Direct determination of unbound lipophilic ligands in aqueous solutions

Due to their hydrophobic nature, lipophilic compounds are always bound to proteins when transported in the organism. The transfer of such compounds between their binding proteins and cells as well as intracellular trafficking is mediated by a very low water-phase concentration of monomers. The use of protein filled resealed red cell membranes (erythrocyte ghosts) as semipermeable bags enables us to determine directly such water-phase concentrations in a biological system where the lipophilic compound is in equilibrium with the compound bound to its binding protein. Equilibrium dissociation constants (K_d ’s) and number of binding sites are determined by regression analyses of data. We describe the method with the hydrophobic anion arachidonate and the neutral N-arachidonoylethanolamide as examples.