Volume shifts and protein binding estimates using equilibrium dialysis: application to prednisolone binding in humans

Sizable volume shifts can occur during equilibrium dialysis. This net movement of water, presumably caused by the osmotic effect of plasma proteins, reduces the concentration of binding proteins. In this paper the theory of protein binding estimation is extended, equations are developed for calculating the unbound and bound drug concentrations at dialysis equilibrium by correcting for the dilution of the proteins, and the equations are applied to a study of prednisolone. To demonstrate the importance of correcting for the volume shift, the parameters of a model in which prednisolone binds to corticosteroid-binding globulin, a protein with a limited capacity, and albumin were estimated. Unbound and bound concentrations were determined by correcting for both volume shifts (average 31%) and loss of drug to the buffer side, by correcting only for loss of drug to buffer side, and by making no correction at all (the usual method of treating equilibrium dialysis data). The error introduced by neglecting volume shifts was analyzed by comparing the parameter values obtained using the three methods. The results confirm the need to adjust for volume shifts and imply that reported binding constants obtained by equilibrium dialysis may be in error for many substances.