T4-binding Globulin Deficiency Research Articles

Improving the Dutch Newborn Screening for Central Congenital Hypothyroidism by Using 95% Reference Intervals for Thyroxine-Binding Globulin

Introduction: Newborn screening (NBS) for congenital hypothyroidism (CH) in the Netherlands consists of thyroxine (T4), thyroid-stimulating hormone (TSH), and T4-binding globulin (TBG) measurements to detect thyroidal CH and central CH (CH-C). CH-C is detected by T4 or a calculated T4/TBG ratio, which serves as an indirect measure of free T4. TSH and TBG are only measured in the lowest 20 and 5% of daily T4 values, respectively. A recent evaluation of the Dutch NBS for CH showed that the T4 and T4/TBG ratio contribute to the detection of CH-C but also lead to a low positive predictive value (PPV). Dried blood spot (DBS) reference intervals (RIs) are currently unknown and may contribute to improvement of our NBS algorithm. Materials and Methods: RIs of T4, TSH, TBG, and the T4/TBG ratio were determined according to Clinical & Laboratory Standards Institute guidelines in heel puncture cards from routine NBS in both sexes and at the common NBS sampling ages. Scatter plots were used to compare the healthy reference population to previously published data of CH-C patients and false positives. Results: Analyses of 1,670 heel puncture cards showed small differences between subgroups and led to the formulation of total sample DBS RIs for T4 (56–118 nmol/L), TSH (<2.6 mIU/L), TBG (116–271 nmol/L), and the T4/TBG ratio (>20). 46% of false-positive referrals based on T4 alone had a TBG below the RI, indicating preventable referral due to partial TBG deficiency. One case of CH-C also had partial TBG deficiency (TBG 59 and T4 12 nmol/L blood). Discussion/Conclusion: Established DBS RIs provided possibilities to improve the PPV of the Dutch CH NBS algorithm. We conclude that by taking partial TBG deficiency into account, approximately half of T4 false-positive referrals may be prevented while maintaining NBS sensitivity at the current level.

TSH and FT4 Concentrations in Congenital Central Hypothyroidism and Mild Congenital Thyroidal Hypothyroidism.

In central hypothyroidism (CeH), free thyroxine (FT4) concentrations are low, whereas thyrotropin (TSH) concentrations may be low, normal, or even slightly elevated due to reduced bioactivity. Congenital CeH (CCeH) may be isolated or part of multiple pituitary hormone deficiencies (MPHD). We tested our hypotheses that (1) TSH concentrations have a more U-shaped distribution in children with CCeH compared with children with a normally functioning hypothalamic-pituitary-thyroid axis and (2) TSH concentrations in children with CCeH with MPHD are higher compared with children with isolated CCeH. We also studied whether FT4 levels are helpful in distinguishing CCeH from mild congenital hypothyroidism of thyroidal origin (CH-T). Dutch neonatal screening TSH and first diagnostic TSH and FT4 were analyzed in all children diagnosed with permanent CCeH between 1995 and 2012. Controls were children with T4-binding globulin deficiency. FT4 concentrations in CCeH were compared with those in CH-T with TSH values in the same range as those of CCeH. We studied 120 children with CCeH (isolated CCeH, n = 50; MPHD, n = 70) and 350 control subjects. Screening TSH concentrations were not significantly different (P = 0.055), but diagnostic TSH values were significantly different between the CCeH group and the control group (P = 0.037). TSH was significantly higher in MPHD compared with isolated CCeH (P = 0.004). FT4 concentrations were significantly lower in CCeH compared with mild CH-T (P < 0.0005). TSH values in CCeH have a more U-shaped distribution compared with controls with the highest TSH concentrations in MPHD. FT4 levels were significantly lower in CCeH compared with CH-T.

Detecting Congenital Central Hypothyroidism by Newborn Screening: Difficulty in Distinguishing from Congenital Thyroxine-Binding Globulin Deficiency

Background/Aims: Congenital central hypothyroidism (CH-C) can be detected on newborn screening (NBS) by programs using thyroxine (T4)-reflex thyroid-stimulating hormone (TSH) test approach. CH-C must be distinguished from T4-binding globulin (TBG) deficiency. We sought to determine whether thyroid function tests reliably separate CH-C from TBG deficiency. Methods: We analyzed NBS and serum free and total T4, T3 resin uptake (T3RU) or TBG, and TSH for infants in the Northwest Regional NBS Program (NWRSP) between the years 2008 and 2015 with either CH-C or TBG deficiency. Results: We discovered a significant overlap in T3RU and TBG levels amongst 21 cases of CH-C and 250 cases of TBG deficiency. Mean serum TBG levels were lower in CH-C cases (20.3 µg/mL, range 14.2–33.3) than what is reported in healthy infants (28.6 µg/mL, range 19.1–44.6). Serum free T4 was lower in CH-C cases than TBG deficiency but did not always differentiate between the two conditions. Conclusion: CH-C benefits from detection on NBS but must be distinguished from TBG deficiency. The diagnosis of CH-C rests solely on subnormal serum free T4, but is supported by the demonstration of other pituitary hormone deficiencies. As an overlap exists, serum TBG (or T3RU) levels do not play a role in the diagnosis of CH-C.

A Novel Mechanism of Inherited TBG Deficiency: Mutation in a Liver-Specific Enhancer

T4-binding globulin (TBG), a protein secreted by the liver, is the main thyroid hormone (TH) transporter in human serum. TBG deficiency is characterized by reduced serum TH levels, but normal free TH and TSH and absent clinical manifestations. The inherited form of TBG deficiency is usually due to a mutation in the TBG gene located on the X-chromosome. Among the 75 families with X-chromosome-linked TBG deficiency identified in our laboratory, no mutations in the TBG gene were found in four families. The aim of the study was to identify the mechanism of TBG deficiency in these four families using biochemical and genetic studies. Observational cohort, prospective. University research center. Four families with inherited TBG deficiency and no mutations in the TBG gene. Clinical evaluation, thyroid function tests, and targeted resequencing of 1 Mb of the X-chromosome. Next-generation sequencing identified a novel G to A variant 20 kb downstream of the TBG gene in all four families. In silico analysis predicted that the variant resides within a liver-specific enhancer. In vitro studies confirmed the enhancer activity of a 2.2-kb fragment of genomic DNA containing the novel variant and showed that the mutation reduces the activity of this enhancer. The affected subjects share a haplotype of 8 Mb surrounding the mutation, and the most recent common ancestor among the four families was estimated to be 19.5 generations ago (95% confidence intervals, 10.4-37). To our knowledge, the present study is the first report of an inherited endocrine disorder caused by a mutation in an enhancer region.

TRH Testing in Its Infancy

In contrast to primary hypothyroidism (1:3000 to 1:4000 births), central hypothyroidism due to pituitary or hypothalamic dysfunction is rare in newborns, with a prevalence of 1:25,000 to 1:100,000 births (1, 2). Although less common, central hypothyroidism is important to recognize because it is often accompanied by other pituitary hormone deficiencies that can cause life-threatening hypoglycemia. The hypothalamic-pituitary-thyroid axis undergoes dramatic changes in the transition from the fetal to newborn period (3). Thirty minutes after birth, newborns undergo a TSH surge to above 50 IU/liter, followed by a slow decrease to adult levels by 5–7 d of life. The TSH surge is accompanied by a rapid, 50% increase in total T4 levels that fall to normal infant levels by 3–5 d of life. However, the intrinsic variability of a maturing thyroid axis results in normal ranges for TSH and T4 values that vary among neonatal screening programs. Moreover, the trend toward an earlier discharge of infants and mothers from the hospital means that blood samples are being obtained earlier after birth. Considering these changes, interpreting the newborn screen must be done with sensitivity to the time of collection. When the newborn screen identifies a patient with a low T4 and elevated TSH level ( 40 IU/liter), the diagnosis of primary hypothyroidism is certain and therapy is begun. Although less common, other combinations of abnormal thyroid function tests are possible and usually result in additional testing. For example, if a screen is collected after 4–5 d, a case of mild primary hypothyroidism may have only a slightly elevated TSH and low to low-normal T4, mimicking central hypothyroidism. In contrast, patients with central hypothyroidism could experience a modified TSH surge followed by an increase in total T4 over the first 48–72 h of life, enough to push their T4 values into the normal or slightly elevated range. We know very little about the TSH surge in these patients. T4-binding globulin deficiency, euthyroid sick syndrome, and the hypothyroxinemia of prematurity can also cause low T4 associated with a normal TSH level. Importantly in these entities, free T4 values are usually normal. Unfortunately, immunoassays for free T4 are affected by low binding protein concentrations, resulting in falsely low free T4 levels (4), which further complicates their use in infants. To distinguish among these possibilities, additional testing is needed such as repetitive thyroid and pituitary hormone measurements and imaging of the thyroid and pituitary gland. Testing is an expensive and time-consuming approach and does not always result in a certain diagnosis. Therefore, alternative strategies may aid in the evaluation of these infants. In this issue of JCEM, van Tijn et al. (5) present their experience using the TRH stimulation test in a cohort of infants with low total T4 and normal TSH on newborn screen (6) and in whom they later confirmed multiple pituitary hormone deficiencies and pituitary dysmorphology with stimulation testing and imaging. After the synthesis of TRH in 1969, TRH stimulation testing was used to detect subtle states of thyroid gland dysfunction, acting as an amplifier to exaggerate underlying abnormalities in TSH secretion. As third and fourth generation TSH assays became more sensitive, however, the TRH stimulation test fell out of favor. Normal adults experience a maximum increase in serum TSH 30 min after TRH administration, and TSH levels gradually fall to basal levels by 120 min (7). In adults with pituitary or hypothalamic disease, the rise in TSH follows a different pattern. Often there is a delayed rise in TSH after TRH stimulation followed by a sustained increase. Sometimes the TSH rise is blunted or may be absent, which is more likely to be seen with pituitary disease in adults (8). Finally, an exaggerated TSH rise to TRH stimulation is occasionally noted in patients with central hypothyroidism (9, 10). This effect is thought to be due to the release of bioinactive TSH that is equally detected by immunoassays (11). These patterns have been verified in normal children and in children with hypopituitarism (12). Experience with the TRH test in newborns is extremely limited. There are significant differences in T4 metabolism in younger vs. older infants and children, which could produce different TRH stimulation test patterns in health and disease. The few available studies have used different TRH doses, collected TSH values at different times, and studied infants of various ages and clinical conditions (13–15). No other study has

Three Novel Mutations Causing Complete T4-Binding Globulin Deficiency

Inherited T4-binding globulin deficiency is caused by mutations in the T4-binding globulin gene located on the X chromosome. We describe herein three novel mutations in three different families producing complete T4-binding globulin deficiency. The proposita of a family from Harwichport is a female with XO Turner’s syndrome who expressed only the mutant T4-binding globulin allele. Her T4-binding globulin sequence has a 19-nucleotide deletion in the distal portion of exon 4. This causes a frameshift and a premature stop at codon 384 of the mature protein. Structure analysis with the Swiss PDB-Viewer revealed that this mutation removes β-strand s5B from the core of the T4-binding globulin molecule, leading to a severe folding defect that is likely to prevent synthesis and secretion. The propositi of complete T4-binding globulin deficiency 7 and 8 were 7-month-old and 3-wk-old male infants who were identified because of low serum T4 levels detected during neonatal screening. Sequencing of complete T4-binding globulin deficiency 7 revealed a single nucleotide deletion, a G at position 2690 in exon 3. This leads to an alteration of the amino acid sequence starting at codon 283 and a premature stop at codon 301. Complete T4-binding globulin deficiency 8 also has a deletion of the first nucleotide of exon 4, a G at position 3358. This leads to a frameshift and a premature stop at codon 374. As in the case of complete T4-binding globulin deficiency J, which has also a nucleotide deletion but downstream (position 3421) and a stop at codon 374, these two T4-binding globulin mutants undoubtedly have a defect in intracellular transport and therefore fail to be secreted. This explains the lack of T4-binding globulin in the hemizygous affected subjects.

A Novel Mutation Causing Complete Thyroxine-Binding Globulin Deficiency (TBG-CD-Negev) among the Bedouins in Southern Israel

T4-binding globulin (TBG) is the major thyroid hormone transport protein in human serum. Inherited TBG abnormalities do not usually alter the metabolic status and are transmitted in X-linked inheritance. A high prevalence of complete TBG deficiency (TBG-CD) has been reported among the Bedouin population in the Negev (southern Israel). In this study we report a novel single mutation causing complete TBG deficiency due to a deletion of the last base of codon 38 (exon 1), which led to a frame shift resulting in a premature stop at codon 51 and a presumed truncated peptide of 50 residues. This new variant of TBG (TBG-CD-Negev) was found among all of the patients studied. We conclude that a single mutation may account for TBG deficiency among the Bedouins in the Negev. This report is the first to describe a mutation in a population with an unusually high prevalence of TBG-CD.

Follow-up of newborns with low thyroxine and nonelevated thyroid-stimulating hormone–screening concentrations: Results of the 20-year experience in the Northwest Regional Newborn Screening Program

Objectives: To determine the type and frequency of thyroid disorders detected in infants with low thyroxine (T4) and nonelevated thyroid-stimulating hormone (TSH) screening test results in the Northwest Regional Newborn Screening Program (NWRNSP) over the 20-year period from May 1975 to May 1995 and to determine the effect of follow-up of these infants on the overall recall rate. Study design: The NWRNSP requests a serum specimen in infants with an absolute T4 level < 38.6 nmol/L (<3 mg/dl) and in infants with two filter paper T4 concentrations less than the 3%, regardless of the TSH concentration. We conducted a retrospective analysis of infants who were followed up because of low T4 and nonelevated TSH concentrations on newborn screening. To determine the effect of follow-up of infants with low T4 levels, nonelevated TSH concentrations on the recall rate, we selected 1 year (1994) for review. Serum sample requests were evaluated to determine the reason for the request. Results: Over this 20-year period, the NWRNSP detected 450 infants with primary hypothyroidism among 1,747,805 infants screened (1:3,884). Of these, 416 were detected on the basis of low T4 levels and nonelevated TSH screening test results, whereas an additional 34 infants with primary hypothyroidism and 29 infants with hypopituitary hypothyroidism were detected as a result of follow-up of low T4 levels and nonelevated TSH screening test results. This included 25 infants with delayed TSH rise (1:67,226), 9 infants with mild hypothyroidism (TSH levels <25 mU/L) (1:194,212), 29 infants with hypopituitary hypothyroidism (1:60,269), and 434 infants with T4-binding globulin deficiency (1:4,027). Excluding those with T4-binding globulin deficiency, the false-positive rate was 43.5:1. This compares with an overall false-positive rate of 12:1 for our screening program. Conclusion: Follow-up of infants with low T4 and nonelevated TSH concentration on screening led to the detection of 63 additional infants with hypothyroidism, for an overall frequency of 1:27,743. We believe this yield justifies continued follow-up of infants with low T4 levels, nonelevated (TSH) screening test results in our program. (J Pediatr 1998;132:70-4)

Measurement of serum free thyroid hormone concentrations: an essential tool for the diagnosis of thyroid dysfunction.

Free thyroid hormones (free thyroxine, FT4, and free triiodothyronine, FT3) represent a more useful index of thyroid status than total thyroid hormones, because the latter are influenced by variations of thyroid hormone-binding proteins, especially T4-binding globulin (TBG). Thus, increased serum total T4 (TT4) and, in many instances, T3 (TT3) concentrations are encountered in euthyroid subjects with TBG excess, familial dysalbuminemic hyperthyroxinemia and transthyretin-associated hyperthyroxinemia, while decreased serum TT4 and TT3 levels are associated with TBG deficiency: under these circumstances, measurement of serum FT4 and FT3 levels correctly establishes the diagnosis of euthyroidism. In cases of suspected hyperthyroidism, a diagnostic strategy can be suggested based on serum FT3 (and TSH) measurement, since FT4 may occasionally be elevated, also in euthyroid subjects, e.g., in patients under chronic amiodarone or L-T4 treatment. When hypothyroidism is suspected, the most reliable test appears to be FT4 (together with TSH), because FT3 may still be normal in patients with subclinical or mild thyroid failure. In any case, it is essential that reliable free thyroid hormone assays be used, which are devoid of methodological limitations responsible for artifactual results under particular circumstances, such as thyroid hormone-binding protein abnormalities, pregnancy and nonthyroidal illness.

Nucleotide Deletion Resulting in Frameshift as a Possible Cause of Complete Thyroxine-Binding Globulin Deficiency in Six Japanese Families

Complete T4-binding globulin deficiency (TBG-CD) is inherited in an X-linked fashion. A nucleotide substitution has been shown to cause this hereditary condition in caucasians of French Canadian origin. Heterogeneity in molecular mechanisms for TBG-CD has also been reported. Genomic DNA from a Japanese male exhibiting TBG-CD was subjected to polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide deletion was found in the first base of the codon for amino acid 352 of the common-type TBG molecule. This mutation causes a frameshift in translation and premature termination. Compared with common-type TBG, the mutated polypeptide results in 1) 22 different amino acids on its carboxy-terminus, 2) a 22-amino acid truncation, and 3) the absence of a potential N-linked glycosylation site. These alterations may lead to profound changes in the secondary and tertiary structures of the molecule. To ascertain the presence of this nucleotide deletion in the genomic DNA of affected subjects, a mutated primer was designed which together with the nucleotide deletion produced a new endonuclease restriction site in the polymerase chain reaction fragment. Results revealed the presence of the mutation in genomic DNA of the subject, and his mother was shown to have both mutant and normal alleles. The same mutation was also detected in five other unrelated families carrying TBG-CD. This mutation may be frequent in Japanese subjects with TBG-CD.

Drug Competition for Thyroxine Binding to Transthyretin (Prealbumin): Comparison with Effects on Thyroxine-Binding Globulin*

We examined the effect of 26 drugs on T4 binding to transthyretin (TTR; prealbumin) and T4-binding globulin (TBG) by determining their ability to inhibit [125I]T4 binding to TTR isolated from normal human plasma and to serum diluted 1:10,000, respectively. The hierarchies for drug inhibition of T4 binding differed greatly for these two proteins. Relative to T4, the drugs were much more potent inhibitors of [125I]T4 binding to TTR than to TBG. Compounds of the anthranilic acid class, such as flufenamic, meclofenamic, and mefenamic acids, interacted particularly strongly with TTR. Flufenamic acid was more potent than T4 itself in inhibiting [125I]T4 binding [175 +/- 17% (+/- SD); cf. T4; n = 3; P less than 0.001], while mefenamic acid, diflunisal, and meclofenamic acid were 20-26% as potent as T4 in their interaction with TTR. The reactivity of diclofenac, fenclofenac, indomethacin, sulindac, and the diuretic ethacrynic acid was 0.8-2.1% relative to that of T4. In contrast, furosemide, the drug most highly reactive with TBG, was only 0.11 +/- 0.03% (n = 7) as potent as T4, followed by meclofenamic acid greater than mefenamic acid greater than fenclofenac greater than flufenamic acid greater than diflunisal greater than milrinone. Aspirin and sodium salicylate were, respectively, 0.05% and 0.20% as active as unlabeled T4 as inhibitors of [125I]T4 binding to TTR, but these compounds had only 3-4 x 10(-6)% of the activity of T4 for TBG binding. Diphenylhydantoin had no detectable effect on T4 binding to TTR and was 2.9 x 10(-4)% as reactive as T4 with TBG. Amiodarone did not interact with either binding site. Drug interactions with TTR may be important when this protein becomes a major circulating T4-binding protein, as in patients with complete or partial TBG deficiency, or when serum T4 is markedly elevated. Such interactions may also be important where TTR is the dominant tissue T4-binding protein, as in the choroid plexus. In addition, the drug competitors described here may be useful as probes to further define the structural basis for specific ligand interactions with different classes of T4-binding sites.

A Mutation Causing Reduced Biological Activity and Stability of Thyroxine-Binding Globulin Probably as a Result of Abnormal Glycosylation of the Molecule

T4-binding globulin (TBG), a 54-kilodalton glycoprotein, is the major thyroid hormone transport protein in man. The exact nature of the mutations causing X chromosome-linked TBG deficiency, which affect about 1 in 2,500 newborn males, is unknown. Here we report the sequence of a unique variant TBG (TBG-Gary) encoding a protein with severely impaired T4 binding as well as decreased stability at 37 C, resulting in its rapid in vivo denaturation. A single nucleotide substitution in the codon for residue 96 of the mature protein replaces isoleucine with asparagine; this replacement creates an additional site for N-linked glycosylation. The anodal shift of TBG-Gary on isoelectric focusing gel electrophoresis suggests that this new site is likely glycosylated. Since glycosylated is required for TBG to assume its correct tertiary structure, but is not subsequently necessary for maintenance of the biological properties or stability of the molecule, we believe that the likely presence of additional carbohydrate probably affects a higher order structure of the molecule and is thus responsible for the reduced stability and hormone binding activity of TBG-Gary (TBGASN-96).

Detection of the thyroxine-binding globulin (TBG) gene in six unrelated families with complete TBG deficiency.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. Inherited TBG defects in man are X-chromosome linked and are expressed in hemizygotes as complete deficiency, partial deficiency, or excess. Since TBG is not necessary for thyroid hormone action, affected subjects are healthy. Using DNA probes for human TBG, we searched for restriction fragment length polymorphisms in six affected males belonging to 6 unrelated families with inherited complete TBG deficiency and an equal number of normal males. TBG could not be detected in the serum of any of the TBG-deficient males by a specific and sensitive RIA capable of detecting as little as 5 micrograms TBG/L or 0.031% of the average normal serum TBG concentration. DNA isolated from white blood cells was digested with 11 restriction endonucleases, and the digests were submitted to DNA blot analysis using two cloned TBG-DNA probes which together covered the entire protein coding and the 5'-flanking sequences of the TBG gene. A total of 26 different bands were detected on DNA blots, identifying 18 restriction sites located within the 4.2-kilobase TBG gene, which includes intronic, exonic, and 5'-flanking sequences. This analysis, which sampled 2.3% of the total TBG genome, failed to reveal differences in fragment size among the 6 TBG-deficient and 6 normal males examined. One restriction endonuclease (NcoI) identified normal sequences at the putative promoter region of the gene, and four other endonucleases (TaqI, SstII, MspI, and HpaII) recognized the cytosine-guanine dinucleotide phosphate sequences representing potential mutation hot spots. Although C was methylated at these sites, no C to T (thymidine) transitions were found. These data suggest that large deletions, insertions, or rearrangements of the TBG gene, or mutations at sites of methylated cytosine-guanine dinucleotide phosphate dimers are not common mechanisms for inherited complete TBG deficiency in man.

Elevated serum thyroglobulin level in congenital thyroxine-binding globulin deficiency.

Serum thyroglobulin (TG) is normally under TSH control. Serum TG levels are elevated during increased thyroid gland stimulation and suppressed by exogenous thyroid hormone. High serum TG levels are also found with thyroid gland damage and in patients with differentiated thyroid neoplasms. Congenital T4-binding globulin deficiency was found in this study to be an additional condition in which serum TG levels may be elevated. Elevated TG levels were found in 13 of 36 patients (36%) with congenital TBG deficiency compared to 1 of 27 unaffected relatives (4%). Mean TSH and free T4 index values were not significantly different. A postulated mechanism involves transient TSH stimulation of the thyroid after transient small declines in circulating free hormone levels due to the decreased extrathyroidal pool of thyroid hormone associated with T4-binding globulin deficiency.

Dichotomy between serum free triiodothyronine and free thyroxine concentrations in familial thyroxine-binding globulin deficiency.

The mean serum total T4 (3.2 +/- 1.0 micrograms/100 ml) and T3 levels (83 +/- 29 ng/100 ml) in eight euthyroid patients with T4-binding globulin (TBG) deficiency from two families were significantly lower than those in either the unaffected relatives (P less than 0.05) or the control subjects (P less than 0.01). The lowest T4 and T3 values were found in the four hemizygotes (2.4 +/- 0.2 micrograms/100 ml and 69 +/- 25 ng/100 ml, respectively). In contrast to the absolute T4 and T3 levels, the mean percentages of free T4 and free T3 in the affected members were about twice as high as those in either the unaffected relatives or the controls. Despite an elevated percentage of free T4 in each of the TBG-deficient members, the mean absolute free T4 (FT4) level (0.89 +/- 0.20 ng/100 ml) was significantly lower than those in both control groups [1.31 +/- 0.2 ng/100 ml (P less than 0.02) and 1.24 +/- 0.30 ng/100 ml (P less than 0.01), respectively]. Serum FT4 levels were subnormal in three of four hemizygotes. In contrast to FT4, the mean FT3 level in the affected members (0.33 +/- 0.04 ng/100 ml) was significantly higher than that in either the nondeficient relatives (0.25 +/- 0.02 ng/100 ml; P less than 0.02) or the control subjects (0.26 +/- 0.04 ng/100 ml; P less than 0.02), whereas the ratio of FT3 to FT4 was about twice as high. The data suggest that in TBG deficiency, euthyroidism in maintained by a subtle equilibrium between low FT4 and high FT3 levels.

Thyroxine-binding globulin: investigation of microheterogeneity.

Preparations of T4-binding globulin (TBG) from human serum was performed using only two affinity chromatography steps. Purity of the protein was demonstrated by a single band in overloaded disc and sodium dodecyl sulfate electrophoresis, equimolar binding to T4, and linearity in sedimentation velocity run. The molecular weight was calculated to be 60,000 +/- 3,000 daltons (n = 3), the sedimentation coefficient was 3.95S, and the Stokes' radius was 37 A. The amino acid composition was found to be in good agreement with the calculations of other authors. By isoelectric focussing (IEF), pure TBG showed four main bands at pH 4.25, 4.35, 4.45, and 4.55 together with several fainter bands. The N-acetylneuraminic acid (NANA) content of the four TBG bands isolated by preparative IEF was found to decrease from 10.2 mol NANA/mol TBG in the band at pH 4.25 to 4.8 mol NANA/mol TBG in the band at pH 4.55. No significant difference in the affinity constants of the TBG bands to T4 was found. The affinity constants for TBG ranged from 3.1 x 10(9) to 7.2 x 10(9) M-1. Sequential kinetic desialylation of pure TBG resulted in a progressive tendency toward one major band at pH 6.0. In native sera, microheterogeneity of TBG was detected after IEF on polyacrylamide gel plates by immunofixation. The typical TBG patterns shown by pure TBG were also found in normal subjects. Characteristic deviations from this pattern were found in the sera of females during estrogen therapy or pregnancy, where there was a gradual increase in density of the band at pH 4.25 and the appearance of an additional band at pH 4.15. In sera from patients with liver disease and elevated TBG levels, there was a fading of the acidic bands, whereas the more alkaline band at pH 4.55 was intensified. It is therefore proposed that microheterogeneity of TBG is caused by differences in NANA content and that variations of TBG patterns in native sera may reflect altered TBG synthesis or degradation. A genetically related microheterogeneity of TBG could not be demonstrated after examination of 800 sera, including 2 families with quantitative TBG deficiency.