Measurements of Free Thyroxine: Comparison of Per Cent of Free Thyroxine in Diluted and Undiluted Sera

Abstract

The effect of dilution on values of the per cent of free T₄ in serum (%FT4), measured by equilibrium dialysis, was determined by comparing the values for %FT₄ in diluted and undiluted specimens. The specimens studied were sera obtained from 10 normal subjects, 10 patients with hyperthyroidism, 5 with hypothyroidism, and 9 women with normal pregnancy. The method of analysis employed was that described by Sterling and Brenner, except that the ¹²⁵I-thyroxine used was predialyzed to remove non-thyroxine contaminants and that “corrected” values for %FT₄ were calculated by dividing measured values by the numerical extent to which the serum had been diluted. Studies in a normal serum pool revealed that corrected values for %FT₄ decreased with increasing dilution of serum, up to a dilution of 1:12, after which no significant further change occurred. In each of the various patient groups studied, for corrected %FT₄ in sera diluted 1:12 were about 70% of those in undiluted serum, and the correlation coefficient of values obtained in individual specimens by the 2 methods was 0.91 (P < 0.001). These findings indicate that the proportionate relationship between values yielded by the 2 methods remains nearly constant over a wide range of alterations in the thyroid hormone-serum protein-binding interaction. In ancillary studies, indirect indices of absolute free T₄ concentration, i.e., the “T₇” value (Abbott) and the Effective Thyroxine Ratio (Mallinkrodt) were found to correlate closely with those obtained by equilibrium dialysis over the same range of abnormal binding interactions.