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.     

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.     

Anti-triiodothyronine antibodies in patients with rheumatoid arthritis associated with Hashimoto's thyroiditis

Anti-triiodothyronine antibody was found in a case of rheumatoid arthritis associated with Hashimoto's thyroiditis. The patient was a 40 year-old woman who had complained of polyarthralgia, joint-swelling and stiffness for seven years. She had a rheumatoid nodule and showed a positive RA test. Radiographic changes of hands and wrists showed osteoporosis, erosions and narrowing of joint space. Nonsteroidal anti-inflammatory drugs had been used for seven years. The diagnosis of Hashimoto's thyroiditis had been made by open biopsy of the thyroid gland seven years before. Serum T4, TSH, TBG, free T4, free T3 and r-T3 were all normal. On the other hand, serum T3 level was almost unmeasurable by radioimmunoassay. Binding of 125I-T3 to the patient's serum was studied by using polyethylene glycol (PEG) and column chromatography. By using the PEG method, the binding of 125I-T3 to the patient's serum was tenfold compared to control serum. Sephadex G-25 column chromatography (0.9 X 1.5 cm) of 125I-T3 with the patient's serum in the presence of 0.1% ANS showed an early radioactive peak, while control serum did not show an early peak. In the next experiments, the patient's serum was labelled with 125I-T3, mixed with human anti-IgG, IgM, IgA, lambda, kappa, incubated at 4 degrees C for 20 hours and centrifuged for 20 min. Strong binding to the anti-IgG and anti-lambda was detected. The presence of this abnormal T3-binding globulin in the patient's serum may have produced an undetectable T3 level.     

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.     

ANTI-TSH ANTIBODY WITH HIGH SPECIFICITY TO HUMAN TSH IN SERA FROM A PATIENT WITH GRAVES'' DISEASE: ITS ISOLATION FROM, AND INTERACTION WITH, TSH RECEPTOR ANTIBODIES

A patient with thyrotoxic Graves' disease had an apparent measurable level of serum TSH (2.5 microU/ml) by double-antibody radioimmunoassay (RIA). The serum IgG bound with both [125I]human(h)TSH and [125I]bovine(b)TSH. The [125I]hTSH binding was more effectively displaced by human than bovine TSH, whereas [125I]bTSH binding was displaced exclusively by bTSH. Scatchard analyses revealed that [125I]hTSH binding showed two components, whereas [125I]bTSH binding had only one component. Serum TSH determined by RIA became undetectable 21 months after antithyroid drug treatment with a parallel decrease of [125I]hTSH binding IgG activity. Four thyrotrophin binding inhibitory immunoglobulins (TBII) from other patients did not interfere with the binding of the patient's serum to [125I]h- or bTSH. Furthermore, the in-vitro thyroid stimulating activities of three thyroid stimulating antibodies (TSAb) were not affected by the addition of this patient's IgG. On the other hand, this patient's Ig (3 mg/ml) abolished the in-vitro thyroid stimulation by bTSH (100 microU/ml), but did not affect that by hTSH (100 microU/ml). The anti-hTSH antibody, TSH receptor antibody and anti-bTSH antibody in the serum, which contains TSAb as well as anti-TSH antibodies, could be partially purified by hTSH-agarose and subsequently by guinea pig fat cell membrane affinity absorptions. However, the anti-hTSH antibody fraction obtained had both hTSH binding activity and thyroid stimulating activity, and this fraction did not show any inhibitory effect on the in-vitro thyroid stimulation of autologous TSH receptor antibody or hTSH. The possible significance of anti-TSH antibodies is discussed.     

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.     

Simultaneous measurements of free thyroxine and triiodothyronine fractions in human serum by means of equilibrium dialysis (author's transl)]

Simultaneous measurements of serum free thyroxine (T4) and triiodothyronine (T3) fractions were studied using a modification of equilibrium dialysis described by Sterling and Brenner. To 1.2 ml of the serum to be assayed, 131I-T4 and 125I-T3 were added in a concentration of 2 mug/dl and 25 ng/dl, respectively, both of which were preliminarily dialysed according to Schussler and Plager. Half ml of the serum with tracers added was dialysed against 9 ml of phosphate buffer (ionic strength 0.15, pH 7.4) for 18 hours at 37 degrees C and 0.1 ml was reserved from the rest in a counting tube (in duplicate). After the completion of dialysis, the dialysate was mixed with 1 ml of pool serum and the contaminating inorganic iodide (in the form of 131I or 125I) was eliminated by adsorption on anion exchange resin. The radioactivity of 3 ml of the dialysate and 0.1 ml of the preserved serum (with the tracers added) was counted and the free (or dialysable) fractions were expressed as a ratio of the count of the former divided by that of the latter adjusted to an equal volume by calculation. The amount of either T4 or T3 added as tracers had no influence on free T4 or T3 fraction unless either of them was added to a concentration of 10 mug/dl. When 125I-T3 of low specific activity (50 muCi/ug) was used as a tracer, free T3 fraction measured simultaneously with free T4 fraction tended to be higher than that measured with a single tracer. When 125I-T3 of higher specific activity (300 muCi/mug) was employed, free T3 fraction obtained with two methods did not differ significantly. Using serum T4 and T3 concentrations measured by competitive protein binding analysis and radioimmunoassay, respectively, the free T4 and T3 concentrations were estimated with sera of normal, hyperthyroid, hypothyroid and uncomplicated pregnant subjects.     

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.     

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.     

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.     

Selective cytotoxicity of 125I-labeled monoclonal antibody T101 in human malignant T cell lines

The radiolabeled anti-T cell antibody T101 can be used for specific tumor localization, but unlabeled T101 produces limited cytotoxicity in patients. We thus studied the in vitro cytotoxic effects of T101 labeled with 125I, a radionuclide known for its short-range, high-linear-energy electrons. We showed that 125I-T101 could be readily prepared at high specific activity with high immunoreactivity. Human malignant T cell lines HUT 102, MOLT-4, and HUT 78 were found to differ in the number of T65 determinants (the antigen recognized by T101) and the sensitivity to external x-ray radiation, which were of significance for the cytotoxicity of 125I-T101 in vitro. The cytotoxic effects of 125I-T101 were also found to be dose dependent and increased with exposure time under frozen conditions. As controls, unlabeled T101 had no cytotoxic effect, while free Na 125I or the 125I-labeled irrelevant antibody 9.2.27 exerted minor cytotoxicity. In HUT 102 and MOLT-4, more than 3 logs' cell killing was achieved within four weeks. Because considerable cytotoxicity was demonstrated in vitro by 125I-T101 on T65-positive malignant cells, and because low-dose 111In-T101 can be used successfully for tumor localization, future trials using 125I-T101 at high specific radioactivity may improve therapeutic results in patients with T65-positive malignancies.     

Use of 125I-triiodothyroacetic acid to measure nuclear thyroid hormone receptor

125I-Triac was employed to measure hepatic thyroid hormone nuclear receptor (RT) in the rat. The binding properties of 125I-Triac and 125I-T3 were compared in a 0.4 M KCl extract of a liver nuclear preparation. The order in which the stable compounds, Triac, T3, T4 and rT3, competed for 125I-Triac and 125I-T3 binding in liver nuclear extract was similar (Triac greater than T3 greater than T4 greater than rT3), suggesting association of both radioligands with RT. Scatchard plot analysis of specific 125I-Triac and 125I-T3 binding in nuclear extract gave approximately equal estimates of the maximum binding capacity (MBC). However, the binding affinity, as represented by the equilibrium association constant (KA), was higher for 125I-Triac than for 125I-T3 (7-10 X 10(9)M-1 vs 1-3 X 10(9)M-1). To determine the effect of contaminating serum proteins on estimates of MBC and KA, a small amount of dilute rat serum was added to the same nuclear extract preparation. Addition of serum decreased the KA value and markedly increased the MBC values estimated by analysis of 125I-T3 binding data. In contrast, KA and MBC values derived from 125I-Triac binding data were not influenced appreciably by the addition of serum. These data indicate that: 1) both 125I-Triac and 125I-T3 bound to RT in rat liver nuclear extract, 2) the affinity of RT for 125I-Triac is appreciably greater than for 125I-T3, and 3) estimates of RT concentration (MBC) made with 125I-Triac are less sensitive to serum protein contamination than those made with 125I-T3. These properties of 125I-Triac may be useful in efforts to demonstrate RT in tissues that have low RT levels and/or when serum contamination is present.     

Demonstration and some properties of cytosol-binding proteins for thyroxine and triiodothyronine in human liver.

Cytosol-binding proteins for L-thyroxine (T4) and triiodo-L-thyronine (T3) were studied in human liver specimens obtained at autopsy from 5 male and 2 female subjects. The liver cytosol containing 131I-T4 or T3, together with or without added stable hormones, was fractionated by Pevikon thin-layer electrophoresis at pH 8.6, 8.0, and7.4. It was demonstrated in all the specimens that besides a small amount of serum T4-binding globulin, there existed three T4-binding proteins, termed hT4-1, hT4-2 and hT4-3, with the electrophoretic mobilities of alpha2- and beta-globulins, and two T3-binding proteins, termed hT3-1 and hT3-2, with the mobilities of gamma-globulin. Binding of hormones by the cytosol proteins was pH-dependent, and a preliminary dialysis had no effect on the hormone binding. The major band of T4, hT4-2, bound more than half the tracer T4, and possessed the maximal binding capacity of 110 mug/100 ml of 33% cytosol at pH 7.4. However, it showed no apparent affinity for T3, because the bound T4 could not be displaced with a T3 load of 600 mug/100 ml. The major band of T3, hT3-2, bound more than 70% of the tracer T3, and appeared to have a large capacity for the hormone although secondary binding sites on the same molecule might be responsible for the large capacity. The binding sites appeared almost specific for T3, because only a small, insignificant displacement was noted with a T4 load of 600 mug/100 ml. The results provide evidence for distinct binding proteins for T4 and T3 in the human liver cytosol, though their physiological roles remain to be elucidated.     

Clinical studies on autoimmune mechanisms of the thyroid. Part 2. Studies on measurements of serum thyroblobulin concentrations in patients with various thyroid diseases (author's transl)]

The present report deals with the measurement of serum thyroglobulin (Tg) in various thyroid disorders by using radioimmunoassay (RIA). The results were as follows: 1) A specific and simple solid-state RIA for the measurement of Tg in human serum was used. This system was a direct RIA using plactic cups coated with crude anti-thyroglobulin antibodies (anti-Tg) and 125I labeled purified anti-Tg. The purification of anti-Tg was performed by affinity chromatography using Tg-Sepharose, as an immunoadsorbent. 2) Affinity chromatography was carried out using a modification of the method of Cuatrecasas. The immunoadsorbent (Tg-Sepharose conjugate) was used in a column procedure for the isolation of anti-Tg from globulin fractions obtained from Hashimoto's sera. The elution was performed with 4M NaI or 0.17M Glycine-HCl with pH 2.3. The eluted materials contained a very small amount of Tg which was removed by Sephadex G-200 chromatography using the same elution buffer. The high purity of the anti-Tg obtained was demonstrated by the fact that almost all of the final product was bound with Tg by using Sepahdex G-200 chromatography. 3) A direct RIA consisting of two incubation steps was applied for Tg measurement. During the first incubation, standard Tg or Tg in serum was bound to the antibody coated cup. After washing, equal amounts of radioactive purified anti-Tg were incubated with the bound Tg. The cups were then washed again, and counted separately. The radioactive counts thus obtained, increased with the amounts of Tg bound to the anti-Tg by the first incubation. The sensitivity of the assay was 4 ng/ml. T3 and T4 did not cross-react against Tg, and did not interfere with the binding between Tg and 125I-anti-Tg. A dilution curve was constructed using the serum of a patient with Graves' disease; the post-operative serum contained a very high level of Tg. This curve paralleled the standard curve. By adding constant Tg to a normal serum or Graves' serum, Tg recovery was good. However, in regard to Hashimoto's serum, Tg recovery was unsatisfactory. 4) Serum Tg concentrations were measured in patients with various thyroid disorders. ..     

INAPPROPRIATE TRIIODOTHYRONINE (T3) AND THYROXINE (T4) RADIOIMMUNOASSAY LEVELS SECONDARY TO CIRCULATING THYROID HORMONE AUTOANTIBODIES

A 16-year-old boy with chronic lymphocytic thyroiditis was noted to have a low free thyroxine (T4) level, low triiodothyronine resin uptake (T3U), and high serum thyrotropin (TSH) values. Unexpectedly, markedly elevated T3 radioimmunoassay (RIA) and T4 (RIA) values, using a double antibody technique were obtained when performed directly on unextracted serum samples. Extremely low T4 (RIA) values were noted when polyethylene glycol (PEG) was used to separate bound from free hormone. The presence of circulating T3- and T4-binding immunoglobulins was suspected and confirmed with the following special studies. With undiluted serum in a T3 (RIA) system, using dextran-coated charcoal separation, 82% binding of ¹²⁵I-labelled T3 occurred in the absence of specific first antibody, with 55% binding retained at 1: 7 dilution with T3-free serum. Comparable results were obtained in the T4 (RIA) system using polyethylene glycol separation. Following ethanol extraction, low T4 (RIA) and low normal T3 (RIA) values were obtained, using a double antibody technique. There was ten-fold greater binding by the patient's serum to rabbit anti-human IgG in both the T3 and T4 radioassay systems as compared to controls. No preferential binding to rabbit anti-human IgM was noted. Scatchard plot analyses for the antibodies against T3 and T4 showed high affinity constants for these hormones. With adequate l -thyroxine therapy, an appropriate decline in serum TSH to normal was achieved. It is concluded that where RIA determinations of T3 and T4 are inconsistent with other laboratory and clinical indices, the presence of autoantibodies to thyroid hormones should be suspected and appropriate tests undertaken.     

Studies on thyroid hormone autoantibody (THAA) in 2 cases of Graves' disease with spuriously high free thyroxine values

Two patients with Graves' disease treated with methimazole (MMI) showed a discrepancy between serum free T4 (FT4) values and other hormone values (especially total T4) which was due to the presence of potent binding activity to labelled T4 analogue (125I-aT4) in their serum. This activity was demonstrated to be in immunoglobulin G (IgG) with kappa light chain isotype in both patients. The binding of 125I-aT4 to their serum was inhibited by unlabelled T4 in a dose-dependent manner. Autoantibodies had almost identical binding affinity to T4 and aT4, although they precipitated more radioactivity when 125I-aT4 was used. The binding of IgG purified from patients' sera to labelled T4 or aT4 was not greater than the corresponding sera, suggesting that the thyroxine binding proteins did not interfere with the assay. Since the specific radioactivity of 125I-aT4 is almost 10 times higher than that of 125I-T4, autoantibodies can precipitate almost 10 times more radioactivity in the FT4 assay than the total T4 assay, thus leading to the spuriously high FT4 values and large discrepancy between FT4 and TT4 values.     

Further evidence for the presence of thyroxine binding globulin-like protein in human breast adipose tissue. Deiodination of thyroxine and triiodothyronine by the microsomal fraction

The object of the study was to obtain information on how adipose tissue of normal subjects processes thyroid hormones. L-125I-thyroxine(125I-T4) is bound by the cytosol fraction of normal human female breast adipose tissue with high affinity. Computer analysis of the binding data revealed the presence of two saturable systems with Kd values of 3.9 and 29.1 nM and binding capacities of 1.7 and 8.7 pmol/mg of cytosol protein, respectively; a third binding system was non-saturable. The binding of the iodothyronines to the cytosol fraction indicated that L-triiodothyronine (T3) possessed two-fold higher affinity as compared to L-thyroxine (T4) whereas other iodothyronines had relative affinities of less than 3%. Binding of 125I-T4 was optimal at pH 7.0 and was sensitive to the action of pronase and neuraminidase. Affinity chromatography of the cytosol fraction using T3-epoxy-sepharose 6B and con A-sepharose 4B, yielded a 125I-T4 binding component that was purified 150-fold. Isoelectric focusing of the purified fraction yielded six major brands of protein which had pI values comparable to those of human serum thyroxine-binding-globulin (TBG); however, the pattern of separation was different. Incubation of the fraction with 125I-T4 followed by isoelectric focusing and autoradiography revealed that the protein bands bound radioactivity. The microsomal fraction of the adipose tissue deiodinated 125I-T4 to L-125I-triiodothyronine (125I-T3) to an extent of 0-0.8 fmol/(mg of protein X min) which corresponded to about 0.2%; T4 was not deiodinated to L-3,3',5'-triiodothyronine (r-T3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormone antibodies and Hashimoto's thyroiditis in mongrel dogs

Abnormally elevated serum T3 concentrations measured by RIA were observed in 19 clinically euthyroid or hypothyroid mongrel dogs. The serum T4 concentrations in these sera were low, normal, or high. Measurement of the intensity of thyroid hormone binding to serum proteins was determined by equilibrium dialysis. A marked decrease in the percent free T3 was observed in these abnormal sera. Polyacrylamide gel electrophoresis, pH 7.4, of normal dog serum enriched with tracer 125I-labeled thyroid hormones demonstrated binding of [125I]T4 to transthyretin, thyroid hormone-binding globulin, and albumin and of [125I]T3 primarily to thyroid hormone-binding globulin. In all abnormal sera, polyacrylamide gel electrophoresis demonstrated strikingly higher binding of T3 to immunoglobulin (Ig). Eleven of 16 abnormal sera had minimal to moderate binding of T4 to Ig. The percent free T4 was lower only in dogs whose sera demonstrated markedly increased binding of T4 to Ig. All abnormal sera tested had positive antithyroglobulin antibodies, consistent with the diagnosis of autoimmune lymphocytic thyroiditis. As in humans, antibodies to thyroid hormones in dogs are more common in the presence of Hashimoto's thyroiditis and should be considered when elevated serum thyroid hormone concentrations are observed in the absence of clinical thyrotoxicosis. When an antibody to only one thyroid hormone is present, a marked discrepancy in the serum concentrations of T3 and T4 will be observed.     

Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats

The effect of hexachlorobenzene (HCB) (1 g/kg bw) administration for 4 weeks, on thyroxine (T4) and triiodothyronine (T3) metabolism was studied in Wistar rats. The effect on serum binding of T4 has also been studied. Animals were injected with a tracer dose of either labeled hormone and by examining serum L-125I-T4 and L-125-I-T3, kinetics of radiolabeled hormones metabolism were calculated. The T4 metabolic clearance (MCI) as well as the distribution space, were increased by 6 fold. Decreased serum T4 levels result from an increase both in deiodinative and fecal disposal in HCB-treated rats. 125I-T3 metabolism was slightly affected. The enhanced peripheral disposition of thyroxine appears to lead to increased thyroid function, as measured by augmented TSH serum levels and 125I-thyroidal uptake. Serum binding of T4 was not affected.     

旋毛虫成虫排泄分泌抗原检测唾液中抗旋毛虫IgG抗体的研究

目的 评价旋毛虫(Trichinella spiralis)成虫排泄分泌抗原(adult worm excretory-secretory antigen,AWESA)作为诊断抗原检测旋毛虫感染日本大耳兔唾液中抗旋毛虫IgG抗体的可行性. 方法 建立旋毛虫感染日本大耳兔和对照组兔动物模型,采集感染前和感染后1～6周兔唾液和血清以及对照组兔唾液和血清.制备AWESA,建立AWESA作为诊断抗原的间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA),以市售旋毛虫IgG抗体检测试剂盒作为对照,测定感染前和感染后1～6周兔唾液和血清以及对照组兔唾液和血清中抗旋毛虫IgG抗体.AWESA和试剂盒测得的唾液A值和血清A值进行线性相关分析,AWESA和试剂盒测得的唾液阳性率和血清阳性率分别进行x2检验.结果 AWESA检测唾液和血清特异性IgG抗体阳性率依次为0、5%、20%、40%、60%、85%、90%,0、30%、60%、85%、95%、100%、100%,除感染后0、1、2周外其余各周唾液A值与血清A值呈显著线性相关(P＞0.05、P＞0.05、P＞0.05、P＜0.05、P＜0.05、P＜0.05、P＜0.05).市售旋毛虫IgG抗体检测试剂盒检测旋毛虫感染前和感染后兔唾液和血清中特异性IgG抗体阳性率依次为0、15%、20%、40%、55%、75%、90%,0、35%、60%、95%、95%、100%、100%,除0、1、3周外其余各周唾液A值与血清A值呈线性相关(P＞0.05、P＞0.05、P＜0.05、P＞0.05、P＜0.05、P＜0.05、P＜0.05). 结论 AWESA与市售试剂盒检测唾液和血清中抗旋毛虫IgG抗体的阳性率具有一致性.