Influence of chronic alcoholism and oestrogen deficiency on the variation of stoichiometry of hydroxyapatite within alveolar bone crest of rats

Influence

of

chronic

alcoholism

and

oestrogen

deficiency

on

the

variation

of

stoichiometry

of

hydroxyapatite

within

alveolar

bone

crest

of

rats

§

Adriana

M.P.S.

Marchini

*,

Camila

P.

Deco

,

Karina

B.

Lodi

,

Leonardo

Marchini

,

Ana

M.E.

Santo

,

Rosilene

F.

Rocha

a_{DepartmentofBiosciencesandDiagnostics,Sa˜oJose´ dosCamposSchoolofDentistry,Sa˜oPauloStateUniversity–UNESP,Sa˜oJose´ dos}

Campos,Sa˜oPaulo,Brazil

b_{SchoolofDentistry,UniversityofValedoParaı´ba–UNIVAP,Sa˜oJose´ dosCampos,Sa˜oPaulo,Brazil}

c_{DepartmentofDentistry,UniversityofTaubate´ –UNITAU,Taubate´,Sa˜oPaulo,Brazil}

d_{InstituteofResearchandDevelopment–IP&D,LaboratoryofBiomedicalVibrationalSpectroscopy,UniversityofValedoParaı´ba,Sa˜oJose´ dos}

Campos,Sa˜oPaulo,Brazil

e_{DepartmentofMathematicalandEarthSciences,FederalUniversityofSa˜oPaulo–UNIFESP,Diadema,Sa˜oPaulo,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Accepted21April2012

Keywords:

Alcoholism Ovariectomy Periodontium

Fluorescencespectrometry Calcium

Phosphorous

a

b

s

t

r

a

c

t

Objective: Previousfindingssuggestthatchronicalcoholismandoestrogenicdeficiencymay

affectbonesingeneral(includingalveolarbone)andincreaseindividuals’susceptibilityto the developmentof periodontal disease.Theaim ofthis studywasto assesspossible alterations in the chemical composition of alveolar bone in rats subjected to chronic alcoholism,oestrogendeficiencyorboth.

Design: Fifty-fourratswereinitiallydividedintotwogroups:ovariectomized(Ovx),and

Shamoperated(Sham).Amonthaftersurgery,thegroupsweresub-dividedandreceived the following dietary intervention for eight weeks: 20% alcohol, isocaloric diet and adlibitumdiet.Samplesofthemandible,inthealveolarbonecrestregion,wereanalyzed toverifypossiblechangesinthestoichiometriccompositionofbonehydroxyapatite,by measuringtherelationshipbetweentheconcentrationofcalciumandphosphorus(Ca/P ratios),usingmicroX-rayfluorescencespectrometry.

Results: TheadlibitumgroupspresentedthehighestaveragevaluesofCa/Pratios,whilethe

groupswithdietaryrestrictionspresentedthesmallestaveragevalues.TheOvxadlibitum grouppresentedthehighestvaluesofCa/Pratios(2.030.04).However,thesevalueswere notconsideredstatisticallydifferent(p>0.05)fromtheShamadlibitumgroup(2.010.01).

TheOvxalcoholgrouppresentedlowervaluesforCa/Pratios(1.920.06),beingtheonly groupstatisticallydifferent(p<0.001)fromtheShamadlibitumgroup.Potential

confound-ingvariablesarediscussed.

Conclusion: Ovariectomyassociatedwithalcoholconsumptionat20%significantlychanged

thestoichiometrycompositionofhydroxyapatiteinthealveolarbonecrest,leadingtoa reductioninCa/Pratios.

#2012ElsevierLtd.

§

Source of support: Adriana M.P.S. Marchini received a scholarship from the Brazilian governmental research agency, CAPES (Coordenac¸a˜odeAperfeic¸oamentodePessoaldeNı´velSuperior).

*Correspondingauthorat:Av.AdhemardeBarros,1136/153,Sa˜oJose´ dosCampos,SP12245-010,Brazil.Fax:+551239221555. E-mailaddress:adrianampsmarchini@gmail.com(AdrianaM.P.S.Marchini).

Available

online

at

www.sciencedirect.com

journalhomepage:http://www.elsevier.com/locate/aob

0003–9969#2012ElsevierLtd.

http://dx.doi.org/10.1016/j.archoralbio.2012.04.011

Open access under the Elsevier OA license.

1.

Introduction

Bonesarecomposedofmineralizedtissueconstitutingmainly of calcium (Ca) and phosphorous (P). These elements are organizedformingcrystalsofhydroxyapatite.1_Some

condi-tions or pathologies affecting this tissue may alter the quantitativedistributionoftheseelements,andconsequently thestoichiometriccompositionofhydroxyapatite.2–4

Osteoporosisisametabolicbonedisorder,themostfrequent etiologicfactorofwhichisoestrogendeficiency,whichoccurs mainlyin womenafter menopause.5 _{This condition causes}

changesinthepatternofboneremodelling,witha predomi-nanceoftheresorptionprocess,whichcanalterthe homeosta-sisofCaandPanddecreasebonemineraldensity.4,6,7_Despite

the importance of oestrogen deficiency in the aetiology of osteoporosis,itisamulti-factorialdisease,involvingseveral otherriskfactors,includingexcessiveconsumptionofalcohol.8

Theeffectsofabusivealcoholconsumptiononbonequality seemtobemoredramaticinyoungindividuals.9However,a decreaseinbonemineraldensitywhenalcoholisconsumedin large quantities, has also been reported in women after menopause.10

Periodontaldiseaseisaninfectiousimmuneinflammatory alterationthataffectsthestructureswhichsupportteeth.The primary etiological factor of which is bacterial biofilm.11

However,itsprogressionmaybeinfluencedbyawiderangeof variableswhichincludesystemicdiseases(e.g.diabetesand osteoporosis),geneticdisorders,habits(e.g.smokingand/or alcoholism),age,gender,stress,nutritionalproblems, includ-ing other factors, which may influence the way the host respondstoanaggressiveagent.12–18

Literaturereviewshavesuggestedthatosteoporosis asso-ciatedwithbothoestrogendeficiencyandexcessivealcohol consumptioncanbeconsideredpotentialriskfactorsforthe developmentofperiodontaldisease,which,ifnotcontrolled, couldleadtotoothloss.However,theinformationavailablein theliteratureisinsufficientforadefinitiveconsensus,which highlights the need for further research by undertaking a greater number of well controlled and longitudinal stud-ies.15,16,19,20_{Itispossiblethatsystemicbonelossassociated}

with osteoporosis/osteopenia can also affect alveolar bone anditsporositywhichwouldleadtoagreatersusceptibilityof boneresorptionintheregion.15

DespitetheimportanceofCaandPasmajorconstituentsof bonemineralphaseandthepossibleimplicationsofoestrogen deficiencyandexcessivealcoholconsumptiononthe devel-opmentofperiodontaldisease,tothebestofourknowledge thereareno studiesthat haveevaluated concentrationsof thesechemicalelementsundertheseconditions,specifically intheregionofalveolarbonecrest,astructurewhoseintegrity isimportantforthemaintenanceofperiodontalhealth.

Consideringtheabsenceofsuchstudies,thispaperaimsto evaluate the effect of oestrogen deficiency and excessive alcohol consumption on alveolar bone crest. Considering previousfindingsregardingoestrogendeficiencyandalcohol consumption,21–23 _{the hypothesis of this study is that}

oestrogen deficiency associated with alcohol consumption canadverselyinfluencethequalityofalveolarboneandalter itsmineralcomposition.

2.

Materials

and

methods

2.1. Treatmentofanimals

Thepresentstudywasapprovedbytheethicscommitteeof Sa˜oJose´ dosCamposSchoolofDentistry,StateUniversityof Sa˜oPaulo–UNESP(ProtocolNo.021/2008-PA/CEP).

Fifty-four rats (Rattus norvegicus, of the albinus, Wistar variety), aged four-months, were initially divided into two groups:ovariectomized(ratssubjectedtooestrogendeficiency by removing the ovaries), and Sham operated (simulated ovariectomy,ovariesexposedbutnotremoved).Amonthafter surgery, thetwogroupsweresub-divided,andreceivedthe following dietaryinterventionforeight weeks:(a) alcoholic diet: solid diet and a 20% alcohol solution ad libitum, (b) isocaloricdiet:solidandliquiddietswiththesameamountof caloriesconsumedbythealcoholgroupand(c)adlibitumdiet: soliddietandwateradlibitum.Theanimalswererandomized byweightintheirrespectivegroups.

The 20% alcohol solution was obtained by an absolute alcoholdilutioninwater.Theconcentrationoftheisocaloric solutioncontained,inmillilitres,thesameamountofcalories asthe20%alcoholsolution.Itwaspreparedbydissolving266g sucrosein1lofwater.Calculationsweremadetakinginto account the alcohol concentrations (20%), the density of absolutealcohol(0.787g/ml)andthecaloricvaluesofsucrose (4.1kcal/g) and alcohol (7.1kcal/g). The solid diet was a commercialfood(Labina–Purina1_{,Paulı´nia,Brazil).}

Theamount ofcalories (soliddiet and alcohol solution) ingestedbyanimalsinthealcoholgroupswasmeasureddaily. Thefollowingday,adietwiththesameamountofcalories (soliddiet and isocaloricsolution)wasofferedtoisocaloric groups.Doingso,thetreatmentofanimalswiththeisocaloric diet began and finished a day after the groups with the alcoholicdiet.

Topreventdehydration,animalsfromtheisocaloricgroups alsoreceivedwaterad libitum.Theseanimalsreceivedtwo bottles, onecontainingthe sucrosesolutionand theother, solely water. However, in the statistical analysis of fluid consumption,fortheisocaloricgroups,onlytheamount of ingestedsucrosesolutionwasconsidered.Thiswasdone,as ourintentionwastocomparetheamountofcaloriesingested bythedifferentexperimentalgroups.

Insummary,duringthedietarytreatment,theratswere divided into six experimental groups (each one presenting

n=9):Shamoperatedandadlibitumdiet(Sham/adlibitum); ovariectomized and ad libitumdiet (Ovx/adlibitum);Sham operatedand alcoholic diet(Sham/alc);ovariectomized and alcoholic diet (Ovx/alc); Sham operated and isocaloric diet (Sham/iso);andovariectomizedandisocaloricdiet(Ovx/iso). TheSham/isogroupwaspair-fedtoSham/alcgroup,whilethe Ovx/isogroupwaspair-fedtotheOvx/alcgroup.

Eightweeksaftercommencingthedietetictreatment,the rats were sacrificed and their mandibles removed. The ovariectomy success was confirmed, aftersacrifice, by the visualizationofovaryabsenceanduterusatrophy.

ðfinalweightinitialweightÞ100 initialweight

Theaveragevalueofsolidandliquiddietconsumedperrat/ perdaywasrecorded.

2.2. X-rayfluorescencespectrometry

TheamountofCaandPandtherelativeratioofCa/P,present inthealveolarbonecrest,weremeasuredusingan energy-dispersivemicroX-rayfluorescencespectrometer(mEDX1300

–50mm–Shimadzu1,Kyoto,Japan).

Aftersacrifice,themandibleswereplacedinasolutionof 10%bufferedformalinfor24h,washedwithwater,thendried and frozen at 208C. Fixation of biological samples in formaldehyde based solutions prior to the analyses of concentrations of Ca and P in bone had already been undertakenbyotherauthors.24–26_{Toreducepossible}

interfer-ence to the fixationprocedure in the interpretation ofthe results,allsampleswerefixedforthesameperiodoftime.The fixationinformalinwasdonetopreventtheputrefactionof thesamplesduringthespectrometricanalysis.

Theregion of the alveolar bone crest, right side ofthe mandible, between the 1st and 2nd molar, were flattened usingsandpaperno.1200coupledtoanautomaticpolishing machine.Thiswasnecessaryasirregularitiesonthesurfaceof thesamplecouldinfluencetheinteractionofelectronsandthe propagationofX-rays.

Thesamplesweremappedonarectangulararea,including the alveolar bone crest, which led to a window of 0.80mm0.60mm (4030 points with increments of 20mm).Thevoltagewassetat15kVwithautomatic

adjust-mentofthecurrent.Thetimerequiredforthemappingofeach samplewas approximately 260min. The calibration ofthe equipmentusedforreference,wasacommercialreagentof synthetic hydroxyapatite(Ca10(PO4)6(OH)2 –99.999% grade–

Sigma–Aldrich1_{, St. Louis, USA). TheCa/P ratio calculated}

(theoretically), in weight percentage, used to compare the results was 2.16, calculated from the stoichiometry. The calculations were obtained considering 10mol of Ca with molarmassof40.08g/moland 6molofPwithmolarmass 30.97g/mol.

Afterobtainingtheimageofthemap,alineof0.3mmwas drawnatthecentreofthebonecrest,approximately0.1mm belowthetipofthecrest,inwhichtheaverageconcentrations of Ca and P were obtained. These averages were used to performthecalculationoftheCa/Pratios(Fig.1).

2.3. Statisticalanalysis

Dataconcerningtheweightanddietoftheratsshowed non-normaldistributionandwereperformedusing non-paramet-ric tests(Kruskal–Wallisand Mann–Whitney).Nostatistical adjustmentwasappliedtothesamples.

Data obtained byspectrophotometrywere normally dis-tributedandwereanalyzedbyanalysisofvariance(ANOVA) andTukeytest.

Allcomparisonsweremadewithasignificancelevelof5%.

3.

Results

3.1. Dietandweight

Thevaluesofweightchangesbypercentage,soliddiet and liquid diet are comparedand described in Tables 1 and 2

(Kruskal–WallisandMann–Whitney).

Inallgroupstherewasanaverageweightgainintherats duringtheexperiment.Thegroupthatgainedmoreweight,by percentage,wasOvx/adlibitum(averagegainof30.912.6%). Thisgroupwasconsideredstatisticallydifferentfromallother groups (Tables 1and 2). Table 3 shows the weight of the animalsbyabsolutevalue,comparingtheinitialvalues(atthe timeofovariectomyorShamsurgery)tothoseoftheendofthe experiment(sacrifice).

Thegroup thatconsumedthe moresoliddiet was Ovx/ ad libitum(18.461.46g),which was statistically different from all other groups except for the Sham/ad libitum. Although there was no statistical difference between the Sham/adlibitumandOvx/adlibitum,onemustconsiderthat the p-value is very close to the acceptable limit, which indicatesatrendtowardsdifference(p=0.058).Itwasnoted also thatthe isocaloric groupsconsumed all the solid diet offered to them. This meant that solid food consumption

betweenOvx/isoandOvx/alcandSham/isoandSham/alcwas equivalent (12.811.44g and 12.911.58g, respectively)

(Tables1and2).

The following liquids were evaluated: alcohol solution (20%), sucrose solution and water, in relation to alcohol, isocaloricandadlibitumdietgroups,respectively.Although the isocaloric groups were offered a sucrose solution equivalent to the alcohol solution (consumed by alcohol groupsonthepreviousday),theratsdidnotingestallthe

solutionavailable tothem.TheOvx/alc groupingested an averageof16.241.41mlofalcoholsolutionandtheOvx/iso groupingested11.371.38mlofsucrosesolution.TheSham/ alc group ingested 17.131.89ml ofalcohol solution and Sham/isogroupingested10.521.30mlofsucrosesolution

(Table1).

Twoanimalsdiedpriortotheendoftreatment(oneinthe Sham/adlibitumgroupandtheotherintheOvx/adlibitum group).Thecauseofthedeathswasunknown.

Table1–Analysiscomparingweightchangesinpercentage,soliddietandliquiddiet(Kruskal–Wallistest).

Groups Sham/adlibitum Ovx/adlibitum Sham/alc Ovx/alc Sham/iso Ovx/iso

Weight(%gain)

Average 19.9% 30.9% 15.9% 13.9% 11.1% 17.3%

Median 19.8% 27.8% 18.8% 15.0% 11.5% 14.8%

Standarddeviation 6.2% 12.6% 5.1% 9.2% 5.0% 11.3%

Confidenceinterval 4.3% 8.7% 3.4% 6.0% 3.3% 7.4%

Samplesize(n)b _{8 8 9 9 9 9}

pValue 0.003a

Soliddiet(g/day/rat)

Average 16.84 18.46 12.91 12.81 12.91 12.81

Median 16.19 18.48 12.62 12.60 12.62 12.60

Standarddeviation 2.08 1.46 1.58 1.44 1.58 1.44

Confidenceinterval 1.44 1.01 1.09 1.00 1.09 1.00

Samplesize(n)c _{8 8 8 8 8 8}

pValue <0.001a

Liquiddiet(ml/day/rat)

Average 32.02 24.98 17.13 16.24 10.52 11.37

Median 31.69 24.58 16.93 15.90 10.38 11.05

Standarddeviation 3.48 3.16 1.89 1.41 1.30 1.38

Confidenceinterval 2.41 2.19 1.31 0.98 0.90 0.95

Samplesize(n)c _{8 8 8 8 8 8}

pValue <0.001a

a _{Valueconsideredstatisticallysignificant.}

b _{Samplesize(n)–consideringthenumberofanimalsineachgroup.}

c _{Samplesize(n)–consideringtheaveragesolidorliquiddietsingestedduringthe8weeksoftreatment.}

Table2–Statisticalpvaluescomparingweightchangesinpercentage,soliddietandliquiddiet(Mann–Whitneytest).

Groups Ovx/adlibitum Sham/alc Ovx/alc Sham/iso Ovx/iso

Weight(%gain)

Sham/adlibitum 0.036a _NSb _NSb _0.009a _NSb

Ovx/adlibitum 0.004a _0.009a _0.001a _0.027a

Sham/alc NSb _0.070c _NSb

Ovx/alc NSb _NSb

Sham/iso NSb

Soliddiet

Sham/adlibitum 0.058c _0.005a _0.002a _0.005a _0.002a

Ovx/adlibitum 0.001a _0.001a _0.001a _0.001a

Sham/alc NSb _NSb _NSb

Ovx/alc NSb _NSb

Sham/iso NSb

Liquiddiet

Sham/adlibitum 0.002a _0.001a _0.001a _0.001a _0.001a

Ovx/adlibitum 0.001a _0.001a _0.001a _0.001a

Sham/alc NSb _0.001a _0.001a

Ovx/alc 0.001a _0.001a

Sham/iso NSb

a _{Statisticallysignificantdifference.}

b _{Notsignificantpvalues.}

3.2. Analysisofalcoholconsumption

Theaverage amountof20%alcoholsolutionconsumedper dayperratwas16.69ml.Withthisdataitwasabletocalculate theamountofalcoholconsumedinotherunitsof

measure-ment(Table4).

Theamountofabsolutealcoholconsumedingramsper kilogramofanimalweightperdaywasevaluated.Theresults showedthatonaverage,theratsconsumed8.76gofabsolute alcoholperkg/day(Table4).

Thepercentageofcaloriesfromthealcoholdietwasalso calculated. The results showed that on average 37.83% of dietarycaloriescamefromalcoholconsumption(Table4).

3.3. Spectrometricanalysis(concentrationsofCa,Pand Ca/Pratios)

Duringtheexperiment,somesampleswerediscarded(dueto failureofstandardizationduringsamplepreparation),which altered the final number of samples analyzed by the spectrometerperexperimentalgroup(Table5).

TheanalysisoftheconcentrationsofCa,PandCa/Pratios areshown inTables5and6(ANOVAandTukey test).The highest values in the Ca/P ratios were obtained from the analysisoftheOvx/adlibitumgroup(mean2.030.04),which was considered statistically different from other groups, excepttheSham/adlibitumandSham/alc.Thelowestvalues wereobtainedintheOvx/alcgroup(mean1.920.06),which was considered statistically different from other groups, exceptthe Sham/iso.Itshouldbealsonotedthatthe Ovx/ alcgroupwastheonlyonetoshowastatisticallysignificant differencewhencomparedwiththeSham/adlibitumgroup (p<0.001).

4.

Discussion

AnalysisofCa/Pratios,ascomparedtotheconcentrationsof CaandPseparately,showlowervaluesofstandarddeviation andcoefficientofvariation,whichmaybemorereliableforthe diagnosisofbonedisorders.2Inourstudy,theaveragevalues for the Ca/P ratios ranged from 1.920.06 to 2.030.04,

Table3–Weightofanimalsbyabsolutevalue,comparingtheirinitialweightsandthatatsacrifice.

Weight(g) Average Median Standarddeviation Confidenceinterval Numberofanimals

Ovx/alc

Initial 278.9 270 16.9 11.1 9

Sacrifice 317.8 325 32.4 21.2 9

Ovx/iso

Initial 270.6 270 24.3 15.9 9

Sacrifice 316.1 310 28.0 18.3 9

Ovx/adlibitum

Initial 273.1 270 16.2 11.3 8

Sacrifice 356.3 355 25.7 17.8 8

Sham/alc

Initial 262.2 260 21.7 14.2 9

Sacrifice 303.3 310 19.0 12.4 9

Sham/iso

Initial 266.1 270 21.3 13.9 9

Sacrifice 295.0 290 17.7 11.5 9

Sham/adlibitum

Initial 248.1 243 24.0 16.7 8

Sacrifice 297.5 290 31.3 21.7 8

Table4–Analysisofalcoholconsumption.

Alcoholconsumption Sham/alc Ovx/alc Averageofalcoholgroups

Averageintakeofalcoholsolution20%(ml/day/rat) 17.13ml 16.24ml 16.69ml Averageintakeofabsolutealcohol(ml/day/rat) 3.43ml 3.25ml 3.34ml Averageintakeofabsolutealcohol(g/day/rat)a _{2.70g 2.56g 2.63g} Averageintakeofabsolutealcohol(g/kgofanimalweight/day)b _{8.99g 8.52g 8.76g}

Feedintake(g/day/rat) 12.91g 12.81g 12.86g

Feedintake(kcal/day/rat)c _{30.80kcal 30.56kcal 30.68kcal}

Alcoholconsumption(kcal/day/rat)d _{19.17kcal 18.18kcal 18.67kcal} %Caloriedietfromalcohol(averageperday/rat) 38.36% 37.30% 37.83% a _{Consideringthat1mlofalcoholisequivalentto0.787g.}

b _{Consideringananimalwithanaverageweightof300g.}

c _{Consideringthat1gofcommercialfood(Labina–Purina}1_{,Paulı´nia,Brazil)has2.386kcal.}

smaller than the molar ratio of synthetic hydroxyapatite, which is 2.16. These results were expected, as the bone mineral phase is formed by nonstoichiometric carbonated apatiteioniccrystals.7

Alterations on bone quality when ovariectomy was associated with alcohol consumption were previously ob-servedby other authors.21–23,27 Inovariectomized ratswho received20%alcoholsolution(similartothatinthepresent experiment)anexacerbationofbonelossinthealveolarbone crest,21_{decreasedCa/P ratiosinthefemur(associatedwith}

serum hyperphosphataemia)22 _{and negative effects on}

osseointegration23_{was noted.Due tothesefindings,itwas}

hypothesizedabovethatoestrogendeficiencyassociatedwith alcoholconsumptioncanadverselyinfluence thequalityof alveolar bone, and change its mineral composition. This

hypothesis was confirmed bythe presented results, asthe Ovx/alcgroupwastheonlygroupthatpresentedstatistically differentresultsconcerningCa/Pratiowhencomparedtothe control(Sham–adlibitumdiet).

Little isknownabout the influence ofmineralizationin periodontaldiseaseortoothloss.Inastudy21_withthesame

experimentaldesigntothatofthecurrentstudy(withrespect to animal treatment), it was observed, by histological and histomorphometrical analyses (slides stained with haema-toxylin–eosin), an exacerbation of alveolar bone loss and inflammatory process, in periodontaltissues, in ovariecto-mizedratswhoreceived20%alcohol(groupOvx/alc).21_Inour

experiment,itwasverifiedthat,underthesameconditions, therewasadecreaseinthevaluesofCa/Pratiosinalveolar bone.Thus,itseemsreasonabletoassumethatthereisthe

Table5–AnalysisforchemicalelementscontentsofCaandP(inweightpercentage),andtheCa/Pratios.

Groups Samplesize(n) Average Standarddeviation Median CV(%)a

Sham/adlibitumCa 7 30.39 0.76 30.47 2.50

Ovx/adlibitumCa 7 31.21 0.74 31.34 2.37

Sham/alcCa 7 29.92 0.86 29.63 2.87

Ovx/alcCa 8 27.71 1.13 27.63 4.08

Sham/isoCa 7 27.76 0.48 27.69 1.73

Ovx/isoCa 6 28.03 0.75 28.03 2.68

Sham/adlibitumP 7 15.15 0.39 15.11 2.57

Ovx/adlibitumP 7 15.37 0.28 15.41 1.82

Sham/alcP 7 14.97 0.41 14.86 2.74

Ovx/alcP 8 14.44 0.39 14.35 2.70

Sham/isoP 7 14.12 0.25 14.12 1.77

Ovx/isoP 6 14.20 0.22 14.12 1.55

Sham/adlibitumCa/P 7 2.01 0.01 2.01 0.50

Ovx/adlibitumCa/P 7 2.03 0.04 2.02 1.97

Sham/alcCa/P 7 2.00 0.01 2.00 0.50

Ovx/alcCa/P 8 1.92 0.06 1.90 3.13

Sham/isoCa/P 7 1.97 0.02 1.97 1.02

Ovx/isoCa/P 6 1.97 0.03 1.98 1.52

a _{Coefficientofvariationbypercentage.}

Table6–StatisticalpvaluesfortheconcentrationsofCa,PandCa/Pratios(ANOVAandTukeytest).

Groups Ovx/adlibitum Sham/alc Ovx/alc Sham/iso Ovx/iso

Ca

Sham/adlibitumCa NSb _NSb _p

<0.001a p<0.001a p<0.001a

Ovx/adlibitumCa p<0.05a p<0.001a p<0.001a p<0.001a

Sham/alcCa p<0.001a p<0.001a p<0.001a

Ovx/alcCa NSb _NSb

Sham/isoCa NSb

P

Sham/adlibitumP NSb _NSb _p<0.001a _p<0.001a _p<0.001a

Ovx/adlibitumP NSb _p

<0.001a p<0.001a p<0.001a

Sham/alcP p<0.05a p<0.001a p<0.001a

Ovx/alcP NSb _NSb

Sham/isoP NSb

Ca/P

Sham/adlibitumCa/P NSb _NSb _p<0.001a _NSb _NSb

Ovx/adlibitumCa/P NSb _p<0.001a _p<0.05a _p<0.05a

Sham/alcCa/P p<0.001a NSb NSb

Ovx/alcCa/P NSb _p

<0.05a

Sham/isoCa/P NSb

possibility of a reduction in mineralization linked to an increaseinalveolarbonelossandtheseverityofperiodontal disease,which could as a consequence compromise tooth retention.However,thisisonlyahypothesis.

In another experiment, Yan et al.,26 _{also noted the}

influenceofmineralizationintheregionoftheperiodontium (morespecificallyinartificiallycreatedfurcationdefects).The authorscheckedwithhistological/histomorphometrical anal-ysis,andbyenergydispersiveX-ray,thathighervaluesinCa/P couldberelatedtoincreasedratesofperiodontalregeneration. Molecular and cellular mechanisms that led to obtain reducedvaluesinCa/Pratioswhentheoestrogenicdeficiency was linked to alcohol consumption are not as yet well understood.However,bothconditionshavebeenseparately associatedwithincreasedexpressionofimportant osteoclas-togenicscytokinesasIL-1,IL-6andTNF-a.28–31Additionally,it

ispossiblethatthereisalsointerferenceintheregulationof theRANK/RANKL/OPG,whichmayoccurthroughincreased expression of RANKL and decreased expression of OPG, leading to changes in the bone remodelling process with increasedboneresorption.31–33_{Itisalsoimportanttoconsider}

thepossibletoxiceffectsofexcessivealcoholconsumptionon osteoblasticactivity,afactorthatcouldimpairtheprocessof boneformationandmineralization.34,35

Thedegreeofmineralizationcanbemodifiedbychangesin osteoblast and osteoclast activity during the remodelling process. In cases concerning the changes in the rate of remodelling, with a predominance of the reabsorption process,asoccursinosteoporosis,thereisnotenoughtime for osteoblasts to complete the process of mineralization before the bone is reabsorbed prematurely by osteoclasts. Thesefactorscouldaffectthedegreeofbonemineralization andconsequentlytheCa/Pratios.6,7,36

Inthepresentstudy,onlytheassociationofalcoholwith oestrogenicdeficiencywasabletosignificantlydecreasethe Ca/Pratioinalveolarbone.Thealcoholalonewasnotcapable ofpromotingsuchchanges.Similarly,Trevisioletal.37_found

that alcohol consumption (ethanol contributing to 35% of caloricintake)didnotimpair mineralizationina modelof osteoinductioninrats.

Theoretically, it was expected that ovariectomized rats could present a tendency to decrease the percentages of minerals,suchasCaandP,duetoincreasedboneremodelling process,witha predominanceof resorptionand decreased bonemineraldensity.6,7,31,36,38_{Inthepresentstudy,}

ovariec-tomizedratsreceivingacontrolleddiet(Ovx/alcandOvx/iso) presenteddecreasedpercentagesofCaandP.However,itdid not occur with rats Ovx/ad libitum, the group where the highestvaluesofCa/Pratioswerefound.

Inthepresentpaper,theOvx/adlibitumgroupgainedmore weightandconsumedmorefood.Otherauthorsalsoobserved an increase in body weight in ovariectomized rats when comparedto sham operated groups.39–41 _{Ovariectomy may}

increasefoodintakeandweightgain,andstudiesshowthat treatmentwith estradiolreverses theseeffects.39,42 _Studies

with knockout animals (for oestrogen receptor-alpha and aromatase) foundthatoestrogen maybeimportantforthe maintenanceoflipidhomeostasis.43,44

Theresultsofthisexperimentdonotallowforelucidating thereasonsleadingtohighervaluesintheCa/Pratiosinthe

alveolar bone crest of ovariectomized rats who received ad libitum diet, compared to other groups exposed to oestrogen deficiency. However, one possible explanation mayrelyonthefactthattheOvx/adlibitumratsconsumed significantlymorefoodthanthosewithdietarycontrol.There isevidence thatexercisemayberelated toincreasedbone mineral density inpostmenopausal women45_{and likewise,}

withincreasedfoodintake,therewasahigherincidence of bite forces on the alveolarbone,which may haveled toa change inbonemineral densitylocally,as suggested earli-er.38,46_{Patulloetal.,}46_{suggestedthattheincidenceofnormal}

occlusalforcescouldpromoteprotectionagainstthe develop-mentofosteopeniainthemandible.

Additionally,theincreasedfoodintakebytheOvx/adlibitum groupalsoresultedinahigherconsumptionofkeynutrientsto maintainbonequality,includingCaandP.47_{Thisfactmayalso}

helptoexplainthehighvaluesintheCa/Pratiosfoundinthis group.Anotherexplanationmaybetheinfluenceofweightgain on bonetissue. Some researcherssuggest that,after meno-pause, heavier women conserve more bone mass when compared towomenwithlowerbody weights.48,49_Leptin,a

cytokinesecretedbyfatcells,hasbeenstudiedasapotential modulatoroftheprotectiveeffectsoffatmassonbones.49

A possible influence of increased food intake, higher incidenceofbiteforcesandweightgainresultinginthehighest values of Ca/P ratios in the group Ovx/ad libitum can be consideredahypothesisasanexplanationtotheresultwhich was not theoretically expected. However, without further analysis,anequallypossiblehypothesisisthatintheabsence ofotherfactors,oestrogendeficiencyactuallycorrelateswith increased alveolar bone mineralization. It is important to considerthatothernumerousfactorscouldalsoinfluencethe progressionofperiodontaldiseaseandpossiblythequalityof alveolarboneandtoothretention.Someofthesefactorsinclude bacterialbiofilm,systemicdiseases,geneticdisorders,habits, age,gender,stressandnutritionalproblems.11–18

Itisalsoimportanttonotethat,despitetheOvx/adlibitum groupshowingthehighestaverageinCa/Pratios,whichwas statisticallydifferentfromotherovariectomizedgroups(Ovx/ alcandOvx/iso),itwasnotdifferentfromSham/adlibitum. Thus, fromthe resultsofthisstudy,it was notpossibleto conclude that ovariectomy alone (without an associated dietary treatment) was able to significantly change the stoichiometryofhydroxyapatiteonalveolarbone.

Someauthorssuggestthatdietarychangesmightinterfere with the host’s response to periodontal disease progres-sion.17,50,51 Possibly,this is relatedtothe fact that chronic inflammation causes oxidative stress, with an increased demandforamineralandantioxidantvitaminrichdiet.17,50

Inthisstudy,itwasfoundthatthelowestvaluesinCa/Pratios wereobtainedingroupswithdietarycontrol(Ovx/alc,Ovx/iso, Sham/alcandSham/iso),andwerehigheringroupswiththe ad libitum diet (Ovx/ad libitum/Sham/ad libitum). These findingssuggestthatdietmayplayanimportantroleinthe variations of the stoichiometric hydroxyapatite. However, furtherstudiesarenecessarytovalidatethisstatementwith greaterstatisticalreliability.

ofcaloriesintherats’ diet.28,52,53_{Thisisconsideredahigh}

dosageof alcoholconsumption,28,52,53 _{resulting inelevated}

blood ethanolconcetrations.52 _{Inanother study}35 _withrats

treatedwith20%ethanol(indrinkingwater),similarlytothat undertakeninourstudy,bloodalcohollevelswereeighttimes higherinthetreatedrats(0.869gl1_{)thanthoseinthecontrol}

group(0.11gl1_{).The20%concentrationwasadministeredfor}

15dayswhichwasconsideredachronicintake.35_Inourstudy

theratsreceivedalcoholforeightweeks.

A comparison of results suggests that our rats were subjectedtoanexcessiveandchronicconsumptionofalcohol. This is an important factor, as most researchers seem to believethattheharmfuleffectsofalcoholonboneisobserved with abusivealcohol consumption and not withmoderate consumption.54,55

Themethodologyforthetreatmentoftheanimalsisbased onpreviousstudies21–23_{thathaveusedsimilarexperimental}

groups,concentrationofalcohol(20%indrinkingwater)and time ofovariectomy. The standardization ofthe treatment facilitatedthecomparisonofourresultswithotherstudies.21–23 However,potentialconfoundingeffectspertainingtothetypeof diet used in our experiment should be considered when interpretingtheresults.Lieber etal.56_{criticizedthedelivery}

ofalcohol indrinkingwater,asit reduceswaterintake and makesitdifficulttocontrolnutrients.Sincenutritionalchanges couldinterferewiththehost’sresponsetotheprogressionof periodontaldisease,17,50_{otherstudiescouldverifyiftheresults}

of this experiment would be similar if other forms of administration ofalcohol couldbeconsidered,for example, usinganutritionallyadequateliquid dietcontainingalcohol (Lieber–DeCarli liquid diet),28,37,53,56 the administration of alcoholbyintraperitonialinjections27,32_{orbyintubation.}57

The present study has some limitations. One of the limitationswasthatofnotbeingabletocontroltheisocaloric groupingestingexactlythesameamountofcaloriesasthose ofthe alcoholgroup(whenconsidering theliquid diet).To minimize this problem, other ways of administrating the liquiddietcouldbeconsidered.However,thislimitationdid not appear to have directly influenced the results, as no significantdifferencesinCa/Pratioswerefoundbetweenthe isocaloricgroupsandcontrolgroup(Sham/adlibitum). How-ever,inrelationtosolidfood,whichprovidedthenutritional intakeof Caand P, nutritional pairing was achieved.Even thoughcontrollingthe amountofalcoholconsumedbythe animalswasachieved,anotherlimitationofourexperiment wastheabsenceofevaluatingbloodalcoholconcentrations. Otherstudiescouldincludethemeasurementofthisintheir experimentaldesigns.Finally,itisimportanttoconsiderthat ourworkwas limitedtoCa/Pratio analysis.Withoutother parametersofevaluation,itwasonlypossibletocorrelatethe results with other searches. Broader studies are therefore required to better verify the potential relevance of these resultsindentalpractice.

5.

Conclusion

Itcanbeconcludedthatovariectomyassociatedwithalcohol consumptionof20%ledtoasignificantdecreaseinCa/Pratios withintheregionofalveolarbonecrestinrats.

Acknowledgements

TheauthorsacknowledgesupportfromCAPES(Coordenac¸a˜ode Aperfeic¸oamentodePessoaldeNı´velSuperior–BrazilianFederal Agency for Supportand Evaluation ofPostgraduate Educa-tion), nativeEnglish speaker V. Hegenberg and statistician consultant,J.Adans.

Funding: Adriana M.P.S. Marchinireceived a scholarship from the Brazilian governmental research agency, CAPES (Coordenac¸a˜odeAperfeic¸oamentodePessoaldeNı´velSuperior).

Competing interests: The authors report no conflict of interestrelatingtothisstudy.

Ethical approval: This study was approved by the ethics committeeofSa˜oJose´ dosCamposSchoolofDentistry,State UniversityofSa˜oPaulo–UNESP(ProtocolNo.021/2008-PA/CEP).

r

e

f

e

r

e

n

c

e

s

1. ClarkeB.Normalboneanatomyandphysiology.Clinical

JournaloftheAmericanSocietyofNephrology2008;3_:S131–9.

2. ZaichickV,TzaphlidouM.Calciumandphosphorus concentrationsandthecalcium/phosphorusratioin trabecularbonefromthefemoralneckofhealthyhumans asdeterminedbyneutronactivationanalysis.Applied

RadiationandIsotopes2003;58_(6):623–7.

3. TzaphlidouM,SpellerR,RoyleG,GriffithsJ.Preliminary estimatesofthecalcium/phosphorusratioatdifferent corticalbonesitesusingsynchrotronmicroCT.Physicsin

MedicineandBiology2006;51_{(7):1849–55.}

4. MoeSM.Disordersinvolvingcalcium,phosphorus,and magnesium.PrimaryCare2008;35_{(2):215–37.}

5. RaiszLG.Pathogenesisofosteoporosis:concepts,conflicts, andprospects.JournalofClinicalInvestigation

2005;115_{(12):3318–25.}

6. MeunierPJ,BoivinG.Bonemineraldensityreflectsbone massbutalsothedegreeofmineralizationofbone: therapeuticimplications.Bone1997;21_(5):373–7.

7. BoivinG,MeunierPJ.Themineralizationofbonetissue:a forgottendimensioninosteoporosisresearch.Osteoporosis

International2003;14_:S19–24.

8. TempletonK.Secondaryosteoporosis.JournaloftheAmerican

AcademyofOrthopaedicSurgeons2005;13_{(7):475–86.}

9. SampsonHW.Alcoholandotherfactorsaffecting osteoporosisriskinwomen.AlcoholResearchandHealth

2002;26_(4):292–8.

10.HannanMT,FelsonDT,Dawson-HughesB,TuckerKL, CupplesLA,WilsonPW,etal.Riskfactorsforlongitudinal bonelossinelderlymenandwomen:theFramingham osteoporosisstudy.JournalofBoneandMineralResearch

2000;15_{(4):710–20.}

11.TaubmanMA,ValverdeP,HanXZ,KawaiT.Immune response:thekeytoboneresorptioninperiodontaldisease.

JournalofPeriodontology2005;76_{(11):2033–41.}

12.KimJ,AmarS.Periodontaldiseaseandsystemicconditions: abidirectionalrelationship.Odontology2006;94_(1):10–21.

13.TimmermanMF,VanderWeijdenGA.Riskfactorsfor periodontitis.InternationalJournalofDentalHygiene

2006;4_(1):2–7.

14.PeruzzoDC,BenattiBB,AmbrosanoGM,Nogueira-FilhoGR, SallumEA,CasatiMZ,etal.Asystematicreviewofstress andpsychologicalfactorsaspossibleriskfactorsfor periodontaldisease.JournalofPeriodontology

15.KuoLC,PoisonAM,KangT.Associationsbetween periodontaldiseasesandsystemicdiseases:areviewofthe inter-relationshipsandinteractionswithdiabetes, respiratorydiseases,cardiovasculardiseasesand osteoporosis.PublicHealth2008;122_{(4):417–33.}

16.AmaralCS,VettoreMV,Lea˜oA.Therelationshipofalcohol dependenceandalcoholconsumptionwithperiodontitis:a systematicreview.JournalofDentistry2009;37_{(9):643–51.}

17.ChappleILC.Potentialmechanismsunderpinningthe nutritionalmodulationofperiodontalinflammation.Journal

oftheAmericanDentalAssociation2009;140_{(2):178–84.}

18.PizzoG,LoReD,PiscopoMR,PizzoI,GiulianaG.Genetic disordersandperiodontalhealth:aliteraturereview.

MedicalScienceMonitor2009;15_{(8):RA167–78.}

19.MegsonE,KapellasK,BartoldPM.Relationshipbetween periodontaldiseaseandosteoporosis.InternationalJournalof

Evidence-basedHealthcare2010;8_{(3):129–39.}

20.ShepherdS.Alcoholconsumptionariskfactorfor periodontaldisease.Evidence-basedDentistry2011;12_(3):76.

21.AlonsoJMSL.Influenceofchronicconsumptionofethanol 20%ininducedperiodontaldiseaseofratswithestrogen deficiency.PhDthesis.Sa˜oJose´ dosCampos:Sa˜oPauloState University.Availablefrom:http://

www.athena.biblioteca.unesp.br/exlibris/bd/bsj/

33004145081P0/2009/alonso_jmsl_dr_sjc.pdf;2009[cited

02.14.12].

22.LodiKB.Bonemineralimpactinchronicalcoholism associatedwithhormonaldepletioninrats.PhDthesis.Sa˜o Jose´ dosCampos:Sa˜oPauloStateUniversity.Available from:http://www.athena.biblioteca.unesp.br/exlibris/bd/

bsj/33004145081P0/2010/lodi_kb_dr_sjc.pdf;2010[cited

02.14.12].

23.DecoCP,MarchiniAMPS,Ba´rbaraMAM,VasconcellosLMR, RochaRF,MarchiniL.Negativeeffectsofalcoholintakeand estrogendeficiencycombinationonosseointegrationina ratmodel.JournalofOralImplantology2011;37_(6):633–9.

24.TanakaS,ShimizuM,DebariK,FuruyaR,KawawaT,Sasaki T.Acuteeffectsofovariectomyonwoundhealingof alveolarboneaftermaxillarymolarextractioninagedrats.

AnatomicalRecord2001;262_{(2):203–12.}

25.SuvorovaEI,PetrenkoPP,BuffatPA.Scanningand transmissionelectronmicroscopyforevaluationoforder/ disorderinbonestructure.Scanning2007;29_{(4):162–70.}

26. YanXZ,GeSH,SunQF,GuoHM,YangPS.Apilotstudy evaluatingtheeffectofrecombinanthumanbone morphogeneticprotein-2andrecombinanthuman beta-nervegrowthfactoronthehealingofclassIIIfurcation defectsindogs.JournalofPeriodontology2010;81_(9):

1289–98.

27.CallaciJJ,JuknelisD,PatwardhanA,WezemanFH.Binge alcoholtreatmentincreasesvertebralbonelossfollowing ovariectomy:compensationbyintermittentparathyroid hormone.Alcoholism-ClinicalandExperimentalResearch

2006;30_{(4):665–72.}

28.IrieK,TomofujiT,TamakiN,SanbeT,EkuniD,AzumaT, etal.Effectsofethanolconsumptiononperiodontal inflammationinrats.JournalofDentalResearch

2008;87_{(5):456–60.}

29.KhorutsA,StahnkeL,McClainCJ,LoganG,AllenJI. Circulatingtumornecrosisfactor,interleukin-1and interleukin-6concentrationsinchronicalcoholicpatients.

Hepatology1991;13_{(2):267–76.}

30.SzaboG.Consequencesofalcoholconsumptiononhost defence.AlcoholandAlcoholism1999;34_{(6):830–41.}

31.LernerUH.Boneremodelinginpost-menopausal osteoporosis.JournalofDentalResearch2006;85_{(7):584–95.}

32.CallaciJJ,HimesR,LauingK,WezemanFH,BrownsonK. Bingealcohol-inducedbonedamageisaccompaniedby differentialexpressionofboneremodeling-relatedgenesin

ratvertebralbone.CalcifiedTissueInternational

2009;84_{(6):474–84.}

33.HornerKC.Theeffectofsexhormonesonbonemetabolism oftheoticcapsule–anoverview.HearingResearch

2009;252_{(1–2):56–60.}

34.ChakkalakalDA.Alcohol-inducedbonelossanddeficient bonerepair.Alcoholism-ClinicalandExperimentalResearch

2005;29_{(12):2077–90.}

35.RosaML,BelotiMM,PrandoN,QueirozRHC,deOliveiraPT, RosaAL.Chronicethanolintakeinhibitsinvitro

osteogenesisinducedbyosteoblastsdifferentiatedfrom stemcells.JournalofAppliedToxicology2008;28_{(2):205–11.}

36.BurnellJM,BaylinkDJ,ChestnutCH,MathewsMW,Teubner EJ.Bonematrixandmineralabnormalitiesin

postmenopausalosteoporosis.Metabolism-Clinicaland

Experimental1982;31_{(11):1113–20.}

37.TrevisiolCH,TurnerRT,PfaffJE,HunterJC,MenaghPJ, HardinK,etal.Impairedosteoinductioninaratmodelfor chronicalcoholabuse.Bone2007;41_{(2):175–80.}

38.AmesMS,HongS,LeeHR,FieldsHW,JohnstonWM,Kim DG.Estrogendeficiencyincreasesvariabilityoftissue mineraldensityofalveolarbonesurroundingteeth.Archives

ofOralBiology2010;55_{(8):599–605.}

39.DenisR,RochonL,DeshaiesY.Effectsofexercisetraining onenergybalanceofovariectomizedrats.AmericanJournal

ofPhysiology1987;253_{(5):R740–5.}

40.KimuraM,IraharaM,YasuiT,SaitoS,TezukaM,YamanoS, etal.Theobesityinbilateralovariectomizedratsisrelated toadecreaseintheexpressionofleptinreceptorsinthe brain.BiochemicalandBiophysicalResearchCommunications

2002;290_{(4):1349–53.}

41.ZothN,WeigtC,Laudenbach-LeschowskiU,DielP.Physical activityandestrogentreatmentreducevisceralbodyfatand serumlevelsofleptininanadditivemannerinadiet inducedanimalmodelofobesity.JournalofSteroid

BiochemistryandMolecularBiology2010;122_{(1–3):100–5.}

42.GuyardB,FrickerJ,BrigantL,BetoulleD,ApfelbaumM. Effectsofovariansteroidsonenergybalanceinratsfeda highlypalatablediet.Metabolism-ClinicalandExperimental

1991;40_{(5):529–33.}

43.HeinePA,TaylorJA,IwamotoGA,LubahnDB,CookePS. Increasedadiposetissueinmaleandfemaleestrogen receptor-alphaknockoutmice.ProceedingsoftheNational

AcademyofSciencesoftheUnitedStatesofAmerica

2000;97_{(23):12729–34.}

44.JonesMEE,ThorburnAW,BrittKL,HewittKN,WrefordNG, ProiettoJ,etal.Aromatase-deficient(ArKO)micehavea phenotypeofincreasedadiposity.ProceedingsoftheNational

AcademyofSciencesoftheUnitedStatesofAmerica

2000;97_{(23):12735–40.}

45.HoweTE,SheaB,DawsonLJ,DownieF,MurrayA,RossC, etal.Exerciseforpreventingandtreatingosteoporosisin postmenopausalwomen.CochraneDatabaseofSystematic

Reviews2011;(7):CD000333.

46.PatulloIMF,TakayamaL,PatulloRF,JorgettiV,PereiraRMR. Influenceofovariectomyandmasticatoryhypofunctionon mandibularboneremodeling.OralDiseases2009;15_(8):580–6.

47.BonjourJP,GueguenL,PalaciosC,ShearerMJ,WeaverCM. Mineralsandvitaminsinbonehealth:thepotentialvalueof dietaryenhancement.BritishJournalofNutrition

2009;101_{(11):1581–96.}

48.CifuentesM,JohnsonMA,LewisRD,HeymsfieldSB, ChowdhuryHA,ModleskyCM,etal.Boneturnoverandbody weightrelationshipsdifferinnormal-weightcomparedwith heavierpostmenopausalwomen.OsteoporosisInternational

2003;14_{(2):116–22.}

49.PetzelM.Actionofleptinonboneanditsrelationshipto menopause.BiomedicalPapersoftheMedicalFacultyofthe

50.EnwonwuCO,RitchieCS.Nutritionandinflammatory markers.JournaloftheAmericanDentalAssociation

2007;138_(1):70–3.

51.JenzschA,EickS,RassoulF,PurschwitzR,JentschH. Nutritionalinterventioninpatientswithperiodontal disease:clinical,immunologicalandmicrobiological variablesduring12months.BritishJournalofNutrition

2009;101_{(6):879–85.}

52.HunterBE,RileyJN,WalkerDW.Ethanoldependenceinthe rat:aparametricanalysis.PharmacologyBiochemistryand

Behavior1975;3_{(4):619–29.}

53.TurnerRT,KidderLS,KennedyA,EvansGL,SibongaJD. Moderatealcoholconsumptionsuppressesboneturnoverin adultfemalerats.JournalofBoneandMineralResearch

2001;16_{(3):589–94.}

54.BergKM,KuninsHV,JacksonJL,NahviS,ChaudhryA,Harris KA,etal.Associationbetweenalcoholconsumptionand bothosteoporoticfractureandbonedensity.American

JournalofMedicine2008;121_{(5):406–18.}

55.Alvisa-NegrinJ,Gonzalez-ReimersE, Santolaria-FernandezF,Garcia-Valdecasas-CampeloE,VallsMRA, Pelazas-GonzalezR,etal.Osteopeniainalcoholics:effect ofalcoholabstinence.AlcoholandAlcoholism2009;44_(5):

468–75.

56.LieberCS,DeCarliLM,SorrellMF.Experimentalmethodsof ethanoladministration.Hepatology1989;10_{(4):501–10.}

57.SampsonHW,ShipleyD.Moderatealcoholconsumption doesnotaugmentbonedensityinovariectomizedrats.

Alcoholism-ClinicalandExperimentalResearch