Binding of Thyroxine by Serum Proteins Evaluated by Equilibrium Dialysis and Electrophoretic Techniques. Alterations in Non-Thyroidal Illness*

Abstract

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...

Alterations in thyroid hormone economy in patients with hydatidiform mole

“…The percentage and absolute concentrations of free T4 in the serum of patients with normal and molar pregnancy are also shown in Table III. In accord with earlier observations, the percentage of free T4 in serum was significantly decreased during normal pregnancy (9,20). The absolute concentration of free T4 in serum, however, was only slightly, and not significantly, lower than normal.…”

“…In patients with the usual varieties of hyperthyroidism, the absolute concentration of free To in serum is usually quite elevated as a result of increases in both the total concentration of To in serum and the proportion which is free. In most series, the absolute concentration of free To in hyperthyroid patients averages about 5 times the normal value (9,20,29,30); in the patients with molar pregnancy, a smaller increase in the concentration of free T4 was generally seen, since the proportion of free To was usually subnormal. Small to moderate increases in the serum concentration of free To in the absence of thyrotoxicosis are also seen in some patients with nonthyroidal illness (9,20), as well as in patients receiving replacement doses of synthetic thyroxine (35).'…”

“…In most series, the absolute concentration of free To in hyperthyroid patients averages about 5 times the normal value (9,20,29,30); in the patients with molar pregnancy, a smaller increase in the concentration of free T4 was generally seen, since the proportion of free To was usually subnormal. Small to moderate increases in the serum concentration of free To in the absence of thyrotoxicosis are also seen in some patients with nonthyroidal illness (9,20), as well as in patients receiving replacement doses of synthetic thyroxine (35).' Hence, it may be, as earlier studies have also suggested (36), that patients can tolerate moderate increments in the availability of T4 to tissues without developing frank thyrotoxicosis.…”

Differences in primary cellular factors influencing the metabolism and distribution of 3,5,3′-L-triiodothyronine and L-thyroxine

“…The assumption was made that the distribution volume of the T3-binding proteins is the same as that of serum albumin. The relative strength of binding of T4 and T3 to plasma proteins was assessed by simultaneous equilibrium dialysis of tracer quantities of T4-'3'I and T3-'1I added to the same serum samples (13). The ratio of the net strength of plasma binding of T4, b4, to the net strength of plasma binding of T3, b3, was calculated from the expression b4 I -DF4 X DF3 b3 1-DF3 DF4 where DF3 and DF4 are the dialyzable fractions of T3 and T4 as previously defined (13).…”

Studies on human thyroxine-binding globulin (TBG)

“…It has been estimated (1)(2)(3)(4) that 99.95% of circulating thyroxine in normal human serum is bound to the three carrier proteins, thyroxine-binding globulin (TBG), albumin, and thyroxine-binding prealbumin (TBPA).…”