Clinical Biochemistry xxx (xxxx) xxx

Contents lists available at ScienceDirect

Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem

Erythrocyte reduced/oxidized glutathione and serum thiol/disulfide

homeostasis in patients with rheumatoid arthritis
Murat Alisik a, *, Tugba Alisik b, Baris Nacir c, Salim Neselioglu d, Irem Genc-Isik e,
Pinar Koyuncu f, Ozcan Erel d
a
Medical Biochemistry, Abant Izzet Baysal University, Bolu, Turkey
b
Physical Medicine and Rehabilitation, Abant Izzet Baysal University, Bolu, Turkey
c
Physical Medicine and Rehabilitation, Ankara Training and Research Hospital, Ankara, Turkey
d
Medical Biochemistry, Yildirim Beyazit University, Ankara, Turkey
e
Dermatology, Kastamonu University, Kastamonu, Turkey
f
Medical Biochemistry, Ankara Training and Research Hospital, Ankara, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Chronic inflammation and oxidative stress are the most known mechanisms in Rheumatoid Arthritis
Rheumatoid arthritis (RA) pathophysiology, which is still not fully elucidated. In this study, we evaluated oxidative status by deter­
Oxidative stress mining intracellular reduced/oxidized glutathione (GSH/GSSG) homeostasis and serum thiol/disulfide (SH/SS)
Thiol
homeostasis in RA patients.
Glutathione
Disulfide
Methods: A total of 152 RA patient and 89 healthy controls were included in the study. RA patients were sub­
divided according to disease activity score-28 (DAS-28) as active RA and remission RA. Intracellular GSH/GSSG
and serum SH/SS homeostasis parameters were analyzed.
Results: Median (1st–3rd quartile values) SS/SH and GSSG/GSH percent ratio levels were significantly higher in
RA patients (6.94 (6.02–8.54) and 69.8 (44.05–85.29); respectively) compared to controls (4.62 (4.15–5.46) and
34.9 (22.43–62.2); respectively) (p < 0.05 for all). SS/SH and GSSG/GSH percent ratio levels were significantly
higher in active RA patients when compared to remission RA patients and controls (p < 0.05 for all). SS/SH and
GSSG/GSH percent ratios were significantly increased in remission RA group compared to controls (p < 0.05 for
all). DAS28 scores were positively correlated with SS/SH and GSSG/GSH percent ratios (rho = 0.259 and 0.296;
respectively).
Conclusions: These findings suggest that active intracellular and extracellular thiol group oxidation process might
play a role in RA pathogenesis and further work in these areas may be warranted to show potential value of
evaluating intracellular GSSG/GSH and serum SH/SS balances together in disease monitoring.

1. Introduction Increment in oxidative state and decrease in antioxidant defenses cause

Rheumatoid Arthritis (RA) is a debilitating chronic autoimmune
disease damaging the joints and limiting the joint range of motion as a
result of inflammatory synovial hyperplasia and leukocyte infiltration
[1,2]. However, RA pathophysiology is still not fully elucidated. The loss
of balance between the pro-inflammatory and anti-inflammatory fac­
tors, elevation in inflammatory cytokines like tumor necrosis factor-α
(TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) plays the
major role in RA pathophysiology [3,4]. Together with inflammatory
process, oxidative mechanisms also contribute to joint damage.
increased oxidative tissue damage and elevated inflammatory signals in
cells and tissues [5,6].
Oxidative stress occupies an important role in RA pathogenesis.
Therefore, oxidative balance should be maintained to achieve favorable
outcome in RA patients [7]. Thiol groups (SH) of proteins and low
molecular weight compounds like glutathione are oxidized with oxidant
molecules in the medium and establish reversible disulfide bonds (SS).
Formed SS bonds may again be reduced to SH’s and by this means thiol/
disulfide (SH/SS) homeostasis is maintained [8]. Dynamic SH/SS ho­
meostasis plays a critical role in antioxidant defense, detoxification,

* Corresponding author at: Department of Medical Biochemistry, Abant Izzet Baysal University, Golkoy, Bolu 14030 Turkey.
E-mail address: muratalisik@gmail.com (M. Alisik).

https://doi.org/10.1016/j.clinbiochem.2021.04.023
Received 9 December 2020; Received in revised form 13 March 2021; Accepted 25 April 2021
Available online 29 April 2021
0009-9120/© 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Please cite this article as: Murat Alisik, Clinical Biochemistry, https://doi.org/10.1016/j.clinbiochem.2021.04.023
M. Alisik et al.
apoptosis, regulation of enzymatic activity, transcription and cellular
signal transduction [9]. Serum thiol-disulphide homeostasis, which is
the thiol indicator of the extracellular thiol pool, is mainly composed of
cysteine residues of albumin and other proteins. Glutathione constitutes
the intracellular thiol pool. It is present in two forms, which are oxidized
glutathione (GSSG) and reduced glutathione (GSH) [10]. GSH/GSSG
homeostasis is a significant indicator of intracellular thiol state and
oxidation state [11,12].
Apart from being oxidative markers, molecules with dynamic thiols
have many functions. So, evaluating both oxidized and reduced forms of
these molecules would provide information about other functions of
thiols and would be a good indicator of oxidative stress. Therefore,
demonstration of thiol status in RA patients is important. Several studies
have been published that investigate the relationship between RA and
thiol, in which either only extracellular thiol levels or only intracellular
thiol levels, but not both together, were measured [13–15]. Simulta­
neous examination of the differences in intracellular GSH/GSSG levels,
which is the major intracellular antioxidant/oxidant molecule, and dy­
namic SH/SS groups in proteins, which is one of the most important
antioxidant mechanisms in serum, will provide more holistic view about
the thiol status of metabolism. To the best of our knowledge, there are no
studies evaluating both intracellular GSH/GSSG and extracellular SH/SS
homeostasis simultaneously in RA patients. Thus, we aim to evaluate the
changes in intracellular thiol status with intracellular reduced/oxidized
glutathione (GSH/GSSG) homeostasis, extracellular thiol status with
serum thiol/disulfide (SH/SS) homeostasis in RA patients and compare
those parameters with disease activity scores.
2. Material and methods
2.1. Study group
Patients over 18 years of age and who fulfilled the American College
of Rheumatology (ACR)/European League Against Rheumatism
(EULAR) 2010 classification criteria for RA [16] and applied to Ankara
Training and Research Hospital Physical Medicine and Rehabilitation
Clinic Rheumatology Outpatient Clinic were included in the study. RA
group consisted of 152 patients and control group consisted of 89
healthy individuals. This study was performed in line with the principles
of the Declaration of Helsinki. Approval was granted by the Ethics
Committee of Yildirim Beyazit University (Ethics approval number:
26379996/111). Written informed consent was obtained from all of the
individuals. Patients with diabetes, hypertension, bronchial asthma,
cardiac disease (coronary artery disease and chronic heart failure),
neoplasia, active infection, those taking antioxidant medications, and/
or smokers were excluded. Age, gender, disease activity score-28 (DAS-
28), duration of disease, treatment/medications, erythrocyte sedimen­
tation rate were noted for each RA patient and control individuals.
RA patients were divided into two groups by using the RA disease
activity score-28 (DAS28), which is calculated with the number of
swollen joints, number of painful joints, and erythrocyte sedimentation
rate [17]. RA patients who have >2.6 DAS28 scores were included in
active RA groups, ≤2.6 were included in the remission RA group [18].
Medications of RA patients were noted as conventional DMARDs
(cDMARDs) such as methotrexate, leflunomide, sulfasalazine, or
hydroxychloroquine and biological DMARDs (bDMARDs) such as eta­
nercept, adalimumab, or rituximab. And patients were divided into
three groups according to their medications as single cDMARDs group,
combined cDMARDs group, and combined bDMARDs and cDMARDs
group.
2.2. Chemicals
Sodium borohydride (NaBH4), 5,5′ -dithiobis(2-nitrobenzoic acid)
(DTNB, Ellman’s reagent), formaldehyde, methanol, hydrochloric acid
(HCl), ethylenedinitrilotetraacetic acid (EDTA), Trizma base,
2
Clinical Biochemistry xxx (xxxx) xxx
trichloroacetic acid (TCA), sodium hydroxide (NaOH), L-glutathione
reduced (GSH), L-glutathione oxidized (GSSG), and sodium chloride
(NaCl) were purchased from Sigma-Aldrich Inc. (Taufkirchen, Germany)
and Merck Co. (Darmstadt, Germany). All chemicals were of analytical
grade and type-1 reagent-grade deionized water was used.
2.3. Samples
Two blood samples from all individuals included in RA and control
groups were obtained from median ulnar or basilic veins by an experi­
enced phlebotomist. About 3 ml of blood was taken to EDTA anticoag­
ulant tubes for intracellular GSH/GSSG homeostasis parameters and
about 5 ml of blood was taken to serum separating tubes for thiol-
disulfide homeostasis parameters from each individual. Samples in
EDTA anticoagulant tubes were immediately centrifuged and washed
thrice in 0.9% NaCl and lysed with deionized water. Then, 1-part 20%
w/v TCA solution (three parts washed blood lysate and one part 20% w/
v TCA solution, final concentration 5% TCA in the medium) was mixed
with 3-part washed blood lysate to precipitate proteins. Supernatants
were stored at − 80 ◦ C. Serum separating tubes for SH/SS homeostasis
parameters allowed to clot for 15 min and centrifuged. Serum samples
were stored at − 80 ◦ C. All supernatant and serum samples were thawed
on the day of measurement and laboratory analysis were made with an
automated analyzer (Cobas c501, Roche-Hitachi, Mannheim, Germany).
2.4. GSH/GSSG homeostasis parameters determination
GSH/GSSG homeostasis parameters were determined by the method
described by Alisik et al. [10]. GSH levels of supernatant samples were
measured using the Ellman method that was using 500 mM Tris solution
(pH: 8.2). GSSG in the 600 µL supernatant samples were reduced with
150 reagents µL including 3.5 M NaBH4 and 1.5 M NaOH to form GSH.
After the reduction procedure, 70 µL HCl solution was added to remove
the remnant NaBH4 in order to prevent extra-reduction of DTNB mole­
cules and re-oxidation of GSH molecules. Total GSH levels (including
native GSH contents and GSH from reduction of GSSG) were measured
using the Ellman method as measurement of GSH. Thiol residues of GSH
reduced the DTNB to 2-nitro-5-benzoic acid which has an absorbance at
412 nm spectrophotometrically. GSH content was subtracted from the
total GSH (GSH + GSSG) content and divided by two equals to the GSSG
amount. Results were expressed as µmol/L. GSSG/GSH percent ratios
were calculated.
2.5. SH/SS homeostasis parameters determination
Serum samples was used to evaluate SH/SS homeostasis parameters
by the method described by Erel and Neselioglu [19]. Briefly, firstly SH
contents of sample were measured with DTNB. Then, SSs were first
reduced by 10 mM NaBH4 to form free functional SHs. Formaldehyde
was used to remove excess unused sodium borohydride. After that, total
SH levels including both reduced and native SHs were determined with
the reaction of free functional SHs with DTNB. The amount of SS was
determined by taking half of the difference between serum total and
native SH levels. After the determination of SH, total SH, and SS
amounts, SS/SH percent ratios were calculated.
2.6. Statistical analysis
Statistical analysis was performed by IBM SPSS Statistics (Version
20) computer program (IBM, Armonk, NY, USA, 2011). Non-
parametrically distributed variables were expressed as median
(1st–3rd quartile value) and normally distributed variables were
expressed as mean ± standard deviation. Non-parametrically distributed
continuous variables were compared with the Mann-Whitney U test or
Kruskal-Wallis test. Post-hoc analysis of Kruskal-Wallis test was con­
ducted with Dunn-Bonferroni pairwise comparison test. Normally
M. Alisik et al. Clinical Biochemistry xxx (xxxx) xxx

distributed data compared using Student’s t test for two groups or and serum SH/SS homeostasis parameters between 84 RA patients who
ANOVA tests with post-hoc Bonferoni test for more than two groups. were treated with single cDMARDs, 40 combined cDMARDs, and 28 RA
Categorical variables were shown as number (%) and compared with patients who treated with combined bDMARDs and cDMARDs group (p
Pearson Chi-square test or Fisher’s exact test which was appropriate. > 0.05 for all) (Table 3).
Correlation analysis was conducted with Spearman test. P values at Correlation analysis between DAS28 score and intracellular GSH/
<0.05 were considered as significant. GSSG, and serum SH/SS homeostasis parameters and were shown in
Table 4. DAS28 scores were positively correlated with SS, GSSG, and SS/
3. Results SH and GSSG/GSH percent ratios (p < 0.01) and negatively correlated
with intracellular GSH levels (p < 0.001) although there is no correla­
One hundred fifty-two patients with RA and 89 healthy volunteers tion with serum SH levels (p = 0.814). Also, SS/SH percent ratio was
were enrolled in the study. The demographic data, clinical characteris­ significantly correlated with GSSG/GSH percent ratio (rho: 0.675, p <
tics, and GSH/GSSG and SH/SS homeostasis parameters of participants 0.001).
are shown in Table 1. No statistically significant difference was observed
in age and sex between groups (p > 0.05). GSH and SH levels were lower 4. Discussion
in the RA group when compared to controls (p = 0.005 and p = 0.001;
respectively). GSSG, GSSG/GSH percent ratio, SS and SS/SH percent In this study, both intracellular and extracellular oxidized thiol
ratio levels were significantly higher in the RA group (p < 0.001 for all). groups were found to be elevated concomitant with a decrease in
Table 2 shows the comparisons of demographic data, clinical char­ reduced thiols in RA patients. Intracellular GSSG/GSH and serum SS/SH
acteristics, GSSG/GSH and SH/SS homeostasis parameters between 73 ratios were increased in active RA patients compared to both remission
patients in active RA group, 79 patients in remission RA group and 89 RA patients and healthy controls. Likewise, intracellular GSSG/GSH and
controls. GSSG and GSSG/GSH percent ratio levels were significantly serum SS/SH ratios were higher in remission RA patients than healthy
higher and GSH levels were significantly lower in the active RA group volunteers. Along with this, despite low GSH levels in active RA patients
when compared to the remission RA group and control group (p < 0.05 compared to remission RA and control groups and SH levels in active RA
for all). GSSG level and GSSG/GSH percent ratio levels were signifi­ patients compared to control group, there was no significant difference
cantly higher in remission group compared to control group (p < 0.05 between remission RA and control groups in GSH and SH levels. A
for both). SS and SS/SH percent ratios levels were significantly higher in correlation was detected between DAS28 activity scores and GSSG/GSH
the active RA group when compared to the remission RA group and ratios and SS/SH ratios. Moreover, the correlation between GSSG/GSH
control group (p < 0.05 for all); and these levels were also significantly percent ratios and SS/SH percent ratios indicates that both intracellular
higher in the remission RA group than control group (p < 0.05 for all). and extracellular balances were similarly shifted towards the oxidized
There were no significant differences in the intracellular GSH/GSSG side. To the best of our knowledge, this study is the first study to
investigate both intracellular oxidized-reduced glutathione and extra­
cellular thiol-disulfide balance simultaneously, and to evaluate the thiol
Table 1 pool in RA patients in an integrated approach.
Comparison of demographic, clinical and reduced/oxidized glutathione (GSH/
Several mechanisms such as genetic factors, cytokines, cellular and
GSSG) and thiol/disulfide (SH/SS) homeostasis parameters of Rheumatoid
humoral immunity, oxidative stress were blamed in RA pathophysi­
Arthritis (RA) patients and healthy controls.
ology, however, it is not still fully enlightened [14]. Despite CD4+ T
RA (N = 152) Control (N = 89) P value cells, B cells, dendritic cells, mast cells and macrophages are known to
Age, years 51 (43–61) 51 (43–59) 0.419 take part in RA disease [20] neutrophils are thought to be the major
Gender Male 36 (23.7%) 17 (19.1%) 0.407 performer [21]. Neutrophils worsen the severity of the disease via
Female 116 (76.3%) 72 (80.9%)
increasing reactive oxygen species (ROS) production, and intensified
Disease duration, years 10 (7–12)
ESR, mm/h 15 (9–24) disease increase the ROS production forming a vicious circle [6].
Hemoglobin, g/dL 13.47 ± 1.26 13.43 ± 1.22 0.819 Furthermore, increased cytokines like TNF-α, IL-1, IL-6 in RA patients
DAS28 2.46(1.92–3.03) stimulate the ROS production [22]. Veselinovic et al. [23] found
Disease Remission 79 (52%)
significantly elevated levels of hydrogen peroxide superoxide anion in
activity Active 73 (48%)
Treatment cDMARD 84 (56.3%)
plasma of patients with RA. Similarly Kundu et al. [33] reported that
c+ 40 (26.3%) superoxide anion and hydroxyl radical radicals were significantly higher
cDMARDs in neutrophils from peripheral blood of patients with RA [24]. In this
b+ 28 (18.4%) study, similarly both intracellular and extracellular thiol pools shifted
cDMARDs
towards oxidative side and these findings are consistent with the hy­
Total Glutathione, μmol/L 898.3 919.4 0.197
(788.4–1068.4) (815–1104.7) pothesis that the oxidative mechanism may plays an important role in
Reduced Glutathione (GSH), 758.7 869.8 0.005 RA pathophysiology, or RA may cause increased oxidative stress and one
μmol/L (609.5–987.5) (737.8–992.1) of mechanisms of complications in RA may be this increased oxidation.
Oxidized Glutathione 69.8 34.9 <0.001 Imbalances in thiol/disulfide homeostasis are indicated in many
(GSSG), μmol/L (44.05–85.29) (22.43–62.2)
GSSG/GSH ratio, % 9.2 (4.44–14.05) 4.88 (2.37–8.02) <0.001
autoimmune diseases like celiac disease, autoimmune subclinical hy­
Total Thiol, μmol/L 351.2 347.2 0.083 pothyroidism, Familial Mediterranean Fever and RA [14,25–27]. Im­
(309.2–379.3) (325.1–412.4) balances favoring the SS is evaluated as an indicator of oxidative
Native Thiol (SH), μmol/L 309.7 315.9 0.001 damage. Tuzcu et al. reported that RA patients have higher SS values
(269.2–335.6) (296.1–382.4)
compared to control (18.5 ± 7.2 vs. 13.1 ± 6.6 μmol/L) and lower SH
Disulfide (SS), μmol/L 21.75 15.45 <0.001
(19.09–24.5) (14.05–17.63) levels (282.3 ± 50.3 vs 339.8 ± 55.7 μmol/L) and the homeostasis
SS/SH ratio, % 6.94 (6.02–8.54) 4.62 (4.15–5.46) <0.001 shifted towards the SS and this was related to disease scores [14]. In a
study evaluating only SH side of the plasma SH/SS homeostasis, they
Data are expressed as median (1st–3rd quartiles) for continuous variables and n
(%) for categorical variables. Remission: RA patients with ≤2.6 DAS28 score, showed that serum SH groups were likewise significantly lower, and
Active disease: RA patients with >2.6 DAS28 score, cDMARD: Single medication even lower thiol levels in active RA patients [28]. In another study,
with a Conventional Disease Modifying Anti-Rheumatic Drug, c + cDMARDs: decreased plasma GSH levels and negative correlation between GSH
medication with combined at least two cDMARDs, b + cDMARDs: combined levels and DAS28 scores were detected [29]. Similarly, our results
biologic and convantional DMARDs. demonstrated that serum SH/SS homeostasis shifted towards the SS side

3
M. Alisik et al. Clinical Biochemistry xxx (xxxx) xxx

Table 2
Comparison of demographic, clinical and reduced/oxidized glutathione (GSH/GSSG) and thiol/disulfide (SH/SS) homeostasis parameters in Rheumatoid Arthritis
(RA) patients sub-grouped according to the disease activity score-28 (DAS28) and healthy control.
Active RA Remission RA Control p value

Age, years 53 (44.5–61) 50 (42–62) 51 (43–59) 0.326

Gender Male 18 (24.7%) 18 (22.8%) 17 (19.1%) 0.682
Female 55 (75.3%) 61 (77.2%) 72 (80.9%)
Disease duration, years 10 (8–12) 10 (7–12) 0.590
ESR, mm/h 22 (15.5–33.5) 10 (5–15) <0.001
DAS28 3.02 (2.62–3.54) 2.06 (1.54–2.42) <0.001
Treatment cDMARD 37 (50.7%) 47 (59.5%) 0.368
c + cDMARDs 23 (31.5%) 17 (21.5%)
b + cDMARDs 13 (17.8%) 25 (19%)
Hemoglobin, g/dL 13.30 ± 1.23 13.63 ± 1.26 13.43 ± 1.22 0.255
Total Glutathione, μmol/L 840.5 (745–944.7)*† 1020.6 (847.9–1132.1) 919.4 (815–1104.7) <0.001
Reducted Glutathione (GSH), μmol/L 683.3 (541.9–819.1)*† 918.1 (693–1063.4) 869.8 (737.8–992.1) <0.001
Oxidized Glutathione (GSSG), μmol/L 78.3 (62.8–93.5)*† 54.25 (36.3–76.7)† 34.9 (22.43–62.2) <0.001
GSSG/GSH ratio, % 11.5 (7.67–17.91)*† 5.58 (3.47–11.18)† 4.88 (2.37–8.02) <0.001
Total Thiol, μmol/L 342.4 (293.5–377.7) 357.7 (327.8–379.7) 347.2 (325.1–412.4) 0.055
Native Thiol (SH), μmol/L 292.4 (251–326.1)† 316 (284.9–337.9) 315.9 (296.1–382.4) <0.001
Disulfide (SS), μmol/L 23.3 (20.88–25.48)*† 19.75 (18.1–23)† 15.45 (14.05–17.63) <0.001
SS/SH ratio, % 8.18 (6.76–9.59)*† 6.45 (5.64–7.31)† 4.62 (4.15–5.46) <0.001

Data are expressed as median (1st–3rd quartiles) for continuous variables and n (%) for categorical variables. Remission: RA patients with ≤2.6 DAS28 score, Active:
RA patients with >2.6 DAS28 score, cDMARD: Single medication with a Conventional Disease Modifying Anti-Rheumatic Drug, c + cDMARDs: medication with
combined at least two cDMARDs, b + cDMARDs: combined biologic and convantional DMARDs. *: significantly differs from Remission RA group, †: significantly differs
from control group.

with an increase in SS and a decrease in SH for RA patients. Moreover, SS

Table 3
and SS/SH percent ratios positively correlated with DAS28 scores and
Comparison of reduced/oxidized glutathione (GSH/GSSG) and thiol/disulfide
the balance is further deteriorated in active RA patients. This condition
(SH/SS) homeostasis parameters in Rheumatoid Arthritis patients grouped ac­
shows serum protein oxidation in RA patients. Along with this, SS/SH
cording to their medications.
ratios and SS levels being elevated in remission RA group without a
cDMARD c + cDMARDs b + cDMARDs p
significant decrease in SH levels indicate that oxidation persists in
value
remission despite the recovery in the antioxidant side. This is supported
Total 923.2 869.4 935.5 0.335 with similar findings in intracellular GSH/GSSG balance.
Glutathione, (776.6–1073.8) (777.1–1030.3) (816.2–1144.9)
μmol/L
Glutathione is the most important intracellular antioxidant molecule
Reduced 791.1 720.9 807.1 0.384 [10]. There are various findings about intracellular GSH levels in RA
Glutathione (600.1–997.7) (600.7–930.7) (625–1080) patients. Staron et al. stated significantly low erythrocyte reduced GSH
(GSH), levels and thiol groups decreasing with oxidation may convert into di­
μmol/L
sulfide form in membrane proteins and increase the aggregation and
Oxidized 66.03 74.23 64.2 0.455
Glutathione (43.68–84.89) (49.79–88.16) (32.46–85.38) membrane permeability, however, they didn’t evaluate the GSSG levels
(GSSG), [13]. Attia et al. showed that blood GSH levels were significantly lower
μmol/L in RA patients and they also stated increment in GSH levels together
GSSG/GSH 8.35 10.3 7.96 0.462 with an improvement in disease scores with laser acupuncture [30].
ratio, % (4.37–14.2) (5.35–14.68) (3.01–13.67)
Total Thiol, 348.3 348.9 366.8 0.383
Likewise, Mateen et al. demonstrated low blood GSH levels in RA pa­
μmol/L (323.4–369.8) (296.3–373.9) (310.6–399.7) tients and further lower levels in active RA patients [28]. In RA rat
Native Thiol 306.1 301.1 318.1 0.364 models, a decrease in GSH levels was also shown [31,32]. In consistent
(SH), μmol/ (279.7–325.1) (260.6–332.5) (268.8–354.5) with previous studies, intracellular GSSG/GSH homeostasis showed a
L
shift towards GSSG in RA patients, which was in a relation with disease
Disulfide (SS), 21.68 22.18 20.4 0.310
μmol/L (19.21–24.61) (19.46–24.8) (16.21–23.64) scores in the current study. As a result of these findings, the GSH/GSSG
SS/SH ratio, % 6.91 7.23 6.72 0.168 balance deteriorates in correlation with inflammation, suggesting that
(6.05–8.54) (6.36–9.22) (5.43–8.43) an antioxidant treatment approach that will restore intracellular thiol
Data are expressed as median (1st–3rd quartiles). cDMARD: Single medication balance may have the potential to slow the deterioration in the disease.
with a Conventional Disease Modifying Anti-Rheumatic Drug, c + cDMARDs: In addition, although a negative relationship was found between GSH,
medication with combined at least two cDMARDs, b + cDMARDs: combined an intracellular antioxidant molecule, and disease activity scores in our
biologic and convantional DMARDs. study, no such relationship was found between serum antioxidant thiols.
These indicate that although oxidized thiols in both intracellular and
extracellular thiol pools are associated with disease severity, only
intracellular thiols in the antioxidant thiol pool are associated with

Table 4
Correlation analysis between DAS28 score and reduced/oxidized glutathione (GSH/GSSG) and thiol/disulfide (SH/SS) homeostasis parameters in Rheumatoid
Arthritis patients.
Total Glutathione, μmol/L GSH, μmol/L GSSG, μmol/L GSSG/GSH ratio, % Total Thiol, μmol/L SH, μmol/L SS, μmol/L SS/SH ratio, %

DAS28 rho − 0.298 − 0.297 0.290 0.296 0.043 − 0.019 0.355 0.259
p <0.001 <0.001 <0.001 <0.001 0.601 0.814 <0.001 0.001

rho: Spearman correlation coefficient, DAS28: Disease Activity Score-28.

4
M. Alisik et al.
disease severity, and intracellular thiol oxidation becomes more severe
as the disease score increases. These findings suggest that preserving the
intracellular thiol pool is more important due to further impairment. In a
rat study, low GSH levels and high GSSG/GSH ratio were detected in
brain samples of rats with arthritis [22]. On the contrary, another study
stated elevated blood GSH levels without significantly different GSSG
levels in RA patients. However, in that study, active RA patients showed
significantly lower GSH levels compared to patients in remission and
GSSG/GSH ratio shift towards GSSG [33]. Alver et al. also found
erythrocyte GSH levels increased in RA patients [34]. Different results of
GSH levels in the studies may be due to elevation of antioxidant enzymes
like glutathione reductase, with a co-elevation in oxidant levels suffi­
cient or insufficient to saturate these antioxidant mechanisms.
In various studies, DMARDs’ antioxidant activity was stated. Also, it
was suggested to assess oxidative stress as an indicator of treatment
response [35]. Dogru et al., compared neutrophile GSH levels in meth­
otrexate and infliximab treatments and did not find a significant dif­
ference [6]. ROS is known as a second messenger of nuclear factor
kappa-B (NF-κB), that is a transcription factor and involved in the
expression of various inflammatory mediators [36]. Inflammatory me­
diators like TNF-α, IL-1, IL-6 in RA patients stimulate the ROS produc­
tion [22]. Thus, occurs a vicious cycle between ROS and inflammatory
mediators. Also, DMARDs target to recover that inflammation in RA
[37]. In this study, no significant difference was observed in intracel­
lular GSH/GSSG and serum SH/SS homeostasis parameters’ levels in the
treatment groups. It was though that, decreasing inflammatory media­
tors or breaking the vicious cycle between inflammation and oxidative
stress by DMARDs may control the further oxidative stress.
In this study, a decrement in both serum dynamic thiol groups and
dynamic disulfide levels were detected. Batooei et al. conducted a ran­
domized, double blind clinical trial administrating N-acetyl cystein with
an active thiol group, resulting in similar disease activity scores in
intervention and control groups. However, they also established a sig­
nificant amelioration in Global Health, Health Assessment Question­
naire, and Visual Analog Scales [1]. In this trial, despite not assessing
any oxidation markers including thiol or GSH levels, the therapeutic
effect of N-acetyl cysteine (NAC) may be due to its positive effects on the
imbalance in thiol homeostasis as shown in our study.
The study has some limitations. The most important limitation of the
study is that quite active RA patients, a significant part of the RA pop­
ulation, and those with additional systemic diseases (such as diabetes
mellitus, hypertension) that may affect GSH/GSSG and SH/SS homeo­
stasis were not included in the study. Therefore, in this study, in which
we aimed to investigate the GSH/GSSG and SH/SS homeostasis in iso­
lated RA patients, the formation of a selected cohort of participants
limits the generalizability of the study results. The another limitation of
our study is that there is no untreated RA group and therefore the drug
effect cannot be ignored. In addition, the fact that the study had cross-
sectional design, and no comparison with other intracellular and
extracellular oxidant-antioxidant balance markers (such as malondial­
dehyde, total antioxidant capacity, total antioxidant capacity, catalase,
superoxide dismutase) were other limitations.
In conclusion, deteriorated intracellular GSH/GSSG and serum SH/
SS balances may play important role in pathogenesis of RA disease. In
this context, further work in these areas may be warranted to determine
potential value of evaluating intracellular GSSG/GSH and serum SH/SS
balances together in disease monitoring.
5. Consent to participate
Informed consent was obtained from all individual participants
included in the study.
Funding
This research did not receive any specific grant from funding
5
Clinical Biochemistry xxx (xxxx) xxx
agencies in the public, commercial, or not-for-profit sectors.
Ethical approval
This study was performed in line with the principles of the Decla­
ration of Helsinki. Approval was granted by the Ethics Committee of
Yildirim Beyazit University (Ethics approval number: 26379996/111).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
References
[1] M. Batooei, A. Tahamoli-Roudsari, Z. Basiri, F. Yasrebifar, M. Shahdoust,
A. Eshraghi, M. Mehrpooya, S. Ataei, Evaluating the effect of oral N-acetylcysteine
as an adjuvant treatment on clinical outcomes of patients with rheumatoid
arthritis: a randomized, double blind clinical trial, Rev. Rec. Clin. Trials 13 (2)
(2018) 132–138.
[2] S. Luo, H. Li, J. Liu, X. Xie, Z. Wan, Y. Wang, Z. Zhao, X. Wu, X. Li, M. Yang, X. Li,
Andrographolide ameliorates oxidative stress, inflammation and histological
outcome in complete Freund’s adjuvant-induced arthritis, Chem. Biol. Interact. 319
(2020), 108984.
[3] X. Xie, H. Li, Y. Wang, Z. Wan, S. Luo, Z. Zhao, J. Liu, X. Wu, X. Li, X. Li,
Therapeutic effects of gentiopicroside on adjuvant-induced arthritis by inhibiting
inflammation and oxidative stress in rats, Int. Immunopharmacol. 76 (2019),
105840.
[4] J. Alam, I. Jantan, S.N.A. Bukhari, Rheumatoid arthritis: Recent advances on its
etiology, role of cytokines and pharmacotherapy, Biomed. Pharmacother. 92
(2017) 615–633.
[5] J. Reglinski, D.E. Paterson, S. Latimer, J.M. Campbell, R. Wilson, D. Porter, R.
D. Sturrock, W.E. Smith, Myocrisin-mediated oxidative stress, Clin. Chim. Acta 268
(1–2) (1997) 85–99.
[6] A. Dogru, M. Naziroglu, B. Cig, Modulator role of infliximab and methotrexate
through the transient receptor potential melastatin 2 (TRPM2) channel in
neutrophils of patients with rheumatoid arthritis: a pilot study, Arch. Med. Sci. 15
(6) (2019) 1415–1424.
[7] O.S. León Fernández, R. Viebahn-Haensler, G.L. Cabreja, I.S. Espinosa, Y.H. Matos,
L.D. Roche, B.T. Santos, G.T. Oru, J.C. Polo Vega, Medical ozone increases
methotrexate clinical response and improves cellular redox balance in patients
with rheumatoid arthritis, Eur. J. Pharmacol. 789 (2016) 313–318.
[8] P. Nagy, Kinetics and mechanisms of thiol-disulfide exchange covering direct
substitution and thiol oxidation-mediated pathways, Antioxid. Redox Signal. 18
(13) (2013) 1623–1641.
[9] V.I. Lushchak, Glutathione homeostasis and functions: potential targets for medical
interventions, J. Amino Acids 2012 (2012).
[10] M. Alisik, S. Neselioglu, O. Erel, A colorimetric method to measure oxidized,
reduced and total glutathione levels in erythrocytes, J. Lab. Med. 43 (5) (2019)
269–277.
[11] C. Gaucher, A. Boudier, J. Bonetti, I. Clarot, P. Leroy, M. Parent, Glutathione:
antioxidant properties dedicated to nanotechnologies, Antioxidants (Basel,
Switzerland) 7 (5) (2018) 62.
[12] M. Alışık, M.U. Işik, The relationship between choroidal thickness and intracellular
oxidised-reduced glutathione and extracellular thiol–disulfide homeostasis at
different stages of diabetic retinopathy, Curr. Eye Res. (2020) 1–6.
[13] A. Staroń, G. Mąkosa, M. Koter-Michalak, Oxidative stress in erythrocytes from
patients with rheumatoid arthritis, Rheumatol. Int. 32 (2) (2012) 331–334.
[14] A. Tuzcu, R.A. Baykara, A. Omma, G.K. Acet, E. Dogan, M.C. Cure, S.C. Sandikci,
E. Cure, S. Neselioglu, O. Erel, Thiol/disulfide homeostasis in patients with
rheumatoid arthritis, Roman. J. Intern. Med. 57 (1) (2019) 30–36.
[15] J.H. Pedersen-Lane, R.B. Zurier, D.A. Lawrence, Analysis of the thiol status of
peripheral blood leukocytes in rheumatoid arthritis patients, J. Leukoc. Biol. 81 (4)
(2007) 934–941.
[16] D. Aletaha, T. Neogi, A.J. Silman, J. Funovits, D.T. Felson, C.O. Bingham 3rd, N.
S. Birnbaum, G.R. Burmester, V.P. Bykerk, M.D. Cohen, B. Combe, K.
H. Costenbader, M. Dougados, P. Emery, G. Ferraccioli, J.M. Hazes, K. Hobbs, T.
W. Huizinga, A. Kavanaugh, J. Kay, T.K. Kvien, T. Laing, P. Mease, H.A. Menard, L.
W. Moreland, R.L. Naden, T. Pincus, J.S. Smolen, E. Stanislawska-Biernat,
D. Symmons, P.P. Tak, K.S. Upchurch, J. Vencovsky, F. Wolfe, G. Hawker, 2010
Rheumatoid arthritis classification criteria: an American College of Rheumatology/
European League Against Rheumatism collaborative initiative, Arthritis Rheum. 62
(9) (2010) 2569–2581.
[17] J.S. Smolen, F.C. Breedveld, G. Eberl, I. Jones, M. Leeming, G.L. Wylie,
J. Kirkpatrick, Validity and reliability of the twenty-eight-joint count for the
assessment of rheumatoid arthritis activity, Arthritis Rheum. 38 (1) (1995) 38–43.
[18] J. Fransen, M.C. Creemers, P.L. Van Riel, Remission in rheumatoid arthritis:
agreement of the disease activity score (DAS28) with the ARA preliminary
remission criteria, Rheumatology (Oxford, England) 43 (10) (2004) 1252–1255.
M. Alisik et al.
[19] O. Erel, S. Neselioglu, A novel and automated assay for thiol/disulphide
homeostasis, Clin. Biochem. 47 (18) (2014) 326–332.
[20] D.J. Veale, C. Orr, U. Fearon, Cellular and molecular perspectives in rheumatoid
arthritis, Semin. Immunopathol. 39 (4) (2017) 343–354.
[21] N. Thieblemont, H.L. Wright, S.W. Edwards, V. Witko-Sarsat, Human neutrophils
in auto-immunity, Semin. Immunol. 28 (2) (2016) 159–173.
[22] H.V. Pereira-Marostica, L.S. Castro, G.A. Goncalves, F.M.S. Silva, L. Bracht, C.
A. Bersani-Amado, R.M. Peralta, J.F. Comar, A. Bracht, A.B. Sa-Nakanishi, Methyl
jasmonate reduces inflammation and oxidative stress in the brain of arthritic rats,
Antioxidants (Basel Switzerland) 8 (10) (2019).
[23] M. Veselinovic, N. Barudzic, M. Vuletic, V. Zivkovic, A. Tomic-Lucic, D. Djuric,
V. Jakovljevic, Oxidative stress in rheumatoid arthritis patients: relationship to
diseases activity, Mol. Cell. Biochem. 391 (1–2) (2014) 225–232.
[24] S. Kundu, P. Ghosh, S. Datta, A. Ghosh, S. Chattopadhyay, M. Chatterjee, Oxidative
stress as a potential biomarker for determining disease activity in patients with
rheumatoid arthritis, Free Radic. Res. 46 (12) (2012) 1482–1489.
[25] M. Kaplan, I. Ates, M. Yuksel, Y.O. Ozin, M. Alisik, O. Erel, E. Kayacetin, Thiol/
disulphide homeostasis in celiac disease, World J. Gastrointest. Pharmacol. Ther. 8
(2) (2017) 120.
[26] I. Ates, M. Altay, F.M. Yilmaz, C. Topcuoglu, S. Neselioglu, O. Erel, N. Yilmaz,
Dynamic thiol/disulfide homeostasis in patients with autoimmune subclinical
hypothyroidism, Endocr. Res. 41 (4) (2016) 343–349.
[27] A. Omma, S.C. Sandikci, O. Kücüksahin, M. Alisik, O. Erel, Can the thiol/disulfide
imbalance be a predictor of colchicine resistance in familial mediterranean fever?
J. Korean Med. Sci. 32 (10) (2017) 1588–1594.
[28] S. Mateen, S. Moin, A.Q. Khan, A. Zafar, N. Fatima, Increased reactive oxygen
species formation and oxidative stress in rheumatoid arthritis, PLoS One 11 (4)
(2016) e0152925–e0152925.
Clinical Biochemistry xxx (xxxx) xxx
[29] D. Shah, A. Wanchu, A. Bhatnagar, Interaction between oxidative stress and
chemokines: possible pathogenic role in systemic lupus erythematosus and
rheumatoid arthritis, Immunobiology 216 (9) (2011) 1010–1017.
[30] A.M. Attia, F.A. Ibrahim, N.A. Abd El-Latif, S.W. Aziz, A.M. Elwan, A.A. Abdel Aziz,
A. Elgendy, F.T. Elgengehy, Therapeutic antioxidant and anti-inflammatory effects
of laser acupuncture on patients with rheumatoid arthritis, Lasers Surg. Med. 48
(5) (2016) 490–497.
[31] J.M. Zhao, X. Chen, K. Cheng, Q. Shi, K. Peng, Anserine and glucosamine
supplementation attenuates the levels of inflammatory markers in rats with
rheumatoid arthritis, AMB Express 10 (1) (2020) 57.
[32] X. Li, P. Xie, Y. Hou, S. Chen, P. He, Z. Xiao, J. Zhan, D. Luo, M. Gu, D. Lin,
Tangeretin inhibits oxidative stress and inflammation via upregulating Nrf-2
signaling pathway in collagen-induced arthritic rats, Pharmacology 104 (3–4)
(2019) 187–195.
[33] A. Garcia-Gonzalez, R. Gaxiola-Robles, T. Zenteno-Savin, Oxidative stress in
patients with rheumatoid arthritis, Rev. Invest. Clin. 67 (1) (2015) 46–53.
[34] A. Alver, A. Senturk, H. Cakirbay, A. Mentese, F. Gokmen, E.E. Keha, F. Ucar,
Carbonic anhydrase II autoantibody and oxidative stress in rheumatoid arthritis,
Clin. Biochem. 44 (17–18) (2011) 1385–1389.
[35] N.T. Costa, T.M.V. Iriyoda, D.F. Alfieri, A.N.C. Simao, I. Dichi, Influence of disease-
modifying antirheumatic drugs on oxidative and nitrosative stress in patients with
rheumatoid arthritis, Inflammopharmacology 26 (5) (2018) 1151–1164.
[36] S. Mateen, S. Moin, S. Shahzad, A.Q. Khan, Level of inflammatory cytokines in
rheumatoid arthritis patients: correlation with 25-hydroxy vitamin D and reactive
oxygen species, PLoS ONE 12 (6) (2017), e0178879.
[37] Y.-J. Lin, M. Anzaghe, S. Schülke, Update on the pathomechanism, diagnosis, and
treatment options for rheumatoid arthritis, Cells 9 (4) (2020) 880.