An evaluation of serum thyroxine binding globulin as a routine test of thyroid function.

Serum thyroxine binding globulin (TBG) values were correlated with other thyroid function test results and with the clinical condition in 680 patients. The estimation of serum TBG was helpful in the evaluation of thyroid status only in those patients who were either acutely ill, were taking the contraceptive pill, or were pregnant. Further, the derived index, thyroxine: TBG ratio, proved to be a better diagnostic index than the thyroxine:tri-iodothyronine uptake ratio and in many cases would have avoided the use of more expensive and time-consuming tests.     

Methods and clinical evaluation of a new enzyme immunologic method for determination of thyroxin binding globulin (TBG) and T4/TBG quotient: a multicenter study

A heterogeneous enzyme immunoassay for the determination of thyroxine binding globulin (TBG) was developed and assessed in clinical trials in 12 laboratories. The assay is based on the competition principle and employs plastic tubes coated with goat anti-TBG. CV's between 1.4-8.9% for intra-assay precision and 2.9-8.6% for inter-assay precision were found over the concentration range of 4-40 mg/l TBG. In comparative studies using highly purified TBG as standard, values with Enzymun-Test TBG were found to be on average 30% lower than those obtained by various TBG-RIAs. A broad-base study, to determine reference values, was carried out on a group of control persons 18 to 50 years old without previous history of thyroid disease. This study revealed a median of 14.33 mg/l TBG, with 95% of all values between 9.6 and 18.5 mg/l TBG. The median in women of 14.5 mg/l TBG was significantly higher than in men (13.4 mg/l TBG). TBG values in hyperthyroid patients were within the reference range while those in hypothyroid individuals were elevated. Highly elevated TBG values were seen in women receiving oestrogen (median: 22.2 mg/l TBG) and in pregnant women (median: 28.5 mg/l TBG). The T4/TBG ratios made it possible to distinguish between euthyroid, hyperthyroid and hypothyroic subjects (median: 4.9, 11.3 and 1.0, respectively). These ratios were significantly lower in pregnant women (median: 3.1) than in the control persons.     

Serum free thyroxine concentrations in normal euthyroid subjects and ones with high serum thyroxine binding globulin concentration

Serum free thyroxine (fT4) was assayed by a commercial fT4 method in 30 normal euthyroid subjects, 19 pregnant females, 13 euthyroid subjects with high thyroxine binding globulin (TBG) and three with low or undetectable serum TBG concentration. In a number of these fT4 was also calculated on the basis of the application of the law of mass action to the binding situation. In states in which TBG was altered for congenital reasons both the experimentally determined and calculated fT4 were not significantly different from their respective means in the normal euthyroid population. Pregnant females had both lower experimental and theoretical free T4 concentrations. It is inferred from these data that TBG concentration per se is without effect on serum fT4 concentration.     

Reference intervals from birth to adulthood for serum thyroxine (T4), triiodothyronine (T3), free T3, free T4, thyroxine binding globulin (TBG) and thyrotropin (TSH).

Disorders in thyroid function can impair normal development in children. Therefore it was our aim to establish reference intervals for serum triiodothyronine (T3), free T3 (fT3), thyroxine (T4), free T4 (fT4), thyroxine binding globulin (TBG) and thyrotropin (TSH) which are applicable from birth to adulthood by using the non-isotopic automated chemiluminescence immunoassay system, Immulite (DPC Los Angeles, USA). Serum samples from 762 euthyroid newborns, children and adolescents (369 female, 393 male; age 1 day to 19 years) were examined; of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (the central 95% interval) were calculated for each group. The median concentrations of T4, fT4 and TSH were up to 3.2-fold higher during the first 2 weeks, while T4 increased during the first month of life. The concentrations in all age groups showed no sex differences. From 1 year onwards, the concentration of all parameters tended to decrease until adult age, with the exception of TBG which increased by >60% (p<0.02) and reached a maximum at approximately 5 years of age. The findings underscore the fact that thyroid hormones are not associated with sexual development, except for TBG, which decreased slightly (p<0.04) between Tanner stages 1 and 5. However, the reference intervals established here demonstrate that marked changes occur in concentrations of thyroid hormones after the neonatal period. Our findings complement these of earlier studies. The developed reference intervals can be used to assess the thyroid status of patients, particularly if the measurements are done on the Immulite/Immulite 2000 system.     

A comparison of commercially available luminescence enhanced enzyme immunoassays with in-house non-radioisotopic assays for thyroxine binding globulin and total thyroxine

Commercial luminescence enhanced enzyme immunoassays (Amersham-Amerlite) for thyroxine binding globulin (TBG) and total thyroxine (TT4) were compared with the in-house methods (TT4--Abbott TDx, TBG--Immunoluminometric Assay (ILMA)). The experimental groups consisted of 108 healthy euthyroid blood donors, 165 non-selected thyroid outpatients, 44 tumour bearers and 84 haemodialysis patients. Total thyroxine/thyroxine binding globulin quotients were constructed as an index of thyroid function. The luminescence enhanced enzyme immunoassays were precise (interassay coefficients of variation less than 10% in the range 5-45 mg/l thyroxine binding globulin and 20-100 micrograms/l for total thyroxine) performed similarly to the in-house methods in the differentiation of eu-, hypo- and hyperthyroidism on the basis of total thyroxine/thyroxine binding globulin quotients. Although statistically significant differences often occurred in comparisons of the in-house method with the luminescence enhanced enzyme immunoassays, these only gave rise to thyroid status differences in two cases out of 273, where the in-house method gave a hyperthyroid, the luminescence enhanced enzyme immunoassay a euthyroid answer, when taken from the total thyroxine/thyroxine binding globulin quotients. The luminescence enhanced enzyme immunoassays performed as well as the in-house methods, and quality assessment data were comparable with their radioimmunological counterparts.     

Transthyretin--an explanation of "anomalous" serum thyroid hormone values in severe illness?

The following serum analytes were measured in 464 patients with defined carcinomas and other tumours as well as those with chronic obstructive lung disease and under regular haemodialysis, and in 261 healthy controls: thyrotropin (TSH), thyroxine (T4), triiodothyronine (T3), free thyroxine (fT4), thyroxine binding globulin (TBG) and transthyretin (TTR). The following ratios were constructed: fT4 x TTR, defined as the thyroxine availability index fT4/TTR, defined as the thyroid hormone compensation index 100 x T3/TBG as the free T3 index (fT3I) and fT3I x TTR, defined as the triiodothyronine availability index. Significantly elevated thyrotropin values (p = 0.05) were only found in patients with breast cancer when compared with age matched controls, although elevated T4 and fT4 values were found in all experimental groups except the haemodialysis patients (p less than 0.01). The thyroxine availability index and triiodothyronine availability index values were not significantly different from the age matched controls (greater than 60 a) in the cancer groups, showing that the transthyretin concentrations compensated for changes in fT4 or vice versa. These findings are reflected in the euthyroid thyrotropin values. The T4 and fT4 values in the dialysis patient group were significantly lower than in the age matched controls (p less than 0.01), while the transthyretin values were significantly higher (p less than 0.01), which accounted for the normal thyroxine availability index and euthyroid thyrotropin values.     

Clinical and laboratory evaluation of four methods of assessing free thyroxine status in thyroid clinic patients

The clinical value of four laboratory methods of assessing free thyroxine status was compared in 82 consecutive patients newly referred to a thyroid clinic with suspected thyroid dysfunction. The methods of determining free thyroxine used were: (1) free thyroxine index using a thyroid hormone uptake test (FTI (THUT)); (2) a free thyroxine index using thyroxine binding globulin (FTI (TBG)); (3) a kinetic radioimmunoassay (Immophase); and (4) an equilibrium dialysis method. The definitive thyroid status was evaluated by a combination of clinical assessment (including Wayne index), routine tests of thyroid function (total T4, T3, TSH, and TRH tests where appropriate), and by therapeutic trial in one case. The diagnostic efficiency of the tests was markedly dependent upon the method of determining the reference range for euthyroid patients. Best efficiency for each test was achieved using an amended range after excluding outliners. Test efficiency was then 97.6% for FTI (THUT) and the kinetic RIA and 96.8% for FTI (TBG). Misclassification by one or more of these tests occurred in only four patients (mild hypothyroid, euthyroid on phenytoin, euthyroid on oral contraceptive and valium, T3 hyperthyroid). In contrast, free T4 by equilibrium dialysis was much less efficient (86.6%) and was technically the most complex. Overall the kinetic T4 RIA provided similar diagnostic information to the indirect indices. However, further studies of cost benefit in settings other than a thyroid clinic are required to assess whether this method might replace total T4 and/or FTI as a first-line test of thyroid function.     

Salicylate-induced increases in free triiodothyronine in human serum: Evidence of inhibition of triiodothyronine binding to thyroxine-binding globulin and thyroxine-binding prealbumin

Addition of sodium salicylate to human serum at concentrations often obtained during aspirin therapy causes 100-200% increases in free triiodothyronine (T(3)) and free thyroxine (T(4)) as estimated by ultrafiltration. The increase in free T(3) was unexpected since previous data had suggested that salicylate inhibits binding of T(4) only to thyroxine-binding prealbumin (TBPA) and that T(3) is not bound to this protein. Using ultrafiltration techniques, we demonstrated binding of T(3) to TBPA. The affinity constant for T(3)-TBPA binding appears to be slightly greater than that for albumin-T(3) binding. While salicylate inhibits the binding of T(3) (and T(4)) to TBPA, it can be predicted that little change will be observed in the free T(3) (or free T(4)) without inhibition of thyroid hormone binding to thyroxine-binding globulin (TBG). Using a competitive-binding protein displacement technique, it has been shown that sodium salicylate, like diphenylhydantoin (DPH), inhibits the binding of T(3) and T(4) to TBG. The magnitude of the increase in free T(3) and free T(4) induced by salicylates suggests that interference with TBG binding is its major effect.Aspirin was administered orally to two normal subjects in quantities sufficient to obtain serum salicylate levels of 20-25 mg/100 ml. This resulted in a decrease of 20-30% in total serum T(3) and T(4) levels. This decrease in T(4) levels is similar in magnitude to that previously observed in subjects receiving DPH. Unlike what has been observed with DPH treatment, therapeutic salicylate levels are associated with increases of 50-75% in the unbound fraction of both T(3) and T(4) which persist throughout an 8-10 day treatment period.     

The T3/TBG ratio and the biochemical investigation of thyrotoxicosis.

The relationship between serum tri-iodothyronine (T3) and thyroxine-binding globulin (TBG) has been studied in euthyroid subjects. This relationship is similar to that previously described for thyroxine (T4) and TBG. Serum T3, TBG and T4 data are presented for euthyroid subjects of all ages and the T4:T3 and T3:TBG ratios calculated. Similar data has been derived for patients with clinically proven thyrotoxicosis and for patients in which the diagnosis was in doubt. The results suggest the T3:TBG ratio enhances the use of the T3 assay to confirm thyrotoxicosis. The fact that 25% of our patients suffering from thyrotoxicosis had apparently normal serum T4 levels reinforces the contention that a serum TBG assay must be carried out as part of a basic thyroid function screen.     

Differentiating various abnormalities of thyroxin binding to serum proteins by radioelectrophoresis of thyroxin and immunoassay of binding proteins

Using the simple method of protein analysis described here, we could identify thyroxin (T4)-binding-protein abnormalities in euthyroid patients with hyperthyroxinemia or hypothyroxinemia. Serum incubated with [125I]thyroxin was analyzed by agarose gel electrophoresis, with bromphenol blue staining of protein. The relative distribution of radioactive T4 was determined for each binding protein--thyroxin-binding globulin (TBG), transthyretin, albumin, and T4-binding immunoglobulin (when present)--and the mass of T4 bound to each was determined. We also used sensitive immunoassays to quantify TBG, transthyretin, and albumin concentrations, then calculated the mass of T4 (as determined by electrophoresis) bound per unit mass of the respective binding protein. When the concentration of binding proteins was altered (e.g., TBG excess or TBG deficiency), the T4 binding/mass ratio for each protein remained within the expected range; but when the functional affinity of a binding protein was altered--as in dysalbuminemic hyperthyroxinemia and in low-T4 nonthyroidal illness--this ratio was abnormal. This procedure can be used to help identify TBG excess, TBG deficiency, dysalbuminemic hyperthyroxinemia, prealbumin-associated hyperthyroxinemia, variant TBG with reduced affinity for T4, euthyroid sickness, and T4-binding autoantibodies.     

Total and Free Triiodothyronine and Thyroid-Binding Globulin Concentration in Elderly Human Persons

Total thyroxine (TT⁴) and triiodothyronine (TT³) were found to be low in healthy elderly subjects with a preferential decrease of triiodothyronine. In order to determine the importance of these findings 22 healthy elderly subjects were examined.
Free triiodothyronine (FT³), thyroid binding globulin (TBG) concentration and basal thyroid stimulating hormone (TSH) were measured by radioimmunoassay. Liver enzymes, cholesterol and total protein concentration were also assayed. TBG was significantly increased compared to a middle-aged group and did not correlate with TT⁴, TT³ and TSH. Basal TSH values were in the normal range and could be detected in all the elderly subjects in contrast to undetectable values in 40 % of the younger subjects. PT³ determined directly did not correlate with the values calculated according to the law of mass action. According to the FT³ values the elderly subjects could be subdivided into three groups independent of their TT⁴, TT³, TBG and TSH values. FT³ was undetectable in one group, in the low normal to normal range in another and elevated in the third group. Our results suggest that 1) there is no correlation between TT⁴, TT³, elevated TBG and FT³ determined directly or by calculation, 2) basal TSH values seem to indicate possible hypothyroidism in elderly persons which is correlated with elevated cholesterol levels and 3) FT³ measured directly subdivides this metabolic state into three groups possibly depending on the intracellular concentration of T⁴.     

Thyroxine binding globulin radioimmunoassay in dried blood spotted on filter paper

A method is described for the determination of thyroxine binding globulin (TBG) in dried blood spotted on filter paper using reagents from a test kit for the measurement of TBG in plasma. By minor modifications of the recommended procedure it was possible to improve precision, sensitivity and tracer displacement. Appropriate TBG standard samples were prepared in 'artificial blood' consisting of a suspension of erythrocytes in buffer with bovine serum albumin (50 milligrams). There is a good correlation between plasma TBG RIA results and blood spot TBG RIA results (r = 0.93). Attention must be paid to the stability of the TBG in blood: our experiments show a decrease of TBG content if filter paper cards with dried blood are stored longer than one month.     

Study of Four New Kindreds with Inherited Thyroxine-Binding Globulin Abnormalities POSSIBLE MUTATIONS OF A SINGLE GENE LOCUS

Five families with inherited thyroxine-binding globulin (TBG) abnormalities were studied. On the basis of serum thyroxine (T(4))- binding capacity of TBG in affected males, three family types were identified: TBG deficiency, low TBG, and high TBG capacity. In all families evidence for X-linked inheritance was obtained and in one family all criteria establishing this mode of inheritance were met. Only females were heterozygous, exhibiting values intermediate between affected males and normals. Overlap in heterozygotes was most commonly encountered in families with low TBG.QUANTITATIVE VARIATION IN THE SERUM CONCENTRATION OF FUNCTIONALLY NORMAL TBG WAS DEMONSTRATED BY: (a) failure of serum from TBG-deficient subjects to react with anti-TBG antibodies; (b) normal kinetics of T(4) and triiodothyronine-binding to TBG in sera from subjects with low TBG and high TBG capacity; (c) concordance of estimates of TBG concentration by T(4) saturation and by immunological methods; and (d) normal rate of heat inactivation of TBG.No abnormalities in serum transport of cortisol, testosterone, aldosterone, or thyroxine bound to prealbumin could be detected.These observations suggest that all the TBG abnormalities thus far observed reflect mutations at a single X-linked locus involved in the control of TBG synthesis.     

Thyroid status in patients after acute myocardial infarction

Subjects followed serially after acute myocardial infarction demonstrated a rapid and sustained fall in serum total tri-iodothyronine (T3) concentration and a rise in reverse tri-iodothyronine (rT3) concentration. There was a transient fall in total thyroxine (T4) concentration. Thyroxine binding globulin (TBG) levels were unchanged after acute myocardial infarction but prolonged falls were observed in thyroxine binding prealbumin (TBPA) and albumin concentrations. In contrast to the fall in total T4, both measured and calculated free T4 concentrations were unchanged but measured and calculated free T3 concentrations fell as did total T3. Despite the observed fall in T3, basal thyrotrophin (TSH) concentrations did not rise. The reduction in circulating T3 levels after acute myocardial infarction suggests that a hypothyroid state exists. Until tissue thyroid status can be assessed directly, however, this conclusion must remain in doubt.     

An assessment under routine conditions of an immunoelectrophoretic assay for thyroxine-binding globulin

The correct clinical interpretation of serum total thyroxine requires a knowledge of the thyroxine binding globulin (TBG) concentration. An immunoelectrophoretic method for TBG assay has been assessed under routine conditions for precision, sensitivity and recovery. TBG has been compared with T3 Resin Uptake for euthyroid subjects, and patients with myxoedema and thyrotoxicosis. The relationship between TBG and age has also been studied. The method proved to be reliable, sensitive and a technically simple method for the routine determination of TBG and an excellent replacement for the indirect T3 Resin Uptake Test.     

Successful treatment of thyrotoxic crisis with plasma exchange

A 36-yr-old woman with thyroid storm was successfully treated with plasma exchange. After the first plasma exchange, free serum thyroxine (T4) was decreased and serum thyroxine-binding globulin (TBG) was significantly increased. The decrease in the patient's free T4 level after plasma exchange can perhaps be attributed to T4's increased binding capacity. Plasma exchange may be an effective therapy for thyrotoxic crisis and should be performed immediately if conventional therapy fails to improve the patient's condition.     

Binding characteristics of thyroxin binding globulin in serum of normal, pregnant, and severely ill euthyroid patients

Serum from normal, pregnant, and severely ill patients was stripped of endogenous thyroid hormones and diluted 1100-fold in barbital buffer. We then used it to study the binding characteristics of thyroxin binding globulin (TBG), noting significant differences in binding capacities among the groups. The mean (+/- SD) triiodothyronine/thyroxin ratio for binding capacity was 18 +/- 4 for normal subjects. The ratio was significantly increased in pregnant patients, 21 +/- 4 (p less than 0.05), and significantly lower in severely ill patients, 12 +/- 4 (p less than 0.05). When serum was diluted before assay, to give a uniform TBG concentration among groups, these apparent differences in binding characteristics were eliminated. It therefore is unlikely that different molecular species of TBG account for the variations in binding characteristics in these clinical states. Apparently, the distribution of thyroxin and triiodothyronine among the binding sites on TBG changes with variations in TBG concentration. This may explain the discrepancies observed in the concentrations of free thyroid hormones as estimated by various methodologies.     

Raised Free Thyroxine Values in Patients with Familial Elevation of Thyroxine Binding Globulin

Thyroxine (T₄) is carried in the plasma bound preferentiallyto an interalphaglobulin-thyroxine binding globulin (TBG) butalso to other proteins, thyroxine binding prealbumin (TBPA)and albumin. According to current theories of regulation ofmetabolism the protein-bound T₄ is in equilibrium with a smalleramount of free T₄ which is the metabolically active part. Elevation of the level of TBG in response to pregnancy and administrationof oestrogens has been recognized for some time. Much more rarelyelevated TBG levels occur as a familial disorder which is usuallyrecognized during the investigation of discordant protein-boundiodine levels. In these cases of elevated TBG levels associatedwith euthyroid states it has been assumed that the level offree thyroxine is normal. Investigation of the family of a euthyroid, mentally backwardinfant in whom the PB¹²⁷I was raised revealed the presence inthe child and in two of his three sibs of an elevated TBG level.This abnormality was also detected in the maternal grandmotherbut not in either parent. Similar findings were obtained inanother apparently unrelated child. Estimation of the free T₄ surprisingly revealed that the freeT₄ in the child and other members of the family with an elevatedTBG was considerably elevated in contrast to the unaffectedmembers of the family and in contrast to a large series of pregnantpatients and normal subjects receiving oestrogen-containingoral contraceptives. Levels of TBPA binding were low or undetectable in this familyirrespective of the level of TBG binding and free thyroxine. These findings suggest that in individuals with familial elevationof TBG that the TBG is not merely present in increased amountsas is the case when oestrogens are given but that qualitativedifferences in the nature of the TBG may be present.     

Unforeseen effect of thyroxine binding globulin when using the microencapsulated antibody method to determine free thyroxine (FT4): misleading results due to circulating unsaturated thyroxine binding globulin

The effect of varying concentrations (0-52 mg/l) of purified thyroxine binding globulin (TBG) on the microencapsulated antibody method for free thyroxine was investigated. The results demonstrated that the free thyroxine values were strongly influenced by the concentration of thyroxine binding globulin in the samples. The standard curve could no longer be distinguished at a concentration of purified thyroxine binding globulin of 52 mg/l. In the clinical application, we observed that the values obtained using the microencapsulated antibody method were significantly higher than the expected values in patients receiving triiodothyronine treatment after total thyroidectomy (theoretically nil) and in patients with untreated primary hypothyroidism with negligible thyroxine (less than 12.9 nmol/l). These false positive values are considered to be due to the methodological problem mentioned above, i.e. the microcapsule membrane is not efficient and therefore must be improved. Consequently, any data based on this method should be interpreted with caution.     

Radioelectrophoresis for determination of thyroid hormone binding abnormalities in human serum

This paper describes a rapid and accurate method for determining binding abilities of thyroid hormones to their corresponding serum proteins: prealbumin, albumin and thyroxine binding globulin. A tube cell agarose gel electrophoresis is used with radioactive labelled triiodothyronine or thyroxine. The distribution curve shows characteristic peaks for prealbumin, albumin and thyroxine binding globulin. A collective of 60 euthyroid thyroid-healthy persons was examined, and the following reference values for the binding of thyroxine to its binding proteins were found: The percentage binding of thyroxine to the prealbumin fraction is 21 to 33%, to the albumin fraction 10 to 15% and to the thyroxine binding globulin fraction 54 to 66%. In a group of 220 patients with suspected thyroid hormone binding abnormalities there were 42 cases of albumin-associated hyperthyroxinaemia, 5 cases of prealbumin-associated hyperthyroxinaemia and 2 cases of thyroxine binding globulin deficiency. In another group of 35 patients with suspected antibodies against thyroid hormones one patient showed antibodies against triiodothyronine.