Original Article Orijinal Makale

50

Levels of Thyroid Autoantibodies in Patients with

Graves Disease and Graves Ophtalmopathy
Graves Hastalnda ve Graves Oftalmopatisinde Tiroid Otoantikor Dzeyleri
Mihail Boyanov, Deniz Bakalov, Galina Sheinkova
Endocrinology Clinic, Department of Internal Medicine, Alexandrovska University Hospital, Medical University Sofia, Sofia, Bulgaria

Abstract
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
zet
Ama: Graves hastal (GH) ve Graves oftalmopati (GO) hastalar zerinde daha nce yaplm olan almalar, genel olarak TSH-reseptr
antikoru (TRAb) dzeylerine odaklanmtr. Bu almann amac GH ve GO hastalarnn TRAb, tiroid peroksidaz (TPOAb) ve tiroglobulin (TGAb)
dzeylerini aratrmaktr.
Gere ve Yntemler: GH tansyla izlenen 98 hasta, (76 kadn, 22 erkek) bu resrospektif almaya dahil edildi. 39 hastada (28 kadn, 11 erkek)
GO grld. Tiroid stimulan hormon (TSH) ve serbest tiroksin (FT4) dzeyleri kemiluminesan yntemi, TPOAb ve TGAb dzeyleri elektrokemiluminesan
yntemi ile, TRAb dzeyi ise enzim-substrat metodu-ELISA ile lld.
Bulgular: GH+GO hastalarnda medyan serum TSH ve FT4 dzeyleri srasyla 0.079 IU/I ve 37,7 pmol/I iken GO bulunmayan GH hastalarnda
TSH ve FT4 dzeylerinin srasyla 0,420IU/I ve 23,2 pmol/I olduu grlmtr (TSH iin p=0,04, FT4 iin p=0,02). GH+GO hastalarnn TRAb ve
TGAb dzeyleri yalnzca GH mevcut hastalara gre daha yksek iken (srasyla 22,1 e karn 10,4IU/L, p<0,001 ve 412,5 e karn 190.6IU/I,
p<0,001), TPOAb dzeyleri daha dkt (390,6 ya karn 690,4IU/I, p=0,001).
Sonu: GO nun elik ettii GH hastalar oftalmopati bulunmayan GH hastalar ile kyaslandnda, TGAb ve TRAb dzeylerinin daha yksek,
TPOAb dzeyinin daha dk olduu grld. Bu bulgular GO patogenezi zerinde yaplacak almalar iin yeni perspektifler sunabilir. Trk
Jem 2010; 14: 50-3
Anahtar kelimeler: Graves hastal, graves of talmopati, tiroid otoantikor
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: mihailboyanov@yahoo.com Recevied: 28.11.2010 Accepted: 07.12.2010
Turkish Journal of Endocrinology and Metabolism, published by Galenos Publishing.

Boyanov et al.
Thyroid Antibodies in Graves Ophtalmopathy

Turk Jem 2010; 14: 50-3

Introduction
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

Patients
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: 49.710.6 years) and twentytwo were male (mean age: 42.711.6 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.
Methods
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 FukudaDenshi 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

51

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, twosided 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 p0.05.

Results
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
4.81.2.
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.
Table 1. Displayed are the clinical data of the participants and the
corresponding thyroid volume (meansstandard deviation)

Men
Women
Men + women
With GO
Without GO
P-value
(T-tests)

Age,
years
42.111.0
50.210.4

Duration of
GD, years
1.61.2
2.21.8

46.010.3
47.811.1
n.s.

2.42.6
1.44.7
0.03

Current
smoking
50.0%
39.4%

Thyroid
volume, cm3
27.515.3
22.913.3

51.3%
35.6%
0.002

24.911.9
23.212.9
n.s.

52

Boyanov et al.

Turk Jem 2010; 14: 50-3

Thyroid Antibodies in Graves Ophtalmopathy

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 =0.2, p=0.03) or of TPOAb ( Spearman's =0.15,
p=0.04). The correlations of thyroid autoantibody levels with the
grade of GO were not significant.

Discussion
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).

Table 2. Displayed are the hormonal levels and the thyroid autoantibodies of the participants - medians and ranges (in parentheses)
Men
Women
Men + women
With GO
Without GO
P value
(Mann-Whit-ney
tests)

TSH, IU/l
0.010
(0.001-7.22)
0.140
(0.001-8.11)

fT4, pmol/l
28.6
(10.1-42.4)
36.4
(10.8-54.7)

0.079
(0.001-8.11)
0.420
(0.0056.42)
0.04

33.7
(17.2 -54.7)
23.2
(10.1-34.6)
0.02

TRAb, IU/l
13.3
(1.9-40.0)
15.7
(2.2-34.4)
22.1
(3.2- 40.0)
10.4
(1.9-22.1)
<0.001

TPOAb, IU/l
924.4
(15-4500)
700.5
(10-6000)

TGAb, IU/l
486.1
(52-1510)
296.7
(24-820)

390.6
(10-1200)
690.4
(34-6000)
<0.001

412.5
(90-1500)
190.6
(52-520)
<0.001

Turk Jem 2010; 14: 50-3

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 thyroidblocking 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).

Conclusion
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.
Acknowledgments
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.

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