Rev. bras. ortop. vol.50 número3

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w w w . r b o . o r g . b r

Original

Article

Analysis

on

the

serum

levels

of

the

biomarker

CTX-II

in

professional

indoor

soccer

players

over

the

course

of

one

season

夽

Rodrigo

Miziara

Severino

∗

_,

_Pedro

_Baches

_Jorge,

_Mauro

_Olivo

_Martinelli,

Marcos

Vaz

de

Lima,

Nilson

Roberto

Severino,

Aires

Duarte

Junior

SchoolofMedicalSciences,SantaCasadeSãoPaulo,SãoPaulo,SP,Brazil

a

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t

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c

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f

o

Articlehistory:

Received22February2014 Accepted30June2014 Availableonline16April2015

Keywords: Osteoarthritis Jointcartilage

Pharmacologicalbiomarkers

a

b

s

t

r

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c

t

Objective:TheaimofthisstudywastoanalyzethebloodserumlevelsofCTX-IIin profes-sionalindoorsoccerplayers,atthreedifferenttimesduringoneseason:atthestartofthe pre-season,fourmonthslater(atimethatmarksthemiddleoftheseason)andattheend oftheseason.

Methods:Fourteenmalesoccerplayersofmeanage19yearswereincluded.Bloodsamples of3mLwerecollectedfromeachindividual.ThesampleswereanalyzedbymeansofElisa tests.

Results:TherewasasignificantincreaseintheserumlevelofCTX-IIintheindoorsoccer players,fromthebeginningtotheendoftheseason(p<0.01).

Conclusion: Thesedatasuggestthatjointdegradationhadoccurredinthesesoccerplayers, bytheendofthisperiod.Itisevidentthatfurtherstudiesareneeded,withmethodological rigor,soastomakeaneffectivecontributiontowardpreciseelucidationoftheetiologyof thisosteoarthritisanditsrelationshipwiththebiomarkers,asatoolforearlydiagnosis.

Análise

dos

níveis

séricos

do

biomarcador

CTX-II

em

atletas

profissionais

de

futebol

de

salão

durante

uma

temporada

Palavras-chave: Osteoartrite Cartilagemarticular

Biomarcadoresfarmacológicos

r

e

s

u

m

o

Objetivo:AnalisarosníveisséricossanguíneosdeCTX-IIematletasprofissionaisdefutebol desalão,emtrêsmomentosdistintosduranteumatemporada:noiníciodapré-temporada, quatromesesapós(períodoquemarcaomeiodatemporada)enofimdatemporada. Métodos:Foramincluídos14atletasdogêneromasculinoemédiadeidadede19anos.Foram coletados3mLdesanguedecadaindivíduo.Asamostrasforamanalisadaspelotestedotipo Elisa.

夽

WorkdevelopedintheDepartmentofOrthopedicsandTraumatology,SantaCasadeSãoPaulo,SP,Brazil.

∗ _{Corresponding}_author_.

E-mail:pbj1980@hotmail.com(R.M.Severino). http://dx.doi.org/10.1016/j.rboe.2015.04.001

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Resultados: HouveaumentosignificativodosníveisséricosdeCTX-IInosatletasdefutebol desalão,comparando-seoinícioeofimdeumatemporada(p<0.01).

Conclusão:Essesdadossugeremaocorrênciadedegradaçãoarticularnosatletas,aotérmino desseperíodo.Ficaevidenteanecessidadedefuturosestudos,comrigormetodológico,que possamcontribuirefetivamenteparaaelucidaçãoprecisadaetiologiadaOAesuarelação comosbiomarcadorescomoinstrumentodediagnósticoprecoce.

Introduction

Primaryosteoarthritis(OA)isamultifactorialdisease charac-terizedbyirreversiblejointdegeneration,withformationof osteophytesandreductionofthejointspace.Itpresentsthe followingmainsymptoms:progressiveincreaseinpain,lossof function,limitationsonday-to-dayactivitiesandrestrictions onsportspractice.1–7

Sometheorieshavecorrelatedcontinualintensepractice ofphysicalactivitywithdevelopmentofprimaryOAinelite athletes,causedbyjointoverloading.Moreover,therehasbeen speculationregardinghowthejointcartilagerespondstothis overloading.However,therelationshipbetweenjointcartilage damageandintensephysicalactivitystillseemstobeamatter ofcontroversyintheliterature.8,9

Some studies have demonstrated that athletes who practicesportsthat includerapidaccelerationwithinstant decelerationor continuous training witha high impacton joints,orwhocompeteatelitelevelforprolongedperiodsof time,presentgreaterlikelihood ofdevelopingOA.However, thetypesandintensitiesofexercisesthatareharmfultojoint cartilageremainunknown.8–11

OA presents aninitial asymptomatic phase,which may beinfluencedbythelevelofoverloadingtowhichthejoint is exposed in a physical activity. However, identification of this joint damage is difficult, given the limitations of the assessment instruments available. In this light, recent studies have demonstrated that biochemical biomarkers are a potentialoption forearly detection ofasymptomatic OA.9,12,13

Underphysiologicalconditions,themetabolismoftypeII collagenisslow.Itsfibrilshaveahalf-lifemeasuredinyears. Intheinitialstagesofcartilagedegeneration,degradationof thesecollagenfibrilsisobserved.Enzymesnamed metallo-proteinases are released, and thesecontribute toward this degradation,especiallycollagenases andaggrecanases. Col-lagenases are responsible forcleaving type IIcollagen and produce fragmentsof this collagen. Specificantibodies for thesefragmentscanbedetectedinsynovialfluid, bloodor urineandhavebeenstudiedaspotentialbiomarkersforthe onsetofjointdegradation.2,5,7

Whenajointcomponentisdegradedandejectedoutofits originaltissue,measuringthisinthejointfluidwouldbethe mostprecisemethod.Onebiomarkerofjointdegradationis theC-telopeptideoftypeIIcollagen(CTX-II).UseofCTX-IIas amarkerforprogressionofcartilaginouslesionsanditsdirect relationshipwithradiologicalgradesandclinical scoresfor OAhavebeenprovenintheliterature.Therefore,assayingof

CTX-IIlevelsseemstobeaneffectivemannerforascertaining typeIIcollagenturnover.14–21

Biomarkersareinstrumentsformeasuringtheprogression ofdiseases or the effectsoftreatment ondisease progres-sion.Thus,theycanserveastoolsforelucidatingtheeffects ofexerciseonjointcartilage andthepossibledevelopment ofprimaryOA.12,13_In_this_light,_the_objective_of_the_present studywastoanalyzeandcomparebloodserumassaylevels ofthebiomarkerCTX-IIinprofessionalindoorsoccerplayers, atthreedifferenttimesduringoneseason.

Methods

Thiswasaprospectivelongitudinalstudythatwasapproved bytheResearchEthicsCommitteeofSantaCasade Misericór-diadeSãoPaulo.

Thestudyincluded14playersinaprofessionalindoor soc-certeam(under-21category).Theplayersweremale,ofmean age19years,allinthesameteam,andweresubjectedtothe sametrainingandmatchload.

Players withpreviousknee surgery(twoplayers)or who were using chondroprotectant medications (three players) wereexcluded.

In addition, four players who were undergoing physio-therapytreatmentforfemoropatellaroverloadduetomuscle imbalanceinthepelvicbeltwereexcludedbecausethey pre-sentedpainofpatellaroriginthatlimitedtheirsportspractice atthebeginningoftheseason.

CTX-IIlevelswereassayedatthreetimes:A–atthe begin-ning ofthe pre-season; B–four months later(atime that markedthemiddleoftheseason);andC–attheendofthe season.

Bloodsamples(3mL)werecollectedfromeachindividual bymeansofsimplepunctureinthenon-dominantarm,using vacuumcollectionkits.Thebloodsampleswerecentrifuged andstoredatatemperatureof−80degrees,untilallthe sam-pleswerereadytobetested.

EachsamplewasanalyzedbymeansofanELISAtestfor detectinghumanCTX-II(HuCTX-IIkit,CusabioBiotech, cata-lognumberCSB–E09323h,batchS20045731,producedinthe UnitedStates).Thiskitpresents100%specificityforhuman CTX-IIalone,withoutcross-reactions,andwithaminimum detectable leveloflowerthan 0.3ng/mL. Thistestwas per-formedinaprivatelaboratoryinthecityofSãoPaulo,with costsentirelybornebytheresearchers.

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Table1–DataonindividualsandtheirCTX-IIassaylevels(inng/mL),observedforeachplayerateachtimeduringthe season.

Age Intensityofpain Beginningofseason Middleofseason Endofseason

Player1 19 Nopain 0.321 0.328 0.328

Player2 18 Nopain 0.201 0.278 0.401

Player3 19 Occasionalnon-limitingpain 0.182 0.331 0.551

Player4 20 Nopain 0.825 1.121 2.537

Player5 20 Occasionalnon-limitingpain 0.312 0.298 0.399 Player6 19 Occasionalnon-limitingpain 0.311 0.315 0.324

Player7 19 Limitingpain 0.299 0.311 0.324

Player9 20 Nopain 0.311 0.315 0.497

Player11 17 Nopain 0.312 0.299 0.395

Player12 19 Nopain 0.287 0.277 0.268

Player14 19 Nopain 0.247 0.317 0.378

Mean(SD) 19 0.366±0.214 0.5±0.485 0.685±0.751

applied(95%confidenceintervalandpvalue<0.05).This non-parametrictestwasthebestoptionbecauseitwasimpossible toassumethatthesamplehadnormaldistribution.

Results

Table1presentsthedataoneachindividualandthemarker levelsobservedforeachofthemateachtimeduringthe sea-son.Itcouldbeseenthat twoplayers(4and 10)presented discrepantdata,withmuchhigherassayedlevelsthanthoseof theotherplayersevaluatedinthisstudy,inalltheevaluations. Nevertheless,theseparticipantsdidnotreportanydeclinein physicalperformance,oranypresenceofsymptomssuchas pain,edemaorinstability.

Individuals3, 5and 10 presented pain at the origin of the patellar tendon in one knee, which became worse at the end ofthe sports practice, improved with physiother-apy and did not limit their participation in training and matches.Individual13presentedaconditionofnon-limiting painabovetheinsertionofthepesanserinus.Also, individ-uals6and8complainedoflateralpainintheirknees,above thelateralepicondyle,duringsportspractice,whichwasalso non-limiting.

Becauseofthetendinousoriginofthepaininthe individ-ualsincludedinthis study(patellartendinopathy,anserine tendinopathyandfrictionoftheiliotibialtract),itwasdecided not to include them separately in statistical comparisons regardingdegradationoftypeII collagenexclusivelyofthe cartilaginoustissue.

ComparisonofCTX-IIassaylevelsbetweendifferenttimes duringtheseason

Becauseofthelimitedsizeofthesample,whichwasselected in a non-randomized manner because this was a closed indoorsoccerteam, thedistribution oftheindividuals and resultsfromthesamplecannotbeconsideredtobenormal. Whenthisoccurs,nonparametricstatisticaltestsneedtobe used.In thegraphicdistribution ofthe data, two individu-alshad values that were very different from those of the

remainderoftheteamandwhichwereconsideredtobe out-liers.

Toavoidtheriskofstatisticalerror,twoseparateanalyses wereperformed:1–comparisonsusingthecompletedata;and 2–comparisonsusingreduceddata(withouttheinformation onplayers4and10)(Table2).

Incomparingthe valuesfrom thefirsttwoanalyses,i.e. beginningoftheseasonversusmiddleoftheseason,no sta-tisticallysignificantdifferencesbetweenthesamplescouldbe seen,ineitherofthecomparisons(1and2).

In the analysisbetween the middle and the endof the season,therewasasignificantdifferenceinassayedlevels, inthecomparisonamongthe14participants(p<0.02). How-ever,whenplayers4and10weretakenoutoftheanalysis, nosignificantdifferencecouldbeseen(analysiswithreduced data).

Incomparingthebeginningandendoftheseason,both analyses(comparisons1and2)indicatedthattherewasa sta-tisticallysignificantincreaseintheCTX-IIbiomarker(p<0.003 andp<0.01,respectively).Inotherwords,betweenthe begin-ningandtheendoftheseason,therewasasignificantincrease inthe levelofthe CTX-IIjoint degradationbiomarker, irre-spective of whether the extreme results were taken into consideration.

Table2–Comparisonofassayedlevelsforthe biomarkerCTX-II(inng/mL)observedbetweenthe differenttimesduringtheseason.Completedata(14 players)andreduceddata(12players:players4and10 weredisregarded).

Comparison Meandifference p

Completedata

Beginningvs.middle 0.134 0.06 Middlevs.end 0.186 0.02 Beginningvs.end 0.320 0.003

Reduceddata

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Discussion

Thisstudyaimedtoanalyzeandcomparebloodserumassay levelsforthebiomarkerCTX-IIamongprofessionalindoor soc-cerplayers,atthreedifferenttimesduringoneseason.From theresultsobserved,itcouldbeseenthattherewasa statis-ticallysignificantincreaseinCTX-II betweenthebeginning and endofthe season.Ithasbeen well establishedinthe literaturethatincreasedlevelsofthebiomarkerCTX-IIarea predictivefactorforjointdegradation.Thus,itisbelievedthat earlyidentificationofCTX-IIlevelswouldbeausefultoolfor makingequallyearlydiagnosesofprimaryosteoarthritis(OA) andtakingpreventiveaction.22,23

Corroborating the findings ofthe present study,O’Kane etal.22_evaluated_different_sports_categories_and_demonstrated through urine analyses that samples from marathon run-nerspresentedhigherCTX-IIlevelsthanthoseofswimmers orrowers.Accordingtotheseauthors,runnersexposetheir lower-limbjointstorepetitiveaxialoverloading,whichmay causeearlydamagetotheirjointcartilage.

The pure and simple increase in CTX-II in the players studiedhere does not necessarilymean that theyare suf-feringorwillsufferosteoarthrosis.Itisknownthatpatients witharthrosishavehighlevelsofthisserumbiomarkerand increasedlevelsaredirectlyrelatedtoradiographicworsening, accordingtotheKelgreen-Laurenceclassification.16_What_we canbesureoffromthesedataisthat,atleastduring competi-tions,chondraldegradationwashigherinthepatientsstudied, sincethebiomarkerCTX-IIcomesfromdestructionoftypeII collagen,exclusivelyinjointcartilage.

Itispossiblethat changes totraining, intensificationof anaerobicexerciseswithmusclestrengthening,compulsory physiotherapeuticfollow-upandevenuseof chondroprotec-tantmedicationsmightbeviablesolutionsforjointprotection. Inordertoprotectthejointhealthofsoccerplayers,teams shouldstarttothinkaboutthistypeofpreventivestrategy.

After all, the aim today should be prevention before treatment. Injury avoidance through practicing preventive medicineshouldbethefocusofexerciseandsportsmedicine, sincecareerscancertainlybeprolongedinthismanner.

Thepossiblelimitationsofthisstudyarethesmallsample size(n=14)andtheabsenceofacontrolgroupforcomparisons betweenfindings.Thesamplesizeisexplainedbythefact thatthisstudyinvestigatedasingleteamwithinthiscategory, whosememberswereallsubjectedtothesametrainingand competitionloads.Itwasdecidednottouseacontrolgroup, becauseouraimwasonlytoascertainwhethertherewould beanyincreaseinjointcollagendegradationoverthecourse ofasingleseasonoftrainingandcompetition.Inotherwords, theindividualsbecametheirowncontrols.

OtherstudiesunderwaywithintheSportsTraumaGroup ofSantaCasadeSãoPauloarecomparingtypesofsportsand controlgroups, includingindoorsoccer.Preliminarystudies indicatethattherearelargedifferencesregardingtypeII col-lagendegradation.

Thediscrepanciesencounteredinoursample,intwoof the individuals analyzed, can be explainedby avariety of theories. These include the notionsthat the markers may havebeeninfluencedbytheindividuals’hormonalstate,diet

or geneticfactors,22 _given_that _neither_of_these _individuals reportedanyreductioninphysicalperformance,orany symp-tomssuchaspain, edemaorinstability. AccordingtoDam etal.,24_the_amount_of_cartilage_degradation_estimated_from thebiomarkerCTX-IIisrelatedtothepresenceofpain.

Conclusion

Therewasasignificantincreaseintheserumlevelsof CTX-IIintheindoorsoccerplayers,comparingthebeginningand endoftheseason.Thesedatasuggestthatincreased degra-dationoftypeIIjointcollagenwasoccurringattheendof this period.Itisclearthatfurtherstudiesareneeded,with appropriatemethodologicalrigor,soastomakeeffective con-tributionstowardpreciseelucidationoftheetiologyofOAand itsrelationshipwithbiomarkersasatoolforearlydiagnosis.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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