Two new inherited defects of the thyroxine-binding globulin (TBG) molecule presenting as partial TBG deficiency.

Serum-denatured TBG (dnTBG) measured in 32 families deficient in native TBG (nTBG) was undetectable in all subjects with complete nTBG deficiency and was high in 2 of 16 families with partial nTBG deficiency. nTBG (in mean micrograms per decaliter +/- SD) in members of the Quebec and Montreal families, respectively were: 258 +/- 54 and 230 in affected men, 747 +/- 190 and 927 +/- 90 in affected women, and 1568 +/- 151 and 1300 +/- 195 in unaffected relatives. Corresponding mean dnTBG levels were: 14.3 +/- 2.9 and 21.3 in affected men, 8.6 +/- 1.0 and 11.6 +/- 3.1 in affected women, and less than 2.1 and less than 2.6 in unaffected relatives. All were euthyroid with normal free thyroxine and thyrotropin levels. In comparison to common type TBG, TBG-Quebec was more heat labile by 10 degrees C and TBG-Montreal by 12 degrees C. The degree of dnTBG elevation and nTBG lability at 37 degrees C were correlated (r = 0.99). Isoelectric focusing showed cathodal shift of all TBG bands: TBG-Quebec by 0.06 isoelectric points (pI) and TBG-Montreal by 0.02 pI. These two TBG variants represent different mutations most likely affecting the polypeptide chain of the molecule. Their inheritance is X-chromosome linked. The instability of these TBGs at 37 degrees C may lead to more rapid degradation in vivo resulting in low nTBG and high dnTBG concentrations in serum.

Studies on human thyroxine-binding globulin (TBG). IX. Some physical, chemical, and biological properties of radioiodinated TBG and partially desialylated TBG.

Thyroxine-binding globulin (TBG) and partially desialylated or slow TBG (STBG) were purified from human serum by affinity chromatography. Purified TBG was identical to TBG present in serum by the criteria of electrophoretic mobility, affinity for thyroxine (T4), and heat-inactivation response. Purified STBG had slower electrophoretic mobility and lower affinity for T4. Both bound T4 in an equimolar ratio, were immunoprecipitable, and had similar inactivation t1/2 at 61 degrees C. TBG and STBG were iodinated by the chloramine-T-catalyzed reaction. An average of from 0.02 to 6 atoms I could be incorporated per molecule of the protein by adjusting the conditions of the reaction (time, protein and iodide concentrations). 125-I, 131-I, and 127-I were used. Iodination increased the anodal mobility of TBG but did not affect the reversible T4-binding, precipitation by antiserum, or the heat-inactivation properties. "Heavily" and "lightly" iodinated TBG had identical disappearance half-times from serum in the rabbit. 15 min after the intravenous administration of [131-I]-STBG and [125-I]TBG mixture to rats, more than 90% of the injected 131-I dose was in the liver, and the liver 131-I/125-I ratio was 32-fold that of serum. Selective uptake of STBG by the liver was also observed in the rabbit and in man. The serum [125-I]STBG/[131-I]TBG ratio declined from 1 to 0.2 in 10 min in the intact rabbit but remained unchanged for 1 h in the acutely hepatectomized animal. In the rabbit, t 1/2 was approximately 3 min for STBG and 0.8-3.4 days for TBG. The radioiodine derived from the iodinated proteins is partly excreted in bile but the bulk was precipitable with specific antibodies. Some isotope in the form of iodide appeared in blood and was excreted in the urine. Since radioiodinated TBG and STBG preserve their biologic and immunologic properties they are useful as tracer materials for metabolic studies. In rat, rabbit, and man STBG is rapidly cleared from serum by the liver. Conversion of TBG to STBG may be the limiting step in the regulation of TBG metabolism.

Bovine thyroxine-binding globulin. Purification and comparison of molecular weight and amino acid composition with human thyroxine-binding globulin.

Bovine and human thyroxine-binding globulin were purified from serum by a three-step purification procedure which comprised affinity chromatography consecutively on thyroxine- and Concanavalin A--Sepharose and finally preparative polyacrylamide gel electrophoresis. The molecular weights of the two proteins were similar (54 000) as well as their carbohydrate contents while some differences in amino acid composition were found. Rabbit antiserum against bovine thyroxine-binding globulin reacted with human thyroxine-binding globulin with no sign of spur formation.

Thyroxine-binding globulin biosynthesis in isolated monkey hepatocytes.

Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyroxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium and increased to 300%, indicating probable thyroxine-binding globulin secretion

Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma.

T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein). The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4). The 27K protein does not appear to be a fragment of TBG since 1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and 2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings: 1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4; 2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and 3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4. The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin. This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG. In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG. The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer. These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.

Thyroid function tests and characterization of thyroxine-binding globulin in the carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome is a newly recognized hereditary disorder that presents with psychomotor retardation, cerebellar ataxia, peripheral sensorimotor neuropathy, and, variably, skeletal abnormalities, lipodystrophy, and retinitis pigmentosa. These abnormalities appear to be produced by a defect that causes reduced carbohydrate content in glycoproteins. We studied seven patients with CDG type I belonging to five unrelated families. The concentration of serum TBG, a glycoprotein of hepatic origin, was measured by RIA and T4 saturation and was found to be below the normal range in three of the seven patients and normal in four of them. More than half of the total serum TBG had reduced sialic acid content and localized on isoelectric focusing (IEF) as two prominent bands cathodal to the three major bands of normal TBG. The latter two bands are responsible for the characteristic IEF pattern or CDG syndrome. TBG in patients with CDG had immunoreactivity indistinguishable from that of normal TBG and had normal affinity for T4, T3, and rT3. Serum total T4, T3, and rT3 were below the normal range in seven, five, and seven patients, respectively. The free T4 index was also below normal in four patients, but the free T4 concentration, measured by equilibrium dialysis at low dilution, and serum TSH were in the midnormal range. The serum total T4 and rT3 levels were disproportionately reduced relative to the serum TBG concentration and compared to the concentrations of these iodothyronines in matched subjects with inherited partial TBG deficiency. Chronic illness cannot explain these changes, because, contrary to patients with nonthyroidal illness, those with CDG had significantly higher serum total T3/T4 and lower rT3/T4 ratios. It is concluded that IEF of TBG is a rapid and simple method for the diagnosis of CDG type I and that the abnormal pattern can be detected as early as 5 days postpartum. Patients with CDG are chemically euthyroid, and it is postulated that the reduction in serum iodothyronine concentrations beyond that explained on the basis of low TBG levels may be due to the interference with binding to TBG by an unidentified substance.

Synthesis and characterization of anti-idiotypic anti-T4 antibodies.

We injected rabbits with purified monoclonal murine immunoglobulin (IgG1) or polyclonal antithyroxine antibodies (anti-T4) and polyclonal anti-triiodothyroacetic acid (anti-Triac) antibodies to stimulate the production of anti-idiotypic antibodies. Purified immunoglobulins from all five rabbits immunized with monoclonal primary antibodies were able to inhibit the interaction between [125I]T4 and the primary antibody. The preimmune sera were inactive. This effect was not due to endogenous T4 contamination or contamination with the injected primary antibody. Half-maximal inhibition of binding of primary antibody with anti-idiotype was between 1.6 and 30 micrograms of total immunoglobulins. Addition of normal mouse IgG1 did not alter the inhibitory effect of the anti-idiotypic antibody, suggesting that this effect is specific. These anti-idiotypic antibodies reacted differently with different polyclonal antibodies, reflecting the heterogeneous nature of polyclonal antibody populations. Polyclonal antibodies were less effective in stimulating anti-idiotypic antibody production. One polyclonal anti-T4 and one anti-Triac antibody produced weak anti-idiotypic antibody that had to be used at a concentration of > 600 micrograms of total immunoglobulins to be inhibitory. Both inhibited the binding of T4 to the monoclonal anti-T4 antibody. However, they were ineffective in inhibiting the function of their own antigen, the polyclonal anti-T4 or anti-Triac antibody. We tested the most potent anti-idiotypic antibodies for their ability to compete with T4 for other T4-binding proteins. Specific inhibition of T4 binding to thyroid-binding globulin was observed with half-maximal effect at approximately 450 micrograms of total IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

The microheterogeneities of thyroxine-binding globulin and alpha 1 protease inhibitor (alpha 1-antitrypsin) are interrelated.

Addition of 125I-thyroxine to serum allows autoradiography for thyroxine-binding globulin microheterogeneity to be carried out after isoelectric focusing has been performed to display (by protein stain) the heterogeneous bands of the alpha 1-antitrypsin (PI) system. Comparison of the protein stain for PI with the autoradiograph for thyroxine-binding globulin indicates that these two systems are interrelated with the major bands of the PI system corresponding to the bands on the autoradiograph. This correspondence holds for PI variants other than the common M type and in particular it holds for the deficient Z type in which the autoradiograph for thyroxine-binding globulin is strikingly different from normal. We conclude that the major cause of microheterogeneity of TBG is due to an association with the PI system under the conditions of isoelectric focusing as normally performed. Precipitation experiments with antisera to PI and TBG suggest that the complex between these biologically important globulins may occur under conditions other than isoelectric focusing, but further work will be needed to examine this possibility.

An improved procedure for isolation of thyroxine-binding globulin from human pregnancy serum.

A convenient method for isolation of TBG from human pregnancy serum is reported. The method is based on batch-wise treatment of serum with hydroxyapatite followed by batch-wise adsorption of TBG to an affinity gel consisting of Sepharose to which thyroxine was bound with a spacer group. After selective desorption the TBG-preparation was practically pure. The small amounts of contaminating proteins were easily removed by affinity chromatography on Concanavalin A-Sepharose. The resulting preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and immunological techniques.

Radioimmunoassay for human thyroxine-binding prealbumin.

A radioimmunoassay (RIA) for human thyroxine-binding prealbumin (PA) is described. It employs highly purified PA, anti-human PA serum at 1:30,000 final dilution, normal bovine serum as a carrier, and polyethyleneglycol to precipitate the immune complexes. This assay is extremely sensitive (limit of detection less than 0.2 micrograms per dL or less than 3.6 X 10(-15) moles per tube), accurate (recovery = 98.7 +/- 9 percent, mean +/- S.D.) and reproducible (intra- and inter-assay coefficients of variation = 3.6 to 6.3 percent and 7.2 to 9.5 percent, respectively). There was a highly significant correlation when the RIA was compared with radical immunodiffusion or with PA maximal binding capacity for thyroxine (r = 0.944 and r = 0.724, respectively, p less than 0.001). Concentration of PA in sera from normal subjects (age range = 20 to 88 years) averaged 27.7 +/- 0.5 mg per dL (mean +/- S.E.M.), with significantly higher values in males than in females in all age groups with the exception of the older subjects (20 to 50 years: males = 26.5 to 37 mg per dL; females = 23.1 to 33.8 mg per dL). Levels of PA progressively declined after the fifth decade of life. Pregnancy, hyperthyroidism, chronic liver diseases, cystic fibrosis, cancer and other non-thyroidal illnesses were associated with decreased levels of serum PA. Untreated hypothyroidism and chronic renal diseases showed widely scattered values of PA. Inherited thyroxine-binding globulin (TBG) abnormalities and bisalbuminemia had no apparent effect on concentrations of serum PA.

[Thyroxine and triiodothyronine]. / Tiroxina e triiodothyronina

The value of measurements of serum thyroxine (T4) and triiodothyronine (T3) concentrations in the clinical evaluation of thyroid function is well established. In the present report the properties of the methods currently available for the assessment of circulating thyroid hormones are examined. Recent data indicate that results of measurements of serum T4 by radioimmunoassay (RIA) or by displacement analysis are qualitatively and quantitatively similar. Evidence has been provided from this and other laboratories that elevated or subnormal serum levels of total tt4 may be observed in several euthyroid physiologicmal serum levels of total T4 may be observed in several euthyroid physiological or pathological conditions associated with altered capacity of the thyroxine-binding protein; in these cases a normal free thyroxine index is usually found indicating its importance in the evaluation of thyroid function. Several physiological observations and the recent recognition of clinical conditions associated with changes in the T4/T3 ratio, justify the present interest in the measurement of serum T3. The advantages of RIA methods with respect to displacement techniques in the determination of this hormone are well documented. The AA. report here their experience with a simple RIA method for total T3, using a single Sephadex G25 column for extraction of T3 from serum and for separation of bound from free hormone. The mean (+/-SD) values of serum T3 found in 78 normal, 23 hyperthyroid and 25 hypothyroid subjects were as follows:160+/37ng/dl, 604+/195ng/dl and 35+/23ng/dl respectively. High values were found in 19 pregnant women at delivery (236+/29ng/dl), but not in 5 subjects on contraceptives (156+/42ng/dl). A relatively large variability is noted when normal values reported from different laboratories are compared. This may be related at least in part to geographical and/or ethnical factors, but methodolgical differences may also be involved. The normal range of circulating T3 should be established in each individual laboratory.

[Radioimmunoassay of serum thyroxin-binding-globulin in euthryoid subjects]. / Dosage radio-immunologique de la "thyroxine-binding-globulin" sérique chez les sujets euthyroidiens.

The authors have set up a thyroxine-binding-globulin radio-immunoassay in blood serum. The standard used has been determined by gravimetry, its maximal thyroxine-binding capacity is 0,96 mole of thyroxine per mole of TBG. Serum concentration of TBG has been measured in 159 euthyroid normals. The mean value of the concentration is 20 mg/l.

Effect of deglycosylation on the binding and immunoreactivity of human thyroxine-binding globulin.

Thyroxine-binding globulin (TBG), prepared from human serum by an improved purification method, was treated with a mixture of neuraminidase, beta-galactosidase, alpha-mannosidase, and beta-N-aectylglucosaminidase, which resulted in the removal of approximately 86% of saccharides. Purification by thyroxine-Sepharose affinity chromatography gave a homogeneous protein as shown by equilibrium sedimentation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Amino acid and NH2-terminal sequence analysis indicated that the protein moiety was intact. Deglycosylation had no effect on the stoichiometry of the binding of L-thyroxine as shown by tryptophanyl fluorescence quenching and equilibrium dialysis at pH 8.6 and 25 degrees C. However, the affinity constant for L-thyroxine was reduced from 1.6 X 10(9) M-1 to 0.58 X 10(9) M-1. Analysis of radioimmunoassay data revealed that deglycosylation resulted in a slight decrease of the affinity constant for anti-TBG antibody from 3.9 X 10(10) M-1 to 1.8 X 10(10) M-1. These results suggest that the polypeptide moiety, rather than the heterosaccharides, contains the antigenic determinants. Removal of the majority of the heterosaccharides of TBG has only a minor effect on its immunoreactivity and on the binding of thyroid hormone.

[Effect of structural changes in thyroxine-binding globulin on its biological activity and immunochemical properties]. / Vliianie strukturnykh izmenenii tiroksinsviazyvaiushchego globulina na ego biologicheskuiu aktivnost' i immunokhimicheskie svoistva.

Using spectroscopic, electrophoretic and microcalorimetric techniques, the changes in the spatial structure of human thyroxine-binding globulin (TBG) induced by exposure of protein solutions to high temperatures (45-90 degrees C) and low pH (2.5-6.0) were studied. Simultaneously the biological activity and immunoreactivity of TBG samples were measured. The structural changes were manifested at 52 degrees C or at pH 4.0 and were then aggravated with a rise in temperature or a decrease of pH. The circular dichroism spectra showed that the molecular ellipticity had a maximum decrease (by 10%) at 218-222 nm. In fluorescence spectra excitable at 280 nm the band half-width increased by 4-6 nm; their intensity decreased by 30-40%, whereas the position of the maxima did not change significantly. After addition of an equimolar amount of thyroxine to inactivated TBG the protein fluorescence was quenched by 25-40%. The electrophoregrams of treated preparations contained additional protein bands possessing no biological activity, whose mobility was less than that of native TBG. Microcalorimetric assays of native TBG revealed a thermoabsorption peak with a maximum at 62.5 degrees C and a half-width of 7.1 degrees C. The thermodynamic parameters of melting of TBG spatial structure were consistent with a model of a two-domain structure of the molecule. The biological activity and immunoreactivity of TBG showed a coordinated decrease with a rise in the degree of protein denaturation, However, the formation of TBG complex with antibodies did not screen the thyroxine-binding center of TBG and did not alter its affinity. Possible mechanisms of structural transition of TBG and its effect on the biological properties of TBG are discussed.

[Use of radioimmunoassay of "thyroxine binding globulin" to determine the concentration of free plasma thyroxine]. / Application du dosage radio-immunologique de la "thyroxine-binding globulin" à la détermination de la concentration de thyroxine plasmatique libre.

A relationship is proposed for calculating the concentration of free serum thyroxine using the measured values of thyroxine and thyroxine-binding globulin total concentrations. This calculation has been performed on a population of 335 patients. A good discrimination of the different thyroid diseases has been obtained.

Immunological quantitation of rat and mouse thyroxine-binding globulins. Ontogenesis and sex-dependence of the circulating levels of the thyroxine-binding globulins.

We describe the preparation of monospecific antisera against a thyroxine-binding globulin partially purified from immature rat sera by affinity chromatography on thyroxine-Sepharose. The antisera are used for the rocket immunoelectrophoresis assay of rat thyroxine-binding globulin and also, owing to their partial cross-reactivity with mouse thyroxine-binding globulin, for the quantitation of this serum binding protein in the mouse. The thyroxine-binding globulin is measured in developing rats and in sexually mature male and female rats and mice. The results of the ontogenetic study confirm the postnatal surge of serum thyroxine-binding globulin levels, formerly demonstrated with binding techniques. They allow further to define the correlations, dependent on age, of the immunoquantitated thyroxine-binding globulin and transthyretin levels with the abilities of the sera to bind thyroxine. In sexually mature rats and mice we demonstrate an opposite sex-dependence of thyroxine-binding globulin levels, characterized by increased levels of the protein in the female rats versus increased levels of the protein in the male mice. This is the first report of immunological quantitation of rat and mouse thyroxine-binding globulins.

The role of thyroid hormone autoantibodies in serum transport.

Eleven sera known to contain thyroid hormone autoantibodies were analysed by reverse-flow electrophoresis for the equilibrium distribution of thyroid hormones between these autoantibodies and the three normal binding proteins found in serum. The binding properties of the autoantibodies determined in vitro did not necessarily predict their contribution to transport in serum of T1 and T3. Some could both bind in vitro and transport in serum. Others were able to bind both hormones but transported only one. However, some autoantibodies could be specific, binding and transporting one hormone only. In some sera, the autoantibody was the dominant transport protein having drawn hormone from thyroxine-binding globulin which is normally the most important. The autoantibodies were not saturated even in euthyroid individuals, indicating that they bind hormone reversibly and are a part of an equilibrium system.

Antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine binding protein of rat pituitary GH3 cells: partial characterization and cross-species immunoreactivity.

To develop antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine (T3) binding protein (M.W. 55,000), rabbits were immunized with formalin-fixed GH3 cells or highly purified plasma membranes from these cells. Antibodies were screened by immunoprecipitation using detergent solubilized N-bromoacetyl-[125I]T3-labeled 55K protein. Among the nine detergents tested, 0.18% CHAPS was found to be the best in its solubilization efficiency and its ability to maintain the integrity of the antigenicity of the 55K protein. The N-bromoacetyl-[125I]T3-labeled 55K protein was also immunoprecipitated by anti-T3 antibodies. The anti-55K protein antibodies cross-reacted with plasma membrane T3 binding proteins from cultured cells and tissues of human and rodent origin. These results indicate that structural similarities exist in human and rodent plasma membrane T3 binding proteins. These antibodies should provide a powerful tool in the characterization and in probing the function(s) of the plasma membrane T3 binding protein in cells.

An evaluation of four different luminescence immunoassay systems: CELIA (chemiluminescent immunoassay), SPALT (solid-phase antigen luminescence technique), ILMA (immunoluminometric assay) and ILSA (immunoluminometric labelled second antibody). A critical study of macro solid phases for use in immunoassay systems, Part III.

The performance of different solid-phase luminescence immunoassays has been documented using four different assay concepts. These are CELIA (chemiluminescence immunoassay), SPALT (solid-phase antigen luminescence technique), ILMA (immunoluminometric assay) and ILSA (immunoluminometric labelled second-antibody assay). CELIA is analogous to a solid-phase radioimmunoassay and uses a labelled antigen, SPALT and ILSA use a labelled second (species-specific) antibody and ILMA a labelled substance-specific antibody, i.e. analogous to the immunoradiometric assay. Both bioluminescent and chemiluminescent labels have been used. Pyruvate kinase was used for bioluminescence and diazoluminol and N-(4-amino-butyl)-N-ethyl isoluminol hemisuccinamide for chemiluminescence. Relevant quality-control parameters and reference ranges have been given for the optimised assays. Assays described are: thyroxine, thyroxine binding globulin, cortisol, caeruloplasmin, ferritin and C-reactive protein. Luminescence immunoassays with coefficients of variation comparable with radioimmunoassay have been designed, values of under 5% being obtainable within the working range of the assay.