Clinical and Experimental Medicine (2019) 19:245–254 https://doi.org/10.1007/s10238-018-00542-7 ORIGINAL ARTICLE Efect of antithyroid drugs on the occurrence of antibodies against type 2 deiodinase (DIO2), which are involved in hyperthyroid Graves’ disease infuencing the therapeutic efcacy Ildikó Molnár1 · József A. Szentmiklósi2 · Rudolf Gesztelyi2 · Éva Somogyiné‑Vári1 Received: 30 September 2018 / Accepted: 12 December 2018 / Published online: 4 January 2019 © Springer Nature Switzerland AG 2019 Abstract Graves’ disease is an organ-specifc autoimmune disease with hyperthyroidism, difuse goiter and autoantibodies against TSH receptor, thyroid peroxidase (TPO) and/or thyroglobulin (Tg). Graves’ hyperthyroidism is characterized by ­T3 domi- nance due to the conversion of ­T4 into ­T3 through type 1 and 2 deiodinase enzymes (DIO1, DIO2). Methimazole (MMI) and propylthiouracil (PTU) therapies inhibit thyroid hormone synthesis blocking the activity of deiodinase and TPO enzymes. The study investigated the occurrence of autoantibodies against DIO2 peptides (cys- and hom-peptides) with the efect of antithyroid drugs on their frequencies in 78 patients with Graves’ disease and 30 controls. In hyperthyroidism, the presence of DIO2 peptide antibodies was as follows: 20 and 11 cases out of 51 for cys- and hom-peptide antibodies, respectively, of whom 8 cases possessed antibodies against both peptides. Antithyroid drugs diferently infuenced their frequencies, which were greater in PTU than in MMI (3/6 vs 13/45 cases, P < 0.016 for cys- and 0/6 vs 2/45 cases for hom-peptide antibodies). Antibodies against both peptides demonstrated more reduced levels of anti-TPO (P < 0.003) and anti-Tg antibodies (P < 0.002) compared with those without peptide antibodies. PTU compared with MMI increased the levels of TSH receptor antibodies (32.5 UI/l vs 2.68 IU/l, P < 0.009). MMI treatment led to more reduced FT­ 3 levels and FT­ 3/FT4 ratios in hyperthyroid Graves’ ophthalmopathy (P < 0.028 for ­FT3, P < 0.007 for ­FT3/FT4 ratio). In conclusion, the presence of DIO2 peptide antibodies is connected to Graves’ hyperthyroidism infuencing the levels of antibodies against TPO, Tg and TSH receptor, as well as the therapeutic efcacy of antithyroid drugs. Keywords Antithyroid drugs · Hyperthyroidism · Graves’ disease · Antithyroid antibodies · Type 2 deiodinase Introduction Graves’ disease is a member of autoimmune thyroid disease (AITD) characterized by hyperthyroidism, difuse goiter and antibodies mainly against TSH receptor, thyroid peroxidase (TPO) and/or thyroglobulin (Tg) [1]. TSH receptor antibod- * Ildikó Molnár ies play a crucial role in the development of hyperthyroidism [email protected] [2]. The causes of antithyroid antibody productions are con- József A. Szentmiklósi sidered as a breaking of the central and peripheral tolerance [email protected] against thyroidal antigens due to genetic, environmental, stress Rudolf Gesztelyi or other unknown factors [3]. The increase in thyroid hormone [email protected] synthesis is associated with the onset of Graves’ disease show- Éva Somogyiné‑Vári ing ­T3 dominance [4]. Type 1 (DIO1) and type 2 (DIO2) deio- somve@t‑online.hu dinase enzymes convert ­T4 into ­T3, of those DIO1 plays the 1 Immunoendocrinology, EndoMed, Bem tér 18/C., main role in Graves’ hyperthyroidism [5]. DIO2 deiodinase is Debrecen 4026, Hungary a common enzyme in thyroid and eye muscle tissues highlight- 2 Department of Pharmacology and Pharmacotherapy, ing its potential role in the thyroid-associated ophthalmopathy University of Debrecen, POBox 12, Debrecen 4012, Hungary (later called Graves’ ophthalmopathy) [6]. Vol.:(0123456789)1 3 246 Clinical and Experimental Medicine (2019) 19:245–254 Antithyroid drugs, methimazole (MMI) and propylthi- DIO2 (GenBank AAD45494-1) aa sequences between ouracil (PTU), are the main, first-choice medicines for 124 and 152 containing the active center of enzyme: inhibition of thyroid hormone synthesis in Hungary. The 124leu-val-val-asn-phe-gly-ser-ala-thr-133cys-pro-pro-phe- antithyroid drugs bound to DIO and TPO enzymes blocked thr-ser-glu-leu-pro-ala-phe-arg144 for hom-peptide and 132 133 136 137 the conversion T 4 into T3 and lead to the inhibition of TPO thr- cyscys-pro-pro- thr- phe-ser-glu-leu-pro-ala- efect on iodination and the phenolic coupling of iodoty- phe-arg-lys-leu-val-glu-glu-phe-ser-ser152 for cys-peptide rosine residues (production of T 4) [7]. Antithyroid drugs, (replaced double cyscys at position 133 and aa reversed at particularly PTU, can induce antineutrophil cytoplasmic positions 136 and 137). Guinea pigs were immunized with antibodies (ANCAs), but very rarely associated with mani- cys-peptide to produce capture antibodies, which were tested fest vasculitis [8]. before with antibody titration using serial dilutions. The pro- In this study, the occurrence of antibodies directed to cedures of peptide synthesis and the guinea pig immuniza- DIO2 peptides was investigated in the presence and absence tion were described in detail in our previous paper [11]. of treatment with antithyroid drugs, as well as their thera- The 96-well plates (Dynatech Immunolon™, USA) were peutic efcacy on thyroid hormone levels. covered with 100 µl/well guinea pig antibodies against cys- peptide in dilution of 1:500 using carbonate bufer (pH Patients and methods 9.6) at 4 °C for overnight. The plates were washed 3 times with Tween PBS (phosphate-bufered saline, pH 7.4, 0.05% Patients Tween 20, Reanal, Hungary). Bovine serum albumin in 1% solution (BSA, SIGMA, USA) was applied for blocking One hundred and eight patients were investigated, of whom the nonspecifc binding at 37 °C for 1 h. When the plates 78 had Graves’ disease [median of age: 46.5 years and inter- had been washed three times, the DIO2 peptides (cys- or quartile range (IQR): 29–53.5 years, 20 males, 38 had oph- hom-peptides) were added to the plates in the concentration thalmopathy] and 30 controls (median age: 50.5 years and of 0.3 µg/100 µl/well at room temperature for 2 h. In the IQR: 35.75–56.25 years, 3 males). The diagnosis of Graves’ end, the plates were washed, and 100 µl/well patient sera disease was based on the presence of hyperthyroidism and in dilution of 1:100 were added to the plates at room tem- difuse goiter with or without eye symptoms. Radioscin- perature for 2 h. The binding reactions of antibodies against tigraphy with technetium99 and ultrasonography were used DIO2 peptides were detected with horseradish peroxidase for the exclusion of toxic adenoma, where hyperthyroidism conjugated with goat antihuman IgG in dilution of 1:5000 was associated with nodular goiter. Fifty-one patients were (SIGMA, USA) at room temperature for 1 h. Chromogenic hyperthyroid, 24 patients euthyroid and 3 patients hypothy- o-phenylenediamine (OPD) substrate (SIGMA, USA) was roid at the time of the study. The duration of Graves’ disease applied for the detection in 0.05 M citrate bufer (pH 5), was given in median: 7 months and IQR: 2–11 months. The and the reaction was stopped with 50 µl/well of 4 N sulfuric diagnosis of ophthalmopathy was based on NOSPECS with acid after 30 min. The optical densities (O.D.) of the wells ophthalmic control [9]. Soft tissue involvement and propto- were measured with ELISA Reader at 492 nm. All tests were sis were found together in the patients with ophthalmopa- performed in duplicates. O.D. values above the control mean thy (n = 37), of whom 18 patients had diplopia due to eye O.D. + 2 SD were regarded as positivity: 106.37 for hom- muscle enlargements. None of the patients showed corneal peptide antibodies and 106.67 for cys-peptide antibodies. ulceration or optic nerve damage. Sixteen patients had the The intra- and interassay coefcients were as follows: 4.11% clinical activity scores for ophthalmopathy, CAS ≤ 4 and 23 and 3.54% for hom-peptide antibodies and 5.9% and 3.8% cases above 4. The median of CAS was 5 and IQR: 4–7 [10]. for cys-peptide antibodies, respectively. Twenty-seven cases have not been treated yet; in 51 cases, the patients received antithyroid drugs (6 cases PTU and 45 cases MMI). Thirty healthy persons formed the control Measurement of thyroid hormone and antibody serum group. levels Methods Thyroid hormone (TSH, FT4, FT3) and antibody (against TPO, Tg) serum levels were measured with chemilumines- The measurement of antibodies against DIO2 peptides cence immunoassay in a fully automated method. The nor- mal ranges of values were the following: 0.3–3 mIU/l for Sandwich enzyme-linked immunosorbent assay (ELISA) TSH, 0.7–1.48 ngl/dl for FT4 and 1.45–3.48 pg/ml for FT3, was used for the detection of antibodies against two and 0–63 IU/ml for anti-TPO and 0–115 IU/ml for anti-Tg DIO2 peptides (cys- and hom-peptides). The amino acid antibodies. Autoantibodies against TSH receptor were meas- (aa) sequences of DIO2 peptides were corresponded to ured with competitive enzyme immunoassay using labeled 1 3 Clinical and Experimental Medicine (2019) 19:245–254 247 TSH (Medizym T.R.A., Medipan, Germany) in ELISA. The The frequency of antibodies against DIO2 peptides values above 1.5 IU/l were regarded as positivity. and their relationships with serum thyroid hormone and antibody levels in hyperthyroidism Statistics Cys-peptide antibodies were found in 18 cases out of 39 without and 15 cases out of 39 with ophthalmopathy, and The descriptive results of measured thyroid hormone 1 case out of 30 controls (Table 1). Hom-peptide antibod- and antibody serum levels were exhibited in arithmetic ies were found in 6 cases out of 39 without and 7 cases out mean ± SD. Data of age and duration of thyroid disease and of 39 with ophthalmopathy, and 1 case out of 30 controls.