The binding of thiopental to serum proteins determined by ultrafiltration and equilibrium dialysis.

An ultrafiltration method is described by which it is possible to estimate the protein bound fraction of a drug at its original concentration in a serum sample. The determination is carried out at 37 degrees and pH 7.4. For thiopental no difference was found between the values of protein binding whether they were determined by the ultrafiltration method or by a less time consuming equilibrium dialysis against an equal volume of phosphate buffered isotonic sodium chloride solution. The equilibrium dialysis was used to measure the concentration of bound and unbound thiopental molecules and the binding parameters in a two class binding model were determined. No evidence was found for binding to other proteins than albumin. About one binding site belonging to the first class was found per 1000 albumin molecules whereas an average of about 5 secondary sites were found for each albumin molecule. The association constants for the primary class of binding sites were 3.4 X 10(6)M-1 for albumin and 13.3 X 10(6)M-1 for serum while the values for the secondary class were 2.2 and 1.2 X 10(3)M-1 for albumin and serum respectively. The estimates of association constants and number of binding sites were based on experiments with total thiopental concentrations ranging from 0.4 to 80 microgram/ml. In a phosphate buffered albumin solution with 2 g albumin per 100 ml, a pH increase from 5 to 8 caused an increase in protein binding from 36 to 76%. A Scatchard plot using data from experiments with increasing albumin concentrations resulted in a "plot" with positive slope. The use of a curve like this is discussed and it is concluded that the binding parameters for thiopental are influenced by the albumin concentration.