Determination of Free Thyroxine in Human Serum: A Theoretical and Experimental Analysis

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Abstract
The effect of dilution of serum on the concentration of free thyroxine was evaluated in an effort to determine the relationship between values previously obtained in a standard dialysis system employing diluted serum (1:150) and the concentration of free thyroxine in undiluted system. A theoretical analysis of thyroxine-binding by serum proteins in terms of a multiple equilibria system suggested that the free thyroxine concentration should be relatively independent of the concentration of serum proteins in the range of dilution considered. In order to test this prediction, experimental techniques were devised to measure free thyroxine in dialysis systems containing higher concentrations of protein. For this purpose, tracer I131 -thyroxine and I125 iodide were used simultaneously. The results of these studies were in general accord with the theoretical formulation. The concentration of free thyroxine was also measured in the ultrafiltrate of 7 sera. Values were in the same order of magnitude as those obtained in the diluted dialysis system. Furthermore, the diminished binding of thyroxine by the serum proteins of patients with wasting nonthyroidal disease was evident at all dilutions of serum studied. The results of these studies are interpreted to indicate 1) that the concentration of free thyroxine obtained in the standard diluted dialysis system is an approximation of that existing in undiluted serum, and 2) that the diminished binding of thyroxine by serum proteins in patients with nonthyroidal disease previously reported is not an artifact due either to dilution of the serum proteins or to the effect of buffer components. The experimental results were also utilized to estimate the apparent association constants of each of the serum thyroxine-binding proteins at 37 C.