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ORIGINAL ARTICLE
doi: 10.5606/ArchRheumatol.2017.5931
Arch Rheumatol 2017;32(2):112-117
Dynamic Thiol/Disulphide Homeostasis in
Patients With Fibromyalgia
Fatma FİDAN,1 Berat Meryem ALKAN, 2 Fatma Gülçin UĞURLU,1 Sinem BOZKURT,2 Nebahat SEZER,1
Cemile BİÇER, 3 Özcan EREL, 3 Özge ARDIÇOĞLU,1 Selami AKKUŞ 1
1Department of Physical Medicine and Rehabilitation, Medical Faculty of Yıldırım Beyazıt University, Ankara, Turkey
2Department of Physical Medicine and Rehabilitation, Atatürk Training and Research Hospital, Ankara, Turkey
3Department of Biochemistry, Medical Faculty of Yıldırım Beyazıt University, Ankara, Turkey
Received: January 28, 2016 Accepted: July 16, 2016 Published online: January 18, 2017
Correspondence: Fatma Fidan, MD. Yıldırım Beyazıt Üniversitesi Tıp Fakültesi Fiziksel Tıp ve Rehabilitasyon Anabilim Dalı, 06830 Bilkent, Ankara, Turkey.
Tel: +90 312 - 291 25 25 e-mail: drfatmafidan@hotmail.com
©2017 Turkish League Against Rheumatism. All rights reserved.
ABSTRACT
Objectives: This study aims to investigate dynamic thiol/disulphide homeostasis in patients with fibromyalgia syndrome (FMS).
Patients and methods: Fifty female patients with FMS (mean age 40.5±7.2 years; range 21 to 55 years) and 40 healthy female controls (mean age
39±9.4 years, range 22 to 55 years) were included in the study. Pain visual analog scale, tender points, Fibromyalgia Impact Questionnaire, and Beck
Depression Inventory were evaluated. Age, body mass index (BMI), and symptom durations were also recorded. Native thiol, disulphide and total
thiol levels were measured with a novel automated method.
Results: Serum disulphide levels were 14.7±3.4 µmol/L and 22.2±3.6 µmol/L in the FMS and control groups, respectively (p<0.001). Native thiol
levels were 452.1±33.8 µmol/L and 433.5±37.6 µmol/L in the FMS and control groups, (p=0.015), while total thiol levels were 481.7±35.6 µmol/L and
477.5±38.9 µmol/L in the FMS and control groups, respectively (p=0.593). In the FMS group, disulphide/native thiol percent ratios and disulphide/
total thiol percent ratios were statistically significantly lower and native/total thiol percent ratios were statistically significantly higher than those
of the control group. There were no correlations between serum thiol/disulphide profiles and pain scores & clinical variables in patients with FMS.
Conclusion: Because of the decreased disulphide and increased native thiol levels, the thiol/disulphide balance has shifted to the reductive side.
This metabolic disturbance may have a role in the pathogenesis of FMS.
Keywords: Disulphide; fibromyalgia; thiol.
Fibromyalgia syndrome (FMS) is a chronic
pain syndrome accompanied by widespread
musculoskeletal pain, fatigue, sleep disturbances,
functional disability, and mood disturbances. 1-3 It
involves several factors, including abnormalities
in the neuroendocrine and autonomic nervous
systems, genetic factors, hormones, immune
system, psychosocial variables, and environmental
stressors.4-6 Information on the physiopathological
mechanisms are limited. There are studies
indicating an imbalance between oxidants and
antioxidants in patients with FMS. 7-10 The results
are controversial. To the best of our knowledge,
no report related to thiol/disulphide homeostasis
in FMS has been published.
Thiols, also known as mercaptans, contain
a sulfhydryl group (-SH) and have numerous
functions, including a central role in coordinating
the antioxidant defense network. 11 The plasma
thiol pool is mainly formed by albumin and protein
thiols and slightly formed by low molecular weight
thiols.12 Thiols can undergo oxidation reaction
via oxidants and form disulphide bonds. The
formed disulphide bonds can again be reduced
to thiol groups; thus, dynamic thiol/disulphide
homeostasis is maintained. 13,14
113Dynamic Thiol/Disulphide Homeostasis in Patients With Fibromyalgia
Dynamic thiol/disulphide homeostasis status
has been shown to play critical roles in antioxidant
protection, detoxification, signal transduction,
apoptosis, regulation of enzymatic activity and
transcription factors, and cellular signalling
mechanisms. 15,16 Abnormal thiol disulphide
homeostasis states are involved in the pathogenesis
of many kinds of diseases, including diabetes, 17
cardiovascular disease, 18 cancer,19 chronic kidney
disease,20 and rheumatoid arthritis. 21
One side of this double-sided balance, namely
thiols, has been measurable since 1979. But
now a new method, developed by Erel and
Neselioglu,25 can measure both variable levels
separately and additively, so they can be evaluated
both individually and holistically. Therefore, in
this study, we aimed to investigate dynamic thiol/
disulphide homeostasis in patients with FMS.
PATIENTS AND METHODS
Fifty female patients (mean age 40.5±7.2 years;
range 21 to 55 years) with FMS diagnosed
according to the American College of
Rheumatology criteria 22 were included in this
study which was conducted between December
2014 and May 2015 at Medical Faculty of Yıldırım
Beyazıd University Ankara Atatürk Training and
Research Hospital. Exclusion criteria were as
follows: diabetes mellitus, inflammatory, renal
and cardiovascular diseases, pregnancy, use of
corticosteroids, and malignancy history. The
control group was composed of 40 healthy
sedentary female volunteers (mean age 39±9.4
years; range 22 to 55 years) who had normal
physical examinations and had no chronic
diseases. The study protocol was approved by
the Medical Faculty of Yıldırım Beyazıd University
Ankara Atatürk Training and Research Hospital
Ethics Committee, and all subjects were informed
about the study and filled written consent forms.
The study was conducted in accordance with the
principles of the Declaration of Helsinki.
Age, body mass index and demographic
characteristics were recorded for all patients and
controls. Musculoskeletal pain was evaluated
using a 10-point graded scale; 0 indicated an
absence of symptoms and 10 indicated the
worst symptoms. FMS patients filled out the
visual analog scale to evaluate pain intensity.
Tenderness was evaluated by applying pressure
over 18 specific body points, and the number of
tender points was recorded.
The Fibromyalgia Impact Questionnaire (FIQ)
was used to evaluate which patients were affected
by FMS. 23 The FIQ is a self-administered tool
comprising 10 items. Participants were also
evaluated by a version of Beck Depression
Inventory (BDI). 24
Venous blood samples were collected in
blood tubes. Serum samples were separated
from cells by centrifugation at 1200 ¥g for
10 minutes. Thiol/disulphide homeostasis
tests were measured by a novel automated
method described by Erel and Neselioglu. 25
Measurements were performed using a
Cobas c501 chemical analyzer (Roche
Diagnostics, Mannheim, Germany). Serum
thiol/disulphide homeostasis values were
presented as mmol/L. In short, disulphide
bonds were first reduced to form-free functional
thiol groups with sodium borohydride. Unused
reductant sodium borohydride was consumed
and removed with formaldehyde to prevent
reduction of 5,5’-dithiobis-(2-nitrobenzoic acid),
and all of the thiol groups, including reduced
and native thiol groups, were determined after
the reaction with 5,5’-dithiobis-(2-nitrobenzoic
acid). All the chemicals were purchased from
Merck Chemicals (Darmstadt, Germany) and
Sigma Aldrich Chemie (Milwaukee, Wisconsin,
USA). Additionally measuring native thiol and
total thiol levels, disulphide levels were calculated
via half of the difference between the total
thiols and native thiols. After the determination
of native (SH) and total thiols (SH+SS),
disulphide amounts (SS), disulphide/total thiol
percent ratios (SS/SH+SS), disulfide/native
thiol percent ratios (SS/SH) and native thiol/
total thiol percent ratios (SH/SH+SS) were
calculated by multiplying with 100.
Statistical analysis
Descriptive statistics were used to describe
demographic characteristics. Comparative
analyses of the parameters were performed using
either the Mann-Whitney U test or student’s
t test depending on the levels of measurement.
Spearman and Pearson correlation tests were
used to determine relationship between variables.
The level of significance was set at p<0.05.
Arch Rheumatol114
Statistical analyses were performed using IBM
SPSS software version 20.0 (IBM Corp., Armonk,
NY, USA).
RESULTS
There were no statistically significant differences
in age and BMI between the groups (p>0.05). The
disease duration, mean FIQ scores, and BDI scores
were 6.7±4.3 years, 66.6±14.0, and 16.1±8.1 in
FMS group, respectively. The demographic and
clinical characteristics of FMS and control groups
are presented in Table 1.
Serum disulphide levels were 14.7±3.4 µmol/L
and 22.2±3.6 µmol/L in the FMS and control
groups, respectively (p<0.001). Native thiol levels
were 452.1±33.8 µmol/L and 433.5±37.6 µmol/L
in the FMS and control groups, respectively
(p=0.015), while total thiol levels were
481.7±35.6 µmol/L and 477.5±38.9 µmol/L
in the FMS and control groups, respectively
(p=0.593). Plasma native thiol levels were higher
and plasma disulphide levels were lower in the
FMS group compared to the control group, while
total thiol levels were not significantly different
between the two groups.
Disulphide/native thiol percent ratios and
disulphide/total thiol percent ratios were 3.3±0.8
and 3.1±0.7, respectively, in the FMS group,
while disulphide/native thiol percent ratios and
disulphide/total thiol percent ratios were 5.2±0.9
and 4.7±0.8, respectively, in the control group.
In patients with FMS, disulphide/native thiol
and disulphide/total thiol percent ratios were
statistically significantly lower and native/total
Table 1. Demographic and clinical characteristics of fibromyalgia syndrome and control groups
Age (year) 40.5±7.2 41.5 12.25 39±9.4 39.5 18.25 0.472
Body mass index 27.5±4.8 26.6±4.1 0.33
Disease duration (year) 6.7±4.3
Visual analog scale pain 7.6±1.8
Tender points 14.5±1.6
Fibromyalgia Impact Questionnaire score 66.6±14.0
Beck Depression Inventory score 16.1±8.1
FMS: Fibromyalgia syndrome; SD: Standard deviation; IQR: Interquartile range.
Patients with FMS (n=50) Healthy controls (n=40)
Mean±SD Median IQR Mean±SD Median IQR p
Table 2. Comparison of thiol/disulphide profiles between fibromyalgia syndrome and control groups
Native thiol (µmol/L) 452.1±33.8 433.5±37.6 0.015*
Disulphide (µmol/L) 14.7±3.4 22.2±3.6 <0.001*
Total thiol (µmol/L) 481.7±35.6 477.5±38.9 0.593
Disulphide/native thiol (%) 3.3±0.8 5.2±0.9 <0.001*
Disulphide/total thiol (%) 3.1±0.7 4.7±0.8 <0.001*
Native thiol/total thiol (%) 93.9±1.4 90.8±1.6 <0.001*
FMS: Fibromyalgia syndrome; SD: Standard deviation; * p value of <0.05 was considered statistically significant.
Patients with FMS (n=50) Healthy controls (n=40)
Mean±SD Mean±SD p
Figure 1. Serum native thiol levels (x± sM) in patients
with fibromyalgia syndrome and healthy controls.
FMS: Fibromyalgia syndrome.
470.00
460.00
450.00
Native thiol (µmol/L)
440.00
430.00
420.00
Patients with FMS Healthy controls
115Dynamic Thiol/Disulphide Homeostasis in Patients With Fibromyalgia
thiol percent ratios were statistically significantly
higher than those of the control group. The data,
including the thiol/disulphide profiles of patients
with FMS and controls, are given in Table 2, and
Figures 1-3.
There were no correlations between serum
thiol/disulphide parameters and disease duration,
tender points, pain visual analog scale, and BDI
and FIQ scores in patients with FMS.
DISCUSSION
In this study, we investigated thiol/disulphide
homeostasis status in patients with FMS for the
first time by a recently developed method of
Erel and Neselioglu. 25 Our findings showed that
serum native thiol levels were higher (p=0.015)
and serum disulphide levels were lower (p<0.001)
in the FMS group compared to the control
group (p<0.05), while total thiol levels were not
statistically significantly different between the
two groups (p=0.593) (Table 2). Because of the
decreased disulphide and increased native thiol
levels, thiol/disulphide balance has shifted to the
reductive side.
The antioxidant defense system comprises
enzymatic and non-enzymatic strategies.
Intracellular antioxidant enzymes are mainly
superoxide dismutase, catalase, and glutathione
peroxidase/glutathione reductase. Non-enzymatic
antioxidants are glutathione (GSH), carotenoids,
tocopherols, and ascorbates. 26,27 GSH is the
most important thiol-containing molecule and
important intracellular antioxidant. It has a very
low concentration in the extracellular milieu. It is
a tripeptide made of three amino acids: cysteine,
glutamic acid, and glycine. GSH protects cells
from toxins, such as free radicals. 28 Sendur
et al. 29 found that serum GSH and catalase
levels were significantly lower in patients with
FMS compared to healthy controls. Furthermore,
Akbas et al. 2 showed that superoxide dismutase
enzyme activity was higher in the FMS group
compared to the control group, while glutathione
peroxidase enzyme activity was not significantly
different between the two groups. Bagis et al. 30
reported decreased serum superoxide dismutase
activity in FMS. The results are controversial.
Studies of oxidant stress and antioxidant
status in patients with FMS have also revealed
different results; some have demonstrated a
decreased total antioxidant status compared with
the control groups, 8,9,31,32 whereas other studies
did not show a significant difference between the
two groups. 10,33 Neyal et al. 9 and Altindag and
Celik31 and Altindag et al. 32 observed decreased
total antioxidant and increased total oxidant
levels in FMS patients. Conversely, Bozkurt et
al.10 reported no significant differences between
patients and controls’ serum total antioxidant
status. Chung et al. 33 concluded in their work
that oxidative stress is not increased in patients
with fibromyalgia. Similarly, our results also
support the notion that oxidative stress is not
increased in FMS patients.
22.50
20.00
Disulphide (µmol/L)
17.50
15.00
12.50
Patients with FMS Healthy controls
Figure 2. Serum disulphide levels (x± sM) on patients
with fibromyalgia syndrome and healthy control groups.
FMS: Fibromyalgia syndrome.
495.00
490.00
485.00
Total thiol (µmol/L)
480.00
475.00
470.00
465.00
Figure 3. Serum total thiol levels (x± sM) in patients
with fibromyalgia syndrome and healthy controls.
FMS: Fibromyalgia syndrome.
Patients with FMS Healthy controls
Arch Rheumatol116
In a study by Tetik et al., 21 plasma thiol levels
were shown to be decreased in rheumatoid arthritis
and primary osteoarthritis patients. Similarly,
Altindag et al. 32 and Sarifakioglu et al. 34 found
that plasma thiol levels were significantly lower
in FMS patients compared with control subjects.
However, due to the current measurement
failure, they were only able to measure the thiol
levels of homeostasis, while they were unable to
measure disulphide and total thiol levels. Hence,
determination of thiol/disulphide homeostasis was
not possible. But thanks to the novel method of
Erel and Neselioglu, 25 we were able to measure
all parameters of homeostasis, and evaluate the
balance entirely and accurately. Our findings
showed that plasma native thiol levels were higher
and plasma disulphide levels were lower in the
FMS group compared to the control group, while
total thiol levels were not statistically significantly
different between both groups.
In our study, we found no correlation between
thiol/disulphide homeostasis results and FMS
duration; number of tender points; fatigue; and
visual analog scale, FIQ and BDI scores in patients
with FMS. Chung et al. 33 found a correlation with
oxidative stress and fatigue, but they found no
correlation between oxidative stress and pain,
quality of life, depression, or number of tender
points. Similarly, Richard and Kahn 35 suggested
that oxidative stress was associated only with
fatigue but not with pain or tender points in
patients with FMS.
In this study, plasma dynamic thiol/disulphide
homeostasis was determined by the recently
developed method by Erel and Neselioglu 25 in
patients with FMS for the first time. In their
preliminary studies, they showed that plasma
disulphide levels were higher in patients with
degenerative diseases, such as diabetes, smoking,
obesity, and pneumonia, and were lower in
patients with proliferative diseases, such as multiple
myeloma, colon cancer, and renal cancer. 36-38 We
found lower disulphide levels in patients with FMS
compared with the healthy controls. However, we
did not observe any difference in total thiol levels
between the two groups.
The facts that the method we used in our study
is new and that the literature lacks studies using
this method on fibromyalgia patients were limiting
in terms of comparing the results.
Although the ethiopathogenesis of FMS is still
unknown, our thiol/disulphide balance results
were observed to fit the pattern of proliferative
disease status rather than degenerative disease
status. We believe that the role of thiol/disulphide
homeostasis on etiopathogenesis of FMS should
be examined by future studies. Our results also
support the notion that oxidative stress is not
increased in FMS patients.
Declaration of conflicting interests
The authors declared no conflicts of interest with
respect to the authorship and/or publication of this article.
Funding
The authors received no financial support for the
research and/or authorship of this article.
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