Fatma FİDAN1, Berat Meryem ALKAN2, Fatma Gülçin UĞURLU1, Sinem BOZKURT2, Nebahat SEZER1, Cemile BİÇER3, Özcan EREL3, Özge ARDIÇOĞLU1, Selami AKKUŞ1

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

Keywords: Disulphide; fibromyalgia; thiol

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.

Introduction

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)

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. 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 4 8 1 . 7 ± 3 5 . 6 μ m o l / L a n d 4 7 7. 5 ± 3 8 . 9 μ m o l / 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 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 Celik(31) 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.

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.

Conflict of Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Financial Disclosure

The authors received no financial support for the research and/or authorship of this article.

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