John W. Barlow

LIMITATIONS OF A NEW FREE THYROXINE ASSAY (AMERLEX® FREE T4)

We have assessed a new method of free T4 measurement (Amerlex®) which uses a novel unidentified T4‐labelled analogue, said to be unreactive with T4 binding proteins in serum, together with an antibody that binds both analogue and T4. Free T4 is assessed by competition with analogue for antibody binding‐sites. The test method has been compared with free T4 measured by equilibrium dialysis and with a technique using an immobilized T4 antibody. All methods gave the expected free T4 levels in normal, hyperthyroid and hypothyroid subjects and normal free T4 levels with high or low levels of T4 binding globulin. However, in autosomal dominant familial euthyroid T4‐excess, where T4 is abnormally bound to albumin, the test method gave apparent high free T4 levels suggestive of hyperthyroidism. In a selected group of severely‐ill euthyroid patients the new method gave apparent low free T4 levels. In view of these discrepancies, binding of labelled analogue was evaluated by dextran‐charcoal separation at 4°C. Familial euthyroid T4‐excess sera showed greater analogue binding and samples with low prealbumin concentration showed less binding than did normal sera. Despite its validity with variations in TBG, it appears that Amerlex® Free T4 is influenced by lower‐affinity, high‐capacity T4 binding sites in serum, so that apparent free T4 concentration may vary with changes in the concentration of such sites.

FAMILIAL DYSALBUMINAEMIC HYPERTHYROXINAEMIA: STUDIES OF ALBUMIN BINDING AND IMPLICATIONS FOR HORMONE ACTION

The abnormal intermediate‐affinity T4 binding to albumin which is characteristic of familial dysalbuminaemic hyperthyroxinaemia (FDH) is dependent on buffer, temperature, and ionic composition. Scatchard analysis of T4‐binding to isolated albumin preparations from FDH subjects showed that half the circulating albumin showed the higher‐affinity T4 binding site, assuming one site per molecule. Using dextran‐charcoal separation at 40C the T4 affinity (Ka) of purified albumin from FDH subjects was 7.5 nmol/l in phosphate and 17 nmol/l in Tris‐CI‐ buffer. T4 binding to FDH albumin was inhibited by a range of substances in the order: 8‐anilino‐l‐naphthalene sulphonic acid > merthiolate>propylthiouracil> methyl‐thiouracil > carbimazole > salicylate > barbitone. Binding of T4 was competitively inhibited by low concentrations of dithiothreitol (DTT). The effect of DTT 0.1–0.5 mmol/l was reversed by removal of DTT by dialysis. Competition with a range of iodothyronines indicated that the 3′, 5′‐iodine atoms are most important for binding to this site. Serum binding of salicylate. frusemide. fenclofenac and barbituric acid, and a range of steroid hormones was similar in FDH and normal sera. Serum levels of sex hormone binding globulin (SHBG), were not significantly different from sex‐matched controls. Nuclear [125I]‐T3 binding sites in circulating lymphocytes from two FDH subjects showed affinities (Kd) of 59 and 79 pmol/l (normal 67±7 pmol/l, n= 6. These findings suggest that the highly specific binding anomaly of FDH is due to a disulphide‐dependent structural change in albumin. The normal T3 affinity of lymphocytic receptors and normal SHBG levels are consistent with a normal relationship between free hormone and tissue response in FDH.

Potentiation of steroid binding to proteins by 7,12-dimethylbenz(α)anthracene

Abstract In cytosols prepared from mammary gland parenchyma of adrenalectomised, ovariectomised 50-day old rats, DMBA (10−6 M) did not affect the binding of [3H]-oestradiol or [3H]-dexamethasone, but increased ~ ten-fold the binding of 10−8 M [3H]-progesterone [3H]-P. Subsequent studies have shown (i) such potential binding is steroid specific, being displaceable by excess radioinert P (ii) no strict protein specificity exists; in addition to mammary gland and uterine cytosol, DMBA potentiates [3H]-P binding to BSA, ovalbumin, catalase and rat plasma (iii) of a range of steroids studied, DMBA potentiates the binding of [ 3 H ]- P > [ 3 H ]- R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) > [3H]-deoxycorticosterone; minimal effects were seen with other [3H]-steroids (iv) 3-methylcholanthrene has a similar but much less marked potentiating effect on steroid binding (v) over the range of protein and DMBA concentrations studied, potentiation of [3H]-P binding increased with increasing DMBA concentrations, and decreased with increasing protein concentrations (vi) binding of [3H]-P as influenced by DMBA involves co-operative phenomena; under given conditions a ten-fold increase in DMBA concentration leads to a hundred-fold increase in bound [3H]-P. Parallel studies on [3H]-DMBA binding demonstrate no departure from linearity. Classically the carcinogenic action of DMBA has been thought to involve an interaction with cell nuclei. Since DMBA may increase albumin binding of progesterone a thousand-fold, it may have activity at a precellular level via modification of the effector actions of progesterone.

Lessons to the learned from TBG deficiency

Once it is secreted, thyroid hormone in the form of thyroxine (T4) will interact with at least three serum proteins before it is deiodinated to the more active T 3. This triumvirate, thyroxine binding globulin (TBG), transthyretin (TTR) and albumin is responsible, to different degrees, for transporting T 3 and T4 to their ultimate sites of action. With the advent of reliable, rapid free T4 estimations, assessment of serum binding is now of reduced diagnostic importance. Even so, there remains a certain degree of mystery surrounding these proteins and there are still important lessons to be learned from understanding their structure and function. As a group the only commonality among the various members is their hepatic synthesis. The proteins have distinct structures and physicochemical properties; even the ability to bind iodothyronines is different. The major carrier is TBG, a single polypeptide chain with five glycosylation sites. TBG is homologous to corticosteroid binding globulin (CBG) and both belong to the serine protease inhibitor (serpin) family of proteins, although neither TBG nor CBG has any protease activity. TTR is the second, but a minor iodothyronine binding protein carrying out 20% of circulating thyroxine. It is also the carrier protein for retinol binding protein. TTR is a tetramer in which identical subunits form a binding channel with two potential thyronine binding sites. In addition to hepatic synthesis, TTR is expressed in the choroid plexus. Albumin also circulates as a single polypeptide chain but is not glycosylated and has a very low affinity for T4. It carries only about 10% of the circulating hormone by virtue of the fact that it is present in such high concentration (Bartelena & Robbins, 1992). Because these proteins are relatively abundant in the serum they have all been readily available for analysis. Consequently, we have a detailed knowledge of their amino acid sequences, binding constants and physical properties. What we do not know is why there is the need for such a high degree of complexity among these proteins or indeed, why there is a need for these proteins at all. To be able to define a role for thyroid hormone binding proteins in human sera we depend on the identification of natural experiments, mutations in which the function of the protein is enhanced or diminished. It is probably fair to say that T 4 binding to albumin is nonspecific. That is not to say it is insignificant, rather the function of keeping lipophilic molecules of all sorts in solution is best performed by albumin. The only mutation concerning thyroxine binding which has been defined for this protein is that which leads to familial dysalbuminaemic hyperthyroxinaemia. Understanding this mutation is probably most informative about the nature of the albumin binding site rather than the function of albumin itself. In humans, a point mutation in the albumin gene results in the substitution of a histidine for arginine at position 218 in the amino acid chain (Petersent al., 1996). This substitution leads to a 20-fold increase in affinity of albumin for T4 binding to the 2A region of the protein, a region which also binds tryptophan, bilirubin and a number of drugs and other ligands. The increase in affinity results in a total T 4 concentration about double normal, although the fraction of the protein occupied by the hormone increases only from 0.0025% to 0.014%. Valid free T4 measurements are normal except where there is coincident thyroid dysfunction. Mutational analysis has probably been most useful for TTR. The occupancy of this protein by T 4 is also very low (0.5%) suggesting that its function as a carrier of retinol-binding protein is of greater importance. Interestingly, while TTR knockout mice are indistinguishable from their normal littermates (Episkapouet al., 1993), the naturally occuring mutations that have been described in human TTR are often associated with varying degrees of neural and/or cardiac amyloid deposits (Saraiva, 1995). These observations are consistent with the site of synthesis of TTR in the choroid plexus and suggest that this protein has a critical role in neurological function, perhaps developmentally. Some mutations lead to a change in affinity for T 4 with a concomitant increase or decrease in total serum T 4 to maintain the free concentration but these appear to be independent of amyloid deposition. Clinically, the albumin mutation is diagnostically more important than the more abundant TTR mutations especially for those free T 4 methods which measure the albumin-bound fraction (Stockigt, 1983). Mutations in TBG are also diagnostically important although because of the high affinitty of TBG and the major contribution of this protein to overall serum binding, evidence of aberrant TBGs can be easily detected. In reality, the primary importance in characterising mutant TBG species lies in understanding the function of the protein. About a dozen TBG mutants have been described which lead to both a partial reduction and a complete absence of the protein from human serum. For most of these mutants the molecular basis of the deficiency has been defined. Invariably, because TBG synthesis arises from a gene on the long arm of chromosome X, the deficiency in males is more Commentary