Relevance of a Hypersaline Sodium-Rich Naturally Sparkling Mineral Water to the Protection against...

Research ArticleRelevance of a Hypersaline Sodium-Rich Naturally SparklingMineral Water to the Protection against Metabolic SyndromeInduction in Fructose-Fed Sprague-Dawley Rats A BiochemicalMetabolic and Redox Approach

Cidaacutelia Dioniacutesio Pereira1 Milton Severo2

Joatildeo Ricardo Arauacutejo1 Joatildeo Tiago Guimaratildees13 Diogo Pestana1 Alejandro Santos4

Rita Ferreira5 Antoacutenio Ascensatildeo6 Joseacute Magalhatildees6 Isabel Azevedo1

Rosaacuterio Monteiro1 and Maria Joatildeo Martins1

1 Department of Biochemistry (U38FCT) Faculty of Medicine University of Porto 4200-319 Porto Portugal2 Department of Clinical Epidemiology Predictive Medicine and Public Health Faculty of MedicineUniversity of Porto 4200-319 Porto Portugal

3 Department of Clinical Pathology Sao Joao Hospital Centre EPE 4200-319 Porto Portugal4 Faculty of Nutrition and Food Sciences University of Porto 4200-465 Porto Portugal5 QOPNA Mass Spectrometry Centre Department of Chemistry University of Aveiro 3810-193 Aveiro Portugal6CIAFEL Research Centre in Physical Activity Health and Leisure Faculty of Sport University of Porto4200-450 Porto Portugal

Correspondence should be addressed to Maria Joao Martins mmartinsmeduppt

Received 5 September 2013 Revised 24 November 2013 Accepted 28 November 2013 Published 5 February 2014

Academic Editor Amelie Bonnefond

Copyright copy 2014 Cidalia Dionısio Pereira et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The Metabolic Syndrome increases the risk for atherosclerotic cardiovascular disease and type 2 Diabetes Mellitus Increasedfructose consumption andor mineral deficiency have been associated withMetabolic Syndrome developmentThis study aimed toinvestigate the effects of 8 weeks consumption of a hypersaline sodium-rich naturally sparkling mineral water on 10 fructose-fedSprague-Dawley rats (Metabolic Syndrome animalmodel)The ingestion of themineral water (rich in sodiumbicarbonate andwithhigher potassium calcium and magnesium content than the tap water used as control) reducedprevented not only the fructose-induced increase of heart rate plasma triacylglycerols insulin and leptin levels hepatic catalase activity and organ weight to bodyweight ratios (for liver and both kidneys) but also the decrease of hepatic glutathione peroxidase activity and oxidized glutathionecontent This mineral-rich water seems to have potential to prevent Metabolic Syndrome induction by fructose We hypothesizethat its regular intake in the context of modern diets which have a general acidic character interfering with mineral homeostasisand are poor in micronutrients namely potassium calcium and magnesium could add surplus value and attenuate imbalancesthus contributing to metabolic and redox health and consequently decreasing the risk for atherosclerotic cardiovascular disease

1 Introduction

TheMetabolic Syndrome (MS) consists of multiple and inter-related risk factors of metabolic origin that appear to directlypromote the development of atherosclerotic cardiovasculardisease The MS strongly associates with type 2 DiabetesMellitus or the risk for this condition Although the exact

etiology of the MS still remains unclear it is known toinvolve complex interactions between geneticmetabolic andenvironmental factors where diet is of central importance[1ndash3]

There has been a substantial increase in fructose con-sumption in the last decades which has been associated withsome adverse metabolic changes similar to those observed

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2014 Article ID 384583 17 pageshttpdxdoiorg1011552014384583

2 International Journal of Endocrinology

in the MS [4ndash7] On the other hand minerals like potas-sium calcium and magnesium proposed as protectiveagainst the MS are generally deficient in MS-inducing diets[3 8ndash10]

Natural mineral waters are waters of underground originprotected from contamination and microbiologically whole-someThey are characterized by their purity at source contentin minerals trace elements and other constituents as wellas by favorable effects on human health [11] Additionallybioavailability ofminerals fromnaturalmineral waters is high[12ndash14]

The fructose-fed rat is an interesting and well-validatedanimal model of diet-induced MS (predominantly acquiredMS model) that is commonly used in MS research [15]Different rat strains with distinct fructose ingestion protocolsare reported in the literature and in all cases fructose hasbeen observed to induce MS features such as moderatehypertension glucose intolerance hyperinsulinemia insulinresistance dyslipidemia (hypertriglyceridemia hypercholes-terolemia) altered cytokine and adipokine status (alteredtumor necrosis factor-alpha (TNF-120572) and leptin levels eg)decreased melatonin production andor increased body fatandor body weight [15ndash20]

Both in humans and rats a strong association has beenfound between MS and oxidative stress [21] and fructose-feeding associates with modification of the hepatic redoxstatus [16 22ndash24]

Beneficial effects of acute or chronic natural mineral-richwaters ingestion on blood pressure (BP) [25ndash27] metabolicprofile (plasma insulin sensitivity [28] fasting serum glucoseconcentration [29] and fasting serum lipid profile [25 29])and plasma oxidative stress markers (reactive oxygen species[30] lipid and protein oxidation product levels total antioxi-dant capacity and total thiol levels [31]) have been publishedbut to our knowledge not in MS individuals or animalmodels The natural mineral waters tested are rich albeit indifferent proportions in bicarbonate calcium magnesiumpotassium andor sodium

We aimed to investigate the possible beneficial effect ofnatural mineral-rich water on MS induction by fructose-feeding In the present work Sprague-Dawley rats (SDR)were fed with 10 fructose in natural mineral-rich water(Pedras Salgadas) for 8 weeks and compared to animals fed10 fructose in tap water The natural mineral-rich watertested has high total mineralization content (2855mgL)being mainly rich in sodium and bicarbonate and withhigher potassium calcium and magnesium content than tapwater

2 Material and Methods

21 Animals and Treatments The study was carried out in21 adult male CD SDR (388ndash483 g) from Charles River Lab-oratories (ChatillonChalaronne France) Telemetry trans-mitters (TA11PA-C40 Data Sciences International (DSI) StPaulMNUSA)were implanted in the abdominal cavity withthe catheter in the abdominal aorta by Charles River Ratswere shipped on the 5th day after surgery Upon arrival rats

were individually housed in an enriched environment andmaintained on a daily photoperiod of 12 h lighting schedule(20ndash22∘C) with free access to standard laboratory pellet food(2014 Teklad Global 14 Protein Rodent Maintenance Dietfrom Harlan Interfauna Iberica SA Barcelona Spain) andtap water Acclimatization took place for 10 days beforestarting the experimental protocol which was authorizedby the Veterinary National Department of the Ministryof Agriculture Rural Development and Fisheries Duringacclimatization rats spent gradually increasing periods oftime inside metabolic cages The handling and care of theanimals were conducted in conformity with the EuropeanCommunity Council guidelines for the use of experimentalanimals (86609EEC) and Act 12992

Animals were randomly divided into 3 groups (7 animalseach) with free access to different drinking solutions (a)tap water (CONT) (b) 10 fructose in tap water (FRUCT)or (c) 10 fructose in natural mineral-rich water (FRUCT-MIN) All experimental groups were fed ad libitum withthe standard laboratory chow diet mentioned above (20 ofenergy derived from protein 13 from fat and 67 fromcarbohydrate) The dietary manipulation lasted 8 weeks A3-week pretreatment period with the natural mineral-richwater was performed to the FRUCTMIN group (while theother rats were drinking tap water) to allow adjustment towater flavor and sparkles This period of time induced nochange in the pattern of food intake or increase of animalsbody weight (data not shown all animals weighed 475ndash597 g at the beginning of the dietary manipulation withfructose) Body weight and food and fluid ingestion valueswere registered weekly from week 0 to week 7 or 8 (as shownin Figures 1(a)ndash1(c) resp) On week 0 these parameters wereevaluated on the same day which occurred 24 to 48 h afterstarting the dietary manipulation and then on every oneweek after the first measurement Each week from week 0 to7 all animals spent 24 h inside metabolic cages for evaluationof food and fluid consumption as well as for urine collection(the latter at 0 2 4 and 6 weeks) At each occasion thethree groups of rats were represented with an equal numberof animals per group Total energy ingestion was calculatedby multiplying food and fluid ingestion values by the corre-sponding reference energy values and then adding these tworesults

The chemical characteristics of tap and natural mineral-rich waters are given in Table 1 The latter is classifiedas a hypersaline sodium-rich naturally sparkling mineralwater in conformity with the European Community Councilguidelines for natural mineral waters (200954EEC) andwas kindly provided by Unicer Bebidas SA (Leca do BalioMatosinhos Portugal)

22 Assessment of Blood Pressure and Heart Rate DataquestART 41 Silver telemetry system (DSI) with RCP-1 receivers(and APR-1 for ambient pressure reference) was used fortelemetric measurement of BP (mm Hg) and heart rate (HRbeatsmin) in the animal cage Dataquest ART acquisitionsoftware (DSI) was used to monitor all rats for 8 weeks Sam-pling was performed everymin and setting segment duration

International Journal of Endocrinology 3

0

50

100

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CONT FRUCT FRUCTMIN

ΔBo

dy w

eigh

t (g)

500

550

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CONTFRUCT

FRUCTMIN

Body

wei

ght (

g)

lowast

0 1 2 3 4 5 6 7 8

Weeks

(a)

CONTFRUCT

FRUCTMIN

lowast

Weeks0 1 2 3 4 5 6 7

Food

inge

stio

n (g

)

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20

(b)

CONTFRUCT

FRUCTMIN

Weeks

00 1 2 3 4 5 6 7

100

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Flui

d in

gesti

on (m

L)

+ $$ $

50

lowastlowast

(c)

Figure 1 (a) Body weight (g 119899 = 7 lowast119875 lt 005 CONT versus FRUCTMIN (Δ body weight (g 119899 = 7) between weeks 8 and 0 in the inset119875 lt 001 FRUCT and FRUCTMIN versus CONT)) (b) food ingestion (g 119899 = 7 lowast119875 lt 005 CONT versus FRUCTMIN and (c) fluidingestion evolution (mL 119899 = 7 +119875 lt 005 CONT versus FRUCT and FRUCTMIN 119875 lt 0001 CONT versus FRUCT and FRUCTMIN$119875 lt 005 CONT versus FRUCT and 119875 lt 001 CONT versus FRUCTMIN $$

119875 lt 001 CONT versus FRUCT and 119875 lt 005 CONT versusFRUCTMIN lowastlowast119875 lt 001 CONT versus FRUCTMIN) during the dietary intervention Results were expressed as mean plusmn standard error ofthe mean CONT control FRUCT 10 fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water

at 20 s 3 different animals (one from each experimentalgroup) were evaluated per day from 400 pm to 800 amseven days per week BP values were not includedconsideredif pulse pressure (the difference between systolic and diastolicBP) was below 20mm Hg and HR values were not used ifpulse pressure values were lower than 10mm Hg [32] Datawere exported from Dataquest ART 40 analysis program(DSI) toMicrosoftExcel 2010 (RedmondWAUSA) and thensubjected to statistical analysis

23 Collection of Samples All chemical substances used in allthe experiments were of analytical grade

At the end of the dietary intervention animals weredeeply anesthetized with sodium pentobarbital (80mgkg ofbody weight) and blood was collected from the left ventricleinto heparinized syringes Then rats were transcardiallyperfused with ice-cold isotonic sodium chloride solutionAfter perfusion liver heart kidneys and epididymal adiposetissuewere rapidly removed from the thoracic and abdominal


Table 1 Chemical characteristics of tap and natural mineral waters

Characteristics Tap waterHypersaline sodium-richnaturally sparkling mineralwatermdashPedras Salgadas

Total mineralization (mgL) 148ndash151 2855pH 65ndash90 616Sodium (mgL) 200 591Calcium (mgL) 305ndash402 925Magnesium (mgL) 36ndash92 262Potassium (mgL) 26 299Copper (mgL) 2 00013Zinc (120583gL) lowast 11Selenium (120583gL) 10 lt20Bicarbonate (mgL) lowast 2013Chloride (mgL) 250 308Sulphate (mgL) 250 64lowast no need for control (Portuguese Act 3062007 from 27th of August)

cavities washed in cold saline solution placed in quali-tative filter paper for excess liquid removal and weighedThe liver was cut into several fragments that were immersedin liquid nitrogen and stored at minus80∘C until further process-ing

24 Assessment of Plasma Biochemical Metabolic Hormo-nal and Inflammatory Markers and Assessment of UrinaryCreatinine and Sodium Plasma concentrations of glucosetriacylglycerols total cholesterol HDL-cholesterol LDL-cholesterol C-reactive protein (CRP) glutamic-oxaloacetictransaminase (GOT) glutamic-pyruvic transaminase (GPT)total bilirubin uric acid urea creatinine total proteinsalbumin ferritin sodium potassium chloride magnesiumcalcium and phosphorus were determined Urinary creati-nine and sodium excretions were also evaluated All thesequantifications were made at the Clinical Pathology Unitof Sao Joao Hospital Centre EPE Porto Portugal usingstandardized methods for human sample routine hospitalmeasurements

Plasma levels of insulin (Mercodia AB 10-1137-01 (Uppsa-la Sweden)) adiponectin (Invitrogen Corporation KRP0041(Camarillo CA USA)) aldosterone (Uscn Life Science IncE0911Ra (Wuhan China)) substance P (RampD Systems IncKGE007SKGE007PKGE007 (Minneapolis MN USA))interleukin-6 (IL-6 Cusabio Biotech Co Ltd CSB-E04640r(Wuhan China)) and TNF-120572 (Cusabio CSB-E11987r) wereevaluated according to the manufacturersrsquo instructions fromthe specific ELISA kits Plasma concentrations of melatonin(RSH69K) nuclear factor kappa-B ligand (RANKL RBN-31 K-1RANKL) leptin and osteoprotegerin (OPG RBN1-31 K) were measured with a Luminex 200 analyzer (LuminexCorporation Austin TX USA) according to protocols (MIL-LIPLEX MAP kits) of Millipore Corporation (Billerica MAUSA) Raw data (mean fluorescence intensity) were analyzedusing ISTM 23 software (Luminex Corporation)

25 Assessment of Hepatic Redox State Markers Oxidativedamage to lipids proteins and DNA was evaluated by mea-suring thiobarbituric acid-reactive substances (viz malondi-aldehyde (MDA)) carbonyls and 8-hydroxy-21015840-de-oxygua-nosine (8-OHdG) levels respectively Catalase total superox-ide dismutase (SOD) glutathione-S-transferase (GST) glu-tathione-peroxidase (GPx) and glutathione-reductase (GR)activities were quantified Reduced (GSH) and oxidized(GSSG) glutathione concentrations were also determined Allthese techniques were performed as described by Assuncaoet al [33] except for the use of Bradford method for proteinquantification and the use of a kit for 8-OHdG quantification(theDNA extraction kit (V-gene) was purchased fromBioronInternational (Ludwigshafen Germany) and the 8-OHdGkit from Japan Institute for the Control of Aging (HaruokaFukuroi Shizuoka Japan))

251 Protein Extraction and Sirtuin 3 Protein Expressionby Western Blot Liver tissue samples (300ndash450mg) werehomogenized with a Teflon-glass homogenizer in an equalvolume of protein extraction buffer (50mM Tris-base150mM NaCl pH 74 1 Triton X-100 05 sodium deoxy-cholate 01 sodium dodecyl sulfate (SDS) 1mM EDTAtablets of protease inhibitors and phosphatase inhibitors(100mM sodium fluoride and 10mM sodium orthovana-date)) with subsequent agitation for 30min at 4∘C Theneach sample was centrifuged at 13 000 g for 20min at 4∘Cand the protein solution under the lipid layer was collectedand kept at minus80∘C until further analysis

Proteins were quantified by using the bicinchoninic acidprotein assay kit (Pierce Rockford IL USA) Proteins weredissolved (1 1) in loading buffer (50mM Tris-HCl pH 68100mM dithiothreitol 2 SDS 001 bromophenol blueand 10 glycerol) and denatured for 5min at 95∘C Then40 120583g of each sample was loaded per well separated by elec-trophoresis in a 12 SDS polyacrylamide gel and transferredto a nitrocellulose membrane (Hybond C-Extra AmershamGE Healthcare Buckinghamshire UK) The membrane wasblocked in Tris-base-buffered saline with 01 Tween 20(vv) (TBST) containing 5 bovine serum albumin (wv)and incubated overnight with the primary antibody againstsirtuin 3 (Sirt3 Cell Signaling Technology Inc Danvers MAUSA) diluted 1 1500 in TBST with gentle agitation at 4∘CThen the membrane was washed in TBST and incubatedwith donkey anti-rabbit polyclonal antibody conjugated tohorseradish peroxidase (Santa Cruz Biotechnology Inc Hei-delberg Germany) diluted 1 5000 in TBST for 1 h at roomtemperature Detection was performed with an enhancedchemiluminescence reagent (Amersham GE HealthcareBuckinghamshire UK) Band intensity was determined usingImage Lab software (version 401 Bio-Rad LaboratoriesHercules CA USA) and normalized for 120573-actin expression(1 1000 and 1 2000 for primary and secondary antibod-ies (Santa Cruz Biotechnology Inc Heidelberg Germany)respectively diluted in 5 (wv) of nonfat dry powderedmilkSveltesse (Nestle Portugal SA Linda-a-Velha Portugal) inTBST)


26 Liver Magnesium and Calcium Content Liver magne-sium and calcium content were measured by inductivelycoupled plasma optical emission spectrometry (ICP-OESActivaM JobinYvon Horiba Scientific Edison NJ USA) at285213 nm and 422673 nm respectively according to ISO11885 (water qualitymdashdetermination of selected elements byICP-OES (httpswwwastandisatshopV5Previewactionjsessionid=CF7234FBAC2BCFDD35A4593A11BD4700preview=ampdokkey=347061ampselectedLocale=en)) after microwaveoven (Mars 5 CEM Corporation Matthews NC USA)assisted acid digestion of liver fragments according to EPA3052 (microwave assisted acid digestion of siliceous andorganically basedmatrices (httpwwwepagovoswhazardtestmethodssw846pdfs3052pdf))

27 Statistical Methods The significance of differences ofeach week cross-sectional statistical analysis among groupsregarding systolic and diastolic BP HR body weight foodand fluid ingestions urine volume urinary sodium andcreatinine excretions total energy ingestion and percentageenergy supplied by fluidtotal energy ingestion was evaluatedusing ANOVA followed by Bonferronirsquos multiple compari-son test or by Kruskal-Wallis followed by Dunnrsquos multiplecomparison test according to their distribution These sta-tistical methods were also used for evaluation of significanceof differences among groups regarding organ weightbodyweight hepatic oxidative stress markers andmineral contentas well as plasma biochemical metabolic hormonal andinflammatory data at the end of the dietary interventionTheassociation between the outcomes (systolic and diastolic BPHR body weight food and fluid ingestions urine volumeurinary sodium and creatinine excretions total energy inges-tion and percentage energy supplied by fluidtotal energyingestion) and the interaction of dietary intervention withtime evolution (evaluated in weeks) longitudinal statisticalanalysis was measured with the interaction terms (120573) whichwere estimated by mixed effects model with random effect inthe interceptThe area under the curve (AUC) was calculatedthrough linear interpolation using the composite trapezoidrule [34]

Statistical analysis was performed using R A languageand environment for statistical computing [34] GraphPadPrism software (version 600 La Jolla CA USA) or IBMSPSS Statistics software (version 200 Armonk NY USA)Values were presented as mean plusmn standard error of the meanand differences considered significant for 119875 lt 005

3 Results

31 Parameters Evaluated over the 8-Week Period ofDietary Intervention

311 Body Weight Food and Fluid Ingestions In generalon each separate week (week by week) body weight (Fig-ure 1(a)) and food ingestion (Figure 1(b)) revealed simi-lar values for the 3 animal groups With time (over thedietary intervention period) a significantly higher and

similar increase of body weight for both fructose groupsversus CONT group was observed (see SupplementaryTable 1 in the supplementary material available onlineat httpdxdoiorg1011552014384583 and inset in Fig-ure 1(a)) With time FRUCTMIN rats decreased food inges-tion significantly more than CONT rats and showed atrend towards a higher decrease with time than FRUCTrats (Supplementary Table 1) Week by week fluid inges-tion showed significantly higher values for both fructose-fed groups versus CONT group without any significantdifference between FRUCT and FRUCTMIN (Figure 1(c))which was in accordance with AUC values (SupplementaryTable 2) Fluid ingestion increased significantly for FRUCT-MIN versus CONT and FRUCT with time (SupplementaryTable 1)

312 Energy Supplied by Fluid to Total Energy Ingestion Ratioand Total Energy Ingestion Week by week no differenceswere observed for fructose ingestion (either from fluidingestion or from both food and fluid ingestions (data notshown)) neither for percentage energy supplied by fluidtotalenergy ingestion between the two intervention groups (thelatter being in accordance with AUC values Figure 2(a) andSupplementary Table 2) in which there was a substantialproportion of energy ingested from fluid (48ndash72 of totalenergy ingestion Figure 2(a)) Total energy ingestionwas sig-nificantly higher everyweek of the protocol for both fructose-fed groups versus CONT group without any significantdifference between FRUCT and FRUCTMIN (Figure 2(b))which was in accordance with AUC (Supplementary Table 2)and body weight (Figure 1(a)) results Total energy ingestiondecreased similarly with time for all groups (SupplementaryTable 3 data not shown for controls)

313 Urine Volume and Urinary Sodium and CreatinineExcretions With time urine volume was significantly higherin FRUCTMIN versus CONT and FRUCT (SupplementaryTable 4) Week by week urinary sodium excretion valueswere expected when taking into consideration the sodiumcontent of tap and natural mineral-rich waters FRUCTMINgroup had significantly higher values than the other twoanimal groups without any significant difference betweenFRUCT and CONT (Figure 3(b)) which also agreed withAUC values (Supplementary Table 2)

314 Blood Pressure and Heart Rate Between weeks 1 and5 FRUCT rats had a significantly higher HR than CONT rats(Figure 4(b)) Interestingly both systolic and diastolic BP andHR evolution over time seemed to be protected from fructoseeffects by the natural mineral-rich water until approximatelyhalf of the dietary intervention period (Figures 4(a)-4(b)resp) A significant increase of systolic BP with time forboth fructose groups versus CONT group was observed(Supplementary Table 5) Diastolic BP in FRUCTMIN groupshowed a tendency to increase with time versus CONTgroup (Supplementary Table 5) A significant increase ofHR with time for FRUCTMIN versus CONT was observed(Supplementary Table 5)


0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

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on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)

Figure 2 (a) Percentage energy supplied by fluidtotal energy ingestion (119899 = 7) and (b) total energy ingestion evolution (kcal 119899 = 7+

119875 lt 005 CONT versus FRUCT and FRUCTMIN ++119875 lt 001 CONT versus FRUCT and FRUCTMIN $119875 lt 0001 CONT versus FRUCT

and FRUCTMIN +$119875 lt 005 CONT versus FRUCT and 119875 lt 0001 CONT versus FRUCTMIN ++$

119875 lt 001 CONT versus FRUCT and119875 lt 0001 CONT versus FRUCTMIN) during the dietary intervention Results were expressed as mean plusmn standard error of the meanCONT control FRUCT 10 fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water

32 Organ Weight to Body Weight Ratios Liver and bothkidneysweight to bodyweight ratioswere significantly higherin FRUCT versus CONT (Figures 5(a) 5(c) and 5(d) resp)Additionally the liver showed a strong trend to an increasein FRUCTMIN versus CONT (119875 = 0053) and a significantincrease in FRUCT versus FRUCTMIN (Figure 5(a)) Naturalmineral-rich water ingestion prevented fructose effects onliver and both kidneys weight to body weight ratios

Epididymal adipose tissue to body weight ratio wasslightly and similarly higher in both fructose-fed animalgroups versus CONT group (Figure 5(b)) No differenceswere found among groups regarding heart weightbodyweight (data not shown)

33 PlasmaHormonal andMetabolic Profiles Triacylglycerollevels significantly increased in FRUCT versus CONT anda tendency to an increase in FRUCTMIN versus CONT(119875 = 0080) was observed (Figure 6(b)) Insulin significantlyincreased (Figure 6(c)) and leptin variation followed the samepattern in FRUCT versus CONT (119875 = 0057) (Figure 6(d))Insulin sensitivity index was also calculated [35] and a strongtendency to a decreasewas observed in FRUCTversusCONT(P and global 119875 = 0055 0247 times 106 plusmn 0032 times 106 0137 times106

plusmn 0009 times 106 and 0211 times 106 plusmn 0030 times 106 for CONT

FRUCT and FRUCTMIN resp) Glucose (Figure 6(a)) andaldosterone (Figure 6(e)) seemed to increase and melatonin(Figure 6(f)) seemed to decrease in FRUCT versus CONTNatural mineral-rich water ingestion appeared to counteract

these fructose-induced metabolic and hormonal effects Novariations were observed for adiponectin levels (data notshown)

34 Plasma Biochemical and Inflammatory Profiles Urea(Table 2) and magnesium (Table 3) levels significantlydecreased in the two fructose-fed groups versus the CONTgroup Total proteins and albumin levels significantly in-creased in both groups of fructose-fed animals versus CONTgroup (except for total proteins in FRUCTMIN versusCONT where a strong tendency was observed) (Table 2)TNF-120572 and IL-6 levels seemed to increase and OPG toRANKL ratio seemed to decrease in FRUCT versus CONT(Table 2) with the natural mineral-rich water improv-ing these parameters CRP and substance P levels slightlyincreased in FRUCTMIN versus the other two animalgroups (Table 2) The replacement of food by fructosesolution as an energy source could explain the similardecreases in plasma urea magnesium GOT GPT ferritinand uric acid levels in both fructose-fed SDR groups ver-sus CONT group (although significantly only for someparameters)

35 Hepatic Redox Status Markers Catalase and SOD activi-ties and GSH to GSSG ratio increased (Figures 7(a) 7(b) and7(e) resp) and GPx activity GSSG level and Sirt3 proteinexpression decreased (Figures 7(c) 7(d) and 7(f) resp) inFRUCT versus CONT (significantly for catalase GPx and


Table 2 Plasma biochemical and inflammatory marker levels at the end of the dietary intervention

CONT FRUCT FRUCTMIN Global P[P between two groups]Mean (SEM) Mean (SEM) Mean (SEM)

GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146

Total bilirubin (mgL)n = 5ndash7 200 (00655) 190 (00447) 207 (0127) 0481

Uric acid (mgL)n = 5ndash7 614 (0662) 472 (0371) 501 (0403) 0161

Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)

lt0001 [(C versus F) lt0001(C versus FM) 0001(F versus FM) 0163]

Creatinine (mgL)n = 5ndash7 514 (0153) 456 (0108) 500 (0162)

0052 [(C versus F) 0019(C versus FM) 0492(F versus FM) 0065]

Total proteins (gL)n = 5ndash7 5957 (0634) 6298 (0881) 6149 (0710)


Albumin (gL)n = 5ndash7 2723 (0342) 2950 (0214) 2901 (0304)


Total cholesterol (gL)n = 5ndash7 0686 (00334) 0666 (0100) 0753 (00390) 0525

HDL-cholesterol (gL)n = 5ndash7 0357 (00224) 03540 (00486) 0397 (00218) 0518

LDL-cholesterol (gL)n = 5ndash7 0201 (00126) 0204 (00227) 0229 (00201) 0505

HDL-cholesteroltotalcholesteroln = 5ndash7

0520 (00140) 0536 (00104) 0527 (000888) 0637

HDL-cholesterolLDLcholesteroln = 5ndash7

182 (0189) 175 (0164) 181 (0171) 0963

Ferritin (UL)n = 5ndash7 2433 (2577) 1918 (0450) 1963 (0374)


OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401

C-reactive protein(mgL)n = 5-6

004167 (00095) 003800 (00058) 005500 (00034) 0216

Substance P (pgmL)119899 = 7

197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514

C or CONT control F or FRUCT 10 fructose in tap water FM or FRUCTMIN 10 fructose in natural mineral-rich water GOT glutamic-oxaloacetictransaminase GPT glutamic-pyruvic transaminase IL-6 interleukin-6 OPG osteoprotegerin RANKL receptor activator of nuclear factor kappa-B ligandSEM standard error of the mean TNF-120572 tumor necrosis factor-alpha


0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um

lowast lowast lowastlowast

excr

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Eq2

4 h)

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16

18

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22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)

Figure 3 (a) Urine volume (mL24 h 119899 = 7 $119875 lt 001 CONT versus FRUCT and 119875 lt 005 CONT versus FRUCTMIN 119875 lt 001 CONT

versus FRUCT and FRUCTMIN) (b) urinary sodium excretion (mEq24 h 119899 = 6-7 lowast119875 lt 001 FRUCTMIN versus CONT and 119875 lt 005 forFRUCTMIN versus FRUCT) and (c) urinary creatinine excretion evolution (mg24 h 119899 = 6-7) every other week during the first 6 weeks ofthe dietary intervention Results were expressed as mean plusmn standard error of the mean CONT control FRUCT 10 fructose in tap waterFRUCTMIN 10 fructose in natural mineral-rich water

GSSG and a strong tendency for GSHGSSG (119875 = 0062global 119875 = 0045)) Natural mineral-rich water ingestioncounteracted all these modifications Regarding catalase andGSSG therewas a strong trend to respectively a decrease andan increase in FRUCTMIN versus FRUCT (119875 = 0065 and119875 = 0055 resp) No significant modifications were observed

for 8-OHdG levels (data not shown) neither for other redoxparameters (Table 4)

36 LiverMagnesium andCalciumContent A slight decreasewas observed in FRUCT versus CONT for both liver mag-nesium and calcium content that was prevented by natural


Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)

lowast lowast lowastlowastlowast

lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)

Figure 4 (a) Systolic blood pressure (mm Hg 119899 = 4ndash7) and diastolic blood pressure (mm Hg 119899 = 4ndash7) and (b) heart rate evolution(beatsmin 119899 = 4ndash7 lowast119875 lt 005 CONT versus FRUCT lowastlowast119875 lt 001 CONT versus FRUCT) during the dietary intervention Results wereexpressed as mean plusmn standard error of the mean CONT control FRUCT 10 fructose in tap water FRUCTMIN 10 fructose in naturalmineral-rich water

Table 3 Plasma electrolyte content at the end of the dietary intervention

CONT FRUCT FRUCTMIN Global P[P between groups]Mean (SEM) Mean (SEM) Mean (SEM)

Sodium (mEqL)n = 5ndash7 14242 (1270) 14360 (0872) 14457 (0719) 0318

Potassium (mEqL)n = 5ndash7 590 (0236) 592 (0208) 548 (0150) 0261

Chloride (mEqL)n = 5ndash7 10000 (0976) 9960 (0600) 10029 (0565) 0836

Magnesium (mEqL)n = 5ndash7 171 (00513) 154 (00385) 155 (00431)

0025[(C versus F) 0020(C versus FM) 0017(F versus FM) 0873]

Calcium (mEqL)n = 5ndash7 532 (00346) 544 (00600) 542 (00359) 0124

Phosphorus (mgL)n = 5ndash7 7611 (3841) 7662 (1964) 7930 (3167) 0756

C or CONT control F or FRUCT 10 fructose in tap water FM or FRUCTMIN 10 fructose in natural mineral-rich water SEM standard error of the mean

mineral-rich water ingestion (Figures 8(a) and 8(b) resp)most particularly for magnesium

4 Discussion

The fructose-fed SDR model mimics a predominantly envi-ronmentally acquired MS model [15] that is commonly usedin MS research Similarly in the present study many of

the alterations observed in different protocols of fructose-induced MS were recapitulated Increased systolic BP adi-posity index and liver and kidney weight to body weightratios as well as modulation of the hepatic redox status andsimilar changes in the plasma levels of hormones except foraldosterone andor energy substrates evaluated in this workhave been reported in different protocols of fructose-inducedMS in SDR [15ndash18 20 22ndash24 36ndash38]

As previously reported [17] fructose intervention in-creases SDR body weight but besides fructose metabolic


CONT FRUCT FRUCTMIN000

001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)

Figure 5 Organ weight to body weight ratios (a) Liver (119899 = 7) (b) epididymal adipose tissue (119899 = 7) (c) right kidney (119899 = 6- 7) and (d)left kidney (119899 = 6-7) at the end of the dietary intervention Results were expressed as mean plusmn standard error of the mean 119875 lt 0001 CONTversus FRUCT 119875 lt 0005 FRUCT versus FRUCTMIN and lowast119875 lt 005 CONT versus FRUCT AT adipose tissue CONT control FRUCT10 fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water G119875 global P

effects two details of our experimental protocol could havecontributed to body weight increase rats were housed indi-vidually which may have reduced their physical activityand were already adult rats at the beginning of the dietarymanipulation (their age was reflected in the high bodyweight values 475ndash597 g) which may have amplified fruc-tose metabolic effects [39] Additionally to the variationsobserved in food and fluid ingestions seen in the CONTgroup with aging (over the 8 weeks of dietary intervention)fructose-fed rats adjusted fluid and food ingestions aimingto maintain the level of energy consumption as previouslydescribed [18] Fructose-fed animals increased their bodyweight similarly between them and more than control rats(associated with a small increase of epididymal body-fat)reflecting the absence of anymajor naturalmineral-richwaterconsumption effect on both food and fructose ingestionsAccordingly effects shown below against MS induction inFRUCTMIN rats related exclusively to natural mineral-richwater ingestion and interestingly natural mineral-rich wateringestion reducedprevented the majority of the fructoseeffects and consequently protected against MS induction

which to our knowledge is described here for the firsttime MS represents a risk for cardiovascular disease (whoseprevalence is increasing worldwide) which together with therecent report of Luo et al on the consumption of low-mineralbottledwater that increases the risk for cardiovascular disease[40] demonstrates the high relevance of our research

The significant increase in plasma insulin levels in theFRUCT versus CONT group could have contributed to thesignificant effects in systolic BP and HR described before(the effect of natural mineral-rich water with time on HRand diastolic BP decreased after body weight adjustment)Hyperinsulinemia may increase BP and HR by increasingthe sympathetic nervous system activity through alterationin the neuronal vascular control andor by enhancement ofkidney sodium reabsorption [6 41 42] Increased sympa-thetic modulation of vessels and heart precedes metabolicdysfunction in mice drinking 10 fructose in tap water forup to 2months [43] HR amarker of autonomic dysfunctionassociates with MS particularly with insulin resistance andinterestingly in Japan the prevalence ofMS increases linearlywith the increase in HR [1 44]



1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)

Figure 6 Metabolic markers and hormonal status in plasma (a) Glucose (gL 119899 = 7) (b) triacylglycerols (gL 119899 = 5ndash7) (c) insulin (120583gL119899 = 7) (d) leptin (pgmL 119899 = 6) (e) aldosterone (ngmL 119899 = 7) and (f) melatonin levels (pgmL 119899 = 6) at the end of the dietaryintervention Results were expressed as mean plusmn standard error of the mean lowast119875 lt 005 CONT versus FRUCT CONT control FRUCT 10fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water G119875 global P

BP correlates with plasma aldosterone levels and anassociation between plasma aldosterone levels and hyperin-sulinemia has been described in obesity [45] Although bothfructose-fed groups in the present study had significantlyincreased body weight versus controls the insulin value inthe FRUCTMIN group (that after body weight adjustmentpresented a strong tendency to decrease versus FRUCT(data not shown)) along with the later BP increase was

in accordance with the unaltered aldosterone levels in theFRUCTMIN group In fructose-fed SDR the absence ofa significant increase in body weight associates with noincrease of aldosterone levels in spite of hyperinsulinemia[46]

Leptin resistance may be an early feature of metabolicdysfunction induced by fructose-feeding since it may pre-cede increased adiposity elevated circulating leptin levels



100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)

GP = 0002 lowast lowastlowast

(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6

lowast lowastlowastO

xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)

Figure 7 Redox state markers in liver (a) Catalase (U catalasemg protein 119899 = 7) (b) superoxide dismutase (U SODmg protein 119899 = 7) (c)glutathione peroxidase activities (U GPxmg protein 119899 = 7) (d) oxidized glutathione content (nmolmg protein 119899 = 7) (e) GSH to GSSGratio (119899 = 7) and (f) sirtuin 3 protein expression (arbitrary units (au) 119899 = 5-6) at the end of the dietary intervention Results were expressedas mean plusmn standard error of the mean lowast119875 lt 005 CONT versus FRUCT lowastlowastlowast119875 lt 0005 CONT versus FRUCT CONT control FRUCT 10fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water G119875 global P GPx glutathione peroxidase GSH reducedglutathione GSSG oxidized glutathione Sirt3 sirtuin 3 SOD superoxide dismutase

and changes in glucose metabolism in rats Despite higherleptin levels (with the comparison versus CONT significantafter adjustment for body weight (data not shown)) whichwould anticipate a reduction in food intake and body fatin healthy conditions FRUCT rats had a lower decrease of

food ingestion with time than FRUCTMIN rats (significantafter adjustment for body weight (data not shown)) as wellas a similar weight gain and amount of epididymal fat Theseresults could reflect a phenomenon of selective leptin resis-tance that together with the activation of the sympathetic



410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)

Figure 8 Liver magnesium (a) and calcium (b) content (120583gg of tissue lyophilize 119899 = 6 for both elements) at the end of the dietaryintervention Results were expressed as mean plusmn standard error of the mean Ca calcium CONT control FRUCT 10 fructose in tap waterFRUCTMIN 10 fructose in natural mineral-rich water G119875 global P Mg magnesium

nerves by hyperleptinemia could have contributed to theearlier development of hypertension in FRUCT rats [5 47ndash49] Melatonin has anti-inflammatory antihyperlipidemicand antihypertensive properties and it is known to influenceinsulin secretion and to enhance its action (it increasesinsulin sensitivity and enhances insulin effects on leptinexpression) [19 50ndash52] The apparent deregulation of leptinmelatonin insulin and aldosterone observed in the FRUCTgroup owing tomodifications in the hormone levels was lessevident in the FRUCTMIN group

Fructose is highly lipogenic as its hepatic metabolismprovides great amounts of triose phosphate precursors forfatty acid synthesis [5 6] Reduced leptin and insulin sensi-tivities contribute to hypertriglyceridemia induced by fruc-tose ingestion [53] The difference in triacylglycerol levelsin the two fructose-fed groups could be explained by theimprovement of leptin insulin and aldosterone levels inFRUCTMIN induced by the natural mineral-rich wateringestion (the magnitude of triacylglycerols increase versusCONT was reduced after body weight adjustment (data notshown)) Normalization of melatonin levels could have alsocontributed [52 54]

The increase in plasma albumin and total protein levelshas been described in fructose-fed rats [55] which couldreflect a combination of undernutrition (also because of thedecrease in food ingestion) some degree of liver disorder(resulting fromMS induction) andor dehydration (owing toloose stools resulting from incomplete fructose absorption)[56 57] Nevertheless the pattern of urine volume mirroredthe pattern of fluid ingestion and we did not observe loosestools which makes dehydration unlikely in the fructose-fedrats

Although we found a significant increase in both kidneysweight to body weight ratios in the FRUCT group we be-lieve that there was no renal functional alteration in thisSDR group taking into consideration the plasma andorurinary profiles of creatinine urea albumin total proteins

magnesium sodium potassium calcium chloride andphos-phorous Despite an increase in the kidney weight to bodyweight ratio Rizkalla et al reported no glomerular basementmembrane thickening in SDR after 10 weeks of 57 fructose-feeding [36]

Fructose-feeding accelerates osteoporosis [58] and ac-cordingly the OPG to RANKL ratio (reflecting the ratio ofosteoblast versus osteoclast activities [58 59]) seemed todecrease in the FRUCT group Interestingly and in accor-dance with FRUCT group results TNF-120572 and IL-6 areimportant mediators in the process of osteoclast differenti-ation and activation [59] and thus the changes observedin their levels might have contributed to the lower OPG toRANKL ratio High levels of leptin and aldosterone have beenlinked to proinflammatory and prooxidant actions [45 47]In the FRUCTMIN group the improvement of the leptinaldosterone TNF-120572 and IL-6 values could have contributedto the improvement of the OPG to RANKL ratio

Taking into consideration all the results obtained forboth fructose-fed SDR groups the slightly increased levels ofsubstance P and CRP in FRUCTMIN rats were unexpectedPlasma substance P levels increase under magnesium defi-ciency and contribute to increase inflammation and proteinand lipid oxidation [60] In fact plasma magnesium levelsof both fructose groups were significantly decreased but theFRUCTMIN group displayed better plasma TNF-120572 and IL-6levels as well as lower hepatic protein oxidation content (afteradjustment for body weight this latter parameter showed atendency to a decrease in FRUCTMIN versus the other twogroups most particularly versus FRUCT (data not shown))Interestingly high CRP levels occur in energy restrictedanimals [61]

In FRUCT animals the absence of oxidative lesions inlipids proteins and DNA (the same as for proteins hap-pened for DNA oxidative lesions (data not shown)) couldbe explained by the significantly increased catalase activityand apparently increased SOD activity Polizio et al describesimilar results for lipid oxidative lesions and catalase and


Table 4 Hepatic redox state characterization at the end of the dietary intervention

CONT FRUCT FRUCTMIN Global PMean (SEM) Mean (SEM) Mean (SEM)

Glutathione-reductase(UGRmg protein)119899 = 7

00297 (00015) 00320 (00011) 00322 (00022) 0507

Glutathione-S-transferase(UGSTmg protein)119899 = 7

03754 (00261) 03738 (00319) 03764 (00263) 0998

Reduced glutathione(nmolmg protein) 119899 = 7 3605082 (164242) 3320008 (90570) 3099596 (295796) 0235

Malondialdehyde (nmolmgprotein) 119899 = 6 00108 (00010) 00100 (00012) 00121 (00013) 0424

Protein carbonyls (nmolmgprotein) 119899 = 7 08465 (00732) 09828 (00808) 07305 (00866) 0113

CONT control FRUCT 10 fructose in tap water FRUCTMIN 10 fructose in natural mineral-rich water GR glutathione-reductase GST glutathione-S-transferase SEM standard error of the mean

Metabolic Syndrome animal modelSprague-Dawley rats with10 fructose in tap water

for 8 weeks

Reduced the effects

Increased heart rateReduced the

effects

Sprague-Dawley rats with10 fructose in a

hypersaline sodium-richnaturally sparkling mineral

water for 8 weeks

Increased

organ weight to body weight ratios (for liver and both kidneys)plasma triacylglycerols insulin and leptin levels

hepatic catalase activity

Decreasedhepatic glutathione peroxidase activity

oxidized glutathione content

Figure 9 Summary of fructose-feeding effects that were reducedprevented by the natural mineral-rich water

SOD activities by 10 fructose ingestion in tap water [24]The significant decrease in GPx activity in FRUCT ratscould be partially compensated by the significant increase incatalase activity since both enzymes can eliminate hydrogenperoxide (converting it to water) Catalase is responsible forthe elimination of high concentrations of hydrogen peroxidewhile GPx does it when concentrations of hydrogen peroxideare low [62] The higher levels of hydrogen peroxide couldhave resulted from the fructose-feeding [7 63] and the smalldecrease of Sirt3 protein expression could have intensifiedreactive oxygen species production in FRUCT rats [64]The significant decrease in hepatic GPx activity could havecontributed to the strong tendency for an increase in theGSHGSSG ratio in FRUCT versus CONT by oxidizingless GSH to GSSG Increased GSSG efflux from hepatocytes

andor increased hepatic GSH synthesis induced by fructosecould also apply However we did not observe an increasein GSH level as it might have been expected from a lowerGSH oxidation andor increased GSH synthesis Althoughcellular ATP depletion induced by fructose prevents ATP-dependent GSSG efflux in freshly isolated rat hepatocytes[65] this phenomenon should not have a strong impacthere since an increase in uric acid formation was notobserved The variations observed for GPx SOD and cata-lase activities in the two fructose-fed groups are in accor-dance with the antioxidant actions of melatonin (probablyits primary function) and Sirt3 Melatonin possesses freeradical-scavenging activity stimulates antioxidant enzymes(eg GPx) and inhibits reactive oxygennitrogen speciesproducing enzymes [66 67] Sirt3 has beneficial effects on


mitochondrial electron transport chain (contributing to areduction in the production of reactive oxygen species) andmitochondrial antioxidant enzymes (probably also on GPxlike melatonin) [64] Reduction of Sirt3 associates with anaccelerated development of metabolic abnormalities similarto the MS [68] which is in agreement with our overallresults

Our results also showed that the natural mineral-richwater could contribute to the preservation of the hepaticintracellular ions namely the magnesium content It is welldocumented that plasma ion levels might not reflect theirtissue levels and here this was evident regarding plasmaand hepatic concentrations of magnesium and calciumMagnesium deficiency is associated with insulin resistanceFor quite some time it was thought that it could be the causeof insulin resistance but very recently it was described thattype 2 Diabetes Mellitus and a lower degree of metaboliccontrol are essential in accounting for the lower levels ofserum magnesium that occur in obese individuals [69]

5 Conclusion

Figure 9 summarizes the significant effects of fructose-feed-ing obtained in this research that were reducedprevent-ed bythe naturalmineral-richwater (taking into consideration thatwhen fructose was coingested with the natural mineral-richwater no significant effects were observed versus the control)

Still although for some of the parameters evaluated inour study the extension of differences among groups didnot achieve statistical significance the variations observedwere consistent with the pattern expected which reinforcestheir biological relevance and justifies their presentation anddiscussion

The results here described suggest that this naturalminer-al-rich water seems to have potential to prevent MS induc-tion We hypothesize that its regular intake in the context ofmodern diets which have a general acidic character interfer-ingwithmineral homeostasis and are poor inmicronutrientsnamely potassium calcium and magnesium could addsurplus value and attenuate imbalances thus contributing tometabolic and redox health and consequently decreasing therisk for atherosclerotic cardiovascular disease

Conflict of Interests

Cidalia Pereira PhD grant (SFRHBDE337982009) was co-financed by Unicer Bebidas SA Other authors declare thatthere is no conflict of interests regarding the publication ofthis article When considering the partial funding by UnicerBebidas SA it is important to mention that the study herepresented was developed in its full extent both in scientificterms and research equipment conditions independently ofthis company

Acknowledgments

The authors kindly thank Unicer Bebidas SA for providingPedras Salgadas and Marco Boscaro (DSI) and Delphine

Bouard (Charles River Laboratories) for the helpful advicesregarding the use of the telemetry software and the handlingof the implanted Sprague-Dawley rats respectively Thiswork was supported by FCT (Fundacao para a Ciencia eTecnologia PEst-OESAUUI00382011) through the Centrode Farmacologia e Biopatalogia Quımica (U38FCT) Fac-ulty of Medicine University of Porto which integrates theDepartment of Biochemistry Faculty ofMedicine Universityof Porto Additional funding was provided by FCT throughthe Fundo Social Europeu Programa Operacional PotencialHumano da UE (SFRHBDE337982009) and by UnicerBebidas SA Portugal

References

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[2] K G M M Alberti R H Eckel S M Grundy et al ldquoHarmo-nizing the metabolic syndrome a joint interim statement of theInternational Diabetes Federation task force on epidemiologyand prevention National Heart Lung and Blood InstituteAmerican Heart Association World Heart Federation Inter-national Atherosclerosis Society and International Associationfor the Study of Obesityrdquo Circulation vol 120 no 16 pp 1640ndash1645 2009

[3] S E Feldeisen and K L Tucker ldquoNutritional strategies inthe prevention and treatment of metabolic syndromerdquo AppliedPhysiology Nutrition and Metabolism vol 32 no 1 pp 46ndash602007

[4] I Aeberli M Hochuli P A Gerber et al ldquoModerate amountsof fructose consumption impair insulin sensitivity in healthyyoung men a randomized controlled trialrdquo Diabetes Care vol36 no 1 pp 150ndash156 2012

[5] M J Dekker Q Su C Baker A C Rutledge and K AdelildquoFructose a highly lipogenic nutrient implicated in insulinresistance hepatic steatosis and the metabolic syndromerdquoAmerican Journal of Physiology Endocrinology andMetabolismvol 299 no 5 pp E685ndashE694 2010

[6] L Tappy and K-A Le ldquoMetabolic effects of fructose and theworldwide increase in obesityrdquo Physiological Reviews vol 90no 1 pp 23ndash46 2010

[7] K A Le and L Tappy ldquoMetabolic effects of fructoserdquo CurrentOpinion in Clinical Nutrition ampMetabolic Care vol 9 no 4 pp469ndash475 2006

[8] H Lee J Lee S S Hwang et al ldquoPotassium intake and theprevalence of metabolic syndrome the korean national healthand nutrition examination survey 2008ndash2010rdquo PLoS ONE vol8 no 1 Article ID e55106 2013

[9] F Fumeron A Lamri and N Emery ldquoDairy products and themetabolic syndrome in a prospective study DESIRrdquo Journal ofthe American College of Nutrition vol 30 no 5 supplement 1pp 454Sndash463S 2011

[10] S L Volpe ldquoMagnesium the metabolic syndrome insulinresistance and type 2 diabetesmellitusrdquoCritical Reviews in FoodScience and Nutrition vol 48 no 3 pp 293ndash300 2008

[11] L Petraccia G Liberati S Giuseppe Masciullo M Grassi andA Fraioli ldquoWatermineralwaters andhealthrdquoClinicalNutritionvol 25 no 3 pp 377ndash385 2006


[12] L Bacciottini A Tanini A Falchetti et al ldquoCalcium bioavail-ability from a calcium-rich mineral water with some observa-tions on methodrdquo Journal of Clinical Gastroenterology vol 38no 9 pp 761ndash766 2004

[13] O Karagulle T Kleczka C Vidal et al ldquoMagnesium absorptionfrommineral waters of different magnesium content in healthysubjectsrdquo Forschende Komplementarmedizin vol 13 no 1 pp9ndash14 2006

[14] R Rylander ldquoDrinking water constituents and diseaserdquo Journalof Nutrition vol 138 no 2 pp 423Sndash425S 2008

[15] M Oron-Herman Y Kamari E Grossman et al ldquoMetabolicsyndrome comparison of the two commonly used animalmodelsrdquo American Journal of Hypertension vol 21 no 9 pp1018ndash1022 2008

[16] H-Y Tsai L-Y Wu and L S Hwang ldquoEffect of aproanthocyanidin-rich extract from longan flower on markersof metabolic syndrome in fructose-fed ratsrdquo Journal ofAgricultural and Food Chemistry vol 56 no 22 pp 11018ndash11024 2008

[17] M H Abdulla M A Sattar N A Abdullah M A H KhanK R L Anand Swarup and E J Johns ldquoThe contribution of1205721B-adrenoceptor subtype in the renal vasculature of fructose-fed Sprague-Dawley ratsrdquoEuropean Journal of Nutrition vol 50no 4 pp 251ndash260 2011

[18] L Vila A Rebollo G S Adalsteisson et al ldquoReduction of liverfructokinase expression and improved hepatic inflammationand metabolism in liquid fructose-fed rats after atorvastatintreatmentrdquo Toxicology and Applied Pharmacology vol 251 no1 pp 32ndash40 2011

[19] A Kitagawa Y Ohta and K Ohashi ldquoMelatonin improvesmetabolic syndrome induced by high fructose intake in ratsrdquoJournal of Pineal Research vol 52 no 4 pp 403ndash413 2012

[20] A Leibowitz E Peleg Y Sharabi Z Shabtai A Shamissand E Grossman ldquoThe role of melatonin in the pathogenesisof hypertension in rats with metabolic syndromerdquo AmericanJournal of Hypertension vol 21 no 3 pp 348ndash351 2008

[21] E Hopps D Noto G Caimi and M R Averna ldquoA novelcomponent of the metabolic syndrome the oxidative stressrdquoNutrition Metabolism and Cardiovascular Diseases vol 20 no1 pp 72ndash77 2010

[22] P K Bagul H Middela S Matapally et al ldquoAttenuation ofinsulin resistance metabolic syndrome and hepatic oxidativestress by resveratrol in fructose-fed ratsrdquo PharmacologicalResearch vol 66 no 3 pp 260ndash268 2012

[23] W Suwannaphet A Meeprom S Yibchok-Anun and S Adis-akwattana ldquoPreventive effect of grape seed extract against high-fructose diet-induced insulin resistance and oxidative stress inratsrdquo Food and Chemical Toxicology vol 48 no 7 pp 1853ndash18572010

[24] A H Polizio S Gonzales M C Munoz C Pena and M LTomaro ldquoBehaviour of the anti-oxidant defence system andheme oxygenase-1 protein expression in fructose-hypertensiveratsrdquo Clinical and Experimental Pharmacology and Physiologyvol 33 no 8 pp 734ndash739 2006

[25] A M Perez-Granados S Navas-Carretero S Schoppenand M P Vaquero ldquoReduction in cardiovascular risk bysodium-bicarbonated mineral water in moderately hyperc-holesterolemic young adultsrdquo Journal of Nutritional Biochem-istry vol 21 no 10 pp 948ndash953 2010

[26] R Rylander and M J Arnaud ldquoMineral water intake reducesblood pressure among subjects with low urinary magnesiumand calcium levelsrdquo BMC Public Health vol 4 article 56 2004

[27] U Schorr A Distler and A M Sharma ldquoEffect of sodiumchloride- and sodium bicarbonate-rich mineral water on bloodpressure and metabolic parameters in elderly normotensiveindividuals a randomized double-blind crossover trialrdquo Journalof Hypertension vol 14 no 1 pp 131ndash135 1996

[28] S Schoppen F J Sanchez-Muniz A M Perez-Granados et alldquoDoes bicarbonated mineral water rich in sodium changeinsulin sensitivity of postmenopausal womenrdquo Nutricion Hos-pitalaria vol 22 no 5 pp 538ndash544 2007

[29] S Schoppen A M Perez-Granados A Carbajal et al ldquoAsodium-rich carbonated mineral water reduces cardiovascularrisk in postmenopausal womenrdquo Journal of Nutrition vol 134no 5 pp 1058ndash1063 2004

[30] M Costantino G Giuberti M Caraglia et al ldquoPossible antiox-idant role of SPA therapy with chlorine-sulphur-bicarbonatemineral waterrdquo Amino Acids vol 36 no 2 pp 161ndash165 2009

[31] S Benedetti F Benvenuti G Nappi et al ldquoAntioxidative effectsof sulfurous mineral water protection against lipid and proteinoxidationrdquo European Journal of Clinical Nutrition vol 63 no 1pp 106ndash112 2009

[32] M F Schreuder M Fodor J A E van Wijk and H ADelemarre-Van de Waal ldquoAssociation of birth weight withcardiovascular parameters in adult rats during baseline andstressed conditionsrdquo Pediatric Research vol 59 no 1 pp 126ndash130 2006

[33] M Assuncao M J Santos-Marques R Monteiro et al ldquoRedwine protects against ethanol-induced oxidative stress in ratliverrdquo Journal of Agricultural and Food Chemistry vol 57 no14 pp 6066ndash6073 2009

[34] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2008

[35] J Cacho J Sevillano J de Castro E Herrera and M P RamosldquoValidation of simple indexes to assess insulin sensitivity duringpregnancy in Wistar and Sprague-Dawley ratsrdquo American Jour-nal of Physiology Endocrinology andMetabolism vol 295 no 5pp E1269ndashE1276 2008

[36] S W Rizkalla J Boillot V Tricottet et al ldquoEffects of chronicdietary fructose with and without copper supplementation onglycaemic control adiposity insulin binding to adipocytes andglomerular basement membrane thickness in normal ratsrdquoBritish Journal of Nutrition vol 70 no 1 pp 199ndash209 1993

[37] P-S Hsieh and Y-H Tai ldquoAqueous extract of Monascus pur-pureus M9011 prevents and reverses fructose-induced hyper-tension in ratsrdquo Journal of Agricultural and Food Chemistry vol51 no 14 pp 3945ndash3950 2003

[38] L G Sanchez-Lozada E Tapia A Jimenez et al ldquoFructose-induced metabolic syndrome is associated with glomerularhypertension and renal microvascular damage in ratsrdquo Amer-ican Journal of Physiology Renal Physiology vol 292 no 1 ppF423ndashF429 2007

[39] R F de Moura C Ribeiro J A de Oliveira E Stevanatoand M A R de Mello ldquoMetabolic syndrome signs in Wistarrats submitted to different high-fructose ingestion protocolsrdquoBritish Journal of Nutrition vol 101 no 8 pp 1178ndash1184 2009

[40] J Luo Q Zhao L Zhang et al ldquoThe consumption of low-mineral bottled water increases the risk of cardiovasculardisease an experimental study of rabbits and young menrdquoInternational Journal of Cardiology vol 168 no 4 pp 4454ndash4456 2013


[41] Y Zamami S Takatori N Hobara et al ldquoHyperinsulinemiainduces hypertension associated with neurogenic vascular dys-function resulting from abnormal perivascular innervations inrat mesenteric resistance arteriesrdquo Hypertension Research vol34 no 11 pp 1190ndash1196 2011

[42] T Fulop D Tessier and A Carpentier ldquoThe metabolic syn-dromerdquo Pathologie Biologie vol 54 no 7 pp 375ndash386 2006

[43] K de Angelis D D Senador C Mostarda M C Irigoyenand M Morris ldquoSympathetic overactivity precedes metabolicdysfunction in a fructose model of glucose intolerance inmicerdquo American Journal of Physiology Regulatory Integrativeand Comparative Physiology vol 302 no 8 pp R950ndashR9572012

[44] G Grassi F Arenare F Quarti-Trevano G Seravalle and GMancia ldquoHeart rate sympathetic cardiovascular influences andthe metabolic syndromerdquo Progress in Cardiovascular Diseasesvol 52 no 1 pp 31ndash37 2009

[45] A Whaley-Connell M S Johnson and J R Sowers ldquoAldos-terone role in the cardiometabolic syndrome and resistanthypertensionrdquo Progress in Cardiovascular Diseases vol 52 no5 pp 401ndash409 2010

[46] I-S Hwang W-C Huang J-N Wu L R Shian and G MReaven ldquoEffect of fructose-induced hypertension on the renin-angiotensin-aldosterone system and atrial natriuretic factorrdquoAmerican Journal of Hypertension vol 2 no 6 part 1 pp 424ndash429 1989

[47] H Katagiri T Yamada and Y Oka ldquoAdiposity and cardiovas-cular disorders disturbance of the regulatory system consistingof humoral and neuronal signalsrdquo Circulation Research vol 101no 1 pp 27ndash39 2007

[48] R J Denver RM Bonett andGC Boorse ldquoEvolution of leptinstructure and functionrdquo Neuroendocrinology vol 94 no 1 pp21ndash38 2011

[49] H J Harwood Jr ldquoThe adipocyte as an endocrine organ in theregulation of metabolic homeostasisrdquo Neuropharmacology vol63 no 1 p 5775 2012

[50] F Radogna M Diederich and L Ghibelli ldquoMelatonin apleiotropic molecule regulating inflammationrdquo BiochemicalPharmacology vol 80 no 12 pp 1844ndash1852 2010

[51] A Korkmaz T Topal D-X Tan and R J Reiter ldquoRole ofmelatonin in metabolic regulationrdquo Reviews in Endocrine andMetabolic Disorders vol 10 no 4 pp 261ndash270 2009

[52] A Agil M Navarro-Alarcon R Ruiz S Abuhamadah M-YEl-Mir and G F Vazquez ldquoBeneficial effects of melatonin onobesity and lipid profile in young Zucker diabetic fatty ratsrdquoJournal of Pineal Research vol 50 no 2 pp 207ndash212 2011

[53] N Roglans L Vila M Farre et al ldquoImpairment of hepaticSTAT-3 activation and reduction of PPAR120572 activity in fructose-fed ratsrdquo Hepatology vol 45 no 3 pp 778ndash788 2007

[54] A Hannemann C Meisinger M Bidlingmaier et al ldquoAssocia-tion of plasma aldosterone with the metabolic syndrome in twoGerman populationsrdquo European Journal of Endocrinology vol164 no 5 pp 751ndash758 2011

[55] Z Mellouk K Louchami E Hupkens et al ldquoThe metabolicsyndrome of fructose-fed rats effects of long-chain polyunsatu-rated omega3 and omega6 fatty acids V Post-mortemfindingsrdquoMolecular Medicine Reports vol 6 no 6 pp 1399ndash1403 2012

[56] M E Latulippe and S M Skoog ldquoFructose malabsorption andintolerance effects of fructose with and without simultaneousglucose ingestionrdquo Critical Reviews in Food Science and Nutri-tion vol 51 no 7 pp 583ndash592 2011

[57] K J Isselbacher Harrisonrsquos Principles of Internal MedicineMcGraw-Hill 13th edition 1994

[58] R Hanayama H Shimizu H Nakagami et al ldquoFluvastatinimproves osteoporosis in fructose-fed insulin resistant modelrats through blockade of the classical mevalonate pathway andantioxidant actionrdquo International Journal ofMolecularMedicinevol 23 no 5 pp 581ndash588 2009

[59] J J Cao ldquoEffects of obesity on bone metabolismrdquo Journal ofOrthopaedic Surgery and Research vol 6 article 30 2011

[60] W B Weglicki I T Mak J H Kramer et al ldquoRole of free radi-cals and substance P in magnesium deficiencyrdquo CardiovascularResearch vol 31 no 5 pp 677ndash682 1996

[61] I Tomada D Fernandes J T Guimaraes H Almeida andD Neves ldquoEnergy restriction ameliorates metabolic syndrome-induced cavernous tissue structural modifications in aged ratsrdquoAge vol 35 no 5 pp 1721ndash1739 2013

[62] B Halliwell and JM C Gutteridge Free Radicals in Biology andMedicine Clarendon Press Oxford UK 4th edition 2007

[63] S Delbosc E Paizanis R Magous et al ldquoInvolvement of oxida-tive stress and NADPH oxidase activation in the developmentof cardiovascular complications in amodel of insulin resistancethe fructose-fed ratrdquo Atherosclerosis vol 179 no 1 pp 43ndash492005

[64] A S Bause and M C Haigis ldquoSIRT3 regulation of mitochon-drial oxidative stressrdquo Experimental Gerontology vol 48 no 7pp 634ndash639 2012

[65] J M Silva L McGirr and P J OrsquoBrien ldquoPrevention ofnitrofurantoin-induced cytotoxicity in isolated hepatocytes byfructoserdquo Archives of Biochemistry and Biophysics vol 289 no2 pp 313ndash318 1991

[66] A Galano D X Tan and R J Reiter ldquoMelatonin as a naturalally against oxidative stress a physicochemical examinationrdquoJournal of Pineal Research vol 51 no 1 pp 1ndash16 2011

[67] R J Reiter D-X Tan and L Fuentes-Broto ldquoMelatonin amultitasking moleculerdquo Progress in Brain Research vol 181 pp127ndash151 2010

[68] M D Hirschey T Shimazu J Y Huang B Schwer and EVerdin ldquoSIRT3 regulates mitochondrial protein acetylation andintermediary metabolismrdquo Cold Spring Harbor Symposia onQuantitative Biology vol 76 pp 267ndash277 2011

[69] A Lecube J A Baena-Fustegueras J M Fort D PelegrıC Hernandez and R Simo ldquoDiabetes is the main factoraccounting for hypomagnesemia in obese subjectsrdquo PLoS ONEvol 7 no 1 Article ID e30599 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

International Journal of

EndocrinologyHindawi Publishing Corporationhttpwwwhindawicom

Volume 2014


Disease Markers

BioMed Research International


OncologyJournal of



Oxidative Medicine and Cellular Longevity

PPARRe sea rch


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice

Parkinsonrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


in the MS [4ndash7] On the other hand minerals like potas-sium calcium and magnesium proposed as protectiveagainst the MS are generally deficient in MS-inducing diets[3 8ndash10]

Natural mineral waters are waters of underground originprotected from contamination and microbiologically whole-someThey are characterized by their purity at source contentin minerals trace elements and other constituents as wellas by favorable effects on human health [11] Additionallybioavailability ofminerals fromnaturalmineral waters is high[12ndash14]

The fructose-fed rat is an interesting and well-validatedanimal model of diet-induced MS (predominantly acquiredMS model) that is commonly used in MS research [15]Different rat strains with distinct fructose ingestion protocolsare reported in the literature and in all cases fructose hasbeen observed to induce MS features such as moderatehypertension glucose intolerance hyperinsulinemia insulinresistance dyslipidemia (hypertriglyceridemia hypercholes-terolemia) altered cytokine and adipokine status (alteredtumor necrosis factor-alpha (TNF-120572) and leptin levels eg)decreased melatonin production andor increased body fatandor body weight [15ndash20]

Both in humans and rats a strong association has beenfound between MS and oxidative stress [21] and fructose-feeding associates with modification of the hepatic redoxstatus [16 22ndash24]

Beneficial effects of acute or chronic natural mineral-richwaters ingestion on blood pressure (BP) [25ndash27] metabolicprofile (plasma insulin sensitivity [28] fasting serum glucoseconcentration [29] and fasting serum lipid profile [25 29])and plasma oxidative stress markers (reactive oxygen species[30] lipid and protein oxidation product levels total antioxi-dant capacity and total thiol levels [31]) have been publishedbut to our knowledge not in MS individuals or animalmodels The natural mineral waters tested are rich albeit indifferent proportions in bicarbonate calcium magnesiumpotassium andor sodium

We aimed to investigate the possible beneficial effect ofnatural mineral-rich water on MS induction by fructose-feeding In the present work Sprague-Dawley rats (SDR)were fed with 10 fructose in natural mineral-rich water(Pedras Salgadas) for 8 weeks and compared to animals fed10 fructose in tap water The natural mineral-rich watertested has high total mineralization content (2855mgL)being mainly rich in sodium and bicarbonate and withhigher potassium calcium and magnesium content than tapwater

2 Material and Methods

21 Animals and Treatments The study was carried out in21 adult male CD SDR (388ndash483 g) from Charles River Lab-oratories (ChatillonChalaronne France) Telemetry trans-mitters (TA11PA-C40 Data Sciences International (DSI) StPaulMNUSA)were implanted in the abdominal cavity withthe catheter in the abdominal aorta by Charles River Ratswere shipped on the 5th day after surgery Upon arrival rats

were individually housed in an enriched environment andmaintained on a daily photoperiod of 12 h lighting schedule(20ndash22∘C) with free access to standard laboratory pellet food(2014 Teklad Global 14 Protein Rodent Maintenance Dietfrom Harlan Interfauna Iberica SA Barcelona Spain) andtap water Acclimatization took place for 10 days beforestarting the experimental protocol which was authorizedby the Veterinary National Department of the Ministryof Agriculture Rural Development and Fisheries Duringacclimatization rats spent gradually increasing periods oftime inside metabolic cages The handling and care of theanimals were conducted in conformity with the EuropeanCommunity Council guidelines for the use of experimentalanimals (86609EEC) and Act 12992

Animals were randomly divided into 3 groups (7 animalseach) with free access to different drinking solutions (a)tap water (CONT) (b) 10 fructose in tap water (FRUCT)or (c) 10 fructose in natural mineral-rich water (FRUCT-MIN) All experimental groups were fed ad libitum withthe standard laboratory chow diet mentioned above (20 ofenergy derived from protein 13 from fat and 67 fromcarbohydrate) The dietary manipulation lasted 8 weeks A3-week pretreatment period with the natural mineral-richwater was performed to the FRUCTMIN group (while theother rats were drinking tap water) to allow adjustment towater flavor and sparkles This period of time induced nochange in the pattern of food intake or increase of animalsbody weight (data not shown all animals weighed 475ndash597 g at the beginning of the dietary manipulation withfructose) Body weight and food and fluid ingestion valueswere registered weekly from week 0 to week 7 or 8 (as shownin Figures 1(a)ndash1(c) resp) On week 0 these parameters wereevaluated on the same day which occurred 24 to 48 h afterstarting the dietary manipulation and then on every oneweek after the first measurement Each week from week 0 to7 all animals spent 24 h inside metabolic cages for evaluationof food and fluid consumption as well as for urine collection(the latter at 0 2 4 and 6 weeks) At each occasion thethree groups of rats were represented with an equal numberof animals per group Total energy ingestion was calculatedby multiplying food and fluid ingestion values by the corre-sponding reference energy values and then adding these tworesults

The chemical characteristics of tap and natural mineral-rich waters are given in Table 1 The latter is classifiedas a hypersaline sodium-rich naturally sparkling mineralwater in conformity with the European Community Councilguidelines for natural mineral waters (200954EEC) andwas kindly provided by Unicer Bebidas SA (Leca do BalioMatosinhos Portugal)

22 Assessment of Blood Pressure and Heart Rate DataquestART 41 Silver telemetry system (DSI) with RCP-1 receivers(and APR-1 for ambient pressure reference) was used fortelemetric measurement of BP (mm Hg) and heart rate (HRbeatsmin) in the animal cage Dataquest ART acquisitionsoftware (DSI) was used to monitor all rats for 8 weeks Sam-pling was performed everymin and setting segment duration


0

50

100

150

CONT FRUCT FRUCTMIN

ΔBo

dy w

eigh

t (g)

500

550

600

650

700

CONTFRUCT

FRUCTMIN

Body

wei

ght (

g)

lowast

0 1 2 3 4 5 6 7 8

Weeks

(a)

CONTFRUCT

FRUCTMIN

lowast

Weeks0 1 2 3 4 5 6 7

Food

inge

stio

n (g

)

5

10

15

20

(b)

CONTFRUCT

FRUCTMIN

Weeks

00 1 2 3 4 5 6 7

100

150

Flui

d in

gesti

on (m

L)

+ $$ $

50

lowastlowast

(c)




















3 Results








0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

gesti

on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)
















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of
















0

50

100

150

CONT FRUCT FRUCTMIN

ΔBo

dy w

eigh

t (g)

500

550

600

650

700

CONTFRUCT

FRUCTMIN

Body

wei

ght (

g)

lowast

0 1 2 3 4 5 6 7 8

Weeks

(a)

CONTFRUCT

FRUCTMIN

lowast

Weeks0 1 2 3 4 5 6 7

Food

inge

stio

n (g

)

5

10

15

20

(b)

CONTFRUCT

FRUCTMIN

Weeks

00 1 2 3 4 5 6 7

100

150

Flui

d in

gesti

on (m

L)

+ $$ $

50

lowastlowast

(c)




















3 Results








0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

gesti

on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)
















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of





























3 Results








0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

gesti

on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)
















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of



















3 Results








0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

gesti

on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)
















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of
















0 1 2 3 4 5 6 740

60

80

Weeks

FRUCTFRUCTMIN

Ener

gy su

pplie

d by

flui

dto

tal e

nerg

y in

gesti

on (

)

(a)

0 1 2 3 4 5 6 70

40

80

120

FRUCTFRUCTMINCONT

Weeks

Tota

l ene

rgy

inge

stion

(kca

l)

++

++ ++

$$

$$ $ $

(b)
















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of


















GOT (UL)119899 = 7

10514 (13062) 8214 (11066) 8471 (7383) 0279

GPT (UL)119899 = 7

5086 (8681) 3857 (1192) 3657 (2626) 0146



Urea (gL)n = 5ndash7 0286 (00104) 0148 (00198) 0186 (00210)












0520 (00140) 0536 (00104) 0527 (000888) 0637


182 (0189) 175 (0164) 181 (0171) 0963



OPG (pgmL)n = 6-7 78957 (226538) 57121 (40265) 66787 (83676) 0606

RANKL (pgmL)n = 6-7 1015 (1694) 1391 (2675) 779 (1470) 0143

OPGRANKLn = 5ndash7 12637 (57324) 4537 (9376) 10938 (41657) 0401


004167 (00095) 003800 (00058) 005500 (00034) 0216


197867 (184475) 196855 (216113) 241607 (158299) 0186

TNF-120572 (pgmL)119899 = 7

71880 (121457) 228275 (1142161) 81877 (241407) 0216

IL-6 (pgmL)n = 5-6 3007 (0412) 3486 (0601) 2681 (0363) 0514



0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of
















0 2 4 60

30

60

90

120

Weeks

FRUCTFRUCTMINCONT

Urin

e vol

ume (

mL24

h)

$ $

(a)

Weeks0 2 4 6

0

1

2

3

4

FRUCTFRUCTMINCONT

Urin

ary

sodi

um


excr

etio

n (m

Eq2

4 h)

(b)

Weeks0 2 4 6

16

18

20

22

24

26

FRUCTFRUCTMINCONT

Urin

ary

crea

tinin

e exc

retio

n (m

g24

h)

(c)







Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of
















Systolic

Diastolic

Weeks

CONTFRUCT

FRUCTMIN

80

100

120

140

0 1 2 3 4 5 6 7 8

Bloo

d pr

essu

re (m

m H

g)

(a)H

eart

rate

(bea

tsm

in)


lowastlowast

310

330

350

370

390

CONTFRUCT

FRUCTMIN

Weeks0 1 2 3 4 5 6 7 8

(b)













4 Discussion






001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of

















001

002

003

004

005Li

ver w

eigh

tbod

y w

eigh

t

GP lt 0001

(a)


0005

0010

0015

0020

0025

Epid

idym

al A

T w

eigh

tbod

y w

eigh

t GP = 0139

(b)


0001

0002

0003

0004

lowast

Righ

t kid

ney

wei

ghtb

ody

wei

ght

GP = 0030

(c)


0001

0002

0003

0004

lowast

Left

kidn

ey w

eigh

tbod

y w

eigh

t

GP = 0033

(d)







1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of

















1

2

3

4

5G

luco

se (g

L)

GP = 0111

(a)

000

065

130

195

260

325

CONT FRUCT FRUCTMIN

lowast

Tria

cylg

lyce

rols

(gL

)

GP = 0046

(b)

0

5

10

15

CONT FRUCT FRUCTMIN

lowast

GP = 0084

Insu

lin (120583

gL)

(c)


5000

10000

15000

20000

25000

Lept

in (p

gm

L)

GP = 0053

(d)

000

001

002

003

004

CONT FRUCT FRUCTMIN

Ald

oste

rone

(ng

mL)

GP = 0343

(e)


5000

10000

15000

20000

25000

Mela

toni

n (p

gm

L)

GP = 0575

(f)







100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of

















100

200

300

400

500Ca

tala

se (U

cata

lase

mg

prot

ein)


(a)


1250

2500

3750

5000

Supe

roxi

de d

ismut

ase

(U S

OD

mg

prot

ein)

GP = 0514

(b)


0100

0200

0300

0400

lowast

Glu

tath

ione

per

oxid

ase

(U G

Pxm

g pr

otei

n)

GP = 0029

(c)


2

4

6


xidi

zed

glut

athi

one

(GSS

G n

mol

mg

prot

ein)

GP = 0002

(d)


100

200

300

GSH

GSS

G

GP = 0045

(e)


0375

0750

1125

1500

Sirt3

prot

ein

expr

essio

n (a

u)

GP = 0135

(f)






410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of

















410

520

630Li

ver M

g (120583

gg)

GP = 0118

(a)


25

50

75

100

Live

r Ca (

120583g

g)

GP = 0249

(b)














00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of



















00297 (00015) 00320 (00011) 00322 (00022) 0507


03754 (00261) 03738 (00319) 03764 (00263) 0998






for 8 weeks

Reduced the effects


effects



water for 8 weeks

Increased











5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of


















5 Conclusion






Acknowledgments



References













































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of
















































































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of


















































of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of




















of





Volume 2014


Disease Markers



OncologyJournal of




PPARRe sea rch




Journal of

ObesityJournal of















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