Biomarkers of oxidation, inflammation and cartilage degradation in osteoarthritis patients undergoing sulfur-based spa therapies

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Clinical Biochemistry 43 (2010) 973–978

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Clinical Biochemistry

j ourna l homepage: www.e lsev ie r.com/ locate /c l inb iochem

Biomarkers of oxidation, inflammation and cartilage degradation in osteoarthritispatients undergoing sulfur-based spa therapies

Serena Benedetti a,⁎, Claudia Canino a, Gaetana Tonti a, Virginia Medda a, Piergiorgio Calcaterra b,Giuseppe Nappi c, Fausto Salaffi d, Franco Canestrari a

a Department of Biomolecular Sciences, Section of Clinical Biochemistry, University of Urbino “Carlo Bo,” Urbino, Italyb Thermal Center of Saturnia, Grosseto, Italyc Study and Research Center of Thermal Medicine, University of Milan, Milan, Italyd Department of Rheumatology, Polytechnic University of Marche, Ancona, Italy

Abbreviations: COMP, cartilage oligomeric matrix proMDA, malondialdehyde; MMP, matrix metalloproteasinflammatory drugs; OA, osteoarthritis; ROS, reactive oTAC, total antioxidant capacity; TNF-α, tumor necrosis f⁎ Corresponding author. University of Urbino “Carlo Bo

Sciences, Section of Clinical Biochemistry, Via Ubaldini 7+39 0722 322370.

E-mail address: [email protected] (S. Bene

0009-9120/$ – see front matter © 2010 The Canadiandoi:10.1016/j.clinbiochem.2010.05.004

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 9 February 2010Received in revised form 3 May 2010Accepted 6 May 2010Available online 20 May 2010

Keywords:OsteoarthritisOxidative stressSpa therapySulfurous waterAntioxidant protection

Objectives: To investigate the effects of sulfur-based spa therapies on oxidation, inflammation andcartilage degradation biomarkers in osteoarthritis (OA) patients.

Design and methods: Analyses were performed before therapy (T0), after therapy (T1) and 1 monthafter its suspension (T2), in OA subjects undergoing mud bath treatments in combination (group A) or not(group B) with hydropinotherapy, and compared with those of patients not subjected to spa therapies(group C).

Results: No modifications in plasma/serum biomarker concentrations were observed throughout thestudy in non-treated patients, while a significant reduction in oxidation, inflammation and cartilagedegradation parameters was evidenced in patients of group A. Group B presented a favorable biochemicalprofile at T1 but not at T2.

Conclusions: To ensure the long term preservation of the chondroprotective effects of sulfur-based

therapies, standard mud bath treatments should be associated with hydropinotherapy in order to maintainreduced oxidative, inflammatory and degradative stimuli longer.

© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Osteoarthritis (OA) is common age-related disabling locomotordisease characterized by the degradation of articular cartilage, focalcartilage loss, and osteocyte formation [1]. The underlying mechan-isms of cartilage matrix degradation are not completely understoodbut it has been widely demonstrated that reactive oxygen species(ROS) are among the contributor factors [2–5]. In fact, when ROSproduction exceeds the antioxidant defense capacities of chondro-cytes, oxidative stress occurs, leading to structural and functionalcartilage damage and to collagen, proteoglycans and hyaluronanoxidation [6]. Degradation products and oxidized molecules may, inturn, contribute to synovial inflammation and form a vicious circle,consisting of new ROS formation and further degradation products.Other than stimulating ROS production, inflammatory mediators are

tein; IL-1β, interleukin-1 beta;es; NSAID, nonsteroidal anti-xygen species; SH, total thiols;actor alpha.,”Department of Biomolecular, 61029 Urbino (PU), Italy. Fax:

detti).

Society of Clinical Chemists. Publish

directly involved in the regulation of cartilage degradation; in fact, thepro-inflammatory cytokines interleukin-1 beta (IL-1β) and tumornecrosis factor alpha (TNF-α) are responsible for the inhibition ofproteoglycan synthesis in chondrocytes, stimulating at the same timethe production of matrix metalloproteases (MMP) [7], whose mainfunction is the remodelling and degradation of the extracellularmatrix since they are also involved in collagen degradation [8].

Dietary antioxidants or antioxidant supplementation might pre-vent or slow-down cartilage degradation in joint diseases; however,to date, epidemiological studies examining the role of antioxidants inhuman OA are few and contradictory [9,10]. Sulfur thermal treat-ments, including mud and bath therapies, are largely employed in themanagement of rheumatic diseases such as OA [11], possibly playingan important role in antioxidant strategies against oxidative cartilageinjury [12–15]. Together with mud and bath treatments, therapiesinvolving the drinking of sulfurous water (hydropinotherapy) arewidely applied in thermal medicine, but principally for their action ongastro-enteric and hepatic functions. Interestingly, we have recentlyobserved a significant decrease in both lipid and protein oxidationproducts in plasma samples obtained from healthy volunteerssubjected to a cycle of hydropinic therapy with sulfurous mineralwater for a period of 2 weeks [16]. Concomitantly, a significantincrement in the total antioxidant capacity of plasma, as well as in

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Table 1Chemical and physical characteristics of the thermal mineral water from Saturnia(Grosseto, Italy).

Temperature (°C) 36.9pH (25 °C) 6.25Conductivity (25 °C) μS/cm) 2996Hardness (°F) 204Fixed residue (180 °C) (mg/L) 2990Sulfidric degree (H2S) (mg/L) 14.5CO2 (mg/L) 674Ca++ (mg/L) 598Mg++ (mg/L) 134Na+ (mg/L) 63.7K+ (mg/L) 9.3HCO3 (mg/L) 675F− (mg/L) 1.9Cl− (mg/L) 71.4NO2

− (mg/L) b0.01P2O5 (mg/L) b0.01SO4

2− (mg/L) 1469NO3

− (mg/L) b0.1NH4

+ (mg/L) 26.8Iron (mg/L) b0.01SiO2 (mg/L) 20.7

974 S. Benedetti et al. / Clinical Biochemistry 43 (2010) 973–978

total plasmatic thiol levels, was evidenced. These findings suggestmajor benefits from sulfurous drinking water consumption due to areduction in bio-molecule oxidation, possibly furnishing validprotection against the oxidative injury commonly associated withaging and age-related degenerative diseases, such as OA.

With this in mind, in the present study we recruited OA patients inorder to investigate the protective antioxidant effects of mud baththerapy in combination or not with hydropinotherapy as compared tonon-treated OA subjects. Subjects were monitored before therapy(T0), immediately after therapy (T1) and 1 month after suspendingthe therapy (T2), in order to evaluate their oxidative status andantioxidant profile as well as some indices of inflammation andcartilage degradation.

Materials and methods

Subjects and study design

The study included 45 patients (M=21, F=24, age 40–76 yearsold, mean 60±11), with symptomatic OA at multiple sites, whosediagnosis was based on the American College of Rheumatologycriteria [17–19]. All these patients gave their informed consent to beenrolled in this study according to the Declaration of Helsinki of 1975(revised 1983). Local ethical committee approval was obtained for thestudy. To be eligible, all patients had to be symptomatic, requiringeither nonsteroidal anti-inflammatory drugs (NSAID) or a pureanalgesic, or both, to control their pain, without any change in thetype or dose of their medications during the thermal therapies. Noneof the subjects received vitamin and/or mineral supplementation forat least 4 weeks before the beginning of the study. Exclusion criteriawere as follows: concurrent systemic inflammatory rheumaticdisease; medical comorbidity that would render the patient unableto participate fully in study procedures (e.g., terminal conditions, suchas end-stage renal disease, heart failure, or malignancy); alcoholabuse or a psychiatric disorder; and previous or planned arthroplastyof the joint to be studied.

After inclusion in the study, participants were randomly dividedinto three groups:

- Group A (n=15): patients subjected to a cycle of mud baththerapy in combination with a cycle of hydropinotherapy;

- Group B (n=15): patients subjected to a cycle of mud baththerapy alone;

- Group C (n=15): patients not subjected to thermal treatments(control group).

As regards to mud bath therapy, subjects of group B underwent atotal of 12 mud pack treatments, with daily frequency, at the ThermalCenter of Saturnia, Grosseto, Italy. The fine soil paste (mud) derivedfrom a mineral water that, for its chemical and physical character-istics, could be considered as sulfurous-sulfate-bicarbonate-calcicwater (Table 1). Mature thermal mud was applied daily for 20 min at46–48 °C, followed by a thermal bath in sulfurous water at 37 °C. Inaddition to mud bath therapy, subjects of group A daily drank 400 mLof sulfurous water having a characteristic smell of rotten egg and asulfidric degree of 14.5 mg/L (Table 1). Water was assumed directlyfrom the mineral spring thus avoiding hydrogen sulfide loss.

Patients were studied at three experimental time points: imme-diately before the beginning of the therapy (T0), at the end of thetherapy, i.e. after 12 days of thermal treatments (T1), and 1 monthafter the suspension of the therapy (T2). Patients of group C wereevaluated on the same days, although not receiving spa treatments. Ateach time, two sets of blood samples were taken from each patientusing Vacutainer® tubes: heparinized tubes were immediatelycentrifuged at 2500 rpm for 10 min and plasma aliquots were storedat −80 °C until assayed; blood samples collected in non-additive

tubes were allowed to clot at room temperature, then centrifuged at2500 rpm for 10 min and serum aliquots were stored at −80 °C.

Joint pain severity was assessed before and after therapy by usingthe Verbal Numeric Scale (VNS, 0–10 scale), with 0 being “no pain”and 10 being “the most intense pain imaginable”.

Biochemical analyses

The following parameters were monitored during the study inplasma samples: hydroperoxides as reactive oxygen metabolites;malondialdehyde (MDA) and protein carbonyls as markers of lipidand protein oxidation, respectively; total thiols (-SH) as mainplasmatic antioxidants and total antioxidant capacity (TAC) of plasmathat takes into account both lipophilic and hydrophilic antioxidantcomponents. In addition, the following markers of inflammation andcartilage degradation were evaluated throughout the study: serumTNF-α as pro-inflammatory cytokine, serum cartilage oligomericmatrix protein (COMP) as an indicator of cartilage turnover andplasma MMP-2 as a protease involved in matrix componentdegradation.

Hydroperoxide determinationHydroperoxides were evaluated in plasma samples using a

commercial kit from Diacron s.r.l. (Grosseto, Italy) as previouslydescribed [16]. Results were expressed in mg of H2O2/dL. Referencevalues of healthy subjects were between 20 and 24 mg H2O2/dL;conditions of low, middle and high oxidative stress were defined byhydroperoxide values of 25–27, 27–32 and 32–40 mg H2O2/dL,respectively [20].

Determination of MDAMDA plasmatic levels were evaluated by reverse-phase HPLC after

sample derivatization with thiobarbituric acid [16,21], using a AlltimaC18 column (4.6 × 250 mm, 5 μm, from Alltech, Milan, Italy). Peakdetection was carried out at 532 nm using Borwin HPLC software(Jasco Corporation, Tokyo, Japan). Reference values from healthyhuman adults were below 1 µmol/L [21].

Protein carbonyl determinationCarbonyls were evaluated using an enzyme immuno-assay kit

from Alexis Biochemicals (San Diego, CA, USA). Proteins from plasmasamples first react with dinitrophenylhydrazine (DNP), then arenonspecifically absorbed onto an ELISA plate. Unconjugated DNPand non-protein constituents are washed away and the absorbed

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proteins are probed with biotinylated anti-DNP antibody followedby streptavidin-linked horseradish peroxidase. The intra- and inter-assay variations were approximately 5%; carbonyl reference valuesfrom healthy human adults, as determined by ELISA, were below 0.1nmol/mg [22].

TAC determinationTAC was evaluated in plasma samples using a commercial kit from

Diacron s.r.l. (Grosseto, Italy) as previously described [16]. Theplasmatic antioxidant power was expressed in μmol/L of vitamin C.The normal value in healthy subjects was approximately 2200 μmol/L; conditions of slight, middle and high antioxidant deficiency statuswere defined by TAC values of 2000–1800, 1800–1600, 1600–1400µmol/L, respectively [23].

Determination of SH levelsTotal thiol groups were evaluated using a commercial kit

distributed by Diacron s.r.l., Grosseto, Italy. The method is based onthe capacity that plasmatic -SH groups have to react with 5,5’-dithiobis-2-nitrobenzoic acid, followed by the development of acolored complex that can be measured photometrically at 405 nm[24]. As reported by the manufacturer, plasmatic values ranged from400 to 600 µmol/L.

Determination of TNF-α levelsSerum levels of TNF-α were measured using the Endogen Human

TNFα ELISA kit from Pierce Biotechnology (Rockford, IL, USA),following the instructions furnished by the manufacturer. Intra- andinter-assay coefficients of variation were 4.2% and 5.2%, respectively.As described in the specifications of the kit, reference values of TNF-αfrom serum of apparently healthy individuals were below the assaydetection limit of 2 pg/mL.

Determination of COMP levelsSerum levels of the cartilage matrix protein COMP were deter-

mined using the WieslabTM hCOMP ELISA kit from Euro-Diagnostica(Malmo, Sweden). The assay serves as an indicator of cartilageturnover; in fact, measurement of intact COMP and fragments inserum has been shown to correlate to cartilage destruction in OApatients [25]. Intra- and inter-assay coefficients of variation were 3.5%and 6.6%, respectively. As reported by the manufacturer, referencevalues of COMP from serum of healthy individuals were 1.35±0.41µg/mL (range 0.99–2.54 µg/mL).

Determination of MMP-2 levelsPlasma levels of MMP-2 were evaluated using the Quantikine

MMP-2 Immunoassay kit from R&D Systems. Intra- and inter-assaycoefficients of variation were 3.4% and 7.4%, respectively. As stated inthe specifications of the kit, reference values of MMP-2 from theheparinized plasma of apparently healthy individuals were 189±29.8ng/mL (range 155–323 ng/mL). The assay recognizes the pro- andmature forms human MMP-2 and no significant cross-reactivity orinterference was observed.

Statistics and data processing

Descriptive analysis included mean and standard deviation (SD).Experimentwise analyses were assessed by ANOVA for repeatedmeasures, pairwise comparisons between time points were madeusing t-test. A linear regression analysis was performed to analyse therelationship between oxidation, inflammation and cartilage degrada-tion biomarkers, and the correlation coefficient was also calculated.Statistics and graphs were obtained using Origin 6.0 (MicrocalSoftware, Inc., Northampton, MA, USA).

Results

Antioxidant profile

Plasma total antioxidant capacity (TAC) and total thiol levels (-SH)are shown in Table 2. TAC evaluation at baseline (T0) revealed that OApatients presented a discreet antioxidant deficiency status as definedby TAC values ranging from 1800 to 2000 µmol/L. No significantdifferences in TAC values were observed in the three groups of OApatients at the three experimental time points. On the contrary,significant modifications (p=0.0265, ANOVA for repeated measures)in SH values were evidenced during the study in patients of group A,due to the increment in SH levels by 10% at T1 and by 17% at T2 whencompared to T0. SH levels did not significantly change in patients ofgroups B and C.

Oxidative stress biomarkers

Plasmatic biomarkers of oxidative stress, namely hydroperoxides,MDA and protein carbonyls, are reported in Table 2. Before thebeginning of the spa therapy (T0), OA patients presented mean levelsof hydroperoxides indicating a condition of medium oxidative stress(defined by hydroperoxide values from 27 to 32 mg H2O2/dL). At thesame time, an accumulation of MDA and carbonyl levels was found atT0 as compared to values generally observed in the healthypopulation (i.e. MDA lower than 1 µmol/L and carbonyls lower than0.1 nmol/mg).

Hydroperoxide values did not significantly change throughout thestudy in the three groups of OA patients. On the contrary, significantmodifications in MDA (p=0.0135, ANOVA for repeated measures)and carbonyls (p=0.0498, ANOVA for repeated measures) wereevidenced during the study in patients of group A. In fact, whencompared to baseline values, MDA decreased by 36% and 27% at T1and T2, respectively; in parallel, carbonyl levels decreased by 48% and37%. Statistical analysis (ANOVA for repeated measures) revealed nosignificant modifications in MDA and carbonyl levels during the studyin patients of group B. This is probably due to the fact that even if asignificant decrease in MDA (p=0.0293, t-test for paired data) andcarbonyls (p=0.0494, t-test for paired data) was evidenced at T1with respect to T0, the same course was not observed at T2. As regardsto patients of group C, no differences in MDA and carbonyl levels werefound at the three experimental time points.

Biomarkers of inflammation and cartilage degradation

TNF-α is a cytokine involved in cartilage inflammation anddegradation. As reported in Table 2, before the beginning of thetherapy (T0), OA patients presented high serum levels of TNF-αwhencompared to values generally found in the healthy population (lowerthan 2 pg/mL). While TNF-α levels remained almost unchanged inpatients of group C throughout the clinical study, significantmodifications in TNF-α values were observed in patients of group A(p=0.0486, ANOVA for repeated measures). In fact, TNF-α dimin-ished by 45% and 39% at T1 and T2, respectively, as compared to T0. Asrevealed from statistical analysis (ANOVA for repeated measures), nosignificant differences in TNF-α levels were observed during the studyin patients of group B. This is possibly due to the fact that even if asignificant decrease in TNF-α was evidenced at T1 with respect to T0(p=0.0370, t-test for paired data), the same course was not found atT2.

Serum levels of COMP as an index of cartilage matrix degradationare shown in Table 2. An accumulation of this protein was observed atT0 in OA patients when compared to values reported for healthysubjects (1.35±0.40 µg/mL). No significant differences in COMPlevels were observed in OA patients of group B and C at the threeexperimental time points. On the contrary, significant modifications

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Table 2Biochemical parameters monitored before therapy (T0), at the end of the therapy (T1) and 1 month after the suspension of the therapy (T2) in OA patients undergoing: mud baththerapy in combination with hydropinotherapy (group A), mud bath therapy alone (group B) and no thermal treatments (group C).

TAC (µmol/L) -SH (µmol/L) Hydroperoxides (mg H2O2/dL) MDA (µmol/L) Carbonyls (nmol/mg) TNF-α (pg/mL) COMP (µg/mL) MMP-2 (ng/mL)

Group A T0 1984±121 453±41 28.5±2.2 1.70±0.39 0.116±.021 14.6±3.5 2.31±0.16 327±68T1 2137±112 498±41 27.0±0.7 1.09±0.19 0.060±0.016 8.0±2.7 2.09±0.17 279±67T2 2099±87 530±56 28.6±1.1 1.24±0.31 0.073±0.014 8.9±2.8 2.09±0.17 310±70

Group B T0 1991±107 462±26 28.1±1.4 1.72±0.23 0.110±0.018 14.6±2.4 2.30±0.13 323±86T1 2065±83 506±40 27.0±1.4 1.33±0.13 0.071±0.015 9.3±2.1 2.15±0.17 311±51T2 1981±99 457±38 29.0±1.3 1.70±0.27 0.092±0.012 14.9±3.1 2.22±0.17 327±82

Group C T0 2094±83 489±27 26.4±2.0 1.68±0.15 0.114±0.014 14.6±0.3 2.29±0.21 324±75T1 2046±102 496±13 26.7±2.0 1.70±0.10 0.115±0.012 15.1±1.3 2.31±0.27 328±80T2 2131±123 498±20 27.2±2.0 1.69±0.16 0.113±0.016 15.7±1.1 2.25±0.20 327±78

For statistical analysis, see the text.

976 S. Benedetti et al. / Clinical Biochemistry 43 (2010) 973–978

in COMP values were evidenced throughout the study in patients ofgroup A (p=0.0128, ANOVA for repeated measures), due to thedecrement in COMP levels by 10% at T1 and T2 with respect to T0.

MMP-2 is an endopeptidase that functions in the breakdown andmodification of extracellular matrix components including collagen.As shown in Table 2, a strong accumulation of this proteinase wasobserved at T0 in all three groups of OA patients when compared tothe values reported for healthy subjects (189±29.8 ng/mL). From astatistical point of view (ANOVA for repeated measures), nosignificant differences in MMP-2 levels were observed during thestudy in the three groups of patients. Interestingly, MMP-2 levelssignificantly decreased at T1 with respect to T0 (p=0.0345, t-test forpaired data) in OA subjects of group A, but the same course was notevidenced at T2.

Biochemical correlations

Linear regression analysis of the biochemical parameters moni-tored throughout the study is reported in Table 3: the evaluation of Rand p values revealed that the indices of oxidative damage to bio-molecules, namely MDA and carbonyls, positively correlated witheach other and with the markers of inflammation and cartilagedegradation TNF-α and COMP. Similarly, TNF-α and COMP werelinked to each other by a direct correlation. Interestingly, a significantpositive correlation was also found between COMP andMMP-2 levels.

Pain severity

At the end of the therapy (T1), a significant decrement (pb0.01, t-test for paired data) in VNS scoreswas observed in both groups A and Bwith respect to baseline (T0); in detail, scores decreased from5.9±0.7to 3.3±0.8 (-44%) in patients of groupA and from5.9±0.6 to 3.7±0.8(−37%) in patients of group B. No significant modifications in the VNSscore were observed in OA subjects of group C.

Table 3Linear regression analysis between plasmatic oxidative biomarkers (i.e. MDA andcarbonyls) and serum indices of inflammation (TNF-α) and cartilage degradation(COMP, MMP-2).

Parameters Correlation index (R) p value

MDA vs. carbonyls 0.348 0.01782MDA vs. TNF-α 0.710 b0.001MDA vs. COMP 0.519 0.00934Carbonyls vs. TNF-α 0.501 0.00054Carbonyls vs. COMP 0.405 0.00472TNF-α vs. COMP 0.307 0.02259MMP-2 vs. COMP 0.373 0.00645

Discussion

In the last few years, numerous studies have documented the roleof ROS in the etiology and pathogenesis of OA [2–6]. In accordancewith literature, our results revealed that, when enrolled in the study,patients suffering from multiple site OA presented a condition ofoxidative stress characterized by a plasmatic accumulation ofhydroperoxides as well as lipid and protein oxidation products ascompared to the healthy population. In parallel, the total antioxidantcapacity of plasma was reduced, indicating a deficiency in theantioxidant defense system. As regards to the markers of inflamma-tion and cartilage degradation, before starting the thermal therapy, anaccumulation of TNF-α COMP and MMP-2 levels was observed in theserum/plasma of OA subjects. These findings confirmed that patientswere characterized by joint cartilage degradation in association withinflammatory processes which may favor chondrocyte catabolicactivities.

In an attempt to investigate new strategies and interventionsaimed at reducing oxidative damage in joint cartilage, in this study wehave evaluated the protective antioxidant role of sulfur-based spatreatments by monitoring, in OA patients, the effects of mud baththerapy in combination (group A) or not (group B) with hydro-pinotherapy as compared to non-treated patients (group C). Otherthan the basal evaluation (T0), subjects were assessed immediatelyafter therapy (T1) and 1month after its suspension (T2). Interestingly,significant modifications in serum/plasma levels of SH, MDA,carbonyls, TNF-α and COMP were evidenced in patients of group A.In fact, SH levels increased at T1 and T2with respect to baseline, whilea decrease in the biochemical markers of oxidation (MDA andcarbonyls), inflammation (TNF-α) and cartilage degradation(COMP) was concomitantly observed at the two experimental timepoints. These findings were not confirmed in patients of group B,which presented a significant decrement in MDA, carbonyl and TNF-αlevels immediately after therapy (T1) but not 1 month after itssuspension (T2). As expected, these biochemical parametersremained almost unchanged throughout the study in patients ofgroup C.

As revealed from statistical analysis (two-way ANOVA for repeatedmeasures), differences between groups A and B were not significant(data not shown), probably due to the small sample size. Neverthe-less, our data strongly suggests that mud bath therapy combined withthe hydropinic treatment may furnish additional protection againstoxidative injury and cartilage degradation in OA patients with respectto mud bath therapy alone, by preserving reduced oxidative,inflammatory and degradative stimuli longer.

In accordance with literature, clearly demonstrating that in OAthere is a close relationship between oxidative stress, inflammationand cartilage degeneration [3], the statistical analysis of the dataobtained in this trial has allowed the identification of significantcorrelations between the markers of oxidative damage and the

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markers of inflammation and cartilage degradation. In particular,MDA and protein carbonyls correlated positively and significantlywith both TNF-α and COMP; similarly, TNF-α and COMP were linkedtogether by a direct correlation, as well as COMP and MMP-2.Interestingly, this is the first report showing a relationship betweenMMP-2 and COMP, probably indicating that the cartilage matrixprotein, belonging to the thrombospondin family [26], could be adirect substrate of this protease. This possibility is currently underinvestigation.

In an attempt to clarify the mechanisms underlying the positivebiochemical effects observed after the spa treatments, it must betaken into account that both mud bath and hydropinic therapies aretreatments based on sulfurous waters, containing a quantity ofhydrogen sulfide (H2S) equal to 14.5 mg/L (Table 1). Literature haswidely demonstrated that H2S is a gas with several protective effectsat the cellular level [27,28]; in particular, it protects cells fromoxidative damage thanks to its ability to counteract the production ofhydrogen peroxide, superoxide anion and peroxynitrite [29–33].These radical molecules are deeply involved in OA cartilagedegeneration [5]; it can therefore be hypothesized that H2S absorbedduring the spa treatments can diminish, at the joint level, ROSformation and oxidative damage to cartilage components, conse-quently leading to a reduction of inflammatory and catabolicprocesses. Moreover, it must be emphasized that, other than beingan effective free radical scavenger, H2S deeply acts at the cellular levelby increasing, on one hand, the synthesis of glutathione [29,30] andother fundamental redox regulators such as tioredoxin-1 [34]; and onthe other, by enhancing the activity of some antioxidant enzymessuch as superoxide dismutase [34,35].

To date, the mechanisms underlying H2S absorption are not fullyunderstood, however it is known that, since the gas is permeable toplasma membranes, once absorbed from skin and intestinal mucosa itcan rapidly diffuse through tissues [27]. Data obtained in this studyappear to demonstrate that the oral ingestion of sulfurouswater in thecourse of hydropinotherapy increases the absorption and bioavail-ability of H2S when compared to mud bath treatments, thus re-enforcing the antioxidant effects of spa therapies not only in the shortperiod, but even 1 month after therapy suspension. As a consequence,our findings strongly support the adjunctive value of hydropinother-apy to standard mud bath therapy in the management of OA.

It should also be noted that the thermal water from Saturniacontains sulfur not only in the form of H2S, but also in the form ofsulfate (SO4

2−) and can therefore have a direct chondroprotectiveeffect on cartilage. In fact, it is well known that chondrocyteproteoglycans are highly sulfated and require inorganic sulfur fortheir synthesis [36]; consequently, thermal treatments with sulfate-rich water can bring direct benefit to the de novo synthesis of thesematrix components, thus countering their loss following oxidativedamage and cartilage degradation.

The clinical beneficial effects of the thermal treatment were alsosustained by the fact that OA patients of both groups A and Bpresented a highly significant reduction in joint pain severity, asevaluated before and after therapy by the VNS score. These evidenceswell agree with the recent literature demonstrating the positiveeffects of spa therapy on the painful symptomatology and a significantimprovement on functional capacities in patients with OA [37–40].

In conclusion, we can affirm that sulfur-based spa therapies surelyrepresent a suitable support in the control of OA so as integrating andcompleting the pharmacological treatments; however, for the longterm preservation of the chondroprotective effects, standard mudbath treatments should be associated with hydropinic therapy inorder to increase the rate of H2S absorption and preserve reducedoxidative, inflammatory and degradative stimuli longer. The factremains that, being OA an age-related degenerative disease, spatreatments should be cyclically repeated during the year so as toguarantee constant antioxidant protection.

Acknowledgments

We thank Dr. Francesca Carducci for advice on the Englishlanguage and Prof. Marco Rocchi for statistical revision.

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