Original Research Article

Transcription

1 IUBMB Life, 51: , 2001 Copyright c 2001 IUBMB /01 $ Original Research Article Antioxidants in the Treatment of Graves Disease Liliana N. Guerra, 2;3 Silvia Moiguer, 1 Mirta Karner, 1 Ma del Carmen R õ os de Molina, 3 Claudia M. Sreider, 2;4 and José A. Burdman 1;2;4 1 Endocrinology Unit, Hospital Israelita Ezrah, Buenos Aires, Argentina 2 Department of Endocrinology, undacion Centro de Investigaciones Medicas Albert Einstein, Buenos Aires, Argentina 3 Department of Biological Chemistry, aculty of Exacts and Natural Science, University of Buenos Aires, Buenos Aires, Argentina 4 Medical Research Center, Universidad Abierta Interamericana; Luis Viale 2831, (1416) Buenos Aires, Argentina Summary An antioxidant mixture (LAROTABE) was evaluated in the treatment of Graves disease. ifty-six hyperthyroid patients were treated with methimazol (MMI) (A), LAROTABE (B), or MMI plus LAROTABE (C). According to a clinical score, improvement was obtained at 8 weeks in A and 4 weeks in B and C. Group A diminished their thyroid hormone concentration to normal levels, while patients with LAROTABE did not reduce T 3 and T 4 unless MMI was introduced. Hyperthyroid patients had increased malondialdehyde (MDA) content and SOD activity and decreased catalase activity compared to controls. Within group A, MDA decreased to control values while SOD was reduced 38.3% and catalase increased 21.6%. Similar results were obtained for MDA and for both enzymes after treatment with LAROTABE. Signs and symptoms of Graves disease might be related to an increase in free radicals; antioxidants could be a new therapeutic tool to improve the clinical manifestation of this illness. IUBMB Life, 51: , 2001 Keywords Hyperthyroidism; antioxidants; Graves disease treatment. INTRODUCTION Graves disease is characterised by elevated levels of T3 and T4 and low levels of TSH hormones. One of the major effects of thyroid hormones (T3, T4) is the increase in mitochondrial respiration, producing an hypermetabolic state with generation of free radicals (1). Although studies in human are scarce, in the last years, metabolic oxidation has been postulated as the origin of signs and symptoms of hyperthyroidism (2). Received 26 January 2001; accepted 19 March Address correspondence to José A. Burdman, Luis Viale 2831, (1416) Buenos Aires, Argentina. ax: (5411) In the synthesis of thyroid hormones, oxidation of thyroidal iodide is mediated by thyroid peroxidase while the mitochondrial system provides a source of hydrogen peroxide. Coupling reactions between the inactive iodotyrosines MIT and DIT are also catalysed by the peroxidase through a mechanism involving a radical-mediated reaction (3). Tissue damage in hyperthyroidism might be mediated by an increase in free radicals. Muscular damage induced by free radicals was described in skeletal and cardiac muscle of hyperthyroid rats by several investigators (1, 3, 4). An increase in superoxide dismutase (SOD) was described in hyperthyroid rats (1). Under physiological conditions, reactive oxygen species (ROS) are generated during normal metabolic events. The damage produced by ROS results of an imbalance between these toxic molecules and the antioxidant capacity (5, 6). Protection involves superoxide dismutase, catalase, and glutathione peroxidase as well as other antioxidant compounds such as vitamin C and vitamin E. An increase in the antioxidant capacity would reduce the damage produced by free radicals. We decided to evaluate the effect of treatment with antioxidants alone or in combination with the antithyroid agent methimazole (MMI) on the clinical and biochemical progression of human hyperthyroidism. EXPERIMENTAL PROCEDURES Patients and Drugs. We studied 56 hyperthyroid patients (Graves Basedow), (22- to 66-year-old), 43 females and 13 males, all of them Caucasian. The antioxidant mixture was composed of: vitamin E 200 mg, -carotene 3 mg, vitamin C 250 mg, Cu 1 mg, Zn 7.5 mg, Mn 1.5 mg, Se 15 ¹g (LAROTABE, ROCHE). Methimazol (MMI) 105

2 106 GUERRA ET AL. Table 1 Effect of treatment on signs and symptoms of hyperthyroidism Before treatment Group A Group B Group C n D 56 n D 23 n D 20 n D 13 4 weeks 8 weeks Nervousness, insomnia Diarrhea Hotness, sweat Weight loss Tremor The results are the percentage of patients presenting the sign or symptom after different treatments. Period of treatment: Group A D MMI 4 or 8 weeks. Group B D Larotabe 4 weeks. Group C D Larotabe C MMI 4 weeks. was from Gador S.A. Levothyroxine sodium tablets were from Quimica Montpellier S.A. The Hospital Ethical Committee has approved these studies. Biochemical Determinations. Hormone concentration (T3, T4, TSH) in sera were determined by R.I.A. using a Diagnostic Products Corporation Kit (DPC). Enzyme activities were investigated in 17 patients. Red blood cells from 2.5 ml blood samples per extraction devoid of leukocytes, were washed and hemolyzed. Supernatants of chloroform/ethanol extracts, devoid of haemoglobin, were assayed in: A) catalase using H 2 O 2 as substrate, absorbance was determined at 240 nm, extinction coef cient D M 1 cm 1 (7), and B) Cu Zn superoxide dismutase (SOD) measuring the ability of red blood cells to inhibit the autooxidation of epinephrine at ph 10.2, 30 ± C (8). One unit of SOD activity was de ned as 50% inhibition of epinephrine oxidation rate. The increase in absorbance at 480 nm was U/min with no added SOD, and 50% inhibition was achieved by 46 ng/ml of bovine SOD (grade II, Sigma, 3.5 U/¹g protein). The concentration of malondialdehyde (MDA) reacting with thiobarbituric acid in the sera of patients was determined using an extinction coef cient of cm 1 M 1 (9). Protein was determined by the method of Bradford et al. (10), with crystalline bovine serum albumin as standard. Normal controls (euthyroid subjects) of similar age and sex were also studied. Clinical Score Diagnosis. A clinical score was established according to the most common symptoms and signs of hyperthyroidism: A) nervousness, insomnia; B) hotness, sweat; C) weight loss, diarrhea; D) tachycardia, and E) tremor. One of the symptoms or signs present in each item was enough to score 1 point. We selected patients with a clinical score of 5. Inclusion of patients in different treatment groups was at random. Treatment. The patients were divided into 3 groups: A) treated with MMI for 8 weeks: 23 patients (18 females and 5 males, years), 4 with exophthalmos and 4 with gioter; B) treated with LAROTABE for 4 weeks: 20 patients (16 females and 4 males, years), 4 with exophthalmos and 4 with goiter; C) treated with MMI plus LAROTABE for 4 weeks: 13 patients (9 females and 4 males, years), 3 with exophthalmos and 6 with goiter; Daily doses: MMI 50 mg/ patient, LAROTABE 2 capsules/patient, one at noon and the other at 9 PM. Statistics. Results are means SEM. Statistical analysis was performed by 1-way analysis of variance (11). RESULTS Clinical Parameters A double-blind approach was carried out to perform this study. Neither the patient nor the observer knew about the patient treatment during the rst 4 weeks. The same physician always determined each patient score. As some signs and symptoms are dif cult to quantify, we decided to evaluate only their presence or absence (Table 1). As shown in Table 2, patients of group A (MMI) diminished the clinical score to 0 2 only after 8 weeks. Meanwhile, groups B (LAROTABE) and C (MMI and LAROTABE) showed a signi cant improvement (P < 0.05) of the score to 0 1 after 4 weeks. The heart rate decreased signi cantly in all groups from pulsation/min before treatment to pulsation/min at 8 weeks for group A and at 4 weeks for Table 2 Effect of treatment on the clinical score in hyperthyroidism 4 weeks treatment 8 weeks treatment clinical score clinical score Group A (n D 23) Group B (n D 20) Group C (n D 13) 0 1 ND All patients had clinical score 5 before treatment. Patients were treated with LAROTABE C MMI after week 4. ND: not determined.

3 ANTIOXIDANTS AND GRAVES DISEASE 107 Table 3 Effect of treatment on thyroid hormones levels in sera of hyperthyroid patients Before treatment 4 weeks treatment 8 weeks treatment T3 T4 TSH T3 T4 TSH T3 T4 TSH nmol/l nmol/l UU/ml Nmol/l nmol/l ¹U/ml nmol/l nmol/l ¹U/ml Group A Group B a a a Group C The results are expressed as: MEANS SEM. a Patients were treated with LAROTABE C MMI after week 4. Normal levels: T3: nmol/l; T4: nmol/l; TSH: up to 5 ¹U/ml. P < 0:05 vs. values before treatment. P < 0:05 vs. values of 4 weeks treatment group A. groups B ( pulsation/min) and C ( pulsation/min) (P < 0.01). Patients with exophthalmos and goiter showed no differences from the rest of the patients, regarding the parameters measured. Biochemical Parameters All patients had high levels of T3 and T4, and low serum levels of TSH before treatment (Table 3). We did not determine TSI. After 4 weeks treatment of group A patients (MMI), the thyroid hormones concentration in sera remained elevated. Only after 8 weeks of treatment did all patients in this group diminish their thyroid hormones concentration to normal levels (Table 3). Group A patients continued treatment with MMI for mo. There were 2 recurrences during this period. Patients treated only with LAROTABE for 4 weeks (group B) showed their serum thyroid hormones levels similar to those found in the patients before treatment. After these 4 weeks, MMI was added to the treatment with LAROTABE, and 4 weeks later thyroid hormonal concentration was signi cantly reduced (Table 3). No recurrences were observed after a follow-up of 24 mo. After 4 weeks of a combined treatment with MMI and LAROTABE from the beginning (group C), thyroid hormone levels in sera decreased to normal levels. Within this group, 4 patients had TSH levels above normal. After 8 weeks, thyroid hormones were below normal in all patients except 3, while TSH was elevated in all of them. rom week 9 and for 24 mo, 11 patients received MMI 10 mg/day together with a daily dose of LAROTABE (2 capsules) and levothyroxine ( ¹g). igure 1. MDA Content. Control: euthyroidism; Before treatment: hyperthyroidism. Group A: MMI, 8 weeks; Group B: LAROTABE, 4 weeks. Malondialdehyde is expressed in mmol MDA/mg protein. Experimental conditions were as described in Experimental Procedures. P < 0.05 vs. values before treatment, P < 0.01 vs. values before treatment.

4 108 GUERRA ET AL. igure 2. SOD Activity. Control: euthyroidism; Before treatment: hyperthyroidism. Group A: MMI, 8 weeks; Group B: LAROTABE, 4 weeks. Superoxide dismutase activity is expressed in enzyme units/mg protein. Experimental conditions were as described in Experimental Procedures. P < 0.05 vs. values before treatment, P < 0.01 vs. values before treatment. All these patients are examined periodically, and so far there are no recurrences. Treatment with MMI normalised TSH levels; combined treatment with LAROTABE produced an increase above normal values (Table 3). MDA (ig. 1) was signi cantly higher in hyperthyroidism than in controls (hyperthyroidism mmol MDA/ mg protein vs. controls mmol MDA/mg protein, P < 0.05) and decreased to values not signi cantly different from controls after MMI administration for 8 weeks. Superoxide dismutase activity (ig. 2) in hyperthyroid patients was signi cantly higher than in controls (hyperthyroidism U/mg protein versus controls U/mg protein, P < 0.05). On the other hand, catalase activity (ig. 3) was lower (hyperthyroidism pmol enzyme/mg protein vs. control pmol enzyme/mg protein, P < 0.005). After MMI treatment (8 weeks), superoxide dismutase activity diminished 38.3% and catalase activity increased 21.6%. Similar results were obtained for both enzyme activities and MDA content when hyperthyroid patients were treated with the antioxidant mixture for 4 weeks (igs. 1, 2, 3). DISCUSSION When hyperthyroid patients were treated with MMI, they became euthyroids clinically and biochemically after approximately 8 weeks. Antioxidant enzymes normalised their values, and plasma MDA decreased to similar levels of euthyroid controls. When hyperthyroid patients were treated with antioxidants alone (4 weeks), a signi cant decrease of the clinical score was observed, and the activities of antioxidant enzymes improved and MDA values returned to normal. However, hormonal igure 3. Catalase Activity. Control: euthyroidism; Before treatment: hyperthyroidism. Group A: MMI, 8 weeks; Group B: LAROTABE, 4 weeks. Catalase activity is expressed in pmol enzyme/mg protein. Experimental conditions were as described in Experimental Procedures. P < 0.05 vs. values before treatment.

5 ANTIOXIDANTS AND GRAVES DISEASE 109 concentrations remained high. Combined treatment with MMI and the antioxidant mixture shortened the period of normalisation of the clinical score as well as the normalisation of hormonal concentrations from approximately 8 to 4 weeks when compared with the administration of MMI alone. According to these results, treatment of hyperthyroidism with antioxidants rapidly improves the patients clinically even with high concentrations of thyroid hormones. It also appears to potentiate the effects of the antithyroid MMI on the synthesis of thyroid hormones. We postulate that elevated thyroid hormones produce signs and symptoms of hyperthyroidism through an increase in free radicals. It has been shown that free radicals are increased in Graves disease (12). When this increase is neutralised with antioxidants, the patients improve clinically, although this treatment has no effect on the production of thyroid hormones. The increased activity of the antioxidant enzyme SOD 1 probably re ects a defence mechanism, trying to preserve the organism from the tissue damage produced by free radicals. According to the clinical course of hyperthyroidism, it is clear that the defence mechanism set up in these patients is not enough to improve the progression of this illness. We have no explanation about the increased effect of MMI when administered together with the antioxidant mixture. Antioxidants could interfere with the action of the peroxidase in one or both reactions, iodide oxidation and/or coupling reaction. MMI acts by inhibiting the peroxidase; in this way a synergetic result could be obtained. However, it does not account for the lack of effect the antioxidant mixture had on the levels of thyroid hormones when administered alone. We can only speculate on the possibility of a dose too low to inhibit thyroidal oxidations and hormonal synthesis by itself. Antioxidants appear to be an effective coadjutant treatment of hyperthyroidism because it induces a rapid clinical improvement evident as soon as 4 weeks after its administration. In erythrocytes of hyperthyroid rats, there was an increase in SOD activity, and the oxidative stress diminished after administration of vitamin E (13). Chronic administration of the antioxidant dibunol diminished the activity of thyroid hormones in rat (14). Thyroid hormones induced modi cations in the electrical activity of the rat heart through an increase of free radicals; these changes were neutralised by the administration of vitamin E (15). The increment of free radicals in heart and soleus of the rat produced by the oxidative stress of hyperthyroidism was also inhibited by vitamin E (16). Antioxidant treatment might be useful in those hyperthyroid patients with a need for rapid clinical improvement such as danger of thyrotoxic crisis, pregnancy, cardiopathy, and MMI intolerance. It might be also an indication before a therapeutic dose of I131 administration or surgery. Clearly, antioxidant therapy does not replace conventional treatment of hyperthyroidism with antithyroid agents such as MMI or I131. Nevertheless, it appears as a new therapeutic tool to improve the clinical manifestation of this illness. ACKNOWLEDGEMENTS These studies were supported by grants from CONICET (Consejo Nacional de Investigaciones Cienti cas y Tecnicas), from Agencia Nacional de Promocion Cienti ca y Tecnologica PICT No , from University of Buenos Aires and by Roche S.A. Jose A. Burdman, Liliana N. Guerra, and M.C. R õ os de Molina are Career Scienti c Researchers in CONICET. The Hospital Ethical Committee has approved these studies. REERENCES 1. Mano, T., Shinohara, R., Sawai, Y., Oda, N., Nishida, T., Mokuno, T., Kotake, M., Hamada, M., Masunaga, R., Nakai, A., and Nagasaka, A. (1995) Effects of thyroid hormone on coenzyme Q and other free radical scavengers in rat heart muscle. J. Endocrinol. 145, Venditti, P., Balestrieri, M., Di Meo, S., and De Leo, T. (1997) Effect of thyroid state on lipid peroxidation, antioxidant defences, and susceptibility to oxidative stress in rat tissues. J. Endocrinol. 155, Doerge D. R., Taurog, A., and Dorris, M. (1994) Evidence for a radical mechanism in peroxidase-catalyse d coupling I. Steady-state experiments with various peroxidases. Arch. Biochem. Biophys. 315, Asayama, K., Dobashi, K., Hayashibe, H., Megata, Y., and Kato, K. (1987) Lipid peroxidation and free radical scavengers in thyroid disfunction in the rat: a possible mechanism of injury to heart and skeletal muscle in hyperthiroidism. Endocrinology 121, Bustamante, J., Guerra, L. N., Bredeston, L, Mordoh, J., and Boveris, A. (1991) Melanin content and hydroperoxid e metabolism in human melanoma cells. Exp. Cell Res. 196, Vilas, G. L., Aldonatti, C., San Martin de Viale, L. C., and Rios de Molina, M. C. (1999) Effect of alpha lipoic acid amide on hexaclorobenzen e porphyria. Biochem. Mol. Biol. Int. 47, Rice-Evans, C. A., Diplock, A. T., and Symons, M. (1991) Techniquesin free radical research. In Laboratory Techiniques in Biochemistry and Molecular Biology (Burdon, R. H. and van Knippengberg P. H., eds.). Volume 22. pp , Elsevier, New York. 8. Misra, H., and ridovich, Y. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, Buege, J. A., and Aust, S. D. (1978) Microsomal lipid peroxidation. In Methods in Enzymology (leisher, E. and Packer, J., eds.). Vol. 52, pp , Academic Press, New York. 10. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, Glantz, S. A. (1992). Primer of Biostatistic: The Program. McGraw Hill, New York. 12. Mano, T., Shinohara, R., Iwase, K., Kotake, M., Kamada, M., Uchimuro, K., Hayakawa, H., Hayashi, R., Nakai, A., Ishizuki, Y., and Nagasaka, A. (1997) Changes in free radical scavengers and lipid peroxide in thyroid glands of various thyroid disorders. Horm. Metab. Res. 29(7), Seven, A., Seymen, O., Hatemi, S., Hatemi, H., Yigit, G., and Candan G. (1996) Antioxidant status in experimental hyperthyroidism: effect of vitamin E supplementation. Clin. Chim. Acta 256, Gorban, E. N. (1991) Effect of the antioxidant dibunol on the hormonal status of adults and old rats. Probl. Endokrinol. 37, Venditti, P., Leo, T., and Meo, S. (1997) Vitamin E administration attenuates the tri-iodothyronine-induce d modi cation of heart electrical activity in the rat. J. Exp. Biol. 200, Asayama, K., Dobashi, K., Hayashibe, H., and Kato, K. (1989) Vitamin E protect against thyroxine-induced acceleration of lipid peroxidation in cardiac and skeletal muscles in rat. J. Nutr. Sci. Vitam., 35,