MASATERU HORIMOTO, MITSUSHIGE NISHIKAWA, CHISATO UNO, NoRIO YOSHIKAWA, NORIMICHI TANIGUCHI AND MITSUO INADA

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1 Relationship among Thyrotropin (TSH), Thyroid Stimulating Immunoglobulins, and Results of Triiodothyronine (T3) Suppression Test in Patients with Graves' Disease MASATERU HORIMOTO, MITSUSHIGE NISHIKAWA, CHISATO UNO, NoRIO YOSHIKAWA, NORIMICHI TANIGUCHI AND MITSUO INADA Second Department of Internal Medicine, Kansai Medical University, 1 Fumizonocho, Moriguchi, Osaka 570, Japan Abstract To investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (TSAb and TBII), and T3 suppression. Out of thirty-three patients with hyperthyroid Graves' disease, twelve patients with subnormal TSH levels were all non-suppressible according to the T3 suppression test results and the detectability of TSAb and/or TBII was as high as 75%. In three out of five patients with euthyroid Graves' disease, the serum TSH level was subnormal. All three showed non-suppressibility in the T3 suppression test and positive action of either TSAb or TBII. One of them became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive, and was therefore diagnosed as having hyperthyroid Graves' disease. The present findings suggest that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low TSH, even if their thyroid hormone concentrations remain normal. Moreover, it is likely that some of the patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism because of the presence of abnormal thyroid stimulator(s). Abnormal thyroid stimulator(s) play an important role in the pathogenesis of hyperthyroidism in Graves' disease. It is reported that thyroid stimulating immunoglobulins are present in the sera of most patients Received April 14, 1988 with hyperthyroid Graves' disease (Clague et al., 1976, Orgiazzi et al., 1976, Zakarija et al., 1980). The triiodothyronine (T3) suppression test may be used before the withdrawal of antithyroid drug therapy in patients with hyperthyroid Graves' disease (Iida et al., 1982). Little or no suppression of thyroid

2 HORIMOTO et al. Endocrinol. Japon. December 1988 uptake suggests that active hyperthyroid Graves' disease is still present and, therefore, that hyperthyroidism might recur if the antithyroid drug treatment were stopped (Utiger 1986). On the other hand, newly developed, highly sensitive assays for TSH can provide a first line test of thyroid function. These assays have the potential to distinguish between normal and subnormal TSH concentrations (Weeks et al., 1984, Spencer et al., 1986). However, TSH concentrations are widely scattered above and below the normal range in clinically euthyroid patients treated with antithyroid drugs for hyperthyroid Graves' disease (Spencer et al., 1986). The relation between abnormal thyroid stimulator(s) and the serum TSH concentration has not been clearly elucidated in these patients. On the other hand, thyroid stimulating immunoglobulins are present in sera from some patients with euthyroid Graves' disease (Teng et al., 1977, Solomon et al., 1977). Despite the presence of thyroid stimulating immunoglobulins, these patients remain clinically euthyroid, and the mechanism responsible for this phenomenon has not yet been identified. In the present study, to investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (thyroid stimulating antibody: TSAb, and thyrotropin binding inhibitor immunoglobulin: TBII), and T3 suppression. Materials and Methods The subjects selected for this study were thirty-three patients with hyperthyroid Graves' disease in whom normal thyroid hormone concentrations in the serum were maintained for more than four months with an antithyroid drug (methimazole or propylthiouracil) therapy and The diagnoses in all subjects were established on the basis of their clinical pictures and the determination of T3, T4, and free T4 concentrations in the sera. The patients with euthyroid Graves' disease had eye changes of Graves' disease appropriate to classes 2-4 of the classification defined by the American Thyroid Association (Werner, 1969), which included exophthalmos of more than 18.5mm, measured with a Hertel exophthalmometer, and enlarged soft tissues or extraocular muscles, demonstrated by CT scan. They had normal serum T4, T3, and free T4, and other possible causes of exophthalmos had been excluded by ophthalmologists. On the basis of careful clinical examination of each patient at the time of the study, none of the patients was considered to be in an overt state of hyperthyroidism. The serum concentration of TSH was determined with a highly sensitive radioimmunometric assay kit provided by Travenol (Tokyo, Japan). The normal TSH concentrations ranged from 0.8 to 2.4ƒÊU/ml. The values below 0.1ƒÊU/ml were expressed as undetectable in this study, the serum TSH in twenty-two patients with untreated hyperthyroid Graves' disease was undetectable. The within-assay coefficients of variation, as tested by more than quadruplicate determinations for three plasma samples from normal subjects, averaged 6.9% and the between-assay coefficients of variation, as tested by more than three consecutive determinations for three plasma samples, averaged 8.2% in the present assay. Serum T4 and T3 were determined with commercial RIA kits purchased from Dainabot (Tokyo, Japan). The normal ranges of T4 and T3 were 4.5 to 13.5ƒÊg/dl and 84 to 174 ng/dl, respectively. The determination of free T4 in serum was carried out with a RIA kit provided by Amersham (Tokyo, Japan). The normal subjects showed free T4 values ranging from 1.07 to 2.44 ng/dl. There were no significant difference in the T4, T3, and free T4 concentrations in the same serum determined in two consecutive assays by the paired Student's t-test. Moreover, the coef- within an assay, for these hormones, were under 11.2% in a normal subject, and in a thyrotoxic and a hypothyroid patient. The T3 suppression test was performed by

3 TSH AND TSAb IN GRAVES' DISEASE measuring the 30 minute uptake of 99 m-tc by the thyroid (99 m-tc uptake: %) before and after the administration of a daily dose of 75 fig of T3 for 7 days. The 99 m-tc uptake before T3 administration ranged from 0.3 to 2.5% in normal subjects. T3 was considered suppressible (T3 suppressible) when the test resulted in both a fall of more than half of the 99 m-tc uptake before T3 and an uptake value after T3 less than 0.9%. TSAb was assayed with cultured porcine thyroid cells and the serum was precipitated by polyethylene glycol, as described by Kasagi et al.(1986). The results were expressed as a percentage of the mean camp accumulation in the presence of serum precipitate from patients, when compared with those from normal subjects. TSAb was considered present when the accumulation was more than 130%. The assay kit provided by Travenol (Tokyo, Japan) was employed for the determination of TBII. The percentage inhibition of 125-I-TSH binding to the TSH receptor averaged-2.6 ( } 5.0: SD) in sera from normal subjects, and, therefore, percentage of the mean camp accumulation in the presence of the serum precipitate from patients, when compared with those from normal subjects, and TSBAb was considered present when the accumulation was less than 85%. The coefficients of variation within the assay for TSAb, TBII, and TSBAb were 15%, 12%, and 13%, respectively. The coefficients of variation between assays for TSAb, TBII, and TSBAb were 12%, 18%, and 19%, respectively. Results Patients with hyperthyroid Graves' disease during treatment with antithyroid to less than 0.1ƒÊU/ml in all patients, re- normal range. The thyroid stimulation-blocking the patients had been receiving antithyroid antibody (TSBAb) was assayed to determine the drug therapy for more than nine months. inhibition of TSH-induced camp increase by immunoglobulins, as described by Konishi et al. (1983). In the present series of experiments, maintenance doses of the drugs. Serum cultured porcine thyroid was employed and the TSH after T3 administration was suppressed immunoglobulins were prepared with polyethylene glycol precipitation from the serum. The gardless of the TSH concentration before cells were exposed to about 3 mg of immunoglobulin in the presence of 0.1mU/ml bovine TSH T3 (basal TSH concentration). (Armour Pharmaceutical, Phoenix, AZ, USA). The results of the TSBAb were expressed as a drugs The T3 suppression test was done in thirty-three patients with hyperthyroid Graves' disease in whom normal thyroid hormone concentrations in the serum were maintained for more than four months by undergoing antithyroid drug therapy. All of During this test, they were placed on Thirty-three patients were divided according to their basal TSH (Table 1). All Table 1. Serum basal TSH concentrations, results of T3 suppression test, and detectability of TSAb and TBII in patients with hyperthyroid Graves' disease who were maintained at normal thyroid hormone concentrations for more than four months when treated with antithyroid drugs TBII: Thyrotropin binding inhibitor immunoglobulin TSAb: Thyroid stimulating antibody

4 HORIMOTO et al. Endocrinol. Japon. December 1988 of the twelve patients who had subnormal basal TSH, ranging from below 0.1 to 0.6 pressibility. In these twelve patients, TSAb was present in six patients and TBII in nine patients. Thus, the prevalence of patients with TSAb and/or TBII was 75% (nine out of twelve patients)(table 1).Out of twenty-one patients who had normal or high basal TSH, eight patients showed T3 suppressibility, while thirteen patients were T3 non-suppressible (Table 1). In eight patients associated with T3 suppressibility, only two patients showed slightly positive TSAb (133 and 176%), and one of these two showed slightly positive TBII (+13.9%). Thus, the occurrence of patients with TSAb and/or TBII was 25% among eight patients (Table 1). On the other hand, in thirteen patients with T3 non-suppressibility, eleven patients (85%) were positive for TSAb and/ or TBII, and the detectability was not significantly different from that in twelve patients with subnormal basal TSH (75%). The basal TSH concentrations (subnormal or not) in the suppressed and the non-suppressed patients who underwent the T3 suppression test as examined by analysis of X2 (p <0.05) were markedly different. Patients with euthyroid Graves' disease Three out of five patients with euthyroid Graves' disease showed T3 non-suppressibility, while the remaining two were T3 suppressible on the first examination (Table 2). Two patients with T3 suppressibility had normal TSH concentrations of 1.0 and 2.4ƒÊU/ml. On the other hand, three patients showing T3 non-suppressibility had low TSH, although serum T4, T3, and free T4 concentrations were all within normal limits. Four out of five patients were positive for either TSAb or TBII, while all were negative for TSBAb (Table 2). In one patient (No. 3 C.H.), the clinical course was observed without any specific

5 TSH AND TSAb IN GRAVES' DISEASE for TSAb (614%) and had a TSH concentration of 0.3ƒÊU/ml, but her TBII was negative on the first examination. After having hyperthyroid Graves' disease and was treated with antithyroid drugs. about two months, she developed a positive TBII (+52.2%), and the TSH concentration was less than 0.1ƒÊU/ml, although her serum free T4 was still within the normal range. After five successive months, her TSAb activity was further increased (1,023 %) and she also became clinically thyrotoxic, associated with a marked increase in serum free T4. She was therefore diagnosed as Fig. 1. Clinical course of one patient with euthyroid Graves' disease who became thyrotoxic. (Patient 3 in Table 2) Discussion Smyth et al.(1983) reported that thyroid stimulating immunoglobulins measured by using the cytochemical bioassay in patients with idiopathic euthyroid goiter caused a modest increase in thyroid hormone secretion, sufficient to blunt the TSH response to TRH, but not enough to cause clinical hyperthyroidism. However, to our knowledge, there have been no detailed reports concerning the relationship between abnormal thyroid stimulator(s) and the basal TSH concentration in patients with hyperthyroid Graves' disease whose thyroid hormone concentration was maintained at normal levels by antithyroid drug therapy or in patients with euthyroid Graves' disease. In the present study, out of 33 patients with hyperthyroid Graves' disease whose normal thyroid hormone concentration was maintained by undergoing antithyroid drug therapy, 12 patients had low basal TSH. All of the twelve revealed T3 non-suppressibility, and in these the prevalence of patients with TSAb and/or TBII was 75% (9 patients). Therefore, it is suggested that the T3 suppression test is unnecessary when serum TSH is subnormal in patients with hyperthyroid Graves' disease during treatment, because their thyroids still seem to be stimulated by abnormal thyroid

6 HORIMOTO et al. Endocrinol. Japon. December 1988 stimulator(s). The results of the basal TSH and the T3 suppression test: are consistent with those of Shishiba et al.(1987) in which medication was discontinued for at least 4 months. On the other hand, in patients who had a normal or high basal TSH concentration, the results of the T3 suppression test could not be predicted on the basis of the basal TSH concentration. It was considered that some of them had normal thyroid hormone concentrations because they were receiving sufficient doses of antithyroid drugs, in spite of the presence of abnormal thyroid stimulator(s), since the T3 suppression test was done during the treatment with maintenance doses of the drugs in the present study. Therefore, it is practical to do the T3 suppression test only in patients with normal or high basal TSH before discontinuing drug use. It was reported that thyrotropin-binding inhibitory activity correlated with T3 suppressibility, whereas thyroid stimulating activity disappeared before T3 suppressibility occurred (Kuzuya et al., 1979). In the present study, TSAb and TBII were almost equally prevalent when normal thyroid hormone concentrations were maintained in the patients for more than four months. The discrepancy between these results might be considered to depend on differences in sensitivity and/or the specificity of each assay system as well as the heterogeneity of the anti-tsh receptor antibody (Shishiba et al., 1982). Solomon et al. reported that euthyroidism in Graves' ophthalmopathy might have more than one cause (1977). A majority of patients with euthyroid Graves' disease show substantial abnormalities in thyroidal anatomy and physiology. In the present study, three out of five patients (60%) with euthyroid Graves' disease showed T3 nonsuppressibility and this finding is compatible with the report that sixty out of ninety-four patients(64%)with euthyroid Graves' disease showed T3 non-suppressibility (Chopra and Solomon, 1986). These three patients with T3 non-suppressibility had low basal TSH, while two with T3 suppressibility had normal TSH. None of the 3 patients with T3 non-suppressibility had both positive TSAb and positive TBII. It is likely from the present findings that the thyroid is stimulated by abnormal thyroid stimulator(s) to a level sufficient to inhibit TSH secretion, but not enough to lead to a thyrotoxic state in some patients with euthyroid Graves' disease, and that patients with euthyroid Graves' disease have relatively weaker action of abnormal thyroid stimulator(s) than those with hyperthyroid Graves' disease, because most patients with hyperthyroid Graves' disease have both positive TSAb and TBII. One of these two patients with T3 suppressibility, who had low normal TSH (1.0ƒÊU/ml), had slightly positive TSAb. It might be possible that this is not sufficient to inhibit TSH secretion, because of the relative weakness of the action or the insensitivity of the individual thyroid. The present study also suggests that TSBAb has little influence on thyroid function in patients with euthyroid Graves' disease. Tamai et al.(1980) reported that thyroid function test results in patients with euthyroid Graves' disease were variable and unstable, and that euthyroid Graves' disease, hyperthyroid Graves' disease, and Hashimoto's disease were closely related to one another and could not be discerned before the clear onset of the disease. In the present study, one patient with euthyroid Graves' disease became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive. This patient was diagnosed as having hyperthyroid Graves' disease. These findings may reflect the evolution of the occurrence of hyperthyroidism in patients with euthyroid Graves' disease. To conclude, it is suggested that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low basal TSH, even, if their

7 TSH AND TSAb IN GRAVES' DISEASE serum thyroid hormone concentrations are within normal limits. Moreover, it is likely that some patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism, because of the presence of abnormal thyroid stimulator(s). Acknowledgments The authors thank Dr. K. Kasagi, Department of Nuclear Medicine, Kyoto University School of Medicine, Kyoto, Japan, for his technical advice. References Chopra, I. J. and D. H. Solomon (1986). Latent Graves' disease: Euthyroid Graves' disease. In The Thyroid (eds. S. H. Ingbar and L. E. Braverman), pp J. B. Lippincott Co., Philadelphia. Clague, R., E. D. Mukhtar, G. A. Pyle, J. Nutt, F. Clark, M. Scott, D. Evered, B. R. Smith and R. Hall (1976). Thyroid-stimulating immunoglobulins and the control of thyroid function. J. Clin. Endocrinol. Metab. 43, Iida, Y., J. Konishi, K. Kasagi, T. Misaki, K. Endo, N. Nesumi, K. Torizuka, K. Tanaka, H. Ishii, K. Naito, M. Nishikawa, M. Inada and H. Imura (1982). The prognosis of Graves' disease after antithyroid drug treatment: Clinical significance of the T3 suppression test and abnormal thyroid stimulators. Folia Endocrinol. Jpn. 58, Kasagi, K., J. Konishi, K. Arai, T. Misaki, Y. Iida, K. Endo and T. Torizuka (1986). A sensitive and practical assay for thyroidstimulating antibodies using crude immunoglobulin fractions precipitated with polyethylene glycol. J. Clin. Endocrinol. Metab. 62, Konishi, J., Y. Iida, K. Endo, T. Misaki, Y. Nohara, N. Matsuura, T. Mori and K. Torizuka (1983). Inhibition of thyrotropin-induced adenosine 3', 5'-monophosphate increase by immunoglobulins from patients with primary myxedema. J. Clin. Endocrinol. Metab. 57, Kuzuya, N., S. C. Chiu, H. Ikeda, H. Uchimura, K. Ito and S. Nagataki (1979). Correlation between thyroid stimulators and 3, 5, 3'-triiodothyronine suppressibility in patients during treatment for hyperthyroidism with thionamide drugs: Comparison of assays by thyroidstimulating and thyrotropin-dispiacing activities. J. Clin. Endocrinol. Metab. 48, Orgiazzi, J., D. E. Williams, I. J. Chopra and D. H. Solomon (1976). Human thyroid adenyl cyclase-stimulating activity in immunoglobulin G of patients with Graves' disease. J. Clin. Endocrinol. Metab. 42, Shishiba, Y., Y. Ozawa, N. Ohtsuki and T. Shimizu (1982). Discrepancy between thyroidstimulating and thyrotropin-binding inhibitory activities of Graves' immunoglobulin Gs assessed in the mouse. J. Clin. Endocrinol. Metab. 54, Shishiba, Y., Y. Ozawa and T. Shimizu (1987). Highly sensitive immunoradiometric assay for TSH and thyroid radioiodine uptake as predictors of thyroid suppression test. Endocrinol. Japon. 34, Smyth, P. P. A., D. Neylan and D. K. O'Donovan (1983). Association of thyroid-stimulating immunoglobulins and thyrotropin-releasing hormone responsiveness in women with euthyroid goiter. J. Clin. Endocrinol. Metab. 57, Solomon, D. H., I. J. Chopra, U. Chopra and F. J. Smith (1977). Identification of subgroups of euthyroid Graves' ophthalmopathy. N. Eng. J. Med. 296, Spencer, C. A., A. O. Lai-Rosenfeld, R. B. Guttler, J. LoPresti, A. O. Marcus, A. Nimalasuriya, A. Eigen, R. C. Doss, B. J. Green and J. T. Nicoloff (1986). Thyrotropin secretion in thyrotoxic and thyroxine-treated patients: Assessment by a sensitive immunoenzymometric assay. J. Clin. Endocrinol. Metab. 63, Tamai, H., T. Nakagawa, N. Ohsako, O. Fukino, H. Takahashi, F. Matsuzuka, K. Kuma and S. Nagataki (1980). Changes in thyroid functions in patients with euthyroid Graves' disease. J. Clin. Endocrinol. Metab. 50, Teng, C. S., B. R. Smith, B. Clayton, D. C. Evered, F. Clark and R. Hall (1977). Thyroidstimulating immunoglobulins in ophthalmic Graves' disease. Clin. Endocrinol.(Oxf) 6, Utiger, R. D.(1986). Tests of the hypothalamic-

8 HORIMOTO et al. Endocrinol. Japon. December 1988 thyrotropic axis: Tests of thyroregulatory mechanism. In the Thyroid (eds. S. H. Ingbar and L. E. Braverman), pp J. B. Lippincott Co., Philadelphia. Weeks, I., M. Sturgess, K. Siddle, M. K. Jones and J. S. Woodhearl (1984). A high sensitivity immunochemiluminometric assay for human thyrotrophin. Clin. Endocrinol.(Oxf) 20, Werner, S. C., Classification of the eye changes of Graves' disease (1969). J. Clin. Endocrinol. Metab. 29, Zakarija, M., J. M. Mckenzie and K. Banovaq (1980). Clinical significance of assay of thyroid-stimulating antibody in Graves' disease. Ann. Intern. Med. 93,