Objective: Previous studies in patients with Graves disease (GD) and Graves ophtalmopathy (GO) have focused mainly on the levels of TSH-receptor antibodies (TRAb). Our aim was to investigate the levels of TRAb, thyroid peroxidase (TPOAb) and thyroglobulin antibodies (TGAb) in patients with GD with and without GO.
Materials and Methods: 98 patients with GD were included in this retrospective study-76 women and 22 men. Thirty-nine patients had manifested GO - 28 women, 11 men. The serum levels of thyroid stimulating hormone (TSH) and free thyroxine (fT4) were measured by a chemiluminescence assay; TPOAb and TGAb - by an electrochemiluminescence method, and TRAb - by an enzymatic-substrate method-ELISA.
Results: Median serum levels of TSH and fT4 were 0.079 IU/l and 37.7 pmol/l in patients with GD + GO versus 0.420 IU/l and 23.2 pmol/l in patients with GD without GO (p=0.04 for TSH and p=0.02 for fT4). In GD + GO, we found higher levels of TRAb (22.1 versus 10.4 IU/L, p<0.001) and TGAb (412.5 vs. 190.6 IU/l, p<0.001), and lower levels of TPOAb (390.6 vs. 690.4 IU/l, p=0.001) than in GD alone.
Conclusion: Higher levels of TGAb and TRAb, and lower levels of TPOAb are found in patients with GD with GO compared to patients without ophtalmopathy. These findings might open new perspectives in studying the pathogenesis of GO. Turk Jem 2010; 14: 50 3
Key words: Graves disease, graves ophtalmopathy, thyroid antibodies
Amaç: Graves hastalığı (GH) ve Graves oftalmopati (GO) hastaları üzerinde daha önce yapılmış olan çalışmalar, genel olarak TSH-reseptör antikoru (TRAb) düzeylerine odaklanmıştır. Bu çalışmanın amacı GH ve GO hastalarının TRAb, tiroid peroksidaz (TPOAb) ve tiroglobulin (TGAb) düzeylerini araştırmaktır.
Gereç ve Yöntemler: GH tanısıyla izlenen 98 hasta, (76 kadın, 22 erkek) bu resrospektif çalışmaya dahil edildi. 39 hastada (28 kadın, 11 erkek) GO görüldü. Tiroid stimulan hormon (TSH) ve serbest tiroksin (FT4) düzeyleri kemiluminesan yöntemi, TPOAb ve TGAb düzeyleri elektrokemiluminesan yöntemi ile, TRAb düzeyi ise enzim-substrat metodu-ELISA ile ölçüldü.
Bulgular: GH+GO hastalarında medyan serum TSH ve FT4 düzeyleri sırasıyla 0.079 IU/I ve 37,7 pmol/I iken GO bulunmayan GH hastalarında TSH ve FT4 düzeylerinin sırasıyla 0,420IU/I ve 23,2 pmol/I olduğu görülmüştür (TSH için p=0,04, FT4 için p=0,02). GH+GO hastalarının TRAb ve TGAb düzeyleri yalnızca GH mevcut hastalara göre daha yüksek iken (sırasıyla 22,1 e karşın 10,4IU/L, p<0,001 ve 412,5 e karşın 190.6IU/I, p<0,001), TPOAb düzeyleri daha düşüktü (390,6 ya karşın 690,4IU/I, p=0,001).
Sonuç: GO nun eşlik ettiği GH hastaları oftalmopati bulunmayan GH hastaları ile kıyaslandığında, TGAb ve TRAb düzeylerinin daha yüksek, TPOAb düzeyinin daha düşük olduğu görüldü. Bu bulgular GO patogenezi üzerinde yapılacak çalışmalar için yeni perspektifler sunabilir. Türk Jem 2010; 14: 50-3
Anahtar kelimeler: Graves hastalığı, graves of talmopati, tiroid otoantikore
Graves ophtalmopathy (GO) is a potentially serious ocular complication of the auto immune thyroid disease (AITD). The treatment of GO is not always successful and the disease can cause constant damage to the anatomy and function of the eye. Close observation of subjects with AITD at high risk of GO would facilitate early preventive measures against this debilitating complication. Little is known about the risk factors for GO such as age, male gender, type of antithyroid treatment and smoking (1). The precise pathological processes, which link both autoimmune diseases are still under debate (2,3). Auto-antibodies to thyroidal antigens might be involved in the disease progress of GO per se. The leading role of TSH-receptor antibodies (TRAb) is now accepted by many thyroidologists and their measurement might be of clinical use (4-7). Other potential orbital antigens include thyroglobulin and cholinesterase epitopes, the flavoprotein subunit of the mitochondrial succinate dehydrogenase, a 55 kDa protein (G2s), calsequestrin and others (8-11). Thyroglobulin (TG) might be produced in small amounts by the orbital fat tissue, so antithyroglobulin antibodies (TGAb) seem to be of practical interest in GO (9-12). However, most publications have been focused on the measurement of TRAb in GO.
The aim of the present study was to investigate thyroid function and auto-antibodies in patients diagnosed with Graves disease (GD) with and without GO.
Materials and Methods
This is a cross-sectional retrospective study, which includes 98 patients with GD treated at the Endocrinology clinic of the Alexandrovska Hospital between 2002 and 2008. Seventy-six patients were female (mean age: 49.7±10.6 years) and twenty-two were male (mean age: 42.7±11.6 years). They had been referred for hospitalization mainly because of fluctuations in their thyroid function during antithyroid drug therapy or development of GO. The mean duration of GD was 1.6 ± 0.8 years. 46 patients had newly discovered hyperthyroidism. At the time of referral, the remaining 52 patients were taking antithyroid drugs. None of them had been treated previously with corticosteroids, radio-iodine or surgery. All procedures described below are part of the routine work-up of GD patients at our Endocrinology clinic and were in accordance with the ethical standards of the Committee on human experimentation at the Alexandrovska Hospital as well as on a national level. All patients gave their informed consent for data processing prior to their hospitalization.
The medical history included family history of thyroid disorders, smoking habits, symptoms of thyroid dysfunction as well as current treatment. A physical examination and anthropometric measurements were then performed. The palpation of the thyroid gland was followed by thyroid ultrasound on a Fukuda-Denshi 5.500 device (Fukuda Corp., Tokyo, Japan). The thyroid volume was calculated according to J. Brunn et al. in milliliters (13). Thyroid hormones-thyroid stimulating hormone (TSH) and free thyroxine (fT4) were measured by a chemiluminescence method (Bayer Diagnostics,Leverkusen, Germany). Anti-peroxidase (TPOAb) and TGAb were measured by an electrochemiluminescence method (Hoffmann-La Roche Ltd., Basel, Switzerland). TRAb were measured by an enzymatic-substrate method-ELISA (DRG International Inc., Mountainside, NJ, USA) and represented thyroid-binding inhibitory immunoglobulins. The upper normal limits for thyroid antibody titers were set as follows: TPOAb < 34 IU/l, TGAb < 115 IU/l and TRAb < 1.5 IU/l.
The diagnosis of GO was based mainly on the clinical picture (eyelid retraction, periorbital swelling, diplopia and others) according to the American Academy of Ophthalmology diagnostic criteria. The grade of the eye disease was estimated according to the NOSPECS classification (1) and the clinical activity score (CAS) according to Mourits et al. (14). All patients were referred for precise work-up by an experienced ophthalmologist at our University Hospital. Grade of exophthalmos, intraocular pressure, ocular motility and visual acuity were recorded.
The statistical analysis was performed on a SPSS 13.0 for Windows package (SPSS Inc., Chicago, IL, USA). Descriptive statistics, two-sided Students t-test, the Mann-Whitney U test, non-parametric Kruskal-Wallis and parametric ANOVA, and Spearmans correlation analysis were performed. Significance was set as p≤0.05.
Thirty-nine study participants had manifested GO-28 women and 11 men. Therefore, the prevalence of GO in our sample of ninety-eight patients with GD was 36.8% in women and 50% in men. According to the NOSPECS classification, three patients (7.7%) had grade 1 GO, seven patients (17.9%) had grade 2, eleven patients (28.2%)-grade 3, fifteen patients (38.4%)-grade 4, two patients (5.1%)-grade 5 and one (2.6%)-grade 6. Twenty-five patients with GO had a CAS score above 4 (an active disease) and the mean CAS score for the GO group as a whole was 4.8±1.2.
The clinical data of the participants including the thyroid volume measured by ultrasound are summarized in Table 1. Smoking was more common in patients with GD + GO than in those without GO. The odds ratio for current smoking in the presence of GO was 1.44. Thyroid volume did not show significant differences between the GO+and the GO-subgroups.
The hormonal and thyroid autoantibody levels of the participants are displayed in Table 2. Fifty-six of all ninety-eight participants (57.1%) were hyperthyroid (low TSH, elevated fT4) at the time of evaluation (48 newly discovered and eight under antithyroid treatment). Another twelve of the fifty treated patients had low TSH despite normal fT4 levels (24%). Five of the fifty treated patients had low normal fT4 levels and TSH<10 IU/l (iatrogenic subclinical hypothyroidism in 10%).
The patients with GO were more hyperthyroid than those without GO. The levels of all three thyroidal antibodies showed significant differences in the subgroups with and without GO. The presence of GO was associated with higher levels of TRAb and TGAb and lower levels of TPOAb. There was no relevant correlation between the CAS and the levels of TRAb and TGAb (Spearman's r=0.2, p=0.03) or of TPOAb ( Spearman's r=0.15, p=0.04). The correlations of thyroid autoantibody levels with the grade of GO were not significant.
Graves ophtalmopathy can develop in 25-40% of hyperthyroid patients with GD and much rarely in euthyroid or hypothyroid patients with autoimmune thyroiditis as well as in euthyroid subjects without evidence of thyroid disease (15). The immune mechanisms underlying the thyroid eye disease imply a possible role of a number of auto-antigens and their specific auto-antibodies. The most likely candidate antigen still remains the TSH-receptor (16). A number of authors have found a positive correlation between the levels of TRAb and the presence or severity of GO (4,6,17-21). The correlations between the levels of TGAb and GO are less well validated. A number of studies reported such a relationship (8-10), while others have not (12). Similarly, data accumulated about the TPOAb levels are also contradictory (11,17,19,22).
Our study was performed in patients with newly discovered GD and in patients already treated with antithyroid drugs. We were able to prove that the presence of GO was associated with higher levels of TRAb and TGAb and lower levels of TPOAb and TSH as compared with patients without GO. The thyroid volume or the duration of AITD showed no association with the presence of GO. A collateral finding was that smokers were more prevalent among patients with GD and GO than among those without GO.
Similar findings have been reported by other authors. A.K. Eckstein et al. assessed 108 patients with GO after steroid therapy or orbital irradiation (23). The simultaneous presence of thyroid-binding inhibitory immunoglobulins and thyroid-stimulating antibodies was associated with significantly higher activity and severity of GO. Only TRAb, but not TPOAb or TGAb medians, demonstrated statistically significant increase with CAS or NOSPECS scores. Another study tested the hypothesis that TRAb are independent risk factors for GO and can help to predict the severity and the outcome of the disease (6). A significant association between elevated initial thyroid-stimulating immunoglobulins and GO was also found in pediatric patients with GD (20). S.Y. Goh et al. studied the autoantibody profile in patients with GD referred to ophthalmologic or thyroid units (17). Patients with dominant GO had significantly higher stimulating TRAb (p=0.003), but lower TPOAb (p= 0.008) and TgAb levels (p<0.001). In contrast, patients with dominant GD had higher fT4 (p =0.048) and higher thyroid-binding inhibitory immunoglobulin (TBII) levels. An association between smoking and low TPOAb levels was also noted. In our study, the levels of TRAb and TgAb did not correlate with the grade or clinical activity of GO. Correlations of different grades have been reported by other investigators (4,6). In the study by M.N. Gerding et al., the authors reported that TBII or thyroid-stimulating immunoglobulin titers did not correlate with thyroidal or orbital disease duration, or with TPOAb levels (4). In contrast, they found a striking and highly significant correlation between the CAS of the eye disease and both types of thyroid antibodies (r = 0.54; p<0.0001, and r=0.50; p<0.0001). TRAb might also be regarded as a surrogate marker for autoimmune activity in GO (21) and their levels are influenced by corticosteroid treatment (24). Bulgarian authors have also investigated the possible link between the levels of TRAb and the presence or severity of GO in GD (25).
We feel that the major contribution of this study lies in the measurement of TGAb and TPOAb levels in GD associated with GO. We were able to show higher levels of TGAb and lower ones of TPOAb in our patients with GO as compared with those without ocular involvement. TGAb and TPOAb appear to be secondary responses to the thyroid injury and are not thought to cause the disease themselves. Our conclusion is that these two auto-antibodies might open new perspectives in studying the pathogenesis of GO.
A link between the serum levels of TG, TGAb and the presence of GO has been investigated in a few studies (9-11,13). T. Kuroki et al. reported that the TG-shared antigen site of ocular connective tissue membranes appeared not to be native thyroglobulin (9). Concerning the anticholinesterase antibodies, J. Geen et al. concluded from their data that the lack of patients with clinically apparent GO militated against a possible causal role of such antibodies (8). Our study has a number of limitations. First, the small study size and cross-sectional design are far from the ideal large prospective study design. Because of low statistical power, we were unable to apply the ROC-analysis and show a threshold of high risk for GO for all three thyroid antibodies. Our correlation data with the disease severity are also inconclusive. Second, our study population consisted of patients referred to a hospital clinic, which might have introduced a bias toward more aggressive forms of GD and GO. Almost half of the patients were currently hyperthyroid, which could be a reason why antibody titers were higher. Previous studies have shown that euthyroid or primarily hypothyroid patients develop milder and more asymmetrical GO (15). Third, we only measured TRAb levels and did not apply any functional assay for testing their thyroid-stimulating or thyroid-blocking activity. There are accumulated data demonstrating that the characteristics of TRAb are of clinical significance for the progression and severity of GD and GO (26,27).
In summary, we performed a pilot study in patients with GD with and without GO and found different levels of TRAb, TGAb and TPOAb in case of presence or absence of GO. Our study should be regarded as an urge for conducting further large prospective studies relating thyroid autoimmunity with the clinical course of GO in GD and for further elucidation of the pathogenesis of GO.
The authors wish to thank Assoc. Prof. V. Christov, former Head of the Endocrinology Clinic, for his encouragement in this work and Dr. L. Wezenkova and Dr. D. Manolov for their help in collecting the patients data.
Address for Correspondence: Mihail A. Boyanov MD, DMSci Endocrinology Clinic, Alexandrovska Hospital 1, G. Sofiiski Str., Sofia 1431 Bulgaria Phone: + 3592 9230 784 E-mail: firstname.lastname@example.org Recevied: 28.11.2010 Accepted: 07.12.2010
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