*5.2.1 Thyroidal, skeletal and eye muscle DIO2 activities with respect to anti-TPO and TSH receptor autoantibodies in FT3 hyperthyroid Graves' Disease*

The effects of autoantibodies against thyroid peroxidase (TPO) and TSH receptor were investigated on thyroidal, skeletal and eye muscle DIO2 activities in FT3 hyperthyroid Graves' Disease. A greater number of patients with ophthalmopathy (n = 11) demonstrated anti-TPO antibodies than those without (n = 4) [42]. Anti-TPO antibody positive patients without ophthalmopathy exhibited 5 times greater DIO2 activities in thyroidal and skeletal muscle cytosol fractions, and even 12 times greater in eye muscle cytosol fraction compared to those with ophthalmopathy (**Figure 3**). DIO2 activities were compared between anti-TPO antibody positive and negative patients. The difference in all DIO2 activities were significant between the patients with and without ophthalmopathy in FT3 hyperthyroid Graves' Disease, as well as between anti-TPO antibody negative and positive patients. DIO2 activities increased 17 times in patients without ophthalmopathy, but decreased by 39% in patients with ophthalmopathy in the presence of anti-TPO antibodies compared to those who were negative for these autoantibodies (**Figure 4**). The alterations could be explained by the greater increased serum FT4 levels in anti-TPO antibody positive patients with Graves' ophthalmopathy in contrast to the patients without ophthalmopathy. The patients without ophthalmopathy showed reduced FT4 levels, which were below the normal range, concomitantly with the elevated serum TSH levels. These result are limited by the small patient number of Graves' Disease without ophthalmopathy.

In FT3 hyperthyroidism, TSH receptor antibody positivity was greater in Graves' ophthalmopathy (n = 11, and 9 out of 11 cases were anti-TPO antibody positive)

#### **Figure 3.**

*The effect of patient sera with Graves' Disease on thyroidal, skeletal and eye muscle DIO2 activities in anti-TPO antibody positive patients between with (GO) and without (G) ophthalmopathy. DIO2: Type 2 deiodinase; TPO: Thyroid peroxidase.*

#### **Figure 4.**

*The effect of patient sera with Graves' Disease on thyroidal, skeletal and eye muscle DIO2 activities between anti-TPO antibody negative and positive patients with (GO) and without (G) ophthalmopathy. DIO2: Type 2 deiodinase; TPO: Thyroid peroxidase.*

than in those who had no ophthalmopathy (n = 2) [42]. DIO2 activities were significantly increased in all cytosol fractions (increased by 3.6 times) for TSH receptor antibody positive patients compared to TSH receptor antibody negative patients with Graves' ophthalmopathy, but the opposite was true for patients without ophthalmopathy (**Figure 5**). Surprisingly, in the absence of ophthalmopathy, TSH receptor antibody positive patients demonstrated relevantly decreased DIO2 activities, which were 10 times lower than those found in TSH receptor antibody negative patients in all cytosol fractions, concomitantly with the increased serum TSH levels.

#### *5.2.2 The effects of IgG and IgM isotype antieye muscle cytosol and membrane autoantibodies on eye muscle DIO2 activity in Graves' ophthalmopathy*

Next, the effects of antieye muscle cytosol and membrane autoantibodies on eye muscle DIO2 activity were examined in Graves' ophthalmopathy. Before the study, the binding reactivity of sera to human eye muscle membrane and cytosol antigens in tissue sections was controlled. In our previous results using immunohistochemistry and immunoblotting methods, antibodies against TCSS peptide, corresponding to amino acid sequence of DIO2 and eye muscle cytosol or membrane antigens (supernatant or pellet fractions of 100 000 x g, separated by centrifugations) were demonstrated, which gave intensive binding reactions to human thyroid, skeletal and eye muscle tissue sections [40].

The binding of guinea pig sera immunized by TCSS peptide could be inhibited by patient sera added in advance, which sera gave positive reactions to eye muscle tissue sections. Toyoda and coworkers investigated the DIO1 enzyme activity in FRTL-5 rat thyroid cells in the presence of IgG type immunoglobulins derived from untreated hyperthyroid Graves' patients and controls [45]. They demonstrated a relevant increased DIO1 activity, which could be completely abolished by the addition of cycloheximide.

Based on the previously mentioned results we wanted to measure the effects of antieye muscle cytosol and membrane autoantibodies on eye muscle DIO2 activity, as well as to compare DIO2 activity to eye muscle enlargements. The hypothesis was, that antieye muscle autoantibodies may affect DIO2 activity, which can lead to eye muscle enlargement. We investigated the role of antieye muscle antibodies in Graves' Disease [46]. In turn, the appearance of these autoantibodies was not

#### **Figure 5.**

*The effect of patient sera with Graves' Disease on thyroidal, skeletal and eye muscle DIO2 activities between TSH receptor antibody negative and positive patients with (GO) and without (G) ophthalmopathy. DIO2: Type 2 deiodinase.*

only connected to ophthalmopathy, but they could also be found in a small part of patients without ophthalmopathy. IgG, IgA and IgM isotype antieye muscle membrane and cytosol autoantibodies were measured with enzyme-linked immunosorbent assay (ELISA) in 32 patients with hyperthyroid Graves' Disease, of whom 20 cases had ophthalmopathy. None of IgA isotype autoantibodies could be detected against membrane and cytosol antigens in any of the patients. A greater number of patients with ophthalmopathy demonstrated IgM (n = 10) and IgG (n = 5) antieye muscle autoantibodies than those without ophthalmopathy, of whom 3 cases had IgG type and 3 cases had IgM type autoantibodies. Surprisingly, the addition of serum containing IgG isotype antieye membrane (EyeM) or cytosol (EyeC) autoantibodies resulted in 6.4 times or 3.9 times increased eye muscle DIO2 activity, respectively compared to those found with IgG negative sera (**Figure 6**). Conversely, the effect of IgM type antieye muscle membrane or cytosol autoantibodies was associated with 3 times or 1.9 times lower eye muscle DIO2 activity, respectively. The presence of IgG type anti-EyeC autoantibodies resulted in 1.5 times greater eye muscle DIO2 activity than anti-EyeM autoantibodies. A similar increase in DIO2 activity could be demonstrated in the presence of IgM type anti-EyeC autoantibodies compared to those with anti-EyeM autoantibodies. In this instance, the increase in eye muscle DIO2 activitiy was 2 times greater. Furthermore, the increase in eye muscle DIO2 activity was 7 times and 5 times higher in the presence of IgG type anti-EyeM and anti-EyeC autoantibodies compared to those in the presence of IgM type anti-EyeM and anti-EyeC autoantibodies, respectively. Eye muscle DIO2 activities strongly correlated with IgG type anti-EyeM and anti-EyeC autoantibody levels. It seems, the autoantibody binding to eye membrane could mediate a signal towards the cytosolic DIO2 enzyme. The findings between eye muscle DIO2 activity and eye muscle enlargement suggest this idea. IgG type anti-EyeM autoantibodies were associated with increased eye muscle enlargement, although the difference was not significant. However, IgM type anti EyeM autoantibodies were associated with a significant decrease in eye muscle enlargement. The fact that IgM type anti-EyeM autoantibodies could play a role in DIO3 activity, could not be excluded. The eye muscle DIO2 activity was more greater in patients with the absence of ophthalmopathy compared to those in the presence of that. Our results suggest that autoantibodies against eye muscle

#### **Figure 6.**

*The effect of patient sera containing IgG and IgM type antieye muscle membrane (EyeM) and cytosol (EyeC) autoantibodies on eye muscle DIO2 activity in Graves' ophthalmopathy. DIO2: Type 2 deiodinase.*

antigens have a role in the development of ophthalmopathy through the eye muscle enlargements. The limitation of this study was the small patient number containing IgM isotype anti-EyeM and IgG isotype anti-EyeC antibodies. Another limitation could be the measurement method of the eye muscle enlargements, which was done using ultrasound in the absence of CT or MRI possibilities.

#### **5.3 The effect of antithyroid drugs on tissue-specific DIO2 activities, as well as their role in the induction of autoantibodies against DIO2 peptides**

#### *5.3.1 The effect of antithyroid drugs on thyroidal, skeletal and eye muscle DIO2 activities*

Antithyroid drugs (ATD) are used very often in the therapy of Graves' hyperthyroidism. Methimazole (MMI) and propylthiouracil (PTU) are the medicines used to block the synthesis of thyroid hormones in Hungary. ATDs are thioamide derivates with the binding reaction to DIO1 enzyme forming an intermediary selenyl-iodide-DIO1 enzyme complex (presumably the same is true for DIO2 also). In addition, they inhibit the activity of TPO enzyme due to the impairment of H2O2 generation and the coupling of iodotyrosines. MMI may be a selective DIO1 blocker and inhibits thyroidal H2O2 generation. However, MMI has no remarkable effect on DIO2 activity. PTU is a very strong inhibitor for DIO1 activity. None of the patients were treated with PTU in the tissue-specific DIO2 activity study.

The difference in thyroidal DIO2 activities was significant between those with and without ophthalmopathy in FT3 hyperthyroidism who did not undergo MMI therapy [42]. MMI therapy was associated with a greater increase in thyroidal, skeletal and eye muscle DIO2 activities in both patients without and with ophthalmopathy (the increase was 17 times and 4 times higher, respectively) compared to the increases in patients who were not treated with MMI. MMI therapy was associated with greater TSH levels and greater ratio of FT3 to FT4 in patients without ophthalmopathy, and greater TSH receptor antibody levels in patients with ophthalmopathy (**Figure 7**).

*Deiodinase Enzymes and Their Activities in Graves' Hyperthyroidism DOI: http://dx.doi.org/10.5772/intechopen.97007*

#### **Figure 7.**

*The effect of patient sera who were treated with methimazole (MMI) on thyroidal, skeletal and eye muscle DIO2 activities in hyperthyroid Graves' Disease with and without ophthalmopathy. DIO2: Type 2 deiodinase.*

#### *5.3.2 Occurrence of antibodies against peptides corresponding to amino acid sequence of DIO2 during antithyroid drug therapy and the efficacy of therapy*

In another study, the occurrence of autoantibodies against DIO2 peptides, such as TCSS (cyspeptide) and LVFR (hompeptide) peptides were investigated in 78 patients with hyperthyroid Graves' Disease [47]. The relationships were examinated among ATD therapies, antibodies against TPO, thyroglobulin (Tg) and TSH receptor, as well as thyroid hormone levels. The appearance of autoantibodies against cys-, hompeptide or both peptides could be detected in 24, 4 or 9 cases, respectively, in Graves' Disease. The appearance of these autoantibodies was not associated with the clinical signs of urticaria or ANCA-associated vasculitis. These anticys- and antihompeptide antibodies could be demonstrated in 12 and 3 cases in hyperthyroidism, and in 10 and 1 cases in euthyroidism. A significant difference was found in the occurrence of anticyspeptide antibodies between PTU (n = 3 out of 3 cases) and MMI (n = 13 out of 42 cases) therapies. The frequency of antipeptide antibodies was smaller in Graves' ophthalmopathy (9 cases for anticyspeptide antibodies and 1 for antihompeptide antibodies). The exact mechanism is not clear, but ATDs are thioamide drugs with the binding feature to DIO and TPO enzymes blocking the T4 conversion to T3, and the iodination with the phenolic coupling of iodothyrosine residues. Their higher binding features are connected to their greater reactivity with free radicals. Not only the asymptomatic occurrence of autoantibodies against cys- and/or hompeptide was surprising in hyperthyroid Graves' Disease, but also their strong relationship with decreasing anti-TPO and increasing TSH receptor antibody levels (**Figure 8**). In hyperthyroidism, two antipeptide antibodies possessed distinct features with relation to the occurrence of anti-TPO, anti-Tg and TSH receptor antibody levels, as well as to the thyroid hormone levels and the ratio of FT3 to FT4. Antibodies against cyspeptide were rather stimulating: Positive correlation could be demonstrated between anticyspeptide antibodies and serum FT4 levels; the ratio of FT3 to FT4 was increased when those antibodies were present compared to when they were absent. In Graves' ophthalmopathy, the serum FT4 and FT3 levels were lower in the presence of antibodies against cyspeptide compared to when those antibodies were absent. The ratio of FT3 to FT4 was increased in patients without ophthalmopathy compared to those when it was present (**Figure 9**). Antibodies against hompeptide and both peptides were rather inhibiting: anti-TPO and anti-Htg antibodies levels were reduced in their presences compared to when they were absent. In hyperthyroid Graves' ophthalmopathy, antibodies against both peptides were associated with reduced antibody levels against TPO and Tg, but with increased TSH receptor antibody levels, particularly when the clinical activity score (CAS) was above 4. In FT4 hyperthyroidism, MMI treated Graves' patients without ophthalmopathy, demonstrated significantly increased FT3 to FT4 ratio with the occurrence of anticyspeptide autoantibodies.

#### **Figure 8.**

*The effect of patient sera containing autoantibodies against peptides (hom – and/or cyspeptide) corresponding to amino acid sequence of DIO2 on the levels of anti-TPO and anti-Tg autoantibodies, as well as on the ratio of FT3 to FT4 in hyperthyroid Graves' Disease. DIO2: Type 2 deiodinase; TPO: Thyroid peroxidase; Tg: Thyroglobulin.*

#### **Figure 9.**

*The effect of patient sera containing autoantibodies against cyspeptide corresponding to amino acid sequence of DIO2 on serum FT4 and FT3 levels, as well as on the ratio of FT3 to FT4 in hyperthyroid Graves' ophthalmopathy and between the presence and absence of ophthalmopathy. DIO2: Type 2 deiodinase.*

*Deiodinase Enzymes and Their Activities in Graves' Hyperthyroidism DOI: http://dx.doi.org/10.5772/intechopen.97007*

#### **Figure 10.**

*The effect of patient sera containing autoantibodies against peptides (hom- and/or cyspeptide) corresponding to DIO2 amino acid sequence on serum FT4 and TSH receptor antibody levels, as well as on the ratio of FT3 to FT4 in hyperthyroid Graves' Disease treated with methimazole (MMI) and propylthiouracil (PTU). DIO2: Type 2 deiodinase.*

The results in thyroid hormone levels supported that the presence of antipeptide antibodies before the treatment and their absence during the treatment with MMI may contribute to the worsening of orbital processes in hyperthyroid Graves' ophthalmopathy. Antibodies against DIO2 peptides may influence the therapeutic efficacy during the treatment (**Figure 10**). In MMI treatment, the presence of antibodies against hompeptide only was connected to increased serum FT4 levels, but when autoantibodies against both hom- and cyspeptide were present, it resulted in a relevant decrease in TSH receptor antibody levels. MMI treatment demonstrated lower TSH receptor antibody levels and lower ratio of FT3 to FT4 in the appearance of anticyspeptide autoantibodies compared to those treated with PTU. The exact role of antipeptide antibodies and their relationship with antithyroid autoantibodies, as well as the possibility of the occurrence of autoantibodies against other amino acid sequence of the whole DIO2 protein need futher investigations.

#### **6. Conclusions**

In hyperthyroid Graves' Disease the thyroid hormone excess is dominantly T3. The thyroidal production of T3 and T4 excess can derive from the thyroidal T4 and T3 formation in the colloid-embedded Tg mediated by thyroidal TPO, and the additional production of T3 due to deiodinase enzymes mediated conversion from T4 resulting in the ratio of 3 to 1 for DIO1 and DIO2 activities in the cytosol, respectively. The results using *in vitro* model for the study of tissue-specific DIO2 activities confirmed the dominance of thyroidal DIO1 activity, but the thyroidal DIO2 activity seemed to be more reduced compared to skeletal and eye muscle DIO2 activities. The degree of DIO2 activities was tissue-specific, but the extent of their decreases in hyperthyroidism and increases in hypothyroidism was identical. The findings highlighted that the increase in tissue-specific DIO2 activity needed an active protein synthesis only in FT3, but not in FT4 hypothyroidism. The appearance of anti-TPO, TSH receptor, and antieye muscle membrane and cytosol autoantibodies modified the tissue-specific DIO2 activities, which manifested in both

increased and decreased serum TSH levels and sometimes in eye muscle enlargements. Besides the effect of ATDs on tissue-specific DIO2 activities, autoantibodies against peptides corresponding to amino acid sequences of DIO2 also appeared asymptomatically in Graves' Disease. Furthermore, they were also detectable before ATD therapy, and the therapy increased their occurrences. The antipeptide autoantibodies were associated with alterations in serum FT4 and FT3 levels, as well as in the levels of autoantibodies against TPO, Tg and TSH receptor. In Graves' ophthalmopathy, the tissue-specific DIO2 activities were much more reduced and they were connected to a lack of appearance of antipeptide autoantibodies. The occurrence of anticyspeptide autoantibodies was associated with lower serum FT4 and FT3 levels compared to those in patients who were negative for these autoantibodies. Although, autoantibodies could be demonstrated against eye muscle cytosol antigens, which inhibited the binding of antipeptide antibodies derived from guinea pig immunization to eye muscle in immunohistochemical studies, these antieye muscle and antipeptide autoantibodies had no pathognomonic role in Graves' ophthalmopathy. These findings above explain why the duality of features causes a greater complexity of hyperthyroidism in Graves' Disease.
