Circulating antitriiodothyronine autoantibodies in two euthyroid patients: apparent lack of interference in total T3 radioimmunoassay based on second antibody or solid phase separation techniques

Two clinically euthyroid patients were noted to have low total T3 levels as assessed by RIA using either dextran-charcoal (DC) or polyethylene glycol (PEG) for separation of bound from unbound T3, in spite of normal free T3, total and free T4 and basal and TRH-stimulated TSH concentrations. The presence of circulating substances binding T3 was suggested by high nonspecific binding in total T3 RIA system using either DC or PEG separation. The presence of anti-T3 autoantibodies was then suspected and confirmed by the presence of [125]-T3 bound to patients' gammaglobulins, precipitated with rabbit anti-human immunoglobulins. Serum T3 concentration determined by extracting T3 from patients' sera with methanol was 166 and 226 ng/dl. Similar or even lower values were unexpectedly obtained in RIA systems with solid phase or second antibody (anti-rabbit) separation and with competitive protein binding assay. To face this paradoxical finding, simulated experiments were carried out by incubating T3- and T4-free sera added with various amounts of stable T3 and T4 in the presence of goat anti-T3 or anti-T4 serum. These samples were then radioimmunoassayed. The DC separation caused a consistent underestimation of the actual T3 and T4 concentration. The second antibody separation caused a T3 and T4 overestimation for actual levels below 200 ng/dl and 10 micrograms/dl, respectively, while at the higher T3 or T4 concentrations, an overlap or, even, an underestimation of actual T3 or T4 levels were found. These data provide evidence that, with second antibody or solid phase separation methods, there could be an apparent lack of interfering effect of endogenously occurring antibodies.     

Triiodothyronine (T3)-binding immunoglobulins in a euthyroid woman: effects on measurement of T3 (RIA) and on T3 turnover.

A 36-year-old woman with nodular goiter, nervousness, and tachycardia was evaluated for T3 toxicosis. Her serum thyroxine level, resin T3 uptake, and thyroidal radioiodine uptake were normal. Her T3 (RIA), by a technique employing charcoal to separate bound and free T3, was reported as indeterminate due to an interfering substance; by a double-antibody method, her T3 (RIA) was 325 ng/dl. Further studies of the patient's serum revealed an abnormal T3-binding protein which misgrated in the beta-gamma globulin zone on paper electrophoresis and gel filtration chromatography (Sephadex G-200), and was precipitated from serum by rabbit anti-human Fab antibody. The gamma globulin fraction of the patient's serum, separated by a standard technique, showed strong binding activity toward [125I]T3, with an association constant of 4.1 X 10(8) 1/mole (Scatchard plot). In a similar system, labeled T4 was not bound. To avoid artefacts which this T3-binding protein might produce in assaying unextracted serum, T3 (RIA) was performed on an ethanol extract of serum and found to be 191 ng/dl, a slight elevation. However, the metabolic clearance rate of injected [125I]T3, estimated by non-compartmental analysis of the serum decay curve or by the specific activity or urinary T3, was about 16 1/day, a low value, so that the T3 production rate, 31 mug/day, was normal. The patient's symptoms disappeared with the resolution of domestic problems, and she appeared clinically euthyroid. Serum TSH was 5.0 uU/ml and antithyroglobulin titer, 1:16. A test for antibodies to thyroid microsomes was negative. We postulate that this subject was euthyroid, but had a concentration of T3-binding immunoglobulin which was sufficient to produce modest slowing of T3 turnover, borderline elevation of extractable T3 (RIA), and a major artefact in the T3 (RIA) measurement of unextracted serum. A similar abnormality may account for other instances of high T3:T4 ratios in serum.     

Anti-thyroxine and anti-triiodothyronine antibodies in three cases of Hashimotos thyroiditis.

Antibodies against thyroxine (T4) and/or triiodothyronine (T3) were detected in 3 patients with Hashimoto's thyroiditis. One of the patients had both anti-T4 and anti-T3 antibodies and the other 2 patients had only anti-T3 antibody. Serum T4 or T3 antibodies and the other 2 patients had only anti-T3 antibody. Serum T4 or T3 values measured by the single antibody radioimmunoassay (RIA), were low or nil in these patients. One patient was mildly hypothyroid. The other 2 patients were clinically euthyroid, but they were considered latent hypothyroid because of a slight elevation in serum TSH. On extraction of the sera with ethanol, high or normal values of T3 were obtained in all cases. Recovery of T4 or T3 added to the patients' sera determined by RIA was significantly low. The binding of [125I]T4 or [125I]T3 to the patients' sera was demonstrated by the polyethylene glycol method and by using RIA kits without adding the antibody provided. The binding activity was localized in the IgG fraction by column chromatography and by immunoprecipitation. T4- or T3-binding protein in two sera migrated in the gammaglobulin region on paper electrophoresis and was found in 7S fraction on Sephadex G-200 chromatography. In one serum containing both anti-T4 and anti-T3 antibodies, the association constants (Ka) for binding of T4 and T3 were 3.8 x 10(8) l/mol and 1.7 x 10(8) l/mol, respectively. The binding capacities in the serum were 8.2 microgram of T4 and 1.9 microgram of T3 per 100 ml of serum. For two sera containing anti-T3 antibody, Ka were 5.5 x 10(8) l/mol and 7.4 x 10(10) l/mol, and the binding capacities were 0.6 microgram/100 ml serum and 0.7 microgram/100 ml serum respectively. The clinical significance of these antibodies is discussed.     

A case report of Hashimoto's disease with anti-thyroxine autoantibody (author's transl)]

In one case of untreated Hashimoto's disease, serum thyroxine (T4) value by radioimmunoassay (RIA) was significantly lower than that by competitive protein binding analysis (CPBA). The discrepancy was found to be due to the presence of antithyroxine autoantibody in the serum. This phenomenon was considered to be of practical importance in interpreting the T4 value by RIA in cases with autoimmune thyroid diseases. The patient was 59-year-old woman with a 30-year history of goiter. A diagnosis of Hashimoto's thyroiditis had been established by open biopsy of the thyroid ten years ago. The patient was judged to be euthyroid on the basis of clinical and laboratory evaluation (mean serum T4 by CPBA (Tetrasorb and Tetratab kit), 5.0 mug/100 ml; serum T3, 165 ng/100 ml; T3 resin uptake, 31.8%; and serum TSH, 2.0 muU/ml). TBG binding capacity was 24 mug/100 ml. Anti-thyroglobulin antibodies (anti-Tg), once positive ten years before, was negative at this time. But the mean T4 in the serum measured by T4 RIA and RIA-Mat T4 kit were 1.7 and 2.9 mug/100 ml, respectively. Recovery of the T4 added to the patient's serum evaluated by RIA-Mat T4 kit, was 71.2%, although the recovery using a control serum was 108%. Binding of 125I-T4 to the serum or fractions of the serum was studied by using polyethylene glycol (PEG) method, column chromatography, and double antibody precipitation. The results were as follows: 1) The binding of 125I-T4 to the patient's serum was detected by using RIA kit system without addition of anti-T4 serum. 2) On Sephadex G-200 chromatography of 125I-T4 incubated with the serum or the rabbit anti-T4 antibody in the presence of ANS, an early radioactive peak was observed by using the patient's serum as in the case of the anti-T4 antibody. When the serum after thermal inactivation of TBG, was incubated with 125I-T4, and was applied to the Sephadex G-200 column, a radioactive peak was observed in the area where 7S fraction was detected by protein peak. 3) The binding of 125I-T4 to the patient's IgG was 9.0% by using double antibody method when the binding to a control IgG was 0.5%. 4) The binding of 125I-T4 to IgG fractions was also proved by PEG method. 5) The binding of 125I-T4 was competitively inhibited by the addition of unlabeled T4. The affinity constant was 1.9 X 10(8) L/mol and its binding capacity was 0.8 mug/100 ml serum. From these data this T4 binding IgG was considered to be anti-T4 autoantibody. The cross reaction with T3 was approximately 8.3%. MIT and DIT did not displace labeled T4 when tested in amounts varying from 0.1 to 100 ng/assay. By using the paper electrophoresis, the binding of 125IT4 to the serum or IgG was not detectable. Therefore this method was considered unsuitable for detecting such anti-T4 antibody. As we couldn't find any significant binding of 125I-T4 to sera in 37 other patients with Hashimoto's disease by using the PEG method, the incidence of this phenomenon was considered to be low...     

Triiodothyronine(T3) autoantibodies in a woman with nonthyroidal disorder: a study on preparation of serum IgG fraction employing protein A column chromatography

A 48-year-old non-goitrous woman, who had undergone cardiac surgery for mitral stenosis under the extracorporeal circulation, showed high levels of serum T3 and free T3 in a recent follow-up study, employing antibody coated-bead RIA for T3 and -Amerlex M particle RIA for free T3. However, other thyroid function tests (T4, free T4, TSH and TBG) were normal. We suspected that thyroid hormone autoantibodies (THAA) in her serum interfered with T3 and free T3 analyses. The presence of THAA was demonstrated by the use of various procedures as follows. Firstly, the patient's serum was directly incubated with 125I-T3 or -T4 analog which did not bind to TBG, followed by B/F separation with polyethyleneglycol, counting the precipitates. Secondly, after the serum was treated with an acid-charcoal solution to remove circulating thyroid hormone, the measurement of THAA was made as stated above. Normal sera were used as controls. Both the non- and acid-charcoal-treated sera showed much higher percentages of 125I-T3 analog precipitation as compared with controls. In the case of 125I-T4 analog, there was no difference between them. In the third study, the presence of IgG antibodies that bound T3 but not T4 was investigated. The IgG fraction of the patient's serum was separated employing a Protein A-Sepharose CL-4B column chromatography. Then, the prepared IgG fraction was purified by a technique of gel filtration chromatography (Sephacryl S 200). Non-purified and purified-IgG fractions both revealed higher binding percentages of 125I-T3 analog than the control IgG fraction and non-IgG fraction of the patient. Furthermore, a good dose response was observed between the binding percentage of 125I-T3 analog and each dose of the patient's serum or IgG fraction. From these observations, it was clarified that this woman had anti-T3 IgG autoantibodies using a Protein A column chromatography with confirmation of gel filtration chromatography.     

Characteristics of auto-antibodies to bovine TSH in the serum of two patients with Graves' disease

In this report we describe the characteristics of auto-antibodies to bovine TSH (bTSH) detected in the serum of 2 females among 102 patients with Graves' disease. These patients had never been injected with bTSH. One patient had high LATS activity and high bTSH binding activity after isotope therapy. The other patient showed no detectable LATS activity. Interestingly, the antibody showed a specifically high binding activity for the labelled TSH preparation purified by receptor. The auto-antibody could be demonstrated by the double antibody method, polyethylene glycol method, and by gel-filtration. The antibody was polyclonal immunoglobulin G (IgG). Because the binding of [125I]bTSH with the patient's antibody was inhibited by pituitary extracts from mammalian species other than human, this antibody may cross-react with bovine, rat, dog, rabbit and whale TSH. Although the incidence of the antibody in Graves' disease is low and the pathological significance remains obscure, the existence of this antibody in the serum of patients may suggest that autoimmune mechanisms may involve not only the thyroid but also the pituitary in Graves' disease.     

Plasma T4 and T3 levels in naturally metamorphosing Eurycea bislineata (Amphibia; Plethodontidae)

We measured the circulating T4 and T3 levels in the plethodontid salamander Eurycea bislineata at various stages of metamorphosis using radioimmunoassay (RIA). Seven distinct metamorphic stages were defined based on specific developmental events concerning the remodeling and differentiation of skeletal elements. Special effort was made to study individual variation in the levels of plasma thyroid hormones. For this reason we did not pool serum from several specimens. The RIA was conducted in aliquots of 2 microliters (T4) and 5 microliters (T3), with minimum detectable levels of 100 ng/dl (T4) and 20 ng/dl (T3). In agreement with previous studies on other amphibians, we found metamorphosis in E. bislineata to be accompanied by a sharp increase in the circulating plasma levels of T3 and T4. No hormones were detectable in the larval and adult stages. Our technique allowed for simultaneous measurement of T3 and T4 levels in some individuals. These data indicated that, although both the onset of the production of the two thyroid hormones is simultaneous, T3 remains in the system longer than T4. However, at all metamorphic stages a large proportion of specimens did not exhibit any measurable levels of T3 and/or T4. These results underscore the need to reassess the mode of operation and production of thyroid hormones in amphibian metamorphosis.     

Thyrotropin (TSH)-releasing hormone-stimulated TSH release and TSH concentration in the guinea pig pituitary, as determined by a heterologous radioimmunoassay

Little is known regarding how the guinea pig (GP) compares with the rat in terms of TSH economy. To develop a heterologous RIA for GP TSH, rabbits were injected with GP TSH. In one rabbit (anti-gpTSH-8), antibodies that bound 125I-labeled bovine (b) TSH and rat (r) TSH but not 125I-labeled bLH or rPRL were generated. The binding of anti-gpTSH-8 to [125I]bTSH was inhibited in a parallel manner by bTSH over a range of 0.047-5.42 ng, rTSH over a range of 0.24-25 ng, and dilutions of GP pituitary extracts. This system, with bTSH as the standard, was employed as the basis for a heterologous TSH RIA (GP TSH RIA). The cross-reactions of rTSH and bLH in the GP TSH RIA were 45% and 7%, respectively. Rat and bovine FSH, GH, and PRL had little or no cross-reaction. GP pituitaries were incubated in vitro and dosed with LHRH and TRH. The GP TSH RIA detected an 11-fold increase in TSH in the medium in response to TRH and no change in immunoreactivity in response to LHRH. In contrast, a RIA for bLH detected a 25-fold increase in LH in the medium in response to LHRH and no increase in response to TRH. The TSH content in GP pituitaries was significantly lower than that in the rat (GP, 16.8 +/- 1.6 ng/mg; rat, 80.3 +/- 6.2 ng/mg; P less than 0.05) as were serum TSH concentrations (GP, 0.8 +/- 0.4 ng/ml; rat, 4.5 +/- 1.1 ng/ml; P less than 0.05). Thyroid hormone administration (T4 Rx) in both GP and rat produced a significant reduction in pituitary TSH content (GP control, 4.8 +/- 0.4 ng/mg; T4 Rx, 2.1 ng/mg; P less than 0.05; rat control, 52.4 +/- 4.0 ng/mg; T4 Rx, 20.5 +/- 1.6 ng/mg; P less than 0.05) and TSH release (GP control, 9.4 +/- 2.3 ng/ml; T4 Rx, 4.3 +/- 1.5 ng/ml; P less than 0.05; rat control, 357 +/- 81 ng/ml; T4 Rx, 133 +/- 27 ng/ml; P less than 0.05) from incubated hemipituitaries. Thyroidectomy in the rat was associated with a decrease in pituitary TSH content, but no change in pituitary content was found in thyroidectomized GPs. These studies demonstrate the feasibility of estimating GP TSH with a heterologous RIA that employs polyvalent antiserum against GP TSH as the first antibody and bTSH as the tracer and standard.(ABSTRACT TRUNCATED AT 400 WORDS)     

Narrow individual variations in serum T(4) and T(3) in normal subjects: a clue to the understanding of subclinical thyroid disease

High individuality causes laboratory reference ranges to be insensitive to changes in test results that are significant for the individual. We undertook a longitudinal study of variation in thyroid function tests in 16 healthy men with monthly sampling for 12 months using standard procedures. We measured serum T(4), T(3), free T(4) index, and TSH. All individuals had different variations of thyroid function tests (P < 0.001 for all variables) around individual mean values (set points) (P < 0.001 for all variables). The width of the individual 95% confidence intervals were approximately half that of the group for all variables. Accordingly, the index of individuality was low: T(4) = 0.58; T(3) = 0.54; free T(4) index = 0.59; TSH = 0.49. One test result described the individual set point with a precision of +/- 25% for T(4), T(3), free T(4) index, and +/- 50% for TSH. The differences required to be 95% confident of significant changes in repeated testing were (average, range): T(4) = 28, 11-62 nmol/liter; T(3) = 0.55, 0.3--0.9 nmol/liter; free T4 index = 33, 15-61 nmol/liter; TSH = 0.75, 0.2-1.6 mU/liter. Our data indicate that each individual had a unique thyroid function. The individual reference ranges for test results were narrow, compared with group reference ranges used to develop laboratory reference ranges. Accordingly, a test result within laboratory reference limits is not necessarily normal for an individual. Because serum TSH responds with logarithmically amplified variation to minor changes in serum T(4) and T(3), abnormal serum TSH may indicate that serum T(4) and T(3) are not normal for an individual. A condition with abnormal serum TSH but with serum T(4) and T(3) within laboratory reference ranges is labeled subclinical thyroid disease. Our data indicate that the distinction between subclinical and overt thyroid disease (abnormal serum TSH and abnormal T(4) and/or T(3)) is somewhat arbitrary. For the same degree of thyroid function abnormality, the diagnosis depends to a considerable extent on the position of the patient's normal set point for T(4) and T(3) within the laboratory reference range.     

Antibody binding serum T3 in a patient with hepatocarcinoma

Thyroid hormone levels were studied in a euthyroid patient with hepatocellular carcinoma. The thyroid gland was normal at autopsy and both antithyroglobulin and antimicrosomal antibodies were undetectable in serum. Serum triiodothyronine (T3) values as measured by different RIA procedures, showed striking discrepancies suggesting the presence of an endogenous T3 binding antibody. The preincubation of the patient's serum with 125I-T3, followed by a precipitation with polyethyleneglycol showed a 74.8% of binding, confirming the presence of an endogenous factor interfering with T3 assays. Agarose electrophoresis of the patient's serum showed that 125I-T3 migrated mainly with the gammaglobulin fraction (60%). When immunoprecipitation tests with different antihuman antiimmunoglobulins were carried out, a positive binding for immunoglobulin G (11.9%), Fab (8.5%) and lambda chain (9.3%) was noted. Scatchard plot analysis showed a binding affinity of 0.77 X 10(9) liter/mol and a binding capacity of 1.02 nmol/liter. These data suggest that the abnormal serum T3 binding was caused by the presence of a T3 antibody which was shown to be an immunoglobulin G specific only for the lambda chain.     

Nuclear 3,5,3'-triiodothyronine (T3) in brown adipose tissue: receptor occupancy and sources of T3 as determined by in vivo techniques

The amount and sources of T3 associated with high affinity, low capacity cellular nuclear receptors in brown adipose tissue (BAT) have been estimated by in vivo pulse-labeling techniques. Maximal binding capacity was measured by in vivo saturation analysis. Nuclear receptor occupancy at endogenous levels of T3 and T4 in euthyroid rats was estimated from the equilibrium nuclear to serum ratio of tracer T3, and the locally generated nuclear T3 to serum T4 ratio after injecting tracer T3 and T4. These ratios were multiplied, respectively, by the endogenous concentrations of T3 and T4 as measured by RIA. The maximal binding capacity was 0.65 ng T3/mg DNA, and saturation was 71%. Fifty-five percent of the nuclear T3 was generated locally, and 45% was derived from circulating T3. BAT is, hence, comparable to the liver in number of receptors (approximately 5000/cell) and to the pituitary with regard to saturation and relative contributions of locally generated T3 and plasma T3 to nuclear T3. These results suggest that BAT may be an important target for thyroid hormones and, along with other data, that alterations in the activity of the type II 5'-deiodinase of this tissue may influence the saturation of nuclear T3 receptors.     

Studies on radioimmunoassays of TSH subunits (author's transl)

In order to establish radioimmunoassay (RIA) systems for the determination of TSH subunits were carried out. Standard TSH supplied from MRC and standard TSH subunits from Calbiochem were identified to be highly purified by gel filtrations through a Sephadex G-100 column. Antibodies of TSH subunits supplied from Calbiochem bound to standard TSH subunits but not to other pituitary hormones except for TSH. Cross reactivity of anti TSH-alpha antibody to standard TSH was 0.000082, and that of TSH-beta to standard TSH was 0.000011. As the standard curve of each subunit and the displacement curve by TSH were parallel in each assay system, serum levels of TSH subunits were computed by the subtraction of the amount of TSH subunits which was overestimated by the cross reaction with native TSH in this assay system. Cross reactivity of anti TSH-alpha antibody to TSH-beta was 0.001, and that of anti TSH-beta antibody to TSH-alpha was 0.002. But in clinical studies, one TSH subunit was not greatly affected by the determination of another TSH subunit. Utilizing these standard TSH subunits, antibodies of TSH subunits and 125I-labelled TSH subunits obtained by the chloramine-T method, the RIA systems for measurement of serum TSH subunits levels without an extraction procedure were developed. In each RIA system of TSH subunit, a good dose response curve was observed in the range from 0.2 to 50 ng/ml of TSH subunits. Recovery and reproducibility of each RIA system were satisfactory. The serum levels of TSH subunits in 13 normal subjects, 12 patients with primary hypothyroidism and 7 patients with hyperthyroidism were determined by the RIA of TSH subunits. In 13 normal subjects, serum levels of TSH-alpha varied from an undetectable level (U.D.: less than 0.2 ng/ml) to 0.3 ng/ml, and those of TSH-beta varied from an undetectable level (U.D.: less than 0.2 ng/ml) to 0.8 ng/ml. In 12 patients with primary hypothyroidism, serum levels of TSH-alpha varied from U.D. to 4.9 ng/ml, and those of TSH-beta varied from U.D. to 13.6 ng/ml. In 7 patients with hyperthyroidism, serum levels of TSH-alpha varied from U.D. to 4.3 ng/ml, and those of TSH-beta were ranged U.D. From the above data, it is suggested that the direct RIA of serum TSH subunits is a useful tool for studying the roles of TSH subunits in peripheral blood.     

Thyroid disease with monoclonal (immunoglobulin G lambda) antibody to triiodothyronine and thyroxine

A man with previous Graves' disease spontaneously developed hypothyroidism. He became euthyroid with T4 therapy, but developed inappropriately elevated serum levels of T3 and, to a lesser extent, T4. Gel filtration analysis (Sephadex G-150) of serum trace-labeled with [125I]T3 revealed binding to a high molecular weight fraction, distinct from normal T3-binding proteins. This abnormal activity cochromatographed with serum immunoglobulin G (IgG) by DEAE-cellulose chromatography and gel filtration, and was retained by the F(ab)2 fragment of IgG, indicating its true antibody nature. By isoelectric focussing, there was restricted heterogeneity of the [125I]T3-antibody complex (pI 9.0-9.1), and the antibody was identified as an IgG (lambda) monoclonal Ig by immune precipitation. Antigenic cross-reactivity with T4 was demonstrated by inhibition of hapten binding. The affinity of the antibody for T3 was high (Ka = 0.9 x 10(9) liter mol-1), and the T3 binding capacity of the antibody in serum was estimated as 1132 ng/dl, equivalent to 1.39 mg T3-specific IgG/liter (0.014% of the total serum IgG). This binding capacity was similar to the serum T3 values (1100-1300 ng/dl) at which transition from hypothyroid to euthyroid states was observed, as judged by clinical examination and measurement of serum TSH levels.     

Studies on thyroid hormone autoantibody in two euthyroid cases with spuriously high value of serum free triiodothyronine]

Spuriously high value of serum free triiodothyronine (FT3: Amerlex free T3 kit, Amersham, UK.) was noted accidentally on routine laboratory examination of two clinically euthyroid patients (case 1: FT3; 18.5 pg/ml, FT4; 1.1 ng/dl, T3; 103 ng/dl, T4; 8.2 micrograms/dl, TSH; 1.74 microU/ml, case 2: FT3; 8.5 pg/ml, FT4; 1.1 ng/dl, T3; 137 ng/dl, T4; 8.9 micrograms/dl, TSH; 1.45 microU/ml), the former with poorly controlled diabetes (FBG 253 mg/dl, HbA1c 12.1%) and the latter with essential hypertension (184/108 mmHg). Although the hypertensive patient showed mild diffuse goiter, there was no evidence that the patients had autoimmune thyroid diseases because anti-thyroglobulin antibody tests measured by radioimmunoassay and MCHA, TGHA or TBII were all negative. Their serum levels of TBG were within the normal range. Further studies revealed that both patients' sera had unusual binding activity to labelled polyaminocarboxy T3 (125I-aT3) but not labelled T3 (125I-T3). Furthermore, this binding protein was precipitated by goat anti-human immunoglobulin G (IgG). The IgG purified from both patients' sera also showed strong binding activity to 125I-aT3, which was inhibited by unlabelled T3 in a dose dependent manner. In conclusion, we found anti-T3 antibody in two clinically euthyroid patients with no apparent evidence of complicating autoimmune thyroid diseases. The stronger binding activity to polyaminocarboxy T3 rather than T3 may lead to the spuriously high value of serum FT3. The mechanisms of the production of such autoantibodies in our cases should be further investigated.     

Coexistence of autoantibody to human thyrotropin (TSH) and autoanti-idiotypic antibody to antihuman TSH antibody in a case with simple goiter

A 70-yr-old woman with simple goiter showed normal serum levels of T4, T3, free T4, TSH receptor antibody (TRAb) and increased TBG. Discrepancy in serum hTSH level was observed by different assay methods. Coexistence of both autoantibodies for hTSH and for anti-hTSH antibody were demonstrated by the reaction of the patient's antibody with both 125I-hTSH and 125I-anti-hTSH (monoclonal antibody; mAb). These two autoantibodies belong to the polyclonal immunoglobulin G (IgG). The autoantibody for hTSH recognized only beta-subunit of hTSH. Neither stimulating type of TRAb in Graves' disease nor blocking type of TRAb in primary hypothyroidism interfered with the binding of the patient's antibody to 125I-hTSH or 125I-anti-hTSH. Anti-idiotypic antibody (anti-ID antibody) for anti-hTSH antibody was purified by anti-hTSH antibody affinity chromatography. The binding reaction of 125I-anti-hTSH (mAb) with this anti-ID antibody could be inhibited by the unlabeled hTSH. This anti-ID antibody might represent the internal image of the nonbiological active site of TSH molecule, because of absence of thyroid stimulating activity. Goiter in this patient may have occurred by the unbound TSH with IgG (free TSH) and the bound TSH with IgG, because TSH levels in both the whole serum and the IgG free serum (the unbound TSH with IgG) were decreased significantly by T4 treatment. Coexistence of these antibodies may participate in the autoimmune mechanism of an idiotype-anti-idiotype network.     

EVALUATION OF T4 AND T3 BINDING KINETICS IN THE THYROXINE BINDING GLOBULIN ABNORMALITY OF AUSTRALIAN ABORIGINES

Low serum total T4 associated with subnormal concentrations of thyroxine binding globulin (TBG) has been reported in up to 40% of euthyroid Australian aborigines. It has been suggested that these subjects show both diminished concentration of TBG and reduced TBG affinity for T4 (Sarne et al., 1985). We have compared 12 euthyroid aborigines with low T4 (total T4 44 +/- 5 nmol/l) and aborigines with normal T4 (T4 99 +/- 9 nmol/l, n = 12) using measurements of free T4 and T3 by equilibrium dialysis. TBG was measured both by RIA (Henning, Berlin, FRG) and a method dependent on T4 binding (Corning Immophase). Aborigines with low T4 showed lower levels of free T4 (12.6 +/- 0.6 cf. 18.7 +/- 1.0 pM), free T4 index (66 +/- 8 cf. 98 +/- 13), total T3 (1.1 +/- 0.2 cf. 1.6 +/- 0.3 nmol/l), TBG RIA (14.0 +/- 0.6 cf. 25.0 +/- 1.2 ng/l), and TBG Immophase (9.0 +/- 0.5 cf. 22.0 +/- 1.2 mg/l) (P less than 0.01), but free T3 (5.3 +/- 0.4 cf. 4.7 +/- 0.4 pM) and TSH (1.9 +/- 0.2 cf. 1.8 +/- 0.2 mU/l) were not significantly different from the values found in aborigines with normal T4. Scatchard analysis of T4 and T3 binding was performed using serum diluted 1 : 20,000 for T4 and 1 : 500 for T3 (barbitone buffer pH 8.6, 4 degrees C, dextran-coated charcoal separation). In euthyroid low T4 aborigines compared to those with normal T4, both T4 capacity (106 +/- 14 cf. 238 +/- 13 nM, P less than 0.01) and affinity (5.05 X 10(10) cf. 8.47 X 10(10) M-1, P less than 0.05) were significantly reduced. Similarly, both T3 capacity (62 +/- 10 cf. 154 +/- 16 nM, P less than 0.01) and affinity (1.67 X 10(9) cf. 2.28 X 10(9) M-1, P less than 0.02) were reduced. A substantial minority of euthyroid Australian aborigines have a TBG variant characterized by both reduced capacity and affinity of T4 and T3. These findings suggest that TBG may be both qualitatively and quantitatively abnormal in these subjects.     

EFFECTS OF THYROID HORMONES (T4, T3), BROMOCRIPTINE AND TRIAC ON INAPPROPRIATE TSH HYPERSECRETION

Inappropriate TSH hypersecretion was diagnosed in a 38-year-old woman (case 1) and in a 38-year-old man (case 2). Both of them had earlier been treated by ablative therapy for hyperthyroidism. The present diagnosis was based on elevated basal serum TSH levels despite elevated serum free thyroid hormone levels. Both of them had exaggerated TSH responses to TRH (peak value 240 mU/l in case 1 and 408 mU/l in case 2). Their albumin and prealbumin levels were normal. The serum TBG level was normal in case 1 but was elevated in case 2. Serum levels of alpha-subunits of TSH, and pituitary CT scans were normal. Despite mild clinical hyperthyroidism, peripheral indices of thyroid hormone action were normal. They had also relatives with apparent resistance to thyroid hormones. In view of the possibility that prolonged pituitary thyrotrophic stimulation is detrimental, various therapeutic approaches to suppress TSH levels were tried. Both T3 and T4 treatments lowered serum TSH levels, but were poorly tolerated. Acute administration of L-dopa or bromocriptine reduced serum TSH levels, but this was not seen during long-term therapy. TRIAC treatment lowered serum TSH levels, and the drug was well tolerated. Serum TSH responses to TRH were not blunted during T3, T4 or TRIAC treatments. Somatostatin also reduced serum TSH levels, but did not potentiate the effect of low dose T3 therapy. Our results suggest that the patients had unbalanced pituitary and peripheral thyroid hormone resistance, predominantly at the pituitary level. Of the drugs studied, TRIAC seemed to be the most suitable therapy.     

Flavonoid administration immediately displaces thyroxine (T4) from serum transthyretin, increases serum free T4, and decreases serum thyrotropin in the rat

Naturally occurring and synthetic plant flavonoids, such as EMD 21388, are potent inhibitors of thyroid hormone 5'-deiodinase (5'-D) in vitro, but not when given in vivo, since they are tightly bound by serum transthyretin (TTR). EMD 21388 also inhibits the binding of T4 to human, dog, and rat serum TTR in vitro and when administered to rats in vivo. In the present studies the administration of EMD 21388 inhibited the binding of T4 to TTR within 3 min, resulting in a decrease in the serum T4 concentration, an increase in the percentage of serum free T4 assessed by equilibrium dialysis, and an increase in the serum total free T4 concentration. Depending upon the dose of EMD 21388 employed, the serum total free T4 concentration was either elevated for at least 60 min or transiently elevated, returning to normal values by 60 min. Although the total serum T3 concentration was decreased and the percent free T3 increased, these changes were modest, and the serum free T3 concentrations remained normal after EMD 21388 administration. The transient elevations of serum free T4 concentrations 10 and 20 min after the administration of 0.3 mumol EMD 21388/100 g BW resulted in a significant decrease in the serum TSH concentration at 60 min. These observations strongly suggest that the serum free T4 concentration and not T4 bound to serum TTR is biologically available to the pituitary to regulate TSH secretion and/or synthesis. The administration of EMD 21388, which rapidly increases the serum free T4, but not the serum free T3, concentration, will now permit studies of the effect(s) of endogenously elevated serum free T4 concentrations, rather than those after the administration of pharmacological quantities of T3 and T4, on various aspects of the biosynthesis and release of pituitary TSH.     

促甲状腺激素受体抗体（TRAb）检测在甲状腺疾病鉴别诊断中的应用

目的分析血清促甲状腺激素受体抗体（TRAb）测定在甲状腺疾病临床诊断中的应用价值。方法采用放射受体分析和放射免疫分析法,测定373例甲状腺疾病患者血清TRAb和T3、T4、TSH、FT3、FT4含量,并根据以上参数的含量为标准,把患者分为单纯性甲状腺肿组、桥本氏病组、原发性甲减组、甲状腺功能亢进（甲亢）组、药物性甲减组,以45例非甲状腺疾病血清中TRAb和T3、T4、TSH、FT3、FT4含量作为对照组。结果单纯性甲状腺肿组、桥本氏病组血清TRAb含量与正常对照组无差异（χ^2=0.462,P〉0.05）;甲亢组、原发性甲减组血清TRAb含量明显高于正常对照组（χ^2=17.035,P〈0.01）;药物性甲减组血清TRAb含量与甲亢组差异显著（χ^2=4.804,P〈0.05）,与正常组比较亦有显著性差异（χ^2=9.071,P〈0.05）;血清TRAb含量与各组间T3、T4、FT3、FT4及TSH浓度之间无显著相关性（χ^2=0.325,P〉0.05）。结论血清TRAb含量的监测对甲状腺疾病的诊断、鉴别诊断、疗效观察等具有临床应用价值。     

Secretion of thyrotropin with reduced concanavalin-A-binding activity in patients with severe nonthyroid illness

Patients with nonthyroid illness (NTI) often have reduced serum T3, free T3, T4, and free T4 concentrations. Paradoxically, serum TSH is usually in the normal range. The data suggest a diagnosis of hypothalamic hypothyroidism, in which TSH may have reduced biological activity because TRH, which is necessary for key steps in the glycosylation of TSH, is deficient. To study the glycosylation of TSH in patients with NTI, we measured the serum TSH concentration in 36 such patients hospitalized on our intensive care units and compared the results with those from a group of 18 normal subjects. Serum TSH was measured in 2 assays: 1) a sensitive TSH RIA of unextracted serum (TSH-RIA) and 2) a RIA of serum TSH after its extraction with Concanavalin-A (Con-A), a lectin which binds glycoproteins containing mannose residues in their oligosaccharide side-chains (TSH-Con-A). The ratio of TSH-Con-A to TSH-RIA was significantly reduced in the NTI patients [0.61 +/- 0.03 (+/- SE) vs. 0.89 +/- 0.05 in the normal subjects] due to reduced binding of the TSH to the Con-A. This change was not dependent on the extent of the abnormalities of thyroid hormone levels. The data suggest that the TSH secreted in NTI has altered glycosylation which is associated with reduced biological activity. This finding may explain in part the low serum T4 level in NTI patients in the face of an apparently normal immunoreactive TSH level.