Considerable evidence suggests that the concentration of free thyroxine, circulating unbound to serum proteins, is more closely relattd to a subject's thyroidal status than is the concentration of total thyroxine (1). Because of the intense binding of thyroxine by the serum proteins, the concentration of free thyroxine is very small and not measurable by conventional chemical methods. Robbins and Rall (2), on the basis of electrophoretic data and the binding characteristics of bovine serum albumin, estimated that the concentration of free thyroxine in normal subjects was in the order of 6 x 10-1" M. By equilibrium dialysis of whole serum, Sterling and Hegedus (3) arrived at a figure approximately twice this. Other techniques for assessing the relative concentrations of free thyroxine in various clinical and physiological states include measurement of the fractional rate of dialysis of I'1"-labeled thyroxine (T4-I131 ) across a semipermeable membrane from one serum compartment to another (4, 5), and simultaneous determination of red-cell uptake of I131-labeled triiodothyronine (T3-"131) and serum protein-bound iodine (PBI) (6).In the present study, we used two independent methods to provide a relative measure of the fraction of thyroxine bound to serum proteins and the concentration of free thyroxine in serum. One method is based on the principle of equilibrium dialysis of diluted serum in an aqueous buffer at pH1 7.4; the other involves determination of critical ratios by filter-paper electrophoresis of whole serum in a glycine-acetate buffer at pH 8.6. Such an electrophoretic system reveals three species of protein-binding sites: thyroxine-binding globulin (TBG), albumin, and thyroxine-binding prealbumin (TBPA). Applied to the study of sera of normal and pregnant subjects and patients with hypothyroidism and hyperthyroidism, these methods gave results that were consistent internally and also with the current view that concentrations of free thyroxine are elevated in hyperthyroidism, depressed in hypothyroidism, and normal in pregnancy. The investigation was thereupon extended to a more intensive study of thyroxine-binding by the serum proteins of patients with nonthyroidal disease. Ingbar and Freinkel (7) have noted that in patients with a variety of diseases, a reduced quantity of thyroxine was associated with TBPA, and this finding suggested to us the possibility that such alterations could influence the level of free thyroxine in serum. METHODS Equilibrium dialysisThe method of these studies is partly based on the dialysis procedures used by Sterling and Tabachnick in their investigation of thyroxine-binding by serum albumin (8). Analogous techniques have been used to study the interaction of certain drugs with thyroxine-binding proteins (9-11). T4-IP`,I tested for chromatographic purity as previously described (9), was added to the serum sample in quantities sufficient to increase the endogenous concentration of thyroxine by 1 ,ug per 100 ml. The mixture was allowed to equilibrate for 30 minutes at room tem...

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