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1 23
International Journal of
Biometeorology
ISSN 0020-7128
Int J Biometeorol
DOI 10.1007/s00484-016-1201-4
Effect of spa therapy with saline
balneotherapy on oxidant/antioxidant
status in patients with rheumatoid
arthritis: a single-blind randomized
controlled trial
Mine Karagülle, Sinan Kardeş, Oğuz
Karagülle, Rian Dişçi, Aslıhan Avcı,
İlker Durak & Müfit Zeki Karagülle
1 23
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ORIGINAL PAPER
Effect of spa therapy with saline balneotherapy
on oxidant/antioxidant status in patients with rheumatoid
arthritis: a single-blind randomized controlled trial
Mine Karagülle
1
&Sinan Kardeş
1
&Oğuz Karagülle
2
&Rian Dişçi
3
&Aslıhan Avcı
4
&
İlker Durak
4
&Müfit Zeki Karagülle
1
Received: 2 March 2016 /Revised: 1 June 2016 / Accepted: 15 June 2016
#ISB 2016
Abstract Oxidative stress has been shown to play a contrib-
utory role in the pathogenesis of rheumatoid arthritis (RA).
Recent studies have provided evidence for antioxidant prop-
erties of spa therapy. The purpose of this study is to investigate
whether spa therapy with saline balneotherapy has any influ-
ence on the oxidant/antioxidant status in patients with RA and
to assess clinical effects of spa therapy. In this investigator-
blind randomized controlled trial, we randomly assigned 50
patients in a 1:1 ratio to spa therapy plus standard drug treat-
ment (spa group) or standard drug treatment alone (control
group). Spa group followed a 2-week course of spa therapy
regimen consisting of a total of 12 balneotherapy sessions in a
thermal mineral water pool at 36–37 °C for 20 min every day
except Sunday. All clinical and biochemical parameters were
assessed at baseline and after spa therapy (2 weeks). The clin-
ical parameters were pain intensity, patient global assessment,
physician global assessment, Health Assessment
Questionnaire disability index (HAQ-DI), Disease Activity
Score for 28-joints based on erythrocyte sedimentation rate
(DAS28–4[ESR]). Oxidative status parameters were
malondialdehyde (MDA), nonenzymatic superoxide radical
scavenger activity (NSSA), antioxidant potential (AOP), and
superoxide dismutase (SOD). The NSSA levels were in-
creased significantly in the spa group (p= 0.003) but not in
the control group (p= 0.509);and there was a trend infavor of
spa therapy for improvements in NSSA levels compared to
control (p= 0.091). Significant clinical improvement was
found in the spa group compared to the control in terms of
patient global assessment (p= 0.011), physician global assess-
ment (p= 0.043), function (HAQ-DI) (p= 0.037), disease
activity (DAS28–4[ESR]) (0.044) and swollen joint count
(0.009), and a trend toward improvement in pain scores
(0.057). Spa therapy with saline balneotherapy exerts antiox-
idant effect in patients with RA as reflected by the increase in
NSSA levels after spa therapy; whether this antioxidant effect
contributes to the clinical improvements observed remains to
be verified.
Keywords Spa therapy .Balneotherapy .Rheumatoid
arthritis .Salt water .Oxidative stress .Antioxidant effect
Abbreviations
AOP Antioxidant potential
CO
2
Carbon dioxide
CRP C-reactive protein
DAS28–4 Disease Activity Score for 28-joints of
4variables
DMARD Disease-modifying antirheumatic drug
ESR Erythrocyte sedimentation rate
H
2
S Hydrogen sulfide
HAQ-DI Health Assessment Questionnaire
Disability Index
MDA Malondialdehyde
NaCl Sodium chloride
NBT Nitroblue tetrazolium
*Mine Karagülle
mkgulle@istanbul.edu.tr
1
Department of Medical Ecology and Hydroclimatology, İstanbul
Faculty of Medicine, İstanbul University, Tıbbi Ekoloji ve
Hidroklimatoloji A.B.D. İstanbul Tıp Fakültesi Fatih, Capa,
34093 İstanbul, Turkey
2
Fachklinik Am Hasenbach, Claustinal-Zellerfed, Germany
3
Department of Biostatistics, İstanbul Faculty of Medicine, İstanbul
University, İstanbul, Turkey
4
Department of Biochemistry, Ankara University Faculty of
Medicine, Ankara, Turkey
Int J Biometeorol
DOI 10.1007/s00484-016-1201-4
Author's personal copy
NSSA Nonenzymatic superoxide
radical scavenger activity
RA Rheumatoid arthritis
RNS Reactivenitrogenspecies
ROS Reactiveoxygenspecies
SD Standard deviation
SOD Superoxide dismutase
TCA Trichloroacetic acid
TNF Tumor necrosis factor
VAS Visual analog scale
Introduction
Rheumatoid arthritis (RA) is an autoimmune inflammatory
disease characterized by synovial inflammation and hyperpla-
sia, cartilage and bone destruction, and any of several system-
ic manifestations including cardiovascular or pulmonary dis-
orders (McInnes and Schett 2011). With regard to the patho-
genesis of RA, several lines of evidence suggest that oxidative
stress plays a contributory role (Hitchon and El-Gabalawy
2004). Oxidative stress is an imbalance between oxidants
and capacity of antioxidant defense systems.Normal metabol-
ic processes in cells generate free radicals, reactive oxygen/
nitrogen species (ROS/RNS), and their derivatives. These ox-
idant products may cause damage to cell membranes, lipids,
nucleic acids, proteins, and matrix components. To protect
cells from these damages, several endogenous antioxidant de-
fense mechanisms have evolved. Once the generation of oxi-
dant products exceeds the capacity of antioxidant defense
mechanisms to neutralize them, oxidative stress occurs and
causes damage to cellular components (Hitchon and El-
Gabalawy 2004). Previous studies have demonstrated in-
creased oxidative enzyme activity, along with decreased anti-
oxidant levels in patients with RA (Oztürk et al. 1999;Cimen
et al. 2000; Ozkan et al. 2007; Seven et al. 2008;Kocabaset
al. 2010; Stamp et al. 2012). Moreover, tumor necrosis factor
(TNF) inhibitors and tocilizumab, which are commonly used
biologic agents in RA, have shown to reduce the oxidative
stress marker levels in patients with RA (Kageyama et al.
2008; Hirao et al. 2012).
Spa therapy involves all medical activities that are originat-
ed and employed in spa resorts and is aimed at health promo-
tion, prevention, therapy, and rehabilitation (Gutenbrunner
et al. 2010). Spa therapy regimens involve balneotherapy,
the immersion in thermal (with a temperature of 36–38 °C)
and/or mineral (with high mineral content) water, as the cen-
tral treatment modality (Karagülle and Karagülle 2015). In
many European countries as well as in Turkey, spa therapy
is widely used as a relevant part of the health care systems for
the treatment and rehabilitation of rheumatic and musculo-
skeletal diseases including RA (Karagülle and Karagülle
2015,2004; Karagülle et al. 2016; Yurtkuran et al. 1999).
Interestingly, in a recent clinical practice guideline for the
non-drug management of RA, spa therapy was recommended
to patients with stable or long-established and non-progressive
RA (grade C) but not indicated when RA is active (profession-
al agreement) (Forestier et al. 2009). An increasing number of
studies have provided evidence—although insufficient
(Verhagen et al. 2015;Santosetal.2015)—for a therapeutic
effect of spa therapy in patients with RA particularly
balneotherapy with radon, sulfur or saline waters (Steiner
et al. 1979; Yurtkuran et al. 1999; Sukenik et al. 1990a,b,
1995; Elkayam et al. 1991; Franke et al. 2000,2007; Codish
et al. 2005; Staalesen Strumse et al., 2009;Caporali et al.
2010; Annegret and Thomas 2013), but very little is known
about the mechanisms of action by which spa therapy im-
proves symptoms of RA (Fioravanti et al. 2011a; Bender
et al. 2005; Grabski et al. 2004; Markovićet al. 2009;
Kloesch et al. 2012).
An in-vitro study investigating the effects of sulfur water in-
cubation on antioxidant enzymes in erythrocytes obtained from
RA patients can be found in the literature (Grabski et al. 2004).
Furthermore, in several clinical studies, the effects of balneo-spa
therapy on oxidant/antioxidant status have been investigated:
sulfur balneotherapy in osteoarthritis patients (Ekmekcioglu et
al. 2002;Jokićet al. 2010; Benedetti et al. 2010; Mourad and
Harzy 2012), carbon dioxide balneotherapy in patients with pe-
ripheral occlusive arterial disease (Dogliotti et al. 2011), and
balneotherapy with alkali-chloridic-hydrogen carbonic and
slightly iodine, alkali-hydrogen carbonic waters in patients with
chronic lumbar complaints (Bender et al. 2007). Mostly an en-
hancement of total antioxidant status and attenuation of oxidant
release have been reported in these studies (Ekmekcioglu et al.
2002;Loosetal.2006;Benderetal.2007;Jokićet al. 2010;
Benedetti et al. 2010;Oláhetal.2010,2011; Dogliotti et al.
2011; Mourad and Harzy 2012). In this context, it is conceivable
that saline balneotherapy might have antioxidant properties in
RA patients, and that this would be associated with improved
clinical outcomes. However, no in vivo or in vitro studies have
yet investigated the effects of saline balneotherapy on oxidant/
antioxidant status in patients with RA.
We aimed to test whether 2-week course of spa therapy
with saline balneotherapy has any influence on the oxidant/
antioxidant status in patients with RA. We also investigated
the clinical effects of spa therapy in RA patients.
Method
Trial design
This was a single-blind randomized controlled clinical trial
and is a part of a trial evaluating the short-and long-term clin-
ical efficacy of spa therapy in patients with RA. It was con-
ducted in accordance with the Declaration of Helsinki and was
Int J Biometeorol
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approved by the Ethics Committee of the Istanbul Faculty of
Medicine. All participants provided written informed consent
before participating in the trial.
Participants
Eligible patients were 18years of age or older, had a diagnosis
of RA according to the American College of Rheumatology
(ACR) 1987 revised criteria (Arnett et al. 1988), and had al-
ready been treated with stable drug regimen (conventional
disease-modifying antirheumatic drug (DMARD), glucocorti-
coids) for 3 months or more. Key exclusion criteria were,
receiving antioxidant supplements, smoking, spa therapy
within the previous 1 year, changes in DMARD or glucocor-
ticoids during the previous 3 months, and general contraindi-
cation to spa therapy.
Randomization and blinding
Eligible patients were randomized to spa therapy plus standard
drug treatment (spa group) or standard drug treatment alone
(control group). Patients were assigned in a 1:1 ratio using a
computer-generated randomization list prepared by an inde-
pendent biostatistician. The clinical assessorand the analyzing
laboratory were blinded to the group assignments. To maintain
the blinding, both groups were assessed on predetermined
days at the Department of Medical Ecology and
Hydroclimatology, Istanbul University, and patients were
instructed not to reveal their group assignment to the assessor.
Interventions
Spa therapy The intervention took place at Tuzla Spa,
Istanbul. Patients in the spa group traveled together to this
spa facility, and stayed there for 2 weeks. They followed a
spa therapy regimen consisting of a total of 12 balneotherapy
sessions in a thermal mineral water pool at 36–37 °C for
20 min every day except Sunday. During balneotherapy ses-
sion, patients were free to move in the pool but were advised
to stay passive and were not allowed to exercise or swim. The
thermal mineral water, which is used in balneotherapy at that
spa facility is saline water-rich in salt (1.9 g/L sodium chloride
(NaCl)) and contains other minerals predominantly magne-
sium and calcium (Table 1).
Standard drug treatment Both groups were instructed to
continue their previous stable drug regimen (conventional
DMARD including methotrexate, hydroxychloroquine,
leflunomide, or sulfasalazine; glucocorticoids). Patients were
allowed to continue their non-steroidal anti-inflammatory
drugs (NSAIDs) whenever they felt it necessary.
Outcome measures
All clinical and biochemical parameters were assessed at base-
line and after spa therapy (2 weeks).
Clinical outcomes
Patient’s assessment of pain, patient global assessment of dis-
ease, and physician global assessment of disease were evalu-
ated with a 100-mm visual analog scale (VAS) where 0
Tabl e 1 Physicochemical properties of water used in balneotherapy at
Tuzla Spa
Characteristic Value
pH 6.94
Conductivity (EC) 4400 μS/cm
Hardness 104.0 °fH
Cations
Sodium (Na
+
) 760.509
Calcium (Ca
2+
) 221.783
Magnesium (Mg
2+
)117.904
Potassium (K
+
) 23.460
Ferrous iron (Fe
2+
)0.590
Manganese (Mn
2+
)0.200
Ammonium (NH
4+
)0.065
Aluminum (Al
3+
)0.000
Zinc (Zn
2+
)0.000
Nickel (Ni
2+
)0.000
Subtotal 1124.510
Anions
Chloride (CI
−
) 1456.286
Bicarbonate (HCO
3
−
) 417.850
Sulfate (SO
4
−
) 345.000
Bromide (Br
−
)1.900
Nitrate (NO
3
−
)0.960
Fluoride (F
−
)0.820
Iodide (I
−
)0.120
Nitrite (NO
2
−
)0.000
Hydrogen phosphate (HPO
42−
)0.000
Carbonate (CO
32−
)0.000
Hydrogen arsenate (HAsO
4
−
)0.000
Sulfide (S
2−
)0.000
Subtotal 2222.936
Carbon dioxide (CO
2
)0.000
Metaboric acid (HBO
2
−
)3.245
Metasilicic acid (H
2
SiO
3
) 16.765
Total 3367.456
Concentrations of chemical constituents are expressed in mg/l. Analysis
was performed at the Balneology Laboratory of the Department of
Medical Ecology and Hydroclimatology, Istanbul Faculty of Medicine
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indicates no pain or best and 100 indicates the most intense
pain imaginable or worst.
Health Assessment Questionnaire disability index
(HAQ-DI) was used to evaluate functional disability sta-
tus. Total scores of 0 to 1 indicate mild to moderate phys-
ical difficulty, 1 to 2 indicate moderate to severe disabil-
ity, and 2 to 3 indicate severe to very severe disability
(Bruce and Fries 2005).
Disease Activity Score for 28-joints of 4 variables
(DAS28–4) was used to evaluate the RA disease activ-
ity. The four variables are tender joint count among 28
specific joints; swollen joint count among the same 28
joints; patient global assessment of disease; and eryth-
rocyte sedimentation rate (ESR) or C-reactive protein
(CRP). Scores less than 2.6 indicate remission; scores
between 2.6 and 3.2 indicate low disease activity; be-
tween 3.2 and 5.1 indicate moderate disease activity and
scores greater than 5.1 indicate high disease activity
(Prevoo et al. 1995).
Biochemical analysis
Bloodsamplesweredrawnfromanantecubitalveininto
anticoagulated tubes, in the morning after an overnight fast.
These were centrifuged at2000 g for 5 min and were stored at
−80 °C until analysis. The samples were analyzed at the
Department of Biochemistry, Ankara University Faculty of
Medicine.
To measure the antioxidant potential (AOP) values of
the samples, they were first preincubated with fish oil and
a xanthine/xanthine oxidase system at room temperature
for 1 h, and then, the thiobarbituric acid-reactive sub-
stances levels were determined; thiobarbituric acid-
reactive substances formation was negatively correlated
with the AOP value in this method [Durak et al. 2000].
Malondialdehyde (MDA) levels were measured by thio-
barbituric acid-reactive substances method, in which the
reactionmediumenrichedwithfishoilsampleswereex-
posed to the superoxide radical (O
2
−
) produced by the
xanthine-xanthine oxidase system for 1 h, and then
MDA levels were determined [Dahle et al. 1962]. Non-
enzymatic superoxide radical scavenger activity (NSSA)
was measured in trichloroacetic acid (TCA)-treated frac-
tions method, in which proteins are first precipitated using
TCA solution (20 %, w/v), and then NSSA assay is per-
formed in the upper clear solution without protein [Durak
et al. 2000]. The superoxide dismutase (SOD) activity
was measured by the nitroblue tetrazolium (NBT) meth-
od: one unit of SOD activity was expressed as the amount
of enzyme protein producing 50 % inhibition in reduction
rate of NBT [Durak et al. 1998]. The AOP levels are
expressed as nmol/ml/h, MDA levels as nmol/ml, and
NSSA and SOD levels as U/ml.
Statistical methods
Descriptive characteristics of patients are presented as
number (percentage) for categorical variables and as
mean ± standard deviation (SD) or median (range) for
continuous variables. Baseline characteristics were com-
pared between the study groups with the use of the
Fisher’s exact test for categorical variables and either the
Student’sttest or Mann-Whitney Utest for continuous
variables. The clinical and biochemical outcome measures
are expressed as mean ± SD. The distribution of these
measures was tested by the Kolmogorov-Smirnov test.
After confirmation of normality with the Kolmogorov-
Smirnov test, parametric paired ttest for comparison of
changes within each group and Student’sttest for com-
parison of differences between groups were used. We used
Benjamini-Hochberg correction for multiple comparisons
for analysis of between-group differences (number of var-
iable was 7; false discovery rate was taken 0.07)
(Benjamini and Hochberg 1995). All statistical analyses
were performed with Statistical Package for the Social
Sciences (SPSS) for Windows version 21.0 (IBM SPSS
Statistics, IBM Corporation, Armonk, NY). pvalues less
than 0.05 were considered statistically significant.
Results
Study population
A total of 50 patients underwent randomization, and 25 were
assigned to each group. Two patients withdrew from the study
before the baseline assessment, eleven patients withdrew be-
fore receiving interventions and these patients were not in-
cluded in the analyses. All remaining patients received allo-
cated intervention and no patients were lost to follow-up
(Fig. 1).
Patient characteristics
The baseline characteristics of patients are summarized in
Table 1. The majority of patients were women (%94.6); the
mean age was 52.7 and the mean duration ofRA was 12.9. All
patients had been treated with conventional DMARD either
monotherapy (62.2 %) or combination. There were no signif-
icant differences in baseline characteristics between the drop-
outs and randomized or analyzed patients, and between ran-
domized and analyzed populations; and baseline characteris-
tics were similar between the two analyzed groups except for
ESR, which was higher in spa group (mean, 38.5 vs 22.0 mm/
h; p=0.005)(Table2).
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Intervention details
The mean number of total balneotherapy sessions was 11.0.
Seven patients had a total of twelve balneotherapy sessions,
four patients had eleven balneotherapy, two patients had ten
balneotherapy, one patient had nine and one patient had eight
balneotherapy sessions.
Clinical outcomes
The changes in clinical outcome measures are detailed in
Table 3.
Pain The VAS pain scores were decreased significantly
compared with baseline in spa group (p= 0.004) but not
in control group (p= 0.101). Although the difference be-
tween groups was not statistically significant, a trend in
favor of spa therapy for improvements in pain score was
found (mean difference −20.15, 95%CI −40.96 to 0.65,
p=0.057)(Table3).
Patient global assessment of disease Both groups showed
significant decrease in VAS patient global assessment scores
compared with baseline (spa therapy p< 0.001; control
p= 0.023), however spa group was superior compared to
control (mean difference −23.33, 95%CI −41.05 to −5.62,
p=0.011)(Table3).
Physician global assessment of disease The VAS physician
global assessment scores were decreased significantly com-
pared with baseline in spa group (p< 0.001) but not in control
group (p= 0.077). The spa group was superior compared to
control (mean difference −17.00, 95%CI −33.42 to −0.58,
p=0.043)(Table3).
Health assessment questionnaire disability index The
HAQ-DI scores were decreased significantly compared with
baseline in spa group (p< 0.001) but not in control group
(p= 0.101). The spa group was superior compared to control
(mean difference −0.34, 95%CI −0.66 to −0.02, p=0.037)
(Table 3).
Disease activity score for 28-joints of 4 variables Both
groups showed significant improvements in DAS28–4
(ESR) scores compared with baseline (spa therapy
p<0.001;controlp= 0.027), however spa group was superior
Assessed for eligibility (n=110)
Excluded (n=60)
Not meeting inclusion criteria (n= 38)
Declined to participate (n=13)
Other reasons (n= 9)
Analysed (n=15)
Excluded from analysis (n=0)
Lost to follow-up (n=0)
Discontinued intervention (n=0)
Allocated to spa therapy plus usual care(n=25)
Received allocated intervention (n=15)
Did not receive allocated intervention (n=10)
(time problem n=4; family problem n=3;
personal reason n=2; newly diagnosed
breast cancer n=1)
Lost to follow-up (n=0)
Discontinued intervention (n=0)
Allocated to usual care (n=25)
Received allocated intervention (n=22)
Did not receive allocated intervention (n=3)
(family problem n=2; personal reason n=1)
Analysed (n=22)
Excluded from analysis (n=0)
Randomized (n=50)
Fig. 1 Flow diagram of the study
population
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compared to control (mean difference −0.74, 95%CI −1.46 to
−0.02, p=0.044)(Table3).
Tender joint count (68 joints) Although mean tender joint
count was decreased significantly compared with baseline in
spa group (p= 0.014) but not in control group (p= 0.055), the
difference between groups was not statistically significant
(mean difference −1.38, 95%CI −10.98 to 8.21, p=0.772)
(Table 3).
Swollen joint count (66 joints) Themeanswollenjointcount
was decreased significantly compared with baseline in spa
group (p< 0.001) but not in control group (p= 0.464). The
spa group was superior compared to control (mean differ-
ence −8.39, 95%CI −14.54 to −2.24, p=0.009)(Table3).
Oxidant/antioxidant status
The changes in oxidant/antioxidant status are detailed in
Table 3.
Malondialdehyde There were no statistically significant
changes in MDA levels compared with baseline in spa group
(p= 0.525) or control group (p= 0.356). Additionally, the
difference between groups was not statistically significant
(mean difference 1.52, 95%CI −1.30 to 4.34, p=0.282)
(Table 4).
Nonenzymatic superoxide radical scavenger activity The
NSAA levels were in spa group (p= 0.003) but not in control
group (p= 0.509). Although the difference between groups
did not reach statistical significance, there was a trend in favor
of spa therapy for improvements in NSSA levels (mean dif-
ference 0.66, 95%CI −0.11 to 1.43, p=0.091)(Table4).
Antioxidant potential There were no statistically signifi-
cant changes in AOP levels compared with baseline in spa
group (p= 0.525) or control group (p=0.356).
Additionally, the difference between groups was not sta-
tistically significant (mean difference 1.52, 95%CI −1.30
to 4.34, p=0.34)(Table4).
Tabl e 2 Baseline characteristics of the study population
Characteristic Randomized patients (n= 50) Analyzed patients (n=37)
Spa group (n=23)
†
Control group(n=25) pVa l u e Sp a g r ou p (n= 15) Control group(n=22) pvalue
Age (years) 50.5 ± 10.6 52.5 ± 12.4 0.562 53.3 ± 11.1 52.3 ± 12.3 0.790
Female sex 21(91.3 %) 25 (100 %) 0.224 13 (86.7 %) 22 (100 %) 0.158
Duration of rheumatoid arthritis (years)
Mean 12.3 ± 11.7 14.1 ± 12.1 0.602 12.3 ± 12.9 13.4 ± 12.0 0.793
Median 8.5 (2–53) 10 (1–43) 0.616 8 (3–53) 10 (1–43) 0.725
Positive for rheumatoid factor 13 (56.5 %) 14 (56.0 %) 1.000 8 (53.3 %) 13 (59.1 %) 0.749
Conventional DMARD treatment
Monotherapy 13 (56.5 %) 18 (72.0 %) 0.367 8 (53.3 %) 15 (68.2 %) 0.493
Double therapy 10 (43.5 %) 7 (28.0 %) 7 (46.7 %) 7 (31.8 %)
Concomitant treatment
Glucocorticoids 9 (39.1 %) 8 (32.0 %) 0.764 6 (40 %) 7 (31.8 %) 0.730
NSAIDs 21 (91.3 %) 20 (80.0 %) 0.419 13 (86.7 %) 19 (86.4 %) 1.000
Tender and swollen joints
‡
Tender 34.5 ± 21.2 42.2 ± 23.2 0.249 37.8 ± 19.8 42.9 ± 23.4 0.489
Swollen 15.0 ± 10.3 15.6 ± 12.0 0.857 17.9 ± 9.7 16.2 ± 11.5 0.631
Pain VAS 63.2 ± 28.7 58.3 ± 25.0 0.537 64.3 ± 25.6 60.5 ± 23.1 0.634
Patient’s global assessment VAS 62.4 ± 23.7 60.7 ± 24.5 0.817 67.5 ± 18.4 61.5 ± 22.4 0.399
Physician’s global assessment VAS 57.4 ± 22.6 57.4 ± 22.7 0.994 64.3 ± 18.0 60.1 ± 21.4 0.533
HAQ-DI 1.4 ± 0.8 1.4 ± 0.7 0.782 1.3 ± 0.7 1.4 ± 0.8 0.676
DAS28–4 (ESR) 6.4 ± 1.0 6.0 ± 1.6 0.284 6.5 ± 0.9 5.9 ± 1.6 0.124
ESR (mm/h) 34.5 ± 17.4 22.8 ± 11.4 0.015 38.5 ± 18.0 22.0 ± 11.6 0.005
Categorical variables are n(%); continuous variables are mean ± SD or median (range). Means were compared with Student’sttest, medians with Mann-
Whitney Utest, and proportions with Fisher’s exact test. DMARD disease-modifying antirheumatic drug, NSAID non-steroidal anti-inflammatory drugs,
VAS visual analog scale, HAQ-DI Health Assessment Questionnaire Disability Index DAS28–4Disease Activity Score for 28-joints of 4 variables, ESR
erythrocyte sedimentation rate
†
Two patients withdrew from the study before the baseline assessment
‡
A total of 68 joints were evaluated for tenderness, and 66 joints were evaluated for swelling
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Superoxide dismutase There was no statistically signifi-
cant change in SOD levels after spa therapy (p= 0.062),
whereas control group showed significant decrease in
SOD levels compared with baseline (p=0.003).
However, this worsening was not statistically significant
between groups (mean difference 4.74, 95%CI −2.93 to
12.41, p=0.218)(Table4).
Safety
A total of three patients reported adverse events during spa
therapy. Two patients had flu-like symptoms; which caused
interruption of spa therapy for one day in these two patients.
One patient had headache that improved with NSAID and did
not cause the interruption of therapy.
Tabl e 3 The clinical outcomes of
the study population Outcome Spa group n= 15 Control group n= 22 Treatment difference pValue
§
Pain VAS
Before 64.33 ± 25.62 60.45 ± 23.07
After 34.00 ± 19.77 50.27 ± 29.94
Change −30.33 (−49.37 to −11.29) −10.18 (−22.5 to 2.15) −20.15 (−40.96 to 0.65) 0.057
pvalue
b
0.004 0.101
Physician global assessment VAS
Before 67.47 ± 18.41 61.50 ± 22.36
After 31.13 ± 21.45 48.50 ± 29.48
Change −27.00 (−39.43 to −14.57) −10.00 (−21.18 to 1.18) −17.00 (−33.42 to −0.58) 0.043
pValue
b
<0.001 0.077
Patient global assessment VAS
Before 64.33 ± 17.98 60.09 ± 21.44
After 37.33 ± 20.60 50.09 ± 27.90
Change −36.33 (−51.64 to −21.03) −13.00 (−24.07 to −1.93) −23.33 (−41.05 to −5.62) 0.011
pvalue
b
<0.001 0.023
HAQ-DI
Before 1.33 ± 0.68 1.43 ± 0.76
After 0.79 ± 0.64 1.23 ± 0.75
Change −0.53 (−0.74 to −0.32) −0.19 (−0.42 to 0.04) −0.34 (−0.66 to −0.02) 0.037
pvalue
‡
<0.001 0.101
DAS28–4(ESR)
Before 6.52 ± 0.88 5.88 ± 1.59
After 5.24 ± 1.32 5.34 ± 1.75
Change −1.28 (−1.85 to −0.71) −0.54 (−1.01 to −0.69) −0.74 (−1.46 to −0.02) 0.044
pvalue
‡
<0.001 0.027
Tender Joints
†
Before 37.80 ± 19.78 42.95 ± 23.42
After 29.60 ± 19.01 36.14 ± 23.09
Change −8.20 (−13.79 to 0.16) −6.82 (−13.79 to 0.16) −1.38 (−10.98 to 8.21) 0.772
pValue
‡
0.014 0.055
Swollen joints
†
Before 17.93 ± 9.68 16.18 ± 11.50
After 8.00 ± 5.22 14.64 ± 11.47
Change −9.93 (−14.32 to −5.86) −1.55 (−5.86 to 2.77) −8.39 (−14.54 to −2.24) 0.009
pvalue
‡
<0.001 0.464
Before and after variables are expressed as mean ± standard deviation; change and difference variables as mean
(95 % confidence interval); proportion variables as n (%).VAS visual analog scale, HAQ-DI Health Assessment
Questionnaire disability index, DAS28–4Disease Activity Score for 28-joints of 4 variables, ESR erythrocyte
sedimentation rate
†
A total of 68 joints were evaluated for tenderness, and 66 joints were evaluated for swelling
‡
Within groups. Paired ttest is used
§
Between groups. Student’sttest is used
Int J Biometeorol
Author's personal copy
Discussion
To our knowledge, this study is the first of its kind aimed to
test the effects of spa therapy with saline balneotherapy on the
oxidant/antioxidant status in patients with RA. We found that
spa therapy with saline balneotherapy significantly improved
the NSSA levels compared with baseline. Additionally, al-
though this improvement did not reach statistical significance
between groups, there was a trend in favor of spa therapy in
increasing NSSA levels compared with control. In addition to
this potential antioxidant effect of spa therapy, significant clin-
ical improvement was found in spa therapy group compared to
control in terms of function (HAQ-DI), disease activity
(DAS28–4 ESR) and swollen joint count, and a trend toward
improvement in pain scores. Similarly, spa therapy group was
superior to control group with respect to patient and physician
global assessment of disease.
Considering not onlybeneficial but alsothe harmful effects
of antioxidants, the clinical improvements observed by spa
therapy cannot necessarily be ascribed to changes in
oxidant/antioxidant status. An increase in ROS plays a con-
tributory role in the pathogenesis of RA (Hitchon and El-
Gabalawy 2004), and antioxidants and antioxidative enzymes
have shown to reduce cartilage damage in animal models of
RA (De Bandt et al., 2002; Zwerina et al. 2005; Wruck et al.
2011). On the other hand, recent studies have also shown
harmful effects of antioxidant supplements in healthy partici-
pants and patients with various diseases (Bjelakovic et al.
2012,2014).TheCochranesystematicreviewandmeta-
analysis that aimed to analyze the influence of antioxidant
supplements (i.e. beta-carotene,vitamin A, vitamin C, vitamin
E, and selenium) on all-cause mortality concluded that beta-
carotene and vitamin E seem toincrease mortality, and so may
higher doses of vitamin A (Bjelakovic et al. 2012). Therefore,
our findings on antioxidant status of the spa group should be
interpreted cautiously.
After the randomization using a computer-generated ran-
domization list; ESR levels were found to be higherin the spa
group at baseline. On the other hand, mean DAS28–4(ESR)
score was higher in spa group than control group, but this was
not statistically different. It seems that the statistical significant
difference in ESR levels was not as high as to reach statistical
significance in DAS28–4(ESR) scores. Furthermore, it also
seems that the scores of other three variables used in
Tabl e 4 Oxidant and antioxidant
parameters Parameter Spa glroup (n= 15) Control group (n= 22) Treatment difference pvalue
§
MDA
Before 2.91 ± 1.91 2.67 ± 1.44
After 3.82 ± 5.12 2.06 ± 2.76
Change 0.91 (−2.10 to 3.91) −0.60(−1.94 to 0.73) 1.52 (−1.30 to 4.34) 0.282
pvalue
‡
0.525 0.356
AOP
a
Before 13.27 ± 2.80 11.99 ± 3.18
After 13.24 ± 2.52 12.84 ± 2.69
Change −0.03(−1.44 to 1.39) 0.86(−1.14 to 2.85) −0.88 (−3.46 to 1.69) 0.490
pvalue
‡
0.968 0.381
NSSA
Before 7.92 ± 0.66 8.41 ± 0.79
After 8.77 ± 1.12 8.60 ± 1.17
Change 0.85 (0.33 to 1.37) 0.20 (−0.41 to 0.80) 0.66 (−0.11 to 1.43) 0.091
pvalue
‡
0.003 0.509
SOD
Before 41.65 ± 8.20 44.57 ± 11.78
After 37.37 ± 7.3 35.45 ± 6.11
Change −4.39(−9.02 to 0.25) −9.12(−14.83 to −3.42) 4.74 (−2.93 to 12.41) 0.218
pvalue
‡
0.062 0.003
MDA levels are expressed as nmol/ml, AOP levels as nmol/ml/h, NSSA and SOD levels as U/ml. Before and after
variables are expressed as mean ± standard deviation; change and difference variables as mean (95 % confidence
interval).MDA malondialdehyde, AOP antioxidant potential, NSSA nonenzymatic superoxide radical scavenger
activity, SOD superoxide dismutase
†
Data were available for 21 patients in the control group
‡
Within groups. Paired ttest is used
§
Between groups. Student’sttest is used
Int J Biometeorol
Author's personal copy
DAS28–4(ESR) calculation compensated the statistical sig-
nificant difference in ESR levels. However, we were not able
to exclude the probability that the higher inflammatory status
of RA patients who underwent spa therapy has influenced our
results on antioxidant activity since a correlation between the
ESR and oxidant/antioxidant status have been documented in
the literature (Sarban et al. 2005); in short, we could not ex-
clude the possibility of that high ESR levels would explain the
higher values in antioxidant activity.
Over the last decades, several randomized controlled stud-
ies have examined the effectiveness of spa therapy in patients
with RA, and in general beneficial therapeutic results in short
and long-term reported particularly with balneotherapy with
radon, sulfur or saline waters (Yurtkuran et al. 1999;Sukenik
et al. 1990a,b,1995; Elkayam et al. 1991; Franke et al. 2000,
2007; Staalesen Strumse et al., 2009;Caporalietal.2010).
Although our results are in accordance with these earlier stud-
ies, direct comparisons are limited by differences in study
design, in type-intensity-period of spa therapy and especially
in main chemical compositions of waters used in
balneotherapy. By taking into account saline balneotherapy
studies, our study is comparable to Sukenik et al.’s two trials
(1990b;1995), in which the therapeutic effects of several
balneotherapy regimens in RA patients were investigated in-
cluding Dead Sea salts, sodium chloride and sulfur baths. The
beneficial clinical effects observed in our study are consistent
with those of Dead Sea salts and sodium chloride
balneotherapy in Sukenik et al.’strials(1990b;1995).
Several systematic reviews addressing the effectiveness of
spa therapy and balneotherapy for the management of RA
have been published recently (Verhagen et al. 2015; Santos
et al. 2015;Katzetal.2012). The authors of these reviews in
general conclude that most of the studies they included report
positive findings of spa therapy and balneotherapy in patients
with RA, but evidence is insufficient because of heterogeneity
in design and methodological flaws of the studies.
Although, nearly all studies provide promising evidence
for a therapeutic effect of spa therapy and balneotherapy in
patients with RA, very little knowledge is available about the
mechanisms of action by which spa therapy improves symp-
toms of RA (Fioravanti et al. 2011a;Benderetal.2005;
Grabskietal.2004; Markovićet al. 2009; Kloesch et al.
2012). The clinical benefits of balneotherapy have been wide-
ly attributed to result of a combination of various effects:
thermal, mechanical and chemical (Sukenik et al. 1999;
Fioravanti et al. 2011a;Benderetal.2005). The well-
documented mechanisms of thermal effect include: vasodila-
tation, gate control theory, neuroendocrine reactions, neurobi-
ological responses and immune mechanisms (Sukenik et al.
1999; Fioravanti et al. 2011a; Bender et al. 2005; Tarner et al.
2009; Martins et al. 2015; Leicht et al. 2015). The mechanical
effect, which is the result of hydrostatic pressure and buoyan-
cy, may induce several physiological reactions including
increased diuresis, natriuresis and cardiac output (Fioravanti
et al. 2011a; Lange et al. 2006; Tenti et al. 2015;O’Hare et al.,
1984,1985;Halletal.1996; Pendergast and Lundgren 2009;
Carter et al. 2014). The chemical effects of balneotherapy
were much less documented than the thermal and mechanical
effects (Fioravanti et al. 2011a; Bender et al. 2005; Fortunati et
al. 2016). Indeed, each water used in balneotherapy has a
unique chemical composition, so theoretically has its own
specific chemical effects.Recently, among the chemical ingre-
dients of thermal mineral waters salt (NaCl), carbon dioxide
(CO
2
), radon and hydrogen sulfide (H
2
S) are gaining specific
interest since the knowledge on their specific biological ef-
fects when used balneotherapy (bathing) and balneological
treatments(inhalation and drinking) and spa therapy regimens
are increasing. This knowledge comes from balneotherapy
trials both in-vivo human (Shehata et al. 2006; Dogliotti
et al. 2011; Boros et al. 2013; Dönmez et al. 2000;
Leibetseder et al. 2004; Ardiç et al. 2007; Vareka et al.
2009; Nugraha et al. 2011;Bazzichietal.2013;Fioravanti
et al. 2011b,2015a,b) and experimental animal model studies
(Karagulle et al. 1996; Yamamoto and Hashimoto 2007a,b;
Kim and Zhilyakov 2008; Boros et al. 2013; Liang et al. 2015)
and partly supported by in-vitro studies (Fioravanti et al. 2013;
Braga et al. 2012,2013; Burguera et al. 2014) and physiolog-
ical investigations (Karagülle et al. 2004; Sato et al. 2009;
Lowry et al. 2009). There have been several attempts to orga-
nize scientific meetings focusing on such specific
balneological agents like salt, radon, sulfur and carbon diox-
ide. Systematic reviews and meta-analysis have also been
published on radon (Falkenbach et al. 2005)andCO
2
(Pagourelias et al. 2011) balneotherapy. Comprehensive infor-
mation can be found also in the Proceedings of the
International Conference on Salt waters (2010). Our study is
an attempt to investigate saline water balneotherapy specific
effects on oxidant/antioxidant status in patients with RA.
Three in-vitro studies specifically focusing mechanisms of
balneotherapeutic agents in RA can be found in the literature
(Markovićet al. 2009; Kloesch et al. 2012; Grabski et al.
2004). Markovićet al. (2009) investigated the effect of hyper-
thermia and sulfur (sodium hydrosulfide {NaHS}) at tran-
scriptional level in several pro-inflammatory genes in
fibroblast-like synoviocytes. They exposed the cells to
30 min of hyperthermia (41–42 °C) or 2 mM NaHS and
showed that both were acting as stressors, inducing a profound
expression of heat shock protein (HSP70). Additionally, they
demonstrated that if the cells were treated with hyperthermia
prior to IL1 beta expression, gene expressions were signifi-
cantly decreased up to 8 h and treatment with NaHS alone
induced expression of observed genes up to 12 h. Their data
indicate that the effect of hyperthermia as balneological treat-
ment is beneficial, but sulfur treatment must be taken in re-
consideration (Markovićet al. 2009). Kloesch et al. (2012)
examined the effect of high concentrations of H
2
S on pro-
Int J Biometeorol
Author's personal copy
inflammatory genes in fibroblast-like synoviocytes derived
from rheumatoid and osteoarthritis patients. They treated
fibroblast-like synoviocytes with NaHS solutions for 20 min
and then they removed this H
2
S-containing medium and re-
placed with fresh, pre-warmed medium. They demonstrated
that high concentrations of H
2
S (above 0.5 mM) elevate the
expression of pro-inflammatory genes such as IL-6, IL-8 and
COX-2 in fibroblast-like synoviocytes and therefore they have
advised caution in patients with active RAwhen taking sulfur
bath therapy (Kloesch et al. 2012). In another in-vitro study,
Grabski et al. (2004) investigated the effect of H
2
S water
balneum on antioxidant status of erythrocytes derived from
RA patients. They obtained erythrocytes from 29 RA patients
and 30 healthy subjects and assessed SOD activity after 5, 10,
15, 20 min of the erythrocytes incubation with H
2
S water.
They showed that erythrocyte SOD activity was higher and
increased significantly in RA patients compared with control
group. This result indicates that hydrogen sulfide water
balneum produces an antioxidant effect on erythrocyte status
in patients with RA (Grabski et al. 2004). Distinct from these
three in-vitro trials with sulfur, our study was an in-vivo hu-
man study and investigated balneological agent was saline
water not sulfur.
Several limitations of our study need to be discussed. The
main limitation of our study was lack of a placebo-controlled
design: true placebo effects caused by the belief in improve-
ment by spa therapy and positive attention may certainly have
contributed to the differences between the intervention groups
and controls [van Tubergen et al. 2001]; however designing
adequate placebo intervention in spa therapy trials is challeng-
ing because of the complexity of the spa therapy course in-
cluding not only balneotherapy but also a stay in spa hotel
leading to changes in environmental and social milieu and,
awareness of the patients about the nature of the thermal min-
eral water pool, which is inherent in such spa therapy trials.
There was a significant dropout of participants after the ran-
domization that might cause selection of a particular subpop-
ulation; however, the baseline characteristics of dropouts were
similar to those of randomized and analyzed populations im-
plying that premature withdrawal did not lead to selection bias
(Reginster et al. 2013). The study was conducted in patients
with RA who had been treated with conventional DMARDs;
therefore our results cannot be generalized to patients with RA
who receive biologic agents. The other limitation is short du-
ration of this part of the study; therefore we could not evaluate
the possible long-term effects of spa therapy on oxidant/
antioxidant status. Despite these several limitations, our
single-blind randomized controlled trial is the first to test the
effects of spa therapy with saline balneotherapy on oxidant/
antioxidant status in patients with RA and has several
strengths. Besides evaluation of oxidant/antioxidant status
with enzymatic or nonenzymatic parameters individually
(i.e. SOD), we also assessed oxidant/antioxidant status with
AOP and NSSA that give a more comprehensive evaluation
into oxidant/antioxidant status of samples (Oztürk et al. 1999).
Conclusion
Spa therapy with saline balneotherapy exerts antioxidant ef-
fect in patients with RA as reflected by the increase in NSSA
levels after spa therapy, whether this antioxidant effect con-
tributes to the clinical improvements observed remains to be
verified.
Compliance with ethical standards
Funding None.
Conflict of interest The authors declare that they have no conflict of
interest.
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