Comparison of new different assay systems for thyrotropin receptor antibodies with reference to thyroid-stimulating antibodies and thyroid stimulation-blocking antibodies in Graves' disease.

The aim of our study was to evaluate the diagnostic sensitivity of the new thyrotropin receptor antibody (TRAb) assays (Cosmic TRAb CT, ELISA and Yamasa DYNOtest TRAb). TRAb was positive in 43 of 44 (97.7%) untreated patients with Graves' disease by both TRAb CT and/or ELISA and NYNOtest TRAb. Thus the new TRAb assays were clearly more sensitive than the conventional assay (positivity: 85%). There was a strong positive correlation between the data obtained in TRAb CT and/or ELISA and those obtained in DYNOtest TRAb (r = 0.942, p < 0.0001). There was a significant correlation between the new TRAb and TSAb (r = 0.696, p < 0.0001). Although there was a significant correlation between the new TRAb and thyroid stimulation-blocking antibody (TSBAb), the correlation coefficient was low (r = 0.605, p < 0.0001). The increased sensitivity of the new TRAb assays for Graves' disease provides an advantage over conventional assay.

Lipid peroxidation levels in rat cardiac muscle are affected by age and thyroid status.

Free radicals, hydroxyperoxides and H(2)O(2) are all known to damage cell components. This study was designed to compare the concentrations of hydroxyperoxide and free radical scavengers in the cardiac muscles of old rats in the hyper- or hypothyroid condition, to determine whether rates of peroxidation would differ with age, thyroid status, or both. Rats were rendered hyper- or hypothyroid by administration of l-thyroxine or methimazole for 4 weeks. Among the old rats, the lipid peroxide (LPO) concentrations, measured as thiobarbituric acid (TBA) reactants, were significantly greater in the hyperthyroid than in the euthyroid state and the LPO concentrations measured as TBA+Fe(3+) reactants, which may be precursors of LPO, were significantly greater in the hyperthyroid state, whereas in young rats, the LPO concentrations measured by TBA or TBA+Fe(3+) methods did not differ significantly in the hyperthyroid state. In the euthyroid state, the concentration of LPO measured as TBA+Fe(3+) reactants was significantly reduced with age. Xanthine oxidase (XOD) activity also was markedly increased with age, being more pronounced in the hyperthyroid than in the euthyroid state. The Mn and Cu/Zn superoxide dismutase activities were greater in the hyperthyroid than in the euthyroid state. Glutathione peroxidase activity decreased with age in the euthyroid and, particularly, in the hyperthyroid state. Catalase activity was not affected in the old rats. Concentrations of alpha-tocopherol in the old rats were high in the hyperthyroid state and low in the hypothyroid state, whereas the levels of beta- and gamma-tocopherols in these rats were unchanged in both conditions as compared with the euthyroid state findings. Data suggest that the site of free radical generation differs in older rats, with additional shifts in the location of intracellular lipid peroxidation being noted during hyperthyroidism. Thus, as rats age, the reduction of the free radical scavenger system and the increase in LPO and XOD activities might induce myocardial dysfunction.

Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders.

To clarify whether the changes of free radicals and its scavengers are induced by thyroid disorders, we measured levels of free radical scavengers and checked O2 radical generating systems in the human thyroid gland. Thyroid specimens from patients with Graves' disease, follicular adenoma, and papillary and follicular carcinomas contained significantly higher concentrations of xanthine oxidase (XOD) and gluthathione peroxidase (GSH-PX), compared to those in the normal thyroid tissue. Catalase concentration was significantly lower in thyroid specimens from patients with Graves' disease and significantly lower in thyroid specimens from patients with follicular adenoma, compared to those in the normal thyroid tissue. Cu/Zn superoxide dismutase (Cu/Zn SOD) concentration was significantly lower in the specimens from follicular adenoma and papillary carcinoma and Mn SOD concentration was significantly higher in the specimens from papillary carcinoma than those in the normal thyroid tissue. The lipid peroxide concentration, expressed as malondialdehyde (MDA) concentration, was significantly higher in the specimens from papillary carcinoma than those in the normal thyroid tissue. These findings suggest that the levels of free radicals are increased and are scavenged and catalyzed in the thyroid of Graves' disease, whereas free radicals and lipid peroxide are not completely scavenged in papillary carcinoma tissues, suggesting that these substances affect some role in cell function of thyroid tumors.

[The variation of Japanese urinary excretion of iodine in different decades of age].

In order to evaluate whether or not iodine intake in Japanese is variable among different aged subjects and also whether this variation is concordant with the age distribution in patients with chronic thyroiditis, we tried to examine the urinary iodine excretion of euthyroid people in various decades of age. One hundred and twenty outpatients without thyroid disorders, aged 22 to 77 y.o., were selected at random and were divided into 6 groups according to age. Mean total urinary iodine excretion (UIT) and concentration (UIC) were 336.1 micrograms/day and 31.4 micrograms/dl/cr, respectively, and they were in good correlation (r = +0.82, p < 0.001). A straight line on log normal probability was recognized between the ratio of cumulative frequency and values of UIT. UIT and UIC were significantly correlated with serum nonhormonal iodine, respectively (r = +0.21, p < 0.01; r = +0.28, p < 0.01). These indicators in the 3rd decade were lower than those in the 6th decade (p < 0.01, p < 0.05). UIT increased with age up to the 6th decade and then decreased gradually to the 8th decade. Therefore, UIT in all the subjects formed a bell-shaped distribution with a significant peak in the 6th decade (H = 12.1, p < 0.05). Rates of renal iodine clearance (UIC/SNI) in the 6th decade increased significantly more than those in the 4th decade (p < 0.01), and the distribution of those rates in the 6 groups were similar to that of UIT.(ABSTRACT TRUNCATED AT 250 WORDS)

[The prevalence of antithyroid antibodies and long term follow-up in patients with lipiodol iodine-induced goiter].

We have carried out a follow-up study on iodine-induced goiter to clarify whether or not iodine could be a factor in the progression as well as the promotion of thyroid autoimmunity. We selected 143 women of child-bearing age without any previous thyroid disorders who had received hysterosalpingography (H.S.G.). 45 Sex and age-matched healthy subjects were chosen as controls. Serum nonhormonal iodine (S.N.I.) levels, frequency of goiter and antimicrosomal antibody (MCHA) in all the Lipiodol-cases were significantly higher than those in the controls (p < 0.001, < 0.01 and < 0.01), respectively. When the subjects were divided into 9 groups according to the duration of each 5 months after H.S.G., serum TSH and S.N.I. levels, incidence of goiter and MCHA in the initial group were significantly higher than those in the other groups (p < 0.05). The S.N.I. levels became normalized in 30 months after H.S.G. and the goiters disappeared in almost the same duration, while the incidence of higher MCHA titers declined gradually but significantly around 40 months after H.S.G. compared with that in the first 5 months after H.S.G. (p < 0.05). The frequency of goiter and MCHA in 44 cases after a 6-39 month follow-up decreased significantly compared to that in the initial group (p < 0.05). Therefore, we tried an individual longitudinal follow-up study on MCHA titer in 12 cases for 35-103 months, resulting in a significant reduction or negativeness of the titer in 6 cases. Likewise, MCHA titers in all cases decreased significantly (p < 0.05) on later evaluation. The present data suggest that iodine in Lipiodol administered via the vagina will act not only as the promoting factor, but as an aggravating agent for thyroid autoimmunity.

Deregulated production of interleukin-4 (IL4) in autoimmune thyroid disease assayed with a new radioimmunoassay.

A sensitive, reproducible and specific radioimmunoassay for human interleukin-4 (IL4) has been developed. Using 125I-labeled IL4 and polyclonal rabbit antisera raised against recombinant human IL4, a competitive inhibition assay was developed which could detect 5 pg/ml of human IL4. Other interleukins, growth factors, hormones, peptides and lectins did not affect the assay. IL4 was measured in supernatants of culture media of stimulated human peripheral blood mononuclear cells (PBMC). Kinetics of IL4 production in PHA-stimulated PBMC from seven normal subjects revealed that the peak levels of IL4 were seen at 24 h and then declined. Peak IL4 levels in PHA stimulation of PBMC from untreated patients with autoimmune thyroid diseases (Graves' disease and chronic thyroiditis) were significantly higher than normal controls. However, after treatment, IL4 production decreased to normal. The present study demonstrates the usefulness of quantitating human IL4 produced by PBMC and that there exists a deregulated production of IL4 in autoimmune thyroid diseases.

Production of bioactive rabbit antibodies against the mutated receptor peptide TSH-R-HIS17 and detection of hypertriiodothyroninemia in the rabbit.

We produced rabbit antibodies against mutated thyrotropin (TSH) receptor peptide TSH-R-HIS17 in comparison with non-mutated TSH receptor peptide TSH-R-ASP17. All the antibodies raised against TSH-R-HIS17 showed thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and/or TSH binding inhibiting immunoglobulin (TBII) activities and also those serum T3 levels were all in thyrotoxic levels associated with TSAb activities. However, all the antibodies raised against TSH-R-ASP17 had no activities of TSAb, TSBAb and/or TBII and those serum T3 levels were all within normal range. The data suggest that the mutation in the TSH receptor gene may have a possible relevance for the pathogenesis of Graves' disease.

Deregulated production of interleukin-8 (IL-8) in autoimmune thyroid disease studied by newly developed IL-8 radioimmunoassay.

We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-8 (IL-8). Using 125I-labelled IL-8 and polyclonal rabbit antisera raised against recombinant human IL-8, a competitive inhibition assay was developed which could detect 5 pg/ml of human IL-8. Other interleukins, growth factors, hormones, peptides and lectins did not affect the assay. IL-8 measured in supernatants of culture media of stimulated human peripheral blood mononuclear cells (PBMC). Kinetics of IL-8 production in SAC-stimulated PBMC from seven normal subjects revealed that the production of IL-8 was detectable within 12 h and reached a plateau at 24 h. IL-8 levels in SAC-stimulation of PBMC from untreated patients with autoimmune thyroid diseases (Graves' disease and chronic thyroiditis) were significantly higher than in normal controls. However, after treatment, IL-8 production decreased to normal. The present study demonstrates the usefulness of quantitating human IL-8 produced by PBMC and the presence of a deregulated production of IL-8 in autoimmune thyroid diseases.

In vivo stimulation of thyroid hormone release by rabbit antibodies with thyroid stimulating activity raised against the mutated receptor peptide TSH-R-HIS17.

The present study was designed to examine whether thyroid stimulating antibodies (TSAb) against the mutated or non-mutated peptide of human TSH receptor stimulates release of thyroid hormones in mice in vivo. We generated antibodies against mutated TSH receptor peptide TSH-R-HIS17 and non-mutated TSH receptor peptide TSH-R-ASP17 immunizing the peptides to rabbits. We isolated immunoglobulin G (IgG) from these antibodies and injected iv into male ddy mice. Control mice were injected iv with bTSH or TSAb-negative normal rabbit IgG. Serum T3 and T4 concentrations were elevated after the administration of bTSH in a dose-dependent manner. Serum T3 and T4 concentrations were significantly elevated after the injection of IgG from the rabbit antibodies with TSAb activity produced against TSH-R-HIS17. However serum T3 and T4 remained within normal levels after the injection of IgG from the rabbit antibodies without TSAb activity toward TSH-R-ASP17. The present data offer in vivo support of the IgG with TSAb activity interaction with the TSH receptor of the thyroid and raise the possibility that TSAb against mutated human TSH receptor peptide might be contributed to induce thyroid hyperfunction.

[A case of chronic thyroiditis with transient painful thyroiditis occurring after the administration of lipiodol].

We reported a female case of painful thyroiditis occurring after hysterosalpingography and investigated whether the destructive thyroiditis was iodine-induced. The patient, aged 28, had TBG deficiency and the previous episode of thyrotoxicosis caused by Graves' disease. Lipiodol (containing 4.8g of iodide in 10ml solution) was administered via vagina for hysterosalpingography. One month after the radiography, serum inorganic iodide and Tg were elevated abnormally, but she was asymptomatic. After the subsequent 3 months she developed a painful and 3rd grade-sized goiter with concomitant marked elevation of thyroid hormones and inorganic iodide and also high titer of MCHA (320(2)X). A dramatic response was obtained with steroid. Thereafter she was treated with acupuncture on the thyroid gland, resulting in a sudden reappearance of tender goiter. This traumatic thyroiditis disappeared successfully in 2 weeks with steroid treatment. The painful thyroiditis subsided in 5 months throughout the course and she remained euthyroid for the ensuing 2 years. Aspiration biopsy was performed twice and revealed lymphocytic thyroiditis. Values of serum Tg varied in good correlation with those of serum inorganic iodide or rT3 throughout the course, respectively (P < 0.01, P < 0.05). Significant correlations between FT4 and FT3, and also T4 and T3 were observed, respectively (P < 0.01, P < 0.05). Serum inorganic iodide was elevated to 316 micrograms/dl at the symptomatic stage of the thyroiditis and decreased to 170 micrograms/dl at the resolving phase 2 months after the inflammation. Iodide disappearance curve showed a diphasic slope. The BHL was calculated as 60.3 days during the symptomatic stage and 6.9 months in euthyroid state.(ABSTRACT TRUNCATED AT 250 WORDS)

[Urinary iodide excretion in Japanese people and thyroid dysfunction].

In order to assess the Japanese dietary iodine intake, we examined the urinary iodine excretion of those on an ordinary Japanese diet chosen at random and observed whether the thyroid function might affect the amounts of urinary iodine excretion. The subjects consisted of cases of untreated hypothyroidism and chronic thyroiditis (CT) and euthyroid controls who were healthy people or had non-thyroidal disorders such as diabetes mellitus or hypertension. Eight cases of hypothyroidism were composed of 3 cases of secondary hypothyroidism with empty sella syndrome and 5 cases of primary hypothyroidism and 32 patients with CT have been maintained in euthyroid states with T4 medication. We selected 32 cases of sex and age-matched healthy people as controls. The mean levels of excreted urinary iodine were 465.6 micrograms/day in the healthy controls and 471.8 micrograms/day in patients with CT, respectively. Urinary iodine excretion was significantly correlated to serum inorganic iodide in both controls and CT patients, of which correlation coefficients were +0.35 and +0.5, respectively. Urinary iodine and serum inorganic iodide ratios (U/S) in hypothyroidism were significantly (p less than 0.05) depressed compared with those in CT. The present study indicated that recent Japanese dietary iodine intake was estimated to be approximately 470 micrograms/day and that the urinary iodine excretion would be influenced not only by iodine intake but also by thyroid function.

[The functional outcome of patients with subacute thyroiditis].

Whether with the passage of time subacute thyroiditis leads to hypothyroidism remains to be determined. Therefore, we evaluated the thyroid function including TRH test of 66 patients with a previous history of subacute thyroiditis and age-matched control subjects with special reference to the measurement of inorganic iodide. The patients were divided into 3 groups according to time lapse since the occurrence. Group 1 consisted of 24 cases followed up for 4 to 24 months. Sixteen cases in group 2 had their courses from 2 to 5 years, and group 3 was composed of 26 cases over the past 5 to 30 years. We selected 169 subjects without history of subacute thyroiditis and divided them into three control groups matched for age, each corresponding to the patient groups (group 1a, 2b and 3c, respectively). 41.7% and 29.2% of cases in group 1 had high basal levels of serum TSH (greater than 3.6 microU/ml) and delta TSH (the increment of TSH after TRH, greater than 46.8 microU/ml). In group 2, levels of serum T3 and T4 returned to normal ranges. However, in group 3, significant higher elevations in TSH and delta TSH than those in group 3c were observed, and the T4, FT4, T3 and FT3 levels were lower than those of group 3c (p less than 0.01 and p less than 0.05, respectively). 42.3% of cases in group 3 showed high TSH, and there were 4 cases with clinical hypothyroidism. Among the cases studied, a significant negative correlation (p less than 0.01) between levels of TSH and T4 was observed, while a correlation between TSH and delta TSH was positive (p less than 0.001). High levels of serum inorganic iodide were observed in 6.1% of cases and a correlation between inorganic iodide and TSH was significantly positive (p less than 0.01) not only in patients with subacute thyroiditis, but also in the control subjects. Antithyroid autoantibodies were detected in 42.4% of all the cases with subacute thyroiditis and also in 45.6% of all the controls. In group 3, MCHA was detected in 64.5% of the cases, and the frequency was higher than that of group 3c as well as that of 25% in group 2 (p less than 0.01), respectively. In 15.4% of the cases in group 3, the titers of MCHA were more than 40(2), and the titers of MCHA were significantly higher in patients with a long-term period after the onset of subacute thyroiditis than those with a short-term period.(ABSTRACT TRUNCATED AT 400 WORDS)

[The effects on the thyroid gland of soybeans administered experimentally in healthy subjects].

To elucidate whether soybeans would suppress the thyroid function in healthy adults, we selected 37 subjects who had never had goiters or serum antithyroid antibodies. They were given 30g of soybeans everyday and were divided into 3 groups subject to age and duration of soybean administration. In group 1, 20 subjects were given soybeans for 1 month. Groups 2 and 3 were composed of 7 younger subjects (mean 29 y.o.) and 10 elder subjects (mean 61 y.o.) respectively, and the subjects belonging to these groups received soybeans for 3 months. The Wilcoxon-test and t-test were used in the statistical analyses. In all groups, the various parameters of serum thyroid hormones remained unchanged by taking soybeans, however TSH levels rose significantly although they stayed within normal ranges. The TSH response after TRH stimulation in group 3 revealed a more significant increase than that in group 2, although inorganic iodide levels were lowered during the administration of the soybeans. We have not obtained any significant correlation between serum inorganic iodide and TSH. Hypometabolic symptoms (malaise, constipation, sleepiness) and goiters appeared in half the subjects in groups 2 and 3 after taking soybeans for 3 months, but they disappeared 1 month after the cessation of soybean ingestion. These findings suggested that excessive soybean ingestion for a certain duration might suppress thyroid function and cause goiters in healthy people, especially elderly subjects.

[Transient thyrotoxicosis induced by Japanese kombu].

Iodine-induced thyrotoxicosis (ITT) has not been reported in Japan. We found that excessive intake of Japanese kombu could elicit ITT. Two Japanese women, 42 and 59 years old, developed thyrotoxicosis one month and one year, respectively, after having eaten foods containing 28-140 mg/day of iodine, calculated from their daily diet. Both patients had high concentrations of serum T3, low ratios of serum T3/T4 or T4/r-T3 compared with untreated Graves' disease, and high concentrations of serum inorganic iodine compared with the mean (M +/- SD: 2.05 +/- 0.99 micrograms/dl) plus 2SD in people eating common foods. Their thyrotoxic signs and symptoms disappeared, and their serum T4, T3, r-T3 and T4/r-T3 normalized one month after the prohibition of kombu intake. To clarify the source of iodine, the iodine content of the kombu and the iodine concentration in water in which the kombu was immersed were measured. Ninety-nine % of the iodine was found in water after 15 min boiling. These findings suggest that a daily intake of more than 28 mg/day of iodine in a diet containing kombu might induce ITT.

Enzyme immunoassay for antibodies to thyroxine in human serum using synthesized antibody as a calibrator.

We prepared human IgG labeled with rabbit antibodies to thyroxine, and used it for the assay of antibodies to thyroxine in human serum as a calibrator. The assay system consisted of thyroxine labeled with beta-D-galactosidase and a microcolumn containing goat antibodies to human IgG immobilized on Sepharose 4B. Each serum sample or standard serum containing human IgG labeled with anti-thyroxine antibodies was incubated with the enzyme-labeled thyroxine, and the reaction mixture was passed through the microcolumn. The column was washed to remove the unbound label, and enzyme activity of the bound label was assayed. The minimum detectable amount of the anti-thyroxine antibodies was 0.3 ng/assay tube. The analytical recoveries of human IgG labeled with the antibodies added to human serum were from 103% to 108%. We measured anti-thyroxine antibodies in human sera from 187 patients with autoimmune thyroid diseases. The antibodies were detected in 32 serum samples, and the values in positive samples varied from 0.31 to 2.31 micrograms/ml. On the other hand, in 58 sera from patients with non-thyroid diseases, the antibodies were detected in only two samples.

Evidence for thyrotropin (TSH)-blocking activity in goitrous Hashimoto's thyroiditis with assays measuring inhibition of TSH receptor binding and TSH-stimulated thyroid adenosine 3',5'-monophosphate responses/cell growth by immunoglobulins.

There are two forms of autoimmune thyroiditis that may cause hypothyroidism: autoimmune atrophic thyroiditis (primary idiopathic hypothyroidism or primary myxedema) and autoimmune goitrous thyroiditis (Hashimoto's disease). Patients with the former have impalpable thyroid glands, and those with the latter have goiters. We studied TSH binding inhibitory immunoglobulins (TBII), TSH-stimulated cAMP response inhibitory immunoglobulins (TSII), and TSH-stimulated cell growth inhibitory immunoglobulins (TGII) in 42 patients with the former (group 1) and 115 patients with the latter (group 2). Porcine thyroid cells in primary culture and rat thyroid cells in continuous culture (FRTL-5 cells) were used to study TSII and TGII activities, respectively; TSII was expressed as percent inhibition of 0.1 mU/ml TSH-stimulated cAMP response by the patient's immunoglobulin (IgG; 1 mg/ml) during 2-h incubation, and TGII was expressed as percent inhibition of 10 mU/ml TSH-stimulated [14C]thymidine incorporation by the patient's IgG (1 mg/ml) during 24-h incubation. The new findings in this report are: some patients in both groups had TBII, TSII, and/or TGII; the frequency of the presence of TBII, TSII, and TGII in the patients with autoimmune atrophic thyroiditis was higher than that in the patients with autoimmune goitrous thyroiditis, and TSII and TGII were significantly associated with autoimmune atrophic thyroiditis; no correlation was found between goiter size and TBII, TSII, or TGII activity; and there were good correlations between TBII, TSII, and TGII activities. We also found that TSH-stimulated thymidine incorporation was through cAMP production and that the inhibitory IgGs inhibited TSH-stimulated thymidine incorporation by decreasing cAMP production in FRTL-5 cells, but not in porcine or human thyroid cells.

Studies on human iodide peroxidase: its activity in various thyroid disorders.

Iodide peroxidase activities in the human thyroid gland were measured in various thyroid disorders. The peroxidase activities in the thyroid gland revealed high values in Graves' disease, normal levels in thyroid adenoma and low values in thyroid carcinoma as compared to those in normal thyroid glands. A high activity of this enzyme was observed in toxic adenoma. The same results were observed whether 10% (W/V) homogenate or mitochondrial and microsomal fractions of the thyroid gland were used.