Rev. bras. ortop. vol.50 número2

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

Original

Article

Effects

of

long-term

administration

of

omeprazole

on

bone

mineral

density

and

the

mechanical

properties

of

the

bone

夽

Gabriela

Rezende

Yanagihara

,

Aline

Goulart

de

Paiva

,

Maurílio

Pacheco

Neto

,

Larissa

Helena

Torres

_,

_Antônio

_Carlos

_Shimano

_,

_Mário

_Jefferson

_Quirino

_Louzada

_,

Raquel

Annoni

_,

_Álvaro

_César

_de

_Oliveira

_Penoni

a_{PousoAlegreSchoolofMedicalSciences,UniversidadedoValedoSapucaí,PousoAlegre,MG,Brazil} b_{DepartmentofClinicalPathology,SchoolofMedicine,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil}

c_{DepartmentofClinicalandToxicologicalAnalyses,SchoolofPharmaceuticalSciences,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil} d_{RibeirãoPretoSchoolofMedicine,UniversidadedeSãoPaulo,RibeirãoPreto,SãoPaulo,SP,Brazil}

e_{Arac¸atubaSchoolofVeterinaryMedicine,UniversidadeEstadualPaulistaJúlioMesquitaFilho,Arac¸atuba,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6April2014 Accepted19May2014 Availableonline14March2015

Keywords:

Bone Bonedensity Omeprazole Rats

a

b

s

t

r

a

c

t

Objectives:Epidemiologicalstudieshaveshownarelationship betweenlong-termuseof protonpumpinhibitorsandbonemetabolism.However,thisrelationshiphasnotyetbecome established.Theaimofthepresentstudywastoanalyzethemechanicalpropertiesandbone mineraldensity(BMD)ofratsthatweresubjectedtolong-termomeprazoleuse.

Methods:Fifty Wistar rats weighing between 200 and 240g were divided equally into five groups: OMP300 (omeprazole intake at a dose of 300␮moL/kg/day); OMP200 (200␮moL/kg/day); OMP40(40␮moL/kg/day);OMP10(10␮moL/kg/day);andCont(control group;intakeofdilutionvehicle).Thesolutionswereadministeredfor90consecutivedays. Aftertheratshadbeensacrificed,theirBMD,themechanicalpropertiesofthedissected femursandtheirserumCa++levelswereanalyzed.

Results:TheBMDoftheOMP300groupwaslowerthanthatofthecontrols(p=0.006).There wasnodifferenceoncomparingtheOMP200,OMP40andOMP10groupswiththecontrols. Themaximumstrengthandrigidityofthefemurdidnotdifferintheexperimentalgroups incomparisonwiththecontrols.TheOMP300grouphadastatisticallylowerserumCa++ concentrationthanthatofthecontrols(p=0.049),buttheothergroupsdidnotshowany differenceinrelationtothecontrols.

Conclusion:Dailyintakeof300␮moL/kg/dayofomeprazoledecreasedtheBMDofthefemur, butwithoutchangestotherigidityandstrengthofthefemurinadultrats.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.Allrightsreserved.

夽

WorkdevelopedatthePousoAlegreSchoolofMedicalSciences,UniversidadedoValedoSapucaí,PousoAlegre,MG,Brazil.

∗ _{Correspondingauthor.}

E-mail:[email protected](G.R.Yanagihara).

http://dx.doi.org/10.1016/j.rboe.2015.03.002

Efeitos

da

administrac¸ão

em

longo

prazo

do

omeprazol

sobre

a

densidade

mineral

óssea

e

as

propriedades

mecânicas

do

osso

Palavras-chave:

Osso

Densidadeóssea Omeprazol Ratos

r

e

s

u

m

o

Objetivos: Estudosepidemiológicosmostramumarelac¸ãoentreousoemlongoprazode inibidoresdebombadeprótonseometabolismoósseo,porémessarelac¸ãoaindanãoestá estabelecida.Oobjetivodesteestudofoianalisaraspropriedademecânicaseadensidade mineralóssea(DMO)deratossubmetidosaousodeomeprazolemlongoprazo.

Métodos: Cinquenta ratos Wistar, entre 200 e 240g, foram divididos igualmente em cincogrupos:OMP300(ingestãodeomeprazolnadosede300␮moL/Kg/dia),OMP200(200

␮moL/Kg/dia),OMP40(40␮moL/Kg/dia),OMP10(10␮moL/Kg/dia)eCont(grupocontrole;

ingestãodoveículodediluic¸ão).Aadministrac¸ãodassoluc¸õesocorreudurante90dias segui-dos.Apósaeutanásia,foramanalisadasaDMO,aspropriedadesmecânicasdosfêmures dissecadoseadosagemdeCa++sérico.

Resultados: ADMOdogrupoOMP300foimenordoqueadoCont(p=0,006).Nãohouve diferenc¸anacomparac¸ãoentreosgruposOMP200,OMP40eOMP10emrelac¸ãoaoCont.A forc¸amáximaerigidezdofêmurnãoforamdiferentesnosgruposexperimentaisquando comparadosaoCont.OgrupoOMP300teveconcentrac¸õesséricasdeCa++estatisticamente menoresdoqueogrupoCont(p=0,049)semdiferenc¸aentreosdemaisgruposemrelac¸ão aoCont.

Conclusão: Aingestãodiáriade300␮moL/Kg/diadeomeprazoldiminuiuaDMOdofêmur, porémsemalterac¸õesnarigidezenaforc¸adofêmurderatosadultos.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Todososdireitosreservados.

Introduction

Proton pump inhibitors (PPIs) are the main drugs used for treating diseases such as duodenal ulcers and reflux esophagitis.1,2 _{Because these drugs present few adverse} effectswhen administeredcorrectly,they havecometo be usednot onlyforacute symptoms inclinical practice, but alsoforlong-termpurposes,eventhoughsuchindicationsare highlydebatable.3–5

PPIsactmainlytowardsuppressinggastricacidsecretion bythe parietalcells ofthe stomach,sincethey inhibitthe enzymeH+K+ATPase,andthisacidsuppressionmaylastfor upto48h.6

Epidemiological studies have indicated that there is a relationship between prolonged use of PPIs and bone metabolism,7–9 _{althoughthisrelationshipisstillnottotally} established. Yang et al.4 _{reported that administration of} omeprazole (20mg/day), which is one of the most impor-tantPPIs,iscapableofsignificantlydiminishingbonemineral density(BMD).Itisbelieved that themechanism responsi-bleforthisconsistsofelevationofgastricpH,whichwould interferewithcalciumabsorption.4,10,11_{Thisoccursbecause} somesalts,suchascalcium,are insolubleinbasicpHand wouldthereforebeabsorbedlessreadily.8 _{However,astudy} by Hyun et al.3 _{suggested that using omeprazole would} tendtodiminishbonereabsorptionandimpedethe progres-siontowardosteoporosis.Therefore,therelationshipbetween usingPPIsand bonedemineralizationand the riskof frac-turesassociatedwithprolongeduseofomeprazoleremains unclear.9

GiventhatthereisevidencethatprolongeduseofPPIsmay changethebehaviorofbonecells,ourobjectiveherewasto analyzethebonemineraldensityandmechanicalproperties ofratfemursthatweresubjectedtolong-termuseof omepra-zole.

Materials

and

methods

Typeofstudy

Thiswasanexperimentalstudyusingananimalmodel.

Animals

The procedures used in this study followed the standards describedbytheBrazilianCollegeforAnimalExperimentation (COBEA)in1991andtheInternationalGuidingPrinciplesfor BiomedicalResearchInvolvingAnimals12_{andwereapproved} bytheEthicsCommitteeforAnimalResearchoftheUniversity ofValedoSapucaí.

FiftymaleadultratsofWistarlineage,weighing200–240g, wereusedinthisstudy.Theanimalswerekeptundernormal environmentalandtemperatureconditions(21±2◦_C; humid-ity of55–60%; and light/darkcycles of12h).They received wateradlibitumandfeedsuitableforrats.Fastingof6hwas maintainedduringthedaytimeperiod,beforethedaily proto-colwasstarted.

Administration of omeprazole or dilution vehicle

Start of protocol

Day 1 Day 66 Day 90

Weighing Sacrifice

Fig.1–Experimentaldesignduringtheexposureperiod.

–200␮mol/kg;(3)OMP40–40␮mol/kg;(4)OMP10–10␮mol/kg; and(5)control(Cont)–intakeonlyofthedilutionvehicle.

Solutions

All theexperiments were performedusing gradeI purified water from the Milli-Q system and using analytical grade reagents.Thedrugsolutionswerepreparedasdescribedby Larssonetal.13_{Theomeprazolegranules(8.5%;Pharma} Nos-tra,Lavras, Minas Gerais, Brazil) were ground up with the aidofamortarand weredispersedinavehiclecontaining 0.25%hydroxyethyl cellulose4400(GalenaQuímica,Lavras, MinasGerais,Brazil)ina0.1Msolutionofsodiumbicarbonate (pH≈7.4).Thesuspensionswerepreparedatconcentrations of10,40,200and300␮mol/kg.

Experimentalprotocol

Overa90-dayperiod,theanimalsoftheexperimentalgroups receivedtheirrespectivedosesofomeprazoleandtheanimals ofthecontrolgroupreceivedthedilutionvehicle.Thedoses wereadministered orallybymeans ofgavage.Fig.1shows theexperimentaldesignoverthecourseofthe90daysofthe experiment.

Attheendofthe90daysofreceivingthecompounds,the animalswereagainweighed.Theywerethenanesthetizedby meansofintramuscularinjectionofxylazine hydrochloride (95mg/kg)andketaminehydrochloride(12mg/kg)and sacri-ficedbymeansofexsanguination,usingcardiacpuncture.14,15 Therightandleftfemursofeachanimalwereextractedfrom theirbodysegmentsandthemuscleandconnectivetissues weretotallyremoved.Theanimalsandthespecimenswere weighedsimultaneously.Afterweighingandmeasurement, thestructureswerekeptinchilled salinesolutionuntilthe dayofthedensitometricanalysisandmechanicaltests(not morethan48hafterthedissection).

Densitometricanalysis

Therightfemursweresubjectedtoanalysisinadual-emission X-raydensitometerfor small animals (Lunar® _{DPX Alpha).}

Theimageswere allacquiredwiththefemursinthesame position, immersed in water at a depth of 2cm. The fol-lowingimagingoptions were selected:appendicular;typeI bone; high-resolution mode; 76kVp; 150␮A; collimation at the fine adjustment; and areas of standardized size com-prising40mminwidthand20mminlength.Inaccordance

with the manufacturer’s recommendations, the apparatus was calibrated using a phantom that the manufacturer supplied.

Theanalysesontheimagingexaminationswereperformed withtheaidofthesamecomputersoftwareashadbeenused forimageacquisition.Usingaselectiontool,theboneswere fullydemarcatedandinformationontheirmassandarea,and consequentlytheirmineraldensity,wasgathered.

Mechanicalanalysis

The animals’ left femurs were analyzed mechanically by meansofathree-pointmechanicalflexiontest(Fig.2).To per-formthesemechanicaltests, auniversaltest machinewas used (EMIC®_{, model DL 10000), with a load cell of}

capac-ity 500N (certified by EMIC®_{), both of which belonged to}

theBioengineeringLaboratoryoftheRibeirãoPretoSchoolof Medicine,UniversityofSãoPaulo,alongwithaccessories spe-ciallydevelopedforthree-pointflexiontestsonthebonesof smallanimals.16–18

The mechanical properties analyzed in the three-point flexiontestswerethemaximumstrength(Fmax)andrigidity (Rig).Thevelocityatwhichtheforcewasappliedinthe three-pointflexiontestswas1mm/min.Thespanbetweenthetest pointswas30mmand anaccommodationtimeof30swas used.

Fig.2–Three-pointmechanicalflexiontest:(A)bone

structure;(B)apparatusforapplyingforce.RibeirãoPreto

Bone mineral density in the experimental groups

OMP 300

BMD (g/cm

2)

OMP 200

* P=.006

OMP300 group compared with control group (Cont)

OMP 40 OMP 10 CONT 0.3

0.2

0.1

0.0

Fig.3–Graphshowingthebonemineraldensityresults

fromeachexperimentalgroup.Valuesareexpressedas

mean±standarddeviation.OMP300(omeprazole,

300␮mol/kg),OMP200(omeprazole200␮mol/kg),OMP40

(omeprazole40␮mol/kg),OMP10(omeprazole10␮mol/kg),

Cont(control);BMD,bonemineraldensity.

SerumCa++_assay

Thebloodthathadbeencollectedthroughcardiacpuncture wasusedtoperformaserumcalciumassay,bymeansofa diagnostickitsuppliedbythecompanyDoles(Goiânia,Brazil). Aftercentrifugation(at1000g,for5min)oftheblood col-lectedbymeansofcardiacpuncture,theserumwascollected andtheserumcalciumassayswereperformedbymeansofa diagnostickitsuppliedbythecompanyDoles(Goiânia,Brazil). Themethodwasbasedon formationofa stainedcomplex betweencalciumandcresolphthaleininanalkalinemedium. Fortheanalyses,aFemto650®_{spectrophotometerwasused}

(FemtoInd.eCom.deInstrumentosLtda.,SãoPaulo,Brazil), adjustedtoawavelengthof570nm.

Statisticalanalysis

Thestatisticalanalysisonthedatawasperformedusingthe SPSSsoftware(SPSS,Chicago,USA),version15.0.Totestthe normalityofthesample,theKolmogorov–Smirnovtestwas used.Becausethedatadistributionwasfoundtobenormal, comparisonsofparametersbetweenthecontrolgroupandthe otherexperimentalgroupsweremadeusingStudent’st-test. Tocomparetheanimals’weightsduringtheexperimentand theweightsofthefemurs,theone-wayANOVAtestwasused. Thesignificancelevelof5%(p≤0.05)wasusedandtheresults wereexpressedasmeans±standarddeviations.

Results

Duringtheexperiment,oneanimalbelongingtotheOMP300 groupwaslost.

Serum calcium concentration in the experimental groups

OMP 300

Ca

++

(mmol/L)

OMP 200

* P=.049.

OMP300 group compared with control group (Cont)

OMP 40 OMP 10 CONT

0 1 2 3 4 5 6

Fig.4–Serumcalciumconcentration(Ca++).Valuesare

expressedasmean±standarddeviation.OMP300

(omeprazole,300␮mol/kg),OMP200(omeprazole

200␮mol/kg),OMP40(omeprazole40␮mol/kg),OMP10

(omeprazole10␮mol/kg),Cont(control).

The animals’ mean weight did not differ between the groupsonday1(p=0.52),day66(p=0.74)orday90(p=0.45)of theexperiment(Table1).

ThebonedensityintheOMP300group(0.20±0.008g/cm2₎

was lower than that ofthe Cont group (0.22±0.107g/cm2_;

p=0.006). The other groups did not show any differ-ences in comparison with the control group: OMP200 (0.21±0.019g/cm2_{; p=0.644), OMP40 (0.21±0.015g/cm}2_;

p=0.305)andOMP10(0.21±0.016g/cm2_{;p=0.410)(Fig.3).}

Therewerenodifferencesintheresultsrelatingtothe max-imumstrengthofthefemurs,incomparingtheContgroup with the other groups: OMP300 (p=0.09); OMP200 (p=0.55); OMP40(p=0.11)andOMP10(p=0.62).Likewise,therewereno differencesrelating tothe rigidityvalues,incomparingthe Contgroupwiththeothergroups:OMP300(p=0.98);OMP200 (p=0.84);OMP40(p=0.08)andOMP10(p=0.72).Thestrength andrigidityvaluesarelaidoutinTable1.

The serum concentration of Ca++ _{in the OMP300 group}

(5.03±0.39mmol/L) was lower than that of the Cont group (5.39±0.35mmol/L; p=0.049). The other groups did not show any differences in comparison with the con-trol group: OMP200 (5.37±0.26mmol/L; p=0.860), OMP40 (5.40±0.25mmol/L;p=0.970)andOMP10(5.41±0.26mmol/L;

p=0.910)(Fig.4).

Discussion

Table1–Initial,meanandfinalweightsoftheanimals,maximumforce(Fmax)andrigidity(Rig).

Group Initialweight(g) Meanweight(g) Finalweight(g) Fmax(N) Rig(N/mm)

OMP300 196.88±14.90 357.55±39.61 379.44±43.94 92.35±11.61 154.92±33.52

OMP200 213.80±36.06 365.90±44.71 391.70±49.79 97.19±12.09 152.34±26.73

OMP40 223.40±39.90 355.10±57.83 382.10±59.283 91.22±14.93 130.30±18.50

OMP10 221.90±40.39 378.80±35.04 413.60±33.59 97.01±15.93 149.52±34.91

CONT 215.70±39.20 362.30±22.63 383.50±35.83 99.76±6.10 155.23±37.24

OMP300(omeprazole,300␮mol/kg),OMP200(omeprazole200␮mol/kg),OMP40(omeprazole40␮mol/kg),OMP10(omeprazole10␮mol/kg),Cont

(control).Resultsarepresentedasmean±standarddeviation.

inserum Ca++_{concentrationinthisgroup.However,useof} omeprazolewasnotseentoinfluencethemechanical proper-tiesofthebonetissue.

Osteoporoticfracturesare considered tobeaworldwide

publichealthproblem.Themortalityrateduringthefirstyear afterhipfracturesisaround30%.19_{Ithasbeenestimatedthat} intheUSA,1.5millionpeopleeveryyearareaffectedby frac-turesrelatingtoosteoporosis.Thisisaneventthatmayreduce qualityoflifeandincreasetheriskofdeath.5 _However,the relationshipbetweentherisksofhipfractureandprolonged useofPPIsremainsunclear.

Inastudyconductedin1993,Mizunashietal.20_evaluated theeffectofPPItherapyontheserumlevelsofparathormone (PTH)inasmallgroupofpatientswithgastriculcers(seven menand12women,withameanageof67±13years).The studyshowedthataftereightweeksoftherapywith omepra-zole,thePTH levelhad increasedby28% inrelationtothe baselineamongthesepatients,accompaniedbyincreasesin osteocalcin,alkalinephosphataseandtartrate-resistantacid phosphatase,whicharemarkersforboneformation.Thus,it wassuggestedthatinthatsmallgroup,therewasanincrease inboneformation,contrarytotheresultsfromthepresent study.

However,theurinaryexcretionofhydroxyprolineand cal-ciumamongthepatientsinthestudybyMizunashietal.20 decreased, whichmay suggest thatthere wasless calcium absorption.Thepossibilityforcomparisonwiththepresent studyislimited,giventhatthiswasanexperimentalstudy, whilethestudy byMizunashiet al.was conductedamong humans.Moreover,PTHwasonlymeasuredatonetimepoint, whichmaynotreflectthedynamiceffectofPTHovera24-h period;andalso,theomeprazoledoseand calcium supple-mentationamongthosepatientswasquestionable.

Ontheotherhand,severalpreviousstudieshave demon-stratedassociationsbetweenPPIuseandavarietyoffractures. Yang etal.4 _{stated that usingPPIs could cause deleterious} effectsinbonetissueandcould increasetheriskof osteo-poroticfractures,especiallyafterfouryearsofcontinualuse. Vestergaardetal.21_{corroboratedthepreviousstudyand} con-firmed the relationship between PPI use and osteoporotic fractures.Furthermore,Grayetal.22_{conductedaprospective} analysisthatincluded161,806women,withtheaimof corre-latingPPIusewiththeriskofclinicalfractures.Theauthors concludedthattherewereassociationsbetweenPPIuseand occurrencesofclinicalfractures ofthe wrist,vertebraeand forearm. Thepresentstudy corroboratesthe previous find-ings,giventhatarelationshipbetweenomeprazoleuseand decreasedbonemineraldensitywasdemonstrated.Thiscan

betakentobeapredictivesignindicatingthatfracturesmay occurasaresultoflong-termPPIuse.

KayeandJick23_{conductedaretrospectivestudyinwhich} PPIuseandtheriskofhipfractureswere evaluatedamong patientswithoutsignificantriskfactors.Theseauthors con-cludedthatpatientsintheagegroupof50–79yearswhomade useofPPIsdidnothaveriskfactorsandsowouldnothave higherfrequencyofhipfractures.Inthatstudy,the epidemi-ologicaldatausedcamefromthesamedatasourcethatYang etal.4_{usedin2006.However,Yangetal.}4_{observedthatthe} risksofhipfracturesincreasedwithPPIuse,especiallywhen usedforfouryearsormore.KayeandJicktooktheviewthat theresultsofYangetal.4_{referredonlytopatientswithsome} formofriskfactorforhipfractures,giventhattheydidnot excludepatientswithsignificantriskfactors.

Targowniketal.9_{conductedacross-sectionalretrospective} studyfollowedbyalongitudinalphase.Inthecross-sectional study,casesofhiporlumbarvertebralosteoporosiswere com-paredwithcontrolspresentingnormalbonemineraldensity. Inthelongitudinalanalysis,bonemineraldensityalterations amongpatientswhoeitherwereorwerenotusingPPIswere successivelyevaluated.Theresultsfromtheirstudyindicated thatchronicuseofPPIwasnotassociatedwithanincreased likelihoodofoccurrencesoffractures.Inaddition,theauthors concludedthathigherintensityofexposurewasnot associ-atedwithincreasedriskofpresentingosteoporosis.Inother words,theybelievedthattheriskoffractureswasrelatedto othervariablesthatwereindependentofthosecorrelatedwith osteoporosis.

Our study cannot be fully compared with that of Tar-gowniket al.,9 _{since ours was experimental. Nonetheless,} it isknown that in addition to the mineral characteristics thatcanbeassessedusingDXA,boneshaveimportant vis-coelasticpropertiesthatallowthistissuetofulfillitsspecific functions.24,25 _{These properties are best evaluated through} experimentalstudies,sincetheseenableexvivoanalyses.To ourknowledge,ourstudywasthefirsttoanalyzetheeffects ofPPIs onthe viscoelasticproperties ofthe bonetissueof animalssubjectedtodifferentdosesofomeprazolefora pro-longedtime.

especiallyamongovariectomizedfemalerats.Itcantherefore beconcludedthatthebonesofosteopenicfemaleratspresent alteredmechanicalproperties.

Despite the high dosages of medications and the long periodoverwhichtheanimalsremainedexposedto omepra-zole intake, the mechanism of action of the PPIs did not influenceanyofthemechanicalproperties ofthe bonesin ourstudy.Webelievethatthehighestdosestipulatedorthe lengthofusewasinsufficienttoalterthemechanical proper-tiesofthebones,eventhoughthesefactorsdiminishedthe bonemineralization.

WedidnotdeterminethedoseofPPIsintheseanimals thatcouldbestipulatedforsimulatingtheiruseinhumans. Thedoseofthismedicationthatisgenerallyadministeredto humansis20mgperday,althoughitmayreachupto90mg. However,itisnotknownwhattheequivalentdoseinanimals wouldbe.Itisknownthatomeprazolebindstoaround95%of theplasmainhumans28_{and,accordingtoRegårdhetal.,}29_the proportionofbindingwouldbelowerindogsandrats(90%and 87%,respectively).Thissuggeststhatasimplecalculationof doseperkilogramwouldnotreflectthedoseusedinhumans. Moreover,it hasbeen seenthatwhilethemean half-lifeof thisdrugis1hinhumansanddogs,half-livesintherangeof 5–15minhavebeenrecordedinrats.29

Somedose–responsestudiesonPPIsinanimalshavebeen conducted,butwithobjectivesthatdifferedfromthoseofour study.Londongetal.30_{conductedadose–responsestudyusing} thedosesof30,60and90mg/kgandasecondstudyuseda doseof1–10mg/kg.31_Adhikayetal.32_{usedadoseof3mg/kg} inrats witha view to curing gastric ulcers that had been induced.In2003,Meloetal.33_{subjectedWistarratstointake} 0.2mg/kg,withtheaimofevaluatingtheeffectofthisintake onpartialhepatectomy.Eventhoughmanydosageshavebeen observed,thereisnoconsensusintheliteratureonanimal experimentsregarding the PPI dose that would be equiva-lenttothoseusedinhumans,forthepurposeoflong-term testing.

ThepresentstudywasbasedontheworkbyCuietal.,1 inwhichtheobjectivesweresimilartoours,i.e.tostudythe long-termeffectsofPPIs.Cuietal.1_{treatedtheiranimalsfor} 77days,atadoseof400␮mol.Thehighestdoseofthepresent study(300␮mol)wasstipulatedasslightlylowerthanthedose usedbythoseauthors,butwasusedfor90days,i.e.forslightly longerthaninthestudybyCuietal.1 _{Itcouldbeseenthat} theeffectsweresimilar,sincethoseauthors alsoidentified decreasedbonemineraldensityamongtheiranimals.

Insummary,thepresent study demonstrated that long-termPPIusedidnotalterthemechanicalpropertiesofthe femursofadultrats.However,itwasobservedthatthefemurs ofthe ratssubjected todailyadministrationofomeprazole atadoseof300␮mol/kg/daydevelopedbone demineraliza-tion,whichmaysuggestthatthisgivesrisetopredisposition tobone fractures. Furtherexperimental studies using pro-tocolssimilartoourswouldbeusefulforconfirmingthese results.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

OursincerethankstoMessrs.LúcioIvandeMelo,JoséLopes and Marcos Martins for their assistance in caring for the animals,andtoMrs.NelsiNaraVieiraMarchetteforher con-tributionrelatingtothemedications.Alsotothepostgraduate studentBrunaRezende,forherhelpinthedensitometry anal-yses.

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