MEASUREMENT OF FREE THYROXINE CONCENTRATION IN HUMAN SERUM*

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Abstract
The existence of "free" thyroxine in human serum was repeatedly verified by dialysis through cellophane, with chromatographic identification of thyroxine in the dialysate. The identity of thyroxine was confirmed by two-dimensional paper chromatography. Measurement of the minute amounts of thyroxine in the dialysate was undertaken by paper chromatography and fractionation with a column of the cation exchange resin, Dowex 50W-X8. Evaporation of the dialysate, followed by both butanol and water extraction of the dried residue, made it possible to recover more than 95 per cent of the radioactivity. Descending paper chromatography, strip scanning, and planimetric measurement of the areas under the thyroxine and iodide peaks yielded values agreeing to within 5 per cent of the values obtained by the resin columns. The mean free thyroxine content was 0.11 per cent of the total thyroxine of normal serum, corresponding to a concentration of 1. 3 x 10-l0M. The same values were obtained whether I13l-labeled thyroxine was added to serum, or serum containing endogenously labeled hormone after 1131 therapy was employed. The observed free thyroxine values, expressed as per cent of the total thyroxine content of the sera, were as follows (mean [plus or minus] standard deviation): thyrotoxicosis, 0.23 [plus or minus] 0.044; euthyroidism, 0.11-[plus or minus] 0. 015; hypothyroidism, 0. 070 [plus or minus] 0. 011; pregnancy, 0.058 [plus or minus] 0.014. The product of the per cent free thyroxine and the protein-bound iodine value gave a result for free thyroxine iodine concentration that showed marked deviation from normal in thyrotoxicosis and hypothyroidism. The findings are compatible with the concept that physiological activity of thyroxine depends upon the concentration of unbound hormone.