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Effect of astaxanthin supplementation on muscle damage and oxidative

stress markers in elite young soccer players

Article  in  The Journal of sports medicine and physical fitness · August 2012

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382 THEJOURNALOFSPORTSMEDICINEANDPHYSICALFITNESS August2012

cer training and soccer exercise are associated with excessive production of free radicals and oxidative stress, which might diminish antioxidant system efficiency. Supplementation with Asx could prevent exercise induced free radical produc-tion and depletion of non-enzymatic antioxidant defense in young soccer playersKey words: Astaxanthine-Soccer-Oxidativestress.

Aerobicexerciseofsufficientintensityanddura-tioncanresultinincreasedproductionofreac-

tiveoxygenspecies (ROS) invarious tissues.1Pro-longedexerciseleadstotheincreasedproductionofROSby themitochondrial electron transport chainthroughanincreaseinoxygenconsumption.2Also,xanthineoxidaseisactivatedviatheischemia–reper-fusionprocessduringexercise,resultinginthepro-ductionofROS.3TheimbalancebetweenenhancedROSproductionand theabilityofantioxidant sys-temstorendertheminactive,leadtocellularlossofredoxhomeostasisandtoproneconditionsofoxida-tive damage to cellular lipids, proteins and DNA.4Additionally, the emerging role of ROS in the de-layed-onsetmusclesorenessandmuscle injuryhasbeenrecentlyreported.5ROSmediatedsarcolemmal

1Institute for Bromatology, Faculty of PharmacyUniversity of Belgrade, Belgrade, Serbia

2Institute for Medical BiochemistryFaculty of Pharmacy

University of Belgrade, Belgrade, Serbia 3Sports Medicine Association of Serbia

Outpatient Clinic Vita Maximam, Belgrade, Serbia

B.DJORDJEVIC1,I.BARALIC1,J.KOTUR-STEVULJEVIC2,A.STEFANOVIC2

J.IVANISEVIC2,N.RADIVOJEVIC3,M.ANDJELKOVIC3,N.DIKIC3

Effect of astaxanthin supplementation on muscle damage and oxidative stress markers in elite young soccer players

Aim. The purpose of the current study was to examine the effect of Astaxanthin (Asx) supplementation on muscle en-zymes as indirect markers of muscle damage, oxidative stress markers and antioxidant response in elite young soccer play-ers.Methods. Thirty-two male elite soccer players were randomly assigned in a double-blind fashion to Asx and placebo (P) group. After the 90 days of supplementation, the athletes per-formed a 2 hour acute exercise bout. Blood samples were ob-tained before and after 90 days of supplementation and after the exercise at the end of observational period for analysis of thiobarbituric acid-reacting substances (TBARS), advanced oxidation protein products (AOPP), superoxide anion (O2

•¯), total antioxidative status (TAS), sulphydril groups (SH), su-peroxide-dismutase (SOD), serum creatine kinase (CK) and aspartate aminotransferase (AST). Results. TBARS and AOPP levels did not change throughout the study. Regular training significantly increased O2

•¯ levels (main training effect, P<0.01). O2

•¯ concentrations increased after the soccer exercise (main exercise effect, P<0.01), but these changes reached statistical significance only in the P group (exercise x supplementation effect, P<0.05). TAS lev-els decreased significantly post- exercise only in P group (P<0.01). Both Asx and P groups experienced increase in total SH groups content (by 21% and 9%, respectively) and sup-plementation effect was marginally significant (P=0.08). Ba-sal SOD activity significantly decreased both in P and in Asx group by the end of the study (main training effect, P<0.01). All participants showed a significant decrease in basal CK and AST activities after 90 days (main training effect, P<0.01 and P<0.001, respectively). CK and AST activities in serum significantly increased as result of soccer exercise (main ex-ercise effect, P<0.001 and P<0.01, respectively). Postexercise CK and AST levels were significantly lower in Asx group compared to P group (P<0.05)Conclusion. The results of the present study suggest that soc-

Correspondingauthor:I.Baralic,VatroslavaLisinskog19/17,11000Belgrade,Serbia.E-mail:ivanabaralic111@gmail.com

Anno:2012Mese:AugustVolume:52No:4Rivista:THEJOURNALOFSPORTSMEDICINEANDPHYSICALFITNESSCodRivista:JSPORTSMEDPHYSFITNESS

Lavoro:3592-JSMtitolobreve:EFFECTOFASTAXANTHINSUPPLEMENTATIONONMUSCLEDAMAGEANDOXI-DATIVESTRESSMARKERSprimoautore:DJORDJEVICpagine:382-92

BODYCOMPOSITION,NUTRITION,SUPPLEMENTATIONORIGINAL ARTICLES

JSPORTSMEDPHYSFITNESS2012;52:382-92

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Basedonthesefindings,thepurposeofthecurrentstudywastoexaminetheeffectofAsxsupplementa-tiononmuscleenzymesasindirectmarkersofmus-cledamage,oxidativestressmarkersandantioxidantresponse after forced training.The influence of 90dayssupplementationonthebasallevelsonantioxi-dantdefenses,aswellasonthebasaloxidativestressmarkersandmuscleenzymeswasalsoanalyzed.

Materials and methods

Subjects and protocol

Thirty-twomaleelite soccerplayersparticipatedinthisstudy.Theyaremembersofyoungselectionof soccer club “Partizan”,Belgrade,Serbia, cham-pionamongyoungselectionsofSerbianchampion-ship.All participants were in good health, had noongoingorprevious (during lastyear) injuries,notonanymedicationknowntoaffectoxidativestressandwerenon-smokers.Subjectshad5to7trainingsessionsperweekwithanaverageweekly trainingof10to15hoursandtookparticipationinNationalchampionshipduringtrainingseason.Theyhaddif-ferentaspectsoftrainings,includingstrength,resist-ance,cardio,flexibilityandproprioceptivetraining.Theparticipationinthestudydidnothaveanyeffecton previously determined training and competitionschedule.The physicians in outpatient clinic “VitaMaxima”,Belgrade,Serbiaevaluatedphysicalper-formanceofallparticipantsduringthestudyperiodandsportinjuryratesandincidencewererecorded.

ThestudywasundertakenincompliancewiththeHelsinki Decleration and approved by the ethicalcommitteeofSportsMedicineAssociationofSerbia.Thesoccerplayersandparentswereinformedaboutprocedures,benefitsandpossiblerisksofparticipa-tioninadvanceofthestudy.Verbalconsenttopar-ticipateinthestudywaswitnessedandformallyre-corded.Priortoenrolmentintothestudy,allsubjectscompletedasubmaximaloxygenconsumption(VO2submax) test, body composition assessment, 4-daydiet record and general health-screening question-naire.Themaximaloxygenconsumption(VO2max)wasmeasuredonamotordriventreadmill(Runrace,Techno gym, Italy), using an indirect calorimetrysystem(Quarkb2,Cosmed, Italy)duringan incre-mentalexercisetesttovolitionalfatigue.Theenergy,

phospholipids peroxidation may play a role in theethiologyofexerciseinducedmuscledamage.6Thespecificactivitypatternofsoccertrainingandmatchmayfavoradditionalpro-oxidantredoxalterations.7

Methods to reduce free radical production andsubsequentoxidativedamageduringandfollowingphysical exercisehavebeen apriorityofmuch re-searchactivity.Variousantioxidantsandtheircombi-nationswereinvestigated.However,theuseofanti-oxidantstoattenuateexercise-inducedmuscleinjuryandoxidativestresshasbeenmetwithmixedresults.While some reports suggest a potential beneficialroleofantioxidantsupplementationinrelationtoex-ercise,8,9othersindicatenobenefit.10,11Discrepan-ciesinfindingsmaybeduetothetype,dosage,andtimingofadministrationoftheantioxidants,inaddi-tiontotheexercisestressandthespecificpopulationbeingstudied.

Astaxanthin (3, 3’-dihydroxy – β, β-carotene-4,4’-dione;Asx) is a red carotenoid pigment, whichoccursincertainmarineanimalsandplantssuchasfish,shrimps,andalgae.12Asxhasuniquechemicalpropertiesbasedonitsmolecularstructure.The pres-enceofthehydroxyl(OH)andketo(C=O)moietiesoneachiononeringexplainssomeofitsuniquefea-tures,namely,ahigherantioxidantactivity.Itisabletoscavengeradicalsbothatthesurfaceandinthein-teriorofphospholipidsmembrane.13RecentstudiescontinuetoevidencethemultiplepossibilitiesofAsxapplication in providing benefits to human health.Furthermore,Asxhasalsodemonstratedcardiopro-tective,14 neuroprotective 15 and anti-inflammatoryproperties(Figure1).16.

TherehasbeenlittledatareportedonevaluationofAsxeffectinsportsfield.ThereisevidencethatAsxsupplementationmay increasemuscle strengthandendurance17andreducemuscledamagecausedbyphysicalactivity.18Also,thereseveralinvitrostudiesandinvivoanimalmodelsshowingbeneficialeffectofAsxonreducingoxidativestressbiomarkers.12,19

Figure1.—Chemicalstructureofastaxanthin

O

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0.4mgcopper,2.1mgmanganese,44.5µgseleniumand2.1mgzinc).Thepost-exercisebloodsampleswereobtained15minutesafter2hoursoccerexer-cise.

Sample collection and analysis

Venous blood was collected into heparin evacu-atedtube(forplasma)andsampletubewithserumseparatorgel(forserum)(GreinerBio-one,Krems-münster,Austria).Plasmaandserumwereseparatedbycentrifugationandmultiplealiquotsofeachsam-plewerestoredat-80°Cuntilanalysis.Thefollow-ingparametersweremeasured: thiobarbituricacid-reactive substances (TBARS), advanced oxidationprotein products (AOPP), superoxide anion (O2

•¯),total antioxidative status (TAS), sulphydryl groups(-SH)andsuperoxide-dismutase(SOD).

PlasmaTBARSweremeasuredusingtheTBARSassay employing the molar absorption coefficientof1.56×105M−1cm−1at535nm,aspreviouslyde-scribed.22Inourhandstheintra-assayCVwas4.8%and the inter-assay was CV 7.2%; the referencevaluewas0.975±0.333µmol/L.TheAOPPwerede-terminedintheplasmausingthemethoddescribedby Witko-Sarsat et al.23 This oxidative stress bi-omarkerwasdetectedintheplasmaofchronicure-micpatients.ItwassuggestedthatAOPPlevelsarea measure of highly oxidized proteins, especiallyalbumin. Briefly, AOPP levels were measured byspectrophotometryat340nminacidicconditionandwerecalibratedwithchloramine-Tsolutionsthat,inthepresenceofpotassiumiodide,absorbat340nm.AOPPconcentrationswereexpressedinμmol×L−1ofchloramine-T equivalents.The intra-assay CV was3.27%andtheinter-assayCVwas6.5%;therefer-encevaluewas14.1±4.48µmol/L).Therateofni-troblue tetrazolium reduction was used to measurethelevelofsuperoxideanion24(theintra-assayCVwas5.6%andtheinter-assayCVwas9.5%;theref-erencevaluewas38.9±4.17µmolNBT/min/L).TASlevels of sera were determined using a colorimet-ric, fully automatedmeasurementmethod,25 whichwas optimized and applied on ILab 300+ analyzer(Instrumentation Laboratory, Milan, Italy) in thelaboratoryoftheInstituteforMedicalBiochemistry,FacultyofPharmacy,Belgrade,Serbia. Potent freeradicalreactionswereinitiatedwiththeproductionofhydroxylradical(OH¯),whichoxidizeABTS(2,

macronutrientandmicronutrientintakeswerecalcu-latedusingCosmedFMed2.0software.

Subjects were randomly assigned in a double-blind fashion to one of two treatment groups.TheAsxgroup(N.=18)wassupplementedwith4mgofAstaxanthinfor90days.TheastaxanthinusedinthisstudywasnaturalAsxderivedfrommicroalgaeHae-matococcus pluvialis supplied by Oriflame (Swe-den). Dosage of 4 mg per day and during a studyperiodof90daysseemstobesafeandhence,harm-fulsideeffectswerenotexpected.Theplacebo(P)group (N.=14)wasgivencapsules, identical inap-pearanceandtaste,butcontainingsacharose.Priortoenteringthestudyandduringthestudy,thepartici-pantswereinstructedtoabstainfromanyantioxidantsupplementation.

Determinations of basal antioxidant enzyme ac-tivity,oxidativestressmarkersandmuscledamagemarkersweremadebeforeandafter90daysofsup-plementation.After thisperiod,sportsmanhad twohoursoccerexerciseandsamplesweretakentode-terminesameparametersbeforeandaftertheexer-cise. During the exercise heart rate of each playerwasmonitoredusingapulsometer.Astherelation-ship between the power output, the heart rate andtheoxygenuptakeislinear,wecanindirectlyevalu-atetheworkdoneduringtrainingthroughtheheartrate.20Therearefivemetaboliczones,thataredefinedaccordingtomaximaloxygenuptake:Z1<70%,Z2:70-80%, Z3: 80-90%, Z4: 90-100%, Z5: 100% orhigher(TableI).Bymeasuringheartrateduringthetrainingsession,wecalculatedtheaveragetimeeachplayerworkateachzone.21

Thepre-exercisebloodsampleswereobtainedbe-tween9-10amafter10hovernightfast.Afterthefirstbloodcollection,beforetheexercise,subjectshadastandardizedbreakfast(providing~650kcal:16-17gprotein,131-135gcarbohydrate,and4-5gfat;190IUvitaminA,30mgvitaminC,0.5mgvitaminE,

TableI.—Physical activity performed during the training.

Placebo Astaxanthin

Z1(%) 12.1±3.2 12.7±3.9Z2(%) 25.8±5.4 24.5±5.7Z3(%) 37.7±6.5 38.4±7.3Z4(%) 23.2±8.5 22.8±9.7Z5(%) 1.2±0.9 1.6±1.1

Zvalues representpersentigeof timeexpendedateachmetaboliczone.Metabolic zones are defined according to maximal oxygen uptake:Z1<70%,Z2:70-80%,Z3:80-90%,Z4:90-100%,Z5:100%orhigher.

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repeatedmeasure(ANOVA).DuetothefactthatthedistributionsofAOPP,O2

•¯,SOD,CKandASTwereskewed, logarithmic transformation of the valueswasperformedbefore statistical comparisonsweremade.Two-tailedpvaluesaregiventhroughout.

Results

Subject’sphysicalcharacteristicsarepresentedinTable II.There were no significant differences be-tweentreatmentgroupswithrespecttothesecharac-teristicsatbaseline.

TheestimateddailyenergyandnurientintakeofsoccerplayersarelistedinTableII.TheAsxandPgroups did not differ in the estimated average en-ergetic and nutritional intake. The dietary analysisobtainedfromthe4-dayfooddiaryshowedthatthemean vitaminA and E intakes were below the di-etary reference intake (DRI) recommendations fortheAsxandPgroups.28,29

Regarding oxidative stress parameters, TBARSremainednearlyunchangedinbothgroups,afterob-servationalperiod(TableIII).We,also,didnotdetectanyinfluenceof2hourssoccerexerciseonTBARSproductioninAsxnorinPgroup.BasalAOPPlevelsdidnotchangethroughoutthestudy.SoccerexerciseattheendofsupplementationperioddidnotinducedsignificantchangesinAOPPlevelsinAsxorinP.AsitcouldbeseeninTableIII,regularsoccertrainingover the period of 90 days significantly, increasedlevelsofO2

•¯inbothgroupsofsoccerplayers(maintrainingeffect,P<0.01).Asignificantexerciseeffect(P<0.01)andinteractioneffectamongsupplementa-tionandexercise(P<0.05)onO2

•¯werefound.O2•¯

concentrationsincreasedsignificantlyaftertheexer-ciseinthePgroup,whiletherewasnochangeinAsxgroupatthesametime.

BasalTASlevelsdidnotchangealongthestudy.However,2X2repeatedmeasuresANOVArevealedsignificantexerciseeffect(P<0.001)andinteractioneffectamongexerciseandsupplementation(P<0.05)onTASlevels.TASlevelsdecreasedpost-exerciseinbothgroups,but this changes reached statisticalsignificanceonlyinPgroup(P<0.01).

Atthebeginningofthestudy,wenoticedsignifi-cant difference inSHgroups content betweenAsxand P group (P=0.05).ANOVA repeated measuresrevealedsignificanttrainingeffect(P<0.001)onto-

2’-Azinodi-[3ethylbenzthiazolinesulphonate])toagreen cation radicalABTS+.Antioxidants, presentin the sample, discolorateABTS+ to a degree thatis proportional to their concentrations. The resultswere expressed as vitamin E analogue which isusedasastandard.The intra-assayCVwas1.94%and the inter-assay CV was 4.39%; the referencevalue was 0.859±0.017mmol/L. The concentrationofsulphydrylgroupsinplasmawasdeterminedus-ing0.2mmol/L5,5′-dithiobis(2-nitrobenzoicacid)(DTNB)reportedbyEllmann26(theintra-assayCVwas2.86%and the inter-assayCVwas5.01%; thereferencevaluewas0.523±0.062mmol/L).Plasmasuperoxide-dismutaseactivitywasmeasuredaccord-ingtoapreviouslypublishedmethod.27Oneunitofsuperoxide-dismutaseactivityisdefinedastheactiv-itythatinhibitstheauto-oxidationofadrenalinby50%.Theintra-assayCVwas6.3%andtheinter-assayCVwas9.2%;thereferencevaluewas120±21U/L.

Serum creatine kinase (CK) and aspartate ami-notransferase (AST) were assayed by routine en-zymaticmethodsusinganILab300+analyzerandreagentspurchasedfromBiosystemsS.A.(Barcelo-na,Spain)andBioanalytica(Belgrade,Serbia).Bio-chemical and hematological tests parameters weredeterminedbeforeadministrationandafter90daysofAsxsupplementation.

Statistical analysis

Statistical analyses were performed using theStatgraphics4.2software(STSC,Inc.&StatisticalGraphics Corporation 1985-1989), MS Excel andEduStat 2.01 (2005,Alpha Omnia, Belgrade, Ser-bia).Alldatawereassessedfornormality(Shapiro-Wilk test). The characteristics of the study popu-lation are presented in terms of mean values andstandarddeviations.When thedistributiondifferedfrom a normal distribution, geometric means and95%confidenceintervalsaregiven.Subjects’base-linephysicalcharacteristicsandnutritionalparame-terswerecomparedusingindependent-samplet-test.The effect of theAsx supplementation and regulartrainingonthebasalparameterswastestedusing2(Asxandplacebogroup)X2(beforeandafter thesupplementation)repeatedmeasureanalysisofvari-anceANOVA.Theeffectofantioxidantsupplemen-tationandsoccerexercisewastestedby2(Asxandplacebogroup)X2 (before andafter the exercise)

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386 THEJOURNALOFSPORTSMEDICINEANDPHYSICALFITNESS August2012

WeobservedsignificanttrainingeffectonCKandAST levels during 90 days of study period (maintraining effect, P<0.01 and P<0.001, respectively).AllparticipantsshowedadecreaseinbasalplasmaCK and AST activities (CK decreased from 477[341-667]U/L to 239 [158-363]U/L in Asx groupandfrom520[265-1018]U/Lto388[254-593]U/L,Pgroup;ASTdecreasedfrom37[29-48]U/Lto24[20-28]U/L in Asx group and from 43[36-52]U/Lto 29[24-34]in P group). There was no difference

talSHgroupscontent.BothAsxandPgroupsex-perienced increase in total SH groups content (by21% and 9%, respectively) and supplementationeffect was marginally significant (P=0.08). BasalSODactivitysignificantlydecreasedboth inPandin the supplemented group at the end of the study(main trainingeffect,P<0.01).The soccerexerciseperformedafter90daysofsupplementationdidnotinfluencedSODactivityinthesupplementedandinPgroup(TableIV).

TableII.—Physical characteristics and nutrition analysis of the tested individuals before nutritional intervention.

Characteristic Asx P

Age(year) 18.1±0.7 17.7±0.6Weight(kg) 72.4±8.35 74.1±7.7Height(cm) 177.6±6.9 180.7±6.4Bodymassindex(kg/m2) 22.8±1.4 22.7±1.7Fat(%) 10.5±2.5 10.6±3.6VO2(ml/min/kg) 55.1±5.3 52.1±3.5Nutritionalanalysis(habitualdietaryintake)

Energy(kcal) 2932±657.8 3154±1107.1Protein(g) 125±28.3 117±27.0Fat(g) 101±29.3 96±25.4Carbohydrates(g) 366±102.7 383±102.4

TableIII.—Effect of the Asx supplementation on basal biomarkers of oxidative damage and antioxidative defence parameters.

Initial Final

placebo astaxanthin placebo astaxanthin

MDA(µmol/L) 1.11±0.14 1.08±0.18 1.07±0.18 1.05±0.25AOPP(µmol/L) 22±14 28±20 27±15 28±13O2

•¯(µmol/minL) 45±23 57±45 85±79a 99±94aTAS(mmolvit.Eequiv/L) 0531±0.139 0.551±0.139 0.585±0.113 0.538±0.108SHgroups(mmol/L) 0.557±0.088 0.493±0.060 0.595±0.059 0.598±0.060aaSOD(U/L) 100±50 95±46 38±13a 47±30a

Valuesareexpressedasmean±S.D.ThedifferenceinrelationtobeforethesupplementationwassignificantatP<0.01(aa)andatP<0.05(a).

TableIV.—Effect of the soccer training and Asx supplementation on biomarkers of oxidative damage and anti-oxidative defence parameters.

Before After

Placebo Astaxanthin Placebo Astaxanthin

MDA(µmol/L) 1.07±0.18 1.05±0.25 1.08±0.15 1.14±0.18AOPP(µmol/L) 27±15 28±13 31±11 27±11O2

•¯(µmol/minL) 85±79 99±94 175±137b 115±85TAS(mmolvit.Eequiv./L) 0.585±0.113 0.538±0.108 0.443±0.135bb# 463±0.162#SHgroups(mmol/L) 0.595±0.059 0.598±0.060 0.605±0.082 0.590±0.126SOD(U/L) 38±13 47±30 45.58±20 52±26

Valuesareexpressedasmean±S.D.ThedifferenceinrelationtobeforethetrainingwassignificantatP<0.01(bb)andatP<0.05(b).Theinteractioneffect(trainingxsupplementation)wassignificantatP<0.05(#).M

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under heavy training and competition are not abletomaintainoptimaltissuelevelsofantioxidantvita-mins,eveniftherecommendeddailyallowancesareconsumedthroughtheirdiets.33

inbasalCKandASTlevelsbetweentheAsxandPgroup.CKandASTactivitiesinserumsignificantlyincreasedasresultofsoccerexercise(mainexerciseeffect,P<0.001andP<0.01,respectively).However,the effect of Asx supplementation on exercise in-ducedchangesinCKandASTlevelswasmarginal-lysignificant(mainsupplementationeffect,P=0.067andP=0.062,respectively),withsignificantlylowerpost-exerciseCKandASTlevelsinAsxgroupcom-paredtoPgroup(P<0.05).

Discussion

ExcessiveROSproduction as a result of intensephysical activity and subsequent oxidative stresscertainly has the ability to result in physiologicaldamage,butcertain levelofprooxidantproductionmayactuallyserveasnecessarystimulusfortheup-regulation of antioxidant defenses, thereby provid-ing protection against future ROS attack30. On theotherhand,somestudiesreportdecreaseofantioxi-dantsystemefficiencyandincreaseinthemarkersofoxidativestressintargettissuesandbloodinprofes-sionalathletes subjected tohigh trainingandcom-petitiveload.31,32Ithasbeensuggestedthatathletes

Figure 3.—Plasma aspartate aminotransferase (AST) activity(U/L)beforeandafterexerciseinplaceboandastaxanthingroup.creatinekinase (CK)activity (U/L)beforeandafterexercise inplaceboandastaxanthingroup.Valuesareexpressedasgeometricmeanvalues(95thconfidenceinterval).Thedifferenceinrelationtopre-exercisewassignificantatP<0.01.Thedifferenceinrela-tiontoAsxwassignifficantatP<0.05.

Figure 2.—Plasma creatine kinase (CK) activity (U/L) beforeandafterexerciseinplaceboandastaxanthingroup.Valuesareexpressed as geometric mean values (95th confidence inter-val). The difference in relation to pre-exercise was significantatP<0.001.ThedifferenceinrelationtoAsxwassignifficantatP<0.05.

TableV.—Correlations between creatine kinase (CK) and aspar-tate aminotransferase (AST) levels during observational period

ASTinitial

placebo astaxanthin

CKinitialSpearmancorrelation 0.837** 0.599**significance 0.000 0.003n 18 14

ASTfinal

placebo astaxanthin

CKfinalSperamancorrelation 0.889** 0.573*significance 0.000 0.026n 18 14

ASTafterthetraining

placebo astaxanthin

CKafterthetrainingSpearmancorrelation 0.921** 0.580*significance 0.000 0.015

n 18 14

*p<0,05;**p<0,01

1000

100

CK

(U

/L)

Pre-exercise Post-exercise

Astaxanthin

Placebo

100

10

AST

(U

/L)

Pre-exercise Post-exercise

Astaxanthin

Placebo

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388 THEJOURNALOFSPORTSMEDICINEANDPHYSICALFITNESS August2012

AOPPisnovelbiomarkerthatmayindicatelong-termeffectsofoxidativestresssuchasafterahighvolumetrainingperiod.42Inthepresentstudy,AOPPlevelswerenotdifferentthoughttheseasonbetweenourexperimentalgroups,indicatingthatbothgroupshad same level of oxidatively modified proteins.Also,wedidnotdetectanychangesinAOPPlevelsasresultofsoccerexerciseinbothgroups.Thisisinagreementwithotherstudyonsoccerplayers,whichshowed no increase in protein oxidation inducedbysoccerexercisetrainingormatch.21,36GroupofyoungsoccerplayersanalyzedinthepresentstudyhadlowerAOPPlevelscomparedtothevaluesob-servedinotherstudies,42,43soitispossiblethaten-dogenousantioxidantdefensemechanismwasabletopreventsignificantchangesinAOPPlevelsduringobservationalperiod.

Continuous physical activity over the 90 days,seemstoinducesignificantincreaseinO2

•¯produc-tion insoccerplayers.However,at theendofsup-plementation period, two groups responded differ-ently to treatment, sinceonlyPgroupexperiencedsignificantincreaseinO2

•¯levelsaftertheexercise,suggestingthatAsxcould,atleastinpart,decreaseexcessive production of O2

•¯ during acute exercisebout inyoungsoccerplayers.O2

•¯ is thefirstROSgenerated during different metabolic processes incells and reacts rapidly with a variety of biologi-calcompoundssuchassulphydryl,polyunsaturatedfattyacids,andDNA4.ByneutralizingO2

•¯Asxsup-plementationmaybehelpfulinpreventionoxidativedamageofvariousbiomolecules.Invitrostudiesre-garding antioxidant properties ofAsx also showedthatAsxcanbeeffectiveinreducingO2

•¯andH2O2production.15,19

Non-enzymaticantioxidantlevelsaremodifiedasa result of aerobic exercise, but results are contra-dictory.Athletesoftenshowed increased totalanti-oxidantcapacity inresponseto theoxidativestressimposedbyintensephysicalactivity.44Increasedto-talantioxidantcapacityisaconsequenceofvitaminEandCmobilizationfromtheir respective reserveinordertoprotectbodyagainstROS4orsignificantaugmenteduricacidsynthesisconsecutivetoanen-hanced activation of xanthine oxidase.44, 45 On theotherhand, itappears that theantioxidantcapacitymay be temporarily reduced during and immedi-atelypostexercise,afterwhichtimelevelstypicallyincreaseabovebasalconditionsduringtherecovery

Thepotentialofdietaryantioxidantsasexogenousdefense tominimize theextentofmuscle injuryoroxidativestressinresponsetoexercisehasreceivedincreasingattentioninrecentyears.However,there-sultsofworksarenotunequivocalandconclusive.This study aims to evaluate the effect ofAsx sup-plementationinattenuatingtheoxidativestressandmuscledamageinducedbytrainingprotocolinfieldsituation,whilefollowingtheirhabitualdietarypat-ternandtrainingandcompetitionprogram.

Several studies have reported oxidative stress ismarkedly upregulated by a soccer game, observedthough TBARS,34 MDA levels 21 and serum hy-droperoxide.35 In addition, it was found that regu-lar training and competition sessions resulted inincreasedbasaloxidativestressas indicatedby theincreasedMDAplasmalevelsinsoccerplayers.21,36Inlinewiththis, in thepresent investigationmeas-uredTBARS levels were above normal values, re-flecting the high physical stress soccer players areexposedtoduringregulartrainingandcompetition.

Thebeneficialeffectofantioxidantvitaminsonin-hibitionofperoxidationreactionshasbeenanissueof many research papers.Although several studiesindicatethatantioxidantsupplementationattenuatesoxidative damage to lipids caused by exercise 8, 37thereare,likewise,publishedliteraturethatsuggeststheirineffectiveness10,38orthatevenreportaproox-idativeeffect.39TheresultsofourstudydidnotshowsignificantchangesofbasalTBARSlevelsorTBARSlevelsaftertheforcedexercise.Also,wedidnotob-servedeffectonthisindicatoroflipidperoxidationbyAsx supplementation.Why dietaryAsx did notreducelipidperoxidationisunclear.Astaxanthinhasbeenshowntobeoneofthemosteffectiveantioxi-dantsagainstlipidperoxidationandoxidativestressin invitroand invivo systems.12,19Astaxanthin is100timesmoreactivethanα-tocopherolinprotect-ingtheratmitochondriaagainstFe2+-catalyzedli-pidperoxidationin vivoandin vitro.40TBARSisthemost widely used biomarker of lipid peroxidationbecauseitisinexpensiveandeasytoassay,thereissomeconcernaboutitsspecificityandsensitivity.IthasbeendemonstratedthatantioxidantsupplementssignificantlyinfluencedlipidhydroperoxideandF2-isoprostanelevelsinresponsetoexercise,whereastheydidnotalterMDA,measuredbyTBARS.41Thismayaccountforthelackofasignificanteffectseeninthisstudy.

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in response to exercise interventions in a trainedpopulation.4, 48 On the other hand, there are stud-ies showing no changes or even decrease in SODactivityafterenduranceexercise.49,50Inourgroupofsoccerplayers,therewassignificantdecreaseinSODactivityduringstudyperiod.Atthesametimelevels of O2

•¯ increased in both groups. It is like-ly that continuous, exhaustive training sessions aswell as the frequent competitive matches exposedparticipantstoincreasedoxidativestress(observedthroughincreasedlevelsofO2

•¯)thatoverwhelmedSODactivity.Ingeneral,modificationsofantioxi-dant enzyme activities after exercise characterizeeitheradaptation(anincreaseintheactivityatfirst)orutilization(adecreaseifoxidativestressisover-whelming).4 These decreases had, hypothetically,been attributed to a modification of the catalyticcentersandsubsequentinactivationofenzymesduetoadisturbedredoxbalanceinducedbyaugmentedoxidativestress.51Thisisthefirststudyinvestigat-ingeffectofAsxonendogenousenzymaticantioxi-dantsinvivo.Although,beneficialeffectofAsxonSODactivitywasreportedinseveralinvitrostudies12,19ourconceptofsupplementationinyoungsoc-cerplayerswasnotabletopreventdecreaseinSODactivity.

High degree of variability existed among soccerplayers with regard to oxidative stress biomarkers,indicatingthatsomeindividualswere“responders”,while others were “non-responders”. Individualcharacteristics,dietary intakeofantioxidants,posi-tion in the field might be possible factors causingthesedifferences.

TheeffluxofmuscleenzymeCKisconsideredtoreflectachangeinthenormalmembranestructure,induced by muscle damage, making it permeableto thesemolecules. In this sense, increasedserumactivitiesofCKisconsideredasindirectmarkerofmusclefiber injury.30ASTactivity is significantlyincreasedimmediatelyaftermuscularexertion,re-maining at high levels for 24h.Therefore, in ath-letes, the implications of increased serum ASTshouldbeconsideredincombinationwiththeactiv-ityofCK.52

Inthepresentstudy,increasedCKandASTactiv-ity in the young soccer players above normal val-ues,atalltimepoints,reflectsthehighphysiologicalrequirementsofthesoccerandalsoimpliesmuscledamage.Overtheperiodof90daysofregulartrain-

period.46Inthepresentstudy,soccerexerciseattheendofobservationalperiod inducedsignificantde-crease in TAS levels in P group, possibly indicat-ingthatplasmaantioxidantsareinstantlyutilizedtoeliminate increased levels of ROS. Soccer playersfolloweddietlowinvitaminAandvitaminEforatleast3months.It ispossiblethatendogenousanti-oxidantscouldnotcompensatelowexogenousanti-oxidantintakeforprolongedperiodoftime.

On the contrary, there were no exercise-inducedchangesinTASlevels intheathleteswhoreceivedAsx for90days.DespiteAsx supplementationdidnothaveamelioratedplasmaticantioxidantcapacityat rest, inagreementwithotherworks,8,45itseemedtohelptocounterbalancetheexerciseoxidativein-sult.Inlinewiththis,supplementationwithvariousantioxidants has been shown to significantly aug-ment the exercise-induced TAS increase, whereasanochanges inTASinrestingconditionswasob-served.47

One of the indices of exercise-induced oxidantproduction is blood thiol oxidation. Cellular thiolsare critically important in maintaining the cellularantioxidantdefensenetwork;inaddition,thiolsplayakeyroleinregulatingredox-sensitivesignaltrans-ductionprocess4.SignificantincreaseinSHgroups’contentwith thenumberofyearsof trainingexpe-riencewasobservedinfemalevolleyballplayers.42In accordance, total SH group’s content increasedasaresultofcontinuousphysicalactivityinyoungsoccerplayers.Thesechangesmightbeapartofaself-protectingmechanismagainst training-inducedoxidativestress.TheeffectofAsxsupplementationwasmarginallysignificant.Namely,atthebeginningofourstudy,basalserumSHgrouplevelsappearedsignificantly lower inAsx group than in P group.After3monthsofAsxsupplementation,thisparam-eterwas completely restored inAsxgroup. In linewiththis,Boninaetal.observedthatdietarysupple-mentationwithredorangecomplex,richinphenoliccompounds(anthocyanins,flavanones,andhydrox-ycinnamicacids) andascorbicacid increase serumlevelofSHgroups’after2months.1

SODfunctionsinthecellasoneof theprimaryenzymaticantioxidantdefensesagainstsuperoxideradicals. Increases in SOD enzyme activity corre-spondswithenhancedresistancetooxidativestress.4

The results of several studies suggest that levelsof SOD activity in blood and muscle is increased

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Conclusions

The relationship between exercise and oxidativestressisnolongerconsideredasadetrimentalphe-nomenon, but rather stimulus for upregulation ofantioxidantdefences.55Althoughsomestudiesdocu-mented inhibitory effect of antioxidant substancesonadaptationstoexercise,thepotentialroleofanti-oxidant supplementation should be investigated.56The results of the present study suggest that soc-cerexerciseandsoccertrainingareassociatedwithhighlyincreasedproductionoffreeradicalsandoxi-dative stress (observed through O2

•¯ and TBARS),whichmightdiminishantioxidantsystemefficiency(observedthroughdiminishedSODandTAS),asitscomponentsareusedtoquenchtheharmfulradicalsproduced.SupplementationwithAsxcouldpreventexerciseinducedfreeradicalproductionanddeple-tionofnonenzymaticantioxidantdefenseinyoungsoccerplayers.Observedreductioninbasalmuscleenzymes levels is part of adaptation process to in-tensephysicalactivityover the90daysof trainingregimen. However, Asx supplementation may behelpfulinreducingserumpeakofCKandASTaftertheforcedsoccerexercise.

Because of exercise and training modificationsofantioxidantdefensesystemsandlowantioxidantdietaryintakesinyoungsoccerplayers,antioxidantsupplementationincertainantioxidantnutrientssuchasAsxseemstobereasonable.

References

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ing and supplementation, there was a significantdecrease in basal CK andAST levels.The changeinCKandASTlevelsfollowedthesamepatterninbothAsxandPgroup.Webelievethatthisreductioninbasalmuscleenzymeslevelsispartofadaptationprocesstointensephysicalactivityoverthe90daysof training regimen. The subjects’ muscle tissueswere strengthenedby regular training, so themus-clesbecomemoreresistanttoexercise-relateddam-age.Enzymaticadaptationsconsequenttolong-termtrainingwerereportedpreviously.53

Theuseofdietaryantioxidantstoreduceexercise-inducedmuscleinjuryhasmetwithmixedsuccess.It was shown that antioxidant vitamin supplemen-tation does not appear to prevent exercise-inducedmuscletissuedamage.54Ontheotherhand,dietarysupplementation with antioxidant vitamins can de-crease the exercise-induced increase in the rate oflipidperoxidation,whichcouldhelppreventmuscletissuedamage.8,9

Consideringvarietyofeventsassociatedwithsignsand symptoms of muscle injury, it is unlikely thatanyspecificantioxidantorcombinationofantioxi-dantswouldfunctiontoeffectivelyeliminatemuscleinjury,butrathertoreducethedegreeofdamage.30In the present study although serum CK andASTactivities increased significantlypostexercise in ei-ther group, significantly lowerCKandAST levelswereobservedintheAsxgroupcomparedwiththeP group, suggesting thatAsx supplementation canattenuateexercise inducedmuscledamagetosomeextent.

Thisisinaccordancewiththeresultsofprevious-lypublishedstudiesregardingAsxsupplementationand muscle injury.Aoi et al. showed thatAsx canattenuateexercise-induceddamageinmouseskeletalmuscleandheart,includinganassociatedneutrophilinfiltrationthatinducesfurtherdamage.18Inexperi-mentofIkeuchietal.theexerciseinducedincreasein plasma CK activity was inhibited by treatmentwith astaxanthin.17 The findings of lower CK andAST activity in soccer players supplemented withAsx might represent the ability ofAsx to stabilizesarcolemmaleadingtolessmembranedisruption.

However,CKandASTactivityshouldnotbeusedaloneasreliableindexofmuscleinjury,consideringthat CK andAST do not correlate well with othermarkersofmuscleinjurysuchasmuscleforce,mus-cleperformanceorcytoskeletaldisruption.30

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