and didigitoxosides, digoxin, and digoxigenin competed with digitoxn for its binding site on albumin. The affinity of the mono- and didigitoxosides for the site was equal to that of digitoxin, but that of digitoxigenin was only one-third as great. The ability of the digitoxose residues of the glycosides to enhance binding to albumin was also observed with digoxin, which was more extensively bound by the protein than digoxigenin. At concentrations of 2 mug/ml or less in plasma, only 23% of digoxin was bound. Albumin, which interacted with digoxin with an apparent association constant of 9 x 10(2) liter/mole at 37 degrees C, was entirely responsible for the binding. Lowering the temperature from 37 degrees to 1 degrees C decreased the fraction of digoxin bound to albumin by two-thirds. The marked difference in avidity of digitoxin and digoxin for serum albumin is reflected by the higher plasma concentrations, lower rate of urinary excretion, and longer half-time of digitoxin as compared to those of digoxin when these compounds are administered to man.