Thyroxine-binding proteins measurement

Until recently it was not always possible to measure unbound thyroxine in serum directly because of the minute quantities involved. Therefore a free thyroxine index calculation was often made, since this value is proportional to the free thyroxine concentration. This index can be arrived at by multiplying a measure of thyroxine in the blood (usually the FBI) by a value derived from the measurement of thyroxine-unoccupied binding sites of thyroxine-binding proteins. This was possible since the uptake of thyroxine or triiodothyronine by red cells or resin is inversely proportional to the number of unoccupied binding sites. As thyroxine-binding sites become increasingly saturated, the free thyroxine levels increase. An example of this method of arriving at a free thyroxine index is the work of Clark and Horn (C5). Calculation of the free thyroxine index is valuable since the FBI determination and the resin uptake of Ts are determinations that are routinely made in many clinical laboratories. 

Standard radio-immunoassay procedures are applied, nowadays mainly solid phase assays which can be rapidly performed and evaluated, they are also several enzyme-linked immunoassay (ELISA) procedures from commercial suppliers. It is recommended to perform an internet check for the most appropriate method at the time of the study (for example DSL 2005). Methods have been described for triiodothyronine (Nejad et al. 1975, Chopra etal. 1972a, Larsen 1972a, 1972,) and for thyroxin (Chopra etal. 1971, Chopra 1972). The use of assays based on thyroxin binding globulin (Chopra et al. 1972b) is no longer recommended and cannot be applied to the rat, because the rat does not have this binding protein. However, for the human, measurement... 

TABLE 52-4 Free Thyroxine Measurements in Common Conditions Affecting Thyroid-Binding Proteins ... 

A. Wang R, Nelson JC, Weiss RM, Wilcox RB. Accuracy of free thyroxine measurements across natural ranges of thyroxine binding to serum proteins. Thyroid 2000 10 31-9. 

LBM includes skeletal muscle, somatic protein, and functional proteins such as circulating proteins and the visceral proteins. Biochemically, LBM can be assessed by measuring the serum visceral proteins, albumin (ALB), transferrin (TFN), and prealbumin (thyroxine-binding prealbumin or transthyretin). Other serum proteins, such as retinol-binding protein, flbronectin (an opsonic protein), and somatomedin-C (insulin-like growth factor-1), that have a very short half-life (less than 12 to 24 hours), have been suggested as... 

An indirect measure of the concentration of a specific binding protein can be obtained by adding increasing amounts of thyroxine to serum which has been partitioned by electrophoretic means. Saturation, or the maximal binding capacity is a function of the number and capacity of binding sites available [1/mole (013) in the case of TBPA]. The concentration of binding sites is proportional to the amount of thyroxine necessary to saturate the protein. Such methods have been described (010). Reverse-flow techniques can be used to eliminate trailing albumin from the TBG (E3). 

The competitive principle of immunoassays can be applied to nonimmuno-logic systems. Any substance that can bind specifically to a macromolecule can be measured using the principle of competitive protein binding. The specific binding macromolecule can be a serum protein such as thyroxine binding globulin, a specific receptor, or an antibody. 

It is now well established that clinically euthyroid patients who have abnormalities in the serum Ti-binding proteins also have abnormalities in the serum total T4 level and in Tg resin uptake. Taking the example of the oral contraceptives, TBG is increased, but the abnormalities in total T4 and Tg uptake diverge in opposite directions—i.e., total T4 is elevated and Tg uptake is depressed. Clark and Horn (C17) took advantage of the inverse nature of these abnormalities and devised a free thyroxine index (FTI) from the mathematical product of FBI and Tg uptake, basing this on a similar index constructed from the measurement of the uptake of Tg by red blood cells (012). The theoretical considerations underlying the FTI are as follows (C17) ... 

Considerable controversy currently centers on the free hormone hypothesis of thyroid and steroid hormone delivery. This postulates that hormonal effects are governed by free hormone concentrations as measured in vitro. Meanwhile the physiological role of the specific hormone binding proteins present in serum (such as thyroxine binding globulin [TBG]) remains unknown. 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

Ultrafiltration has been used to determine the protein bound fraction of many drags, such as methadone (Wilkins et al. 1997), phenylacetate and phenylbu-tyrate (Boudoulas et al. 1996), etoposide (Robieux et al. 1997), doxorubicin and vincristine (Mayer and St-Onge 1995), disopyramide (Echize et al. 1995), and ketamine and its active metabolites (Hijazi and Boulieu 2002). Schumacher et al. (2000) have shown the applicability for the determination of erythro-cyte/plasma distribution. The method of UF has been applied in the measurement of free unaltered thyroxin or after displacement by salicylate as well after displacement by heparin in healthy people and in patients with non-thyroidal somatic illness (Faber et al. 1993). The protein binding of tritium labeled, antidiabetic repaglinide and its displacement by warfarin, furosemide, tolbutamide, diazepam, glibenclamide and nicardipine were determined by ultrafiltration (Plumetal. 2000). 

Seligson, H., and Seligson, D., Measurement of thyroxine by competitive protein binding. Clin. Chim. Acta 38,199-205 (1972). 

Radioimmunoassay methods. Specific thyroxine antisera have been raised and can be used to measure serum thyroxine by radioimmunoassay. Most laboratories now probably measure thyroxine by this means. Radioimmunoassays have the following advantages over the competitive protein binding techniques ... 

---