Protein binding of methohexital. Study of parameters and modulating factors using the equilibrium dialysis technique.

This paper describes the parameters that characterize methohexital-albumin binding and the influence of physiological or analytical factors on this binding. Two useful and reproducible methods for measuring the free concentration-equilibrium dialysis (ED) and ultrafiltration (UF)-are described and their performances are compared. Methohexital binds exclusively to albumin according to a two-class binding model. The first is a saturable class site of high affinity constant (KA = 11 200 M-1) and a number of sites per albumin molecule of 1. The second is a non-saturable site of poorer affinity (nKA = 810 M-1). The bound fraction of methohexital in the therapeutic range and at physiological albumin concentration is 86.7 +/- 0.9% in isolated albumin solution. In serum, it ranges from 80 to 84.5%, according to subjects (n = 6). Binding is inhibited by the presence of endogenous compounds of serum (for a given albumin concentration the bound fraction decreases from 90.3% in isolated albumin solution to 82.6% in serum), probably by free fatty acids. An increase in the bound fraction is observed when the pH is increased from 7 to 9. This phenomenon may be explained by a higher affinity of the drug towards the basic (B-form) conformation of the albumin molecule, in analogy with the close barbiturate thiopental. A decrease in the bound fraction against temperature is shown, as though binding forces diminished with increase in temperature. Indeed, the binding modification is less pronounced in the presence of serum endogenous compounds. As expected, there is no evidence of any effect of heparin anticoagulant on the bound fraction. Methohexital binding is strongly modified by the albumin concentration; the bound fractions change from 67 to 91% in the albumin range 150-900 microM.