Thyroid hormone effect is dependent on the quantity of the hormone that reaches the tissues and the availability of unaltered thyroid hormone receptors in the cells nuclei. Since free thyroid hormone is transported across cell membrane, free rather than rather total serum hormone is a more accurate indicator of the activity level of thyroid hormone-dependent processes. Under normal conditions, free hormone levels are maintained constant by appropriate stimulation or suppression of hormone secretion and disposal.

Total hormone concentration in serum is normally kept at a level proportional to the concentration of carrier proteins, and appropriate to maintain a constant free hormone level. Most carrier protein dependent alterations in total hormone concentration in serum are due to quantitative changes in the hormone-binding proteins and less commonly to changes in affinity for the hormone. Since wide fluctuations in the concentration of thyroid hormone carrier proteins do not alter the hormonal economy or metabolic status of the subject (1), their function is open to speculation. They are responsible for the maintenance of a large extrathyroidal pool of thyroid hormone of which only the minute, <0.5 % fraction of free hormone is immediately available to tissues. It can be estimated that in the absence of binding proteins the small extrathyroidal T ₄pool would be significantly reduced, if not completely depleted in a matter of hours following a sudden cessation of hormone secretion. In contrast, in the presence of normal concentrations of T ₄-binding serum proteins, and in particular thyroxine-binding globulin (TBG), a 24-h arrest in hormonal secretion would bring about a decrease in the concentration of T ₄and T ₃in the order of only 10 and 40 per cent, respectively. Thus it seems logical to assume that one of the functions of T ₄-binding proteins in serum is to safeguard the body from the effects of abrupt fluctuations in hormonal secretion. The second likely function of T ₄-binding serum proteins is to serve as an additional protection against iodine wastage by imparting macromolecular properties to the small iodothyronine molecules, thus limiting their urinary loss (2). The Lack of high affinity T ₄-binding proteins in fish (3), for example, may be teleologically attributed to the greater iodine abundance in their natural habitat. Liver perfusion studies suggest a third function, that facilitating the uniform cellular distribution of T ₄, allowing for changes in the circulating thyroid hormone level to be rapidly communicated to all cells within organ tissues (4). Finally, a fourth function, modeled after the corticosteroid-binding globulin, is targeting the amount of hormone delivery by site specific, enzymatic, alteration of TBG (5). Presumably, neutrophil derived elastase cleaves TBG to release T ₄at the site of inflammation (6).

In normal man, approximately 0.03 per cent of the total serum T ₄, and 0.3 per cent of the total serum T ₃are present in free or unbound form (3, 7). The major serum thyroid hormone-binding proteins are TBG (Thyropexin), transthyretin [TTR or thyroxine-binding prealbumin (TBPA)], and albumin (HSA, human serum albumin) (8). Several other serum proteins, in particular high density lipoproteins, bind T ₄and T ₃as well as rT ₃(9, 10), but their contribution to the overall hormone transport is negligible in both physiological and pathological situations. In term of their relative abundance in serum, HSA is present at approximately 100-fold the molar concentration of TTR and 2,000-fold that of TBG. However, from the view point of the association constants for T ₄, TBG has highest affinity, which is 50-fold higher than that of TTR and 7,000-fold higher that of HSA. As a result TBG binds 75 % of serum T ₄, while TTR and HSA binds only 20% and 5%, respectively (Table 1).

The existence of inherited thyroid hormone binding protein abnormalities was recognized 1959, with the report of a family with TBG-excess (11) but it took 30 years before the first mutation in a defective was identified (12). Genetic variants of thyroid hormone binding proteins having different capacity or affinity for their ligands than the common type protein result in euthyroid hyper- or hypo-iodothyroninemia. Polymorphisms and mutations in genes encoding TBG and TTR and HSA have been identified (see chapter on Defects of Thyroid Hormone Transport in Serum).

The molecule, structure and physical properties: TBG is a 54 kDa acidic glycoprotein migrating in the inter-α-globulin zone on conventional paper electrophoresis, at pH 8.6. The term, thyroxine-binding globulin, is a misnomer since the molecule also binds T ₃and reverse T ₃. It was first recognized to serve as the major thyroid hormone transport protein in serum in 1952 (13). Since TBG binds 75% of serum T ₄and T ₃, quantitative and qualitative abnormalities of this protein have most profound effects on the total iodothyronine levels in serum. Its primary structure was deduced in 1989 from the nucleotide sequence of a partial TBG cDNA and an overlapping genomic DNA clones (14). However, it took 17 years to characterize its three dimensional structure by crystallographic analysis (15).

TBG is synthesized in the liver as single polypeptide chain of 415 amino acids. The mature molecule, minus the signal peptide, is composed of 395 amino acids (44 kDa) and four heterosaccharide units with 5 to 9 terminal sialic acids. The carbohydrate chains are not required for hormone binding but are important for the correct post-translational folding and secretion of the molecule (16, 17) and are responsible for the multiple TBG isoforms (microheterogeneity) present on isoelectric focusing (18). The isoelectric point of normal TBG ranges from pH 4.2 to 4.6, however, this increases to 6 when all sialic acid residues are removed.

The protein is very stable when stored in serum, but rapidly loses its hormone binding properties by denaturation at temperatures above 55°C and pH below 4. The half-life of denaturation at 60°C is approximately 7 min but association with T ₄increases the stability of TBG (19-21). TBG can be measured by immunometric techniques or saturation analysis using one of its iodothyronine ligands (21-23).

Recently, the tertiary structure of TBG was solved by co-crystallizing the in-vitro synthesized non-glycosylated molecule with T ₄(15). The molecule caries T ₄in a surface pocket held by a series of hydrophobic interactions with underlying residues and hydrogen bonding of the aminoproprionate of T ₄with adjacent residues. TBG differs from other members of the SERPIN family in having the upper half of the main ß-sheet completely opened. This allows the reactive center peptide loop to move in and out of the sheet, resulting in binding and release of the ligand without cleavage of TBG. Thus the molecule can assume a high-affinity and a low-affinity conformation, a model proposed earlier by Grasberger et al (24). This reversibility is due to the unique presence of P8 proline in TBG, rather than a threonine in all other SERPINs, limiting loop insertion.

Gene structure and transcriptional regulation: The molecule is encoded by a single gene copy located in the long arm of the human X-chromosome (Xq22.2) (25, 26). The gene consists of 5 exons spanning 5.5kbp (Fig. 1). The first exon is a small and non-coding. It is preceded by a TATAA box and a sequence of 177 nucleotides containing an hepatocyte transcription factor-1 (HNF-1) binding motif that imparts to the gene a strong liver specific transcriptional activity (27). The numbers and size of exons, their boundaries and types of intron splice junctions as well as the amino acid sequences they encode are similar to those of other members of the serine protease inhibitor (SERPIN) family, to which TBG belongs (27). These include cortisol-binding globulin and the serine protease inhibitors, α1-antitrypsin (α1AT) and α1-antichymotrypsin (α1ACT).

Biological properties: The TBG molecule has a single iodothyronine binding site with affinity slightly higher for T ₄than for T ₃(28) (Table 1). Optimal binding activity requires the presence of the L-alanine side chain, an unsubstituted 4'-hydroxyl group, a diphenyl ether bridge, and halogen (I or Br) constituents at the 3,5,3' and 5' positions.(29). Compared to L-T ₄, rT ₃binds to TBG with ~40% higher affinity, D-T ₄with half that of the L-isomer and tetraiodothyroacetic acid with ~25%. A number of organic compounds compete with thyroid hormone-binding to TBG. Most notable are: 5,5-diphenylhydantoin (30), 1,8-anilinonaphthalenesulfonic acid and salicylates (31). While reversible flip-flop conformational changes of TBG allow for binding and release of the hormone ligand, cleavage of the molecule by leukocyte elastase. produces a permanent change in the properties molecule. this modified form has reduced T ₄-binding and increased heat stability (32).

Denatured TBG does not bind iodothyronines but can be detected with antibodies that recognize the primary structure of the molecule (21). In euthyroid adults with normal TBG concentration, about one-third of the molecules carry thyroid hormone, mainly T ₄. When fully saturated, it carries about 20 μg of T ₄/dl of serum. The biologic half-life is about 5 days, and the volume of distribution is similar to that of albumin (33, 34) (Table 1). TBG is cleared by the liver. Loss of sialic acid accelerates its removal through interaction with the asialo-glycoprotein receptors reducing the half live by 500-fold (19). However, it is unknown whether desialylation is a required in the normal pathway of TBG metabolism.

The molecule, structure and physical properties: TTR is a 55 kDa tetramer which is highly acidic although it contains no carbohydrate. Formerly known as thyroxine-binding prealbumin (TBPA), for its electrophoretic mobility anodal to albumin, was first recognized to bind T ₄in 1958 (55). Subsequently it was demonstrated that TTR also forms a complex with retinol-binding protein and thus plays a role in the transport of vitamin A (retinol, or trans retinoic acid) (56, 57).

TTR circulates in blood as a stable tetramer of identical subunits, each containing 127 amino acids (58). Although the tetrameric structure of the molecule was demonstrated by genetic studies (59, 60), detailed structural analysis is available through X-ray crystallography (61, 62). Each TTR subunit has 8 ß-strands four of which form the inner sheet and four the outer sheet. The four subunits form a symmetrical ß-barrel structure with a double trumpeted hydrophobic channel that traverses the molecule forming the two iodothyronine binding sites. Despite the apparent identity of the two iodothyronine binding sites, TTR usually binds only one T ₄molecule because the binding affinity of the second site is greatly reduced through a negative cooperative effect (63). The TTR tetramer can bind four molecules of RBP that do not interfere with T ₄-binding, and vice versa (64). TTR can be measured by densitometry after its separation from the other serum proteins by electrophoresis, by hormone saturation, and by immunoassays.

Gene structure and transcriptional regulation: TTR is encoded by a single gene copy located on human chromosome 18 (18q11.2-12.1) (58, 65) (Fig. 1). The gene consists of 4 exons spanning for 6.8 kbp. Knowledge about the transcriptional regulation of the human TTR gene comes from studies of the mouse gene structural and sequence homology of which extends to the promoter region (66, 67)[. In both species a TATAA box and binding sites for HNF-1, 3 and 4 are located within 150 bp from the transcription start site.

Although TTR in serum originates from the liver (68), TTR mRNA is also found in kidney cells the choroid plexus and pancreatic islet cells (69-71). TTR constitutes up to 25% of the total protein present in ventricular cerebrospinal fluid where it binds 80% of T ₄(72).

Biological properties: Despite the 20-fold higher concentration of TTR in serum relative to that of TBG, it plays a lesser role in iodothyronine transport. In the presence of normal levels of TBG, wide fluctuations in TTR concentration or its removal from serum by specific antibodies has little influence on the concentration of free T ₄(73). Some of the properties of TTR are summarized in Table 1.

The first T ₄molecule binds to TTR with a Ka of about 100-fold higher than that for HSA and about 100-fold lesser than that for TBG. Properties necessary for optimal binding activity include iodines at the 3' and 5' positions and a desamino acid side chain which explain the lower T ₃and higher tetraiodoacetic acid affinities relative to that of T ₄(29, 74). Non-iodothyronine ligands are also differentially bound, the most notable example being the flavonoid compounds which have a markedly higher binding affinity for TTR than for TBG (75). Among drugs that compete with T ₄-binding to TTR are ethacrynic acid, salicylates, 2,4-dinitrophenol, and penicillin (76, 77). Barbital also inhibits iodothyronine binding to TTR.

Only 0.5% of the circulating TTR is occupied by T ₄. TTR has a relatively rapid turnover (t1/2 = 2 days) and a distribution space similar to that of HSA and TBG (78, 79) except that it also exists in CSF. Hence, acute diminution in the rate of synthesis is accompanied by a rapid decrease of its concentration in serum.

The molecule, structure and physical properties: HSA is a 66.5 kDa protein synthesized by the liver. It is composed of 585 amino acids with high content of cystines and charged amino acids but no carbohydrate (88). The three domains of the molecule can be conceived as three tennis balls packaged in a cylindrical case.

Gene structure and transcriptional regulation: HSA is encoded by a single gene copy located on human chromosome 4 (4q11-q13) (89). The gene contains 15 exons, 14 of which are coding (90) (Fig. 1). The promoter region of the HSA gene has been most intensive studied. The transcriptional regulation has been best characterized in rodents that share 90% sequence homology with the corresponding human gene, including a distal enhancer element 10 kbp upstream from the promoter region (91).. Binding sites for hepatocyte enriched nuclear proteins, such as HNF-1, C/EBP, and DBP have been identified (92-94).

Biological properties: HSA associates with a wide variety of substances including hormones and drugs possessing a hydrophobic region, and thus the association of thyroid hormone to HSA can be viewed as nonspecific. Of the several iodothyronine-binding sites on the HSA molecule, only one has a relatively high affinity for T ₄and T ₃. Yet these are 10,000-fold inferior to those of TBG (22). Fatty acids and chloride ions decrease their binding to HSA (95). The biologic t1/2 of HSA is relatively long (96). Some of its properties are summarized in Table 1.

More than half of the total protein content in serum is albumin. As a result, it is the principal contributor to the maintenance of the colloid osmotic pressure (88). It has been suggested that HSA synthesis may be, in part, regulated by a feedback mechanism involving alteration in the colloid osmotic pressure. Indeed, down-regulation of albumin gene expression has been recently observed during the infusion of macromolecules in the rat (97).

Supported in part by grants DK-15070 and RR-00055 from the National Institutes of Health (USA) and by the Blum-Kovler and Tivoli Wien Katz research funds.