Reevaluation of Measurement of Serum Free Thyroxine by Equilibrium Dialysis Based on Computational Analysis of the Interaction between Thyroxine and Its Binding Proteins

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Abstract
Scatchard plots were made from data of free T4 concentrations (FT4) measured by equilibrium dialysis of 0.5 ml serum against 9 ml phosphate-buffered saline using a series of serum samples with graded enrichment of T4 up to 2000 μg/100 ml. The resulting hyperbolic curves were regressed by the iterative least square method, and the binding characteristics of T4- binding globulin, T4-binding prealbumin, and albumin (Alb) were estimated. Proper corrections for volume changes in serum during dialysis due to osmosis and for dilution of the concentration of the T4 and binding proteins were needed before computation of FT4. Elimination of chloride ion from the dialysis buffer resulted in an increased T4 binding with Alb. Elimination of magnesium and/or calcium from phosphate-buffered saline or reduction of the concentration of phosphate to the level of the serum free phosphate concentration did not affect the binding characteristics of the T4-binding proteins. Use of a dialysate volume 4-fold greater than the dialysant volume decreased the T4 binding of Alb. The computed T4-binding capacities of T4- binding globulin, T4-binding prealbumin, and Alb were 24.0, 241, and 58,340μg/100 ml; their equilibrium affinity constants were 1.90 × 1010,4.41 ×108, and 7.51 × 105 liters/mol; and the fractions of the endogenous T4 bound by them were 67.2%, 23.8%, and 9.9%, respectively. These results are comparable with those found through other methods.