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ISSN: 1301-2193 E-ISSN: 1308-9846

Turkish
Journal of
Endocrinology
and
Metabolism

Levels of Thyroid Autoantibodies in Patients

with Graves Disease and Graves
Ophtalmopathy - Original Article

A
Abbssttrraacctt
O
Obbjjeeccttiivvee:: 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.
M
Maatteerriiaallss aanndd M
Meetthhooddss:: 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.
R
Reessuullttss:: 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. 
C
Coonncclluussiioonn:: 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
K
Keeyy w
woorrddss:: Graves disease, graves ophtalmopathy, thyroid antibodies

Ö
Özzeett
A
Ammaaçç:: 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.
G
Geerreeçç vvee YYöönntteem
mlleerr:: 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.
B
Buullgguullaarr:: 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).
SSoonnuuçç:: 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
A
Annaahhttaarr kkeelliim
meelleerr:: Graves hastalığı, graves of talmopati, tiroid otoantikore

IInnttrroodduuccttiioonn

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.

M
Maatteerriiaallss aanndd M
Meetthhooddss

PPaattiieennttss
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.
M
Meetthhooddss
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.

R
Reessuullttss

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. (Javascript:ResimGoster('sayilar/67/1-1.jpg'))
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. (Javascript:ResimGoster('sayilar/67/1-2.jpg')) 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.

D
Diissccuussssiioonn

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).

C
Coonncclluussiioonn

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.
A
Acckknnoow
wlleeddggm
meennttss
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.

A
Addddrreessss ffoorr C
Coorrrreessppoonnddeennccee:: Mihail A. Boyanov MD, DMSci Endocrinology Clinic,
Alexandrovska Hospital 1, G. Sofiiski Str., Sofia 1431 Bulgaria Phone: + 3592 9230
784 E-mail: mihailboyanov@yahoo.com (mailto:mihailboyanov@yahoo.com)
Recevied: 28.11.2010 Accepted: 07.12.2010

R
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