Free Thyroxine in Serum by Equilibrium Dialysis: Effects of Dilution, Specific Ions and Inhibitors of Binding

In equilibrium dialysis measurements employing labeled thyroxine, dilution of serum results in a decrease in the percent free thyroxine (PFT) ranging between 25 and 40%. This observation is contrary to the prediction from the law of mass action that the free thyroxine should actually increase slightly. Removal of weakly bound contaminants from the labeled thyroxine does not eliminate the dilution effect. Rather, the dilution phenomenon appears to be largely explained by ionic effects. Both chloride and phosphate ions are shown to inhibit thyroxine binding to serum proteins and raise the free thyroxine concentration. The chloride effect is proportional to increasing concentration over a wide range, but the phosphate effect does not increase beyond 0.05m. Thus when serum is diluted with phosphate buffer, the PFT decreases in a manner predictable from the final equilibrium concentrations of chloride and phosphate in each sample. The PFT is very sensitive to changes in pH. The magnitude of the pH effect is inversely related to the concentration of the buffer used. Free thyroxine is reported to be elevated in renal insufficiency. The present work demonstrates that a number of compounds thought to be present in elevated concentrations in uremia also elevate the PFT. These compounds include indole, several of its natually occurring congeners, and a variety of phenols. The effects of inorganic ions may explain much of the dilution effect in normal serum, but the finding of other naturally occurring small mol wt compounds that affect the PFT is consistent with the possibility that compounds with similar effects are responsible for the elevated free thyroxine of certain disease states.