Free fraction change with differing volume ratios in equilibrium dialysis: cases of non-linear drug-protein binding

When equilibrium dialysis is used to measure drug-protein binding, an increase in the volume ratio (buffer solution/protein solution) causes a decrease in the total drug concentration at equilibrium. When binding is non-linear, this decrease in total drug-concentration also causes a decrease in the free fraction. By use of the same volume ratio (3:1) binding curves were obtained for the binding of prednisolone to plasma proteins, and for the binding of salicylic acid and tolmetin to human serum albumin (HSA). When the volume ratio was changed, the observed free fractions were appreciably different from those expected by taking into account only the changes in the total equilibrium drug concentration. There is evidence that this phenomenon is the result of competition for binding sites between the drug and an impurity in the HSA. The effects are not the result of the presence of free fatty acids.