Rev. bras. ortop. vol.51 número4

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SOCIEDADE BRASILEIRA DE ORTOPEDIA E TRAUMATOLOGIA

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

Original

article

Vitamin

D

status

and

its

relationship

with

bone

mineral

density

in

a

healthy

Iranian

population

Patricia

Khashayar

a,b,∗

,

Hamid

Reza

Aghaei

Meybodi

a

,

Mohsen

Rezai

Hemami

c

,

Abbasali

Keshtkar

d,e

,

Hans

Peter

Dimai

f

,

Bagher

Larijani

c

a_Osteoporosis_Research_Center,_{Endocrinology}_and_Metabolism_Clinical_Sciences_Institute,_Tehran_University_of_Medical_Sciences,

Tehran,Iran

b_Center_for_Microsystems_Technology_(CMST),_Ghent_University,_Ghent,_Belgium

c_{Endocrinology}_and_Metabolism_Research_Center,_{Endocrinology}_and_Metabolism_Clinical_Sciences_Institute,

TehranUniversityofMedicalSciences,Tehran,Iran

d_Department_of_Health,_The_Ministry_of_Health_and_Medical_Education_(MOHME),_Tehran,_Iran

e_Department_of_Nutrition,_The_Ministry_of_Health_and_Medical_Education_(MOHME),_Tehran,_Iran

f_Department_of_Internal_Medicine,_Division_of_{Endocrinology}_and_Metabolism,_Medical_University_of_Graz,_Graz,_Austria

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26August2015 Accepted9September2015 Availableonline2February2016

Keywords:

Bonemineraldensity Osteoporosis Biologicalmarkers

VitaminD

Phosphorus Calcium

a

b

s

t

r

a

c

t

Objectives:Consideringthecontroversialresultsregardingtherelationshipbetweenvitamin Dlevelsandbonemineraldensityindifferentpopulations,thepresentstudywasdesigned toevaluatethiscorrelationinahealthyIranianpopulation.

Methods:Usingarandomclustersampleofapparentlyhealthymenandwomen,this multi-centercross-sectionalstudywascarriedoutamong4450individualslivinginurbanareasof fivemajorcitiesinIran.Bonemineraldensity(BMD)valuesatdifferentsiteswereanalyzed alongwiththeserumlevelsof25(OH)DandPTH.Analysisofvariance(ANOVA)wasusedto estimatethemaineffects,throughcomparingthemeanvaluesofthesemarkersbasedon thebonemineraldensitystatusofthestudygroupineachsex.

Results:25(OH)DlevelswereinverselycorrelatedwithBMDvaluesattotalhip(r=−0.062in menandr=−0.057inwomen)andspine(r=−0.076inmenandr=−0.107inwomen).After adjustingthedataforage,theinversecorrelationwasnolongerstatisticallysignificant. Conclusion:Serum25(OH)Dlevelsareinverselycorrelatedwithbonemassvaluesinboth sexes.

Correlac¸ão

entre

os

níveis

de

vitamina

D

e

densidade

mineral

óssea

em

uma

populac¸ão

iraniana

saudável

Palavraschave:

Densidademineralóssea Osteoporose

r

e

s

u

m

o

Objetivos:Considerandoosresultadoscontroversossobrearelaçãoentreníveisdevitamina Dedensidademineralósseaemdiferentespopulações,opresenteestudofoidesenhado paraavaliarestacorrelaçãoemumapopulaçãoiranianasaudável.

∗ _{Corresponding}_author_.

E-mail:patricia.kh@gmail.com(P.Khashayar).

http://dx.doi.org/10.1016/j.rboe.2015.09.011

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Biomarcadores VitaminaD Fósforo Cálcio

Métodos: Usandoumaamostraaleatóriadehomensemulheresaparentementesaudáveis, esteestudotransversalmulticêntricoconsiderou4450indivíduosquevivememnaregião urbanade cincograndescidadesnoIrã.Osvalores dadensidademineralóssea(DMO) foramanalisadosemconjuntocomosníveisséricosde25(OH)DePTH.Análisedavariação (ANOVA)foiutilizadaparaestimarosprincipaisefeitosatravésdacomparaçãoentreos val-oresmédiosdestesmarcadoreseacondiçãodadensidademineralósseadecadagênero nestaamostradeestudo.

Resultados:Níveisde25(OH)DforaminversamenteproporcionaisaosvaloresdeDMOanível doquadril(r=-0.062emhomenser=-0.057emmulheres)ecolunavertebral(r=-0.076em homenser=-0.107emmulheres).Apósajustedosdadosparaidade,acorrelac¸ãonegativa nãofoimaisestatisticamentesignificante.

Conclusão: Níveisséricosde25(OH)Dsãoinversamentecorrelacionadoscomosvaloresde massaósseaemambososgêneros.

Introduction

Osteoporosisisasystemicdiseasecharacterizedbylowbone

mass and microarchitectural deterioration of bone tissue,

resultinginanincreasedriskoffracture;ifidentifiedearlyin itscourse,however,manyofthefracturescanbeprevented.1

Apartfrommodifiablelifestylefactors,bonemineral den-sity(BMD)isinfluencedbyseveralgenetic,environmental,and hormonalfactors.2,3_While_the_influence_of_biochemical

mark-ersonthefractureriskhasbeenwelldocumentedinprevious

studies,theassociationbetween serum25(OH)D levelsand

BMDindifferentethnicitiesresidingindifferentgeographic areasremainscontroversial.Manyofthemhavereportedno

directrelationshipbetweenserum 25(OH)Dlevelsand BMD

wasobserved.4

Thepresentstudythereforewasdesignedtoevaluatethe relationshipbetweenvitaminDstatus,bonemineraldensity andPTHinahealthyIranianpopulation.

Materials

and

methods

Subjectselection

Thisstudyispartofacomprehensivesurvey(IMOS)assessing

the prevalence of osteoporosis and related factors among

healthyadults(agerange:20–70Years),representativesample ofIranianpopulationlivinginurbanareas,intheurbanareas offivemajorcitiesofIran(Tehran,Tabriz,Mashhad,Shiraz andBooshehr)inlatewinter2001(February–March).

Details on the survey design and methods have been

reportedpreviously.5_Briefly,_the_IMOS_used_a_random_cluster

samplingdesigntodrawfiveprovinciallyrepresentative,

inde-pendent samplesof healthy adults excluding those taking

medicationsthatcouldmodifybonemetabolism,thosewith

hepaticorrenaldisorders,metabolicbonedisease, hypercor-tisolism, malabsorption,sterility, oligomenorrhea, diabetes,

malignancy,and immobility formore than 1weekas well

asthepregnant andlactating women.TheResearchEthics

CommitteeoftheEndocrineandMetabolismResearchCenter

(EMRC) approved the protocol of this study. An informed

consentwasobtainedfromthesubjectsbeforetheyentered thestudy.

All subjects underwent adetailed medicalexamination,

measurementofbonemineraldensityatdifferentsites,and

certain biochemical testing. Apart from demographic data,

thesubjectswereaskedabouttheirmenopausalstatusand

theyearspassedsincetheirmenopause.Menopause,inthis study,wasdefinedaspreviousnaturalorsurgicalcessationof

menstruationformorethan12months.

Biochemicaltests

Afastingblood sample(10cm3 _of_venous_blood) _was_taken

from all participants attheir residence place. Sample

cen-trifuge and serum extraction were done in the field. The

sampleswerethenfrozenandsenttotheEMRClaboratory

forfurtheranalysis.

Serum Caand Plevels were analyzed bya calorimetric

methodusingChem.EnzymeLabKit;Iran.Thenormal

labo-ratoryrangeforserumCawas8.6–10.8mg/dlandforserum

P was 2.3 to 5mg/dl. Serum levels of vitamin D (25 (OH)

D)andPTHweremeasuredwithRIA(Radio-Immuno-Assay)

method (IDS LtdKit; UK) and IRMA (Immuno-Radiometric)

method (Diasorin Kit; USA), respectively. Normal range for

serum25(OH)DandPTHwere23–113ng/mland13–54pg/ml,

correspondingly.Theinter-andintra-assayvariationsforthe markerswere8%/6.8%and8.9%/6.1%,respectively.

Basedon25(OH)Dvalues,subjectswereclassifiedasthose suffering from vitamin D deficiency (≤20ng/ml), – insuffi-ciency(milddeficiency)(20–30ng/ml)and–sufficiency(higher

than30ng/ml).Thecompletemethodusedtodeterminethe

25(OH)Dlevelsforclassifyingtheparticipantsisdescribedin ourpreviousstudies.6,7

Bonemineraldensity

Ineachcity,patientsunderwentanL1–L4anteroposterior

lum-bar spine,hip andits sub-regionsDXA studywitha Lunar

DPXMDdensitometer(Lunar7164,GE,Madison,WI)equipped

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Z-scores.Qualitycontrolprocedureswerecarriedoutin

accor-dance with the manufacturer’s recommendations. In each

city,theinstrumentvariationwasdeterminedregularlybya weeklycalibrationprocedureusingaphantomsuppliedbythe

manufacturer(thephantomequilibriumwassentfromone

citytoanotheraftereach testing).Theinterdevicevariance wascheckedseveraltimesduringthestudyperiod.Therewas anirrelevantsmalldifferencebetweenthereportedmeasures whichwasnegligible.PrecisionerrorforBMDmeasurements was1–1.5%inthelumbarand2–3%inthefemoralregions.

BasedonWorldHealthOrganizationStudyGroup

recom-mendation,BMDvalueswereclassifiedasnormal,osteopenic andosteoporotic.8_Since,_there_is_no_widely_accepted_definition

ofosteoporosisinmales,theWHOcriteriafortesting osteo-porosisinthefemalepopulationwassimilarlyusedforthis group.

Statisticalanalysis

DatawereenteredtoMicrosoftAccessDatabank.Inviewofthe factthatadifferentinstrumentwasusedtoassesstheBMD insubjectswhowererecruitedinBooshehr;datagatheredin thiscitywasassumedtobemissing.Allstatisticalanalyses

wereperformedwithSPSS13.0forWindows(SPSS,Chicago,

IL)basedonapair-wiseapproach,andPvalueslowerthan0.05 wereconsideredstatisticallysignificant.

All analyses were conducted separately for each

gen-der,and then forthe group overall.Means±SD were used

to express standard descriptive statistics.Categorical

vari-ables were expressed as percentages and compared using

Chi-square.Differencesamongmeans wereinvestigatedby

analysisofvariance(ANOVA)withposthoctest.Thetwo-sided

Student’st-testwas used to compare the mean

biochemi-calvaluesindifferentgroups.Theassociationbetweenthe

outcome variable(BMD) and thebiochemical variableswas

examinedbybivariateanalysisandthenbyadjustedstepwise multipleregressionanalyses.

Results

Atotalof4450individualswiththemeanageof42.6±13.9

years were studied; from among them 1900 (42.7%) were

male. Overally some 246 (6.5%) of the total studied

sub-jects(4.8% ofmales and7.7%offemales) werereported to haveosteoporosis.Therewasasignificantdifferencebetween thefrequenciesofosteoporosisamongthetwogenders(P -value<0.001).Osteopenia,onthe otherhand,wasreported among848(58.6%)ofthestudiedfemalesandsome600(41.4%) ofthestudiedmen.

Serumlevels of25(OH)D wassignificantlyhigheramong

thosesufferingfromosteoporosis(Table1).ThePearson’s cor-relationcoefficientsbetweenthestudyvariablesrevealedan inversecorrelationbetween25(OH)DlevelsandBMDvaluesat totalhip(r=−0.062inmenandr=−0.057inwomen)andspine (r=−0.076inmenandr=−0.107inwomen)(Table2).

Thecorrelation between PTH levels and BMDvalues at

allsitesfailedtoremainsignificant afteradjustingdatafor gender.Eachyearincreaseinagewasassociatedwith1.061 and1.141higherriskofdevelopingosteoporosisinmalesand

females, respectively. After adjusting the data forage, the inversecorrelationbetweenage,and25(OH)DlevelsandBMD valuesatallsiteswasnolongerstatisticallysignificant(data

notshown).

Discussion

HypovitaminosisDandosteoporosisisfrequentinindividuals livingincountrieswithabundantsunshinesuchasIran.9–12

The reasonbehind this finding, however, remains unclear.

Many believethe optimal level of25(OH)D, which leadsto

maximumsuppressionofcirculatingiPTHlevels,shouldbe

definedbasedonfunctionalratherthananepidemiological definition.13

Consideringthecorrelationbetweenserum25(OH)Dlevels and BMDvaluescontroversialresultsare found.While cer-tain studies havefailedto reportany correlationsbetween thesetwovariables,others haverevealedapositive associ-ation betweenserum25(OH)D levelsand BMDvalues.4,14–18

Aryaetal.reportedasignificantcorrelationbetweenserum

25(OH)D levels and BMDvalues atproximal femur.11 _They

concludedthatsubclinical25(OH)Ddeficiencyhasanadverse effectonbonemassandthereforeislinkedwithlowBMDin thesesubjects.Villarealetal.similarlysuggestedthatwomen withlowserum25(OH)Dlevelsshouldbereferredfor osteo-porosisscreening,stressingthatlowBMDcouldbetheonly

symptominthesewomen.19

Onthecontrarywiththesestudies,thepresentresearch

demonstrated a significant but negative relation between

25(OH)DlevelsandBMDvaluesatallthestudiedsites.

Apparentdiscrepancies betweenourstudyand previous

ones can be contributed to the fact that many of these

population-basedstudieshaverecruitedsubjectswitha rel-ativelygoodhealthstatus,andthereforelowerprevalenceof severevitaminDdeficiencyandosteoporosis.14_The_probable

associationbetween25(OH)DandBMDmayalsovarybasedon

thevarioussitesusedfordensitometrymeasurementowing

todifferentcompositionoftrabecularandcorticalbone tis-sue.Moreover,samplingfromapopulation-basedstudy,and comparativelylowerprevalenceofseverevitaminDdeficiency mightincreasethegeneralizabilityofourresults.Inaddition, thehigherserum levelsof25(OH)Dinosteoporoticpatients couldbesecondarytothefactthatosteoporosisismore com-monamongtheelderlyandthisagegrouparemorefrequently treatedwithVitaminDsupplements.Consideringour exclu-sioncriteria,individualstakingsuchsupplementsshouldnot havebeen recruitedinthepresentstudy;this comeswhile manypeople,particularly,olderindividuals,havereceivedthe

supplementswithoutbeingawareoftheirname.

Themeanserum25(OH)Dconcentrationofthewholestudy

populationwas35.37±30.3ng/mlandsome67.2%ofthe stud-iedsubjectswerereportedtohavemoderatetoseverevitamin Ddeficiencyregardlessoftheirgender.Exceptfortrochanter, therewasnodifferenceinthecorrelationbetweenthese vari-ablesinthetwogenders.Itshouldbenotedthatthehigher vitaminDlevelsreportedinosteoporoticcasesinthisstudy couldbecontributedtothelowernumberofstudied

osteo-porotic casescompared tothat ofnormalcases.Moreover,

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Table1–Themeanvaluesofthestudiedbiochemicalmarkersbasedonbonemineraldensitycategorization.

Bonemineraldensitycategorization(no.ofcases) Pvalue

Osteoporosis(246) Osteopenia(1448) Normal(2096)

25(OH)D(ng/ml) 42.03±34.59 35.97±26.49 33.04±23.78 <0.001

PTH(pg/ml) 28.12±16.19 28.82±16.18 28.73±17.72 0.856

Ca(mg/dl) 9.44±0.54 9.43±0.56 9.46±0.57 0.454

P(mg/dl) 3.22±0.49 3.15±0.48 3.19±0.47 0.109

Dataexpressedasmean±SD.

Table2–ThepartialcorrelationbetweenbiochemicalmarkersandBMDvaluesaftercontrollingforgender(A–maleand B–female).

25(OH)D PTH Ca P BMD(femoralneck) BMD(trochanter) BMD(totalhip)

A-Male:

PTH −0.101a

Ca 0.053b ₋_0.032a

P 0.077a _0.088a _0.213a

BMD(femoralneck) −0.073a _0.023 _0.070a _0.137a

BMD(Trochanter) 0.000 −0.008 0.044 0.074b _0.770a

BMD(totalHip) −0.062b _0.013 _0.064b _0.090a _0.876a _0.934a

BMD(spine) −0.076a _0.038 _0.022 _0.083a _0.633a _0.616a _0.666a

B-Female:

PTH −0.175a

Ca 0.079a ₋_0.038

P 0.075a ₋_0.072a _0.245a

BMD(femoralneck) −0.095a ₋_0.024 _0.004 _0.004

BMD(Trochanter) −0.074a ₋_0.046 ₋_0.037 ₋_0.054b _0.806a

BMD(totalHip) −0.057b ₋_0.042 ₋_0.021 ₋_0.034 _0.889a _0.926a

BMD(spine) −0.107a ₋_0.009 ₋_0.030 ₋_0.053b _0.699a _0.624a _0.689a

a _Correlation_is_significant_at_the_0.01_level_(2-tailed).

b _Correlation_is_significant_at_the_0.05_level_(2-tailed).

andelderlypopulationinIranspendsmoretimeoutdoors,it

couldbeconcludedthattheyareexposedtomoresunand

thereforehavehigher25(OH)Dlevels.

Certain studies have revealed an inverse correlation

betweenserumPTHlevelsandBMDvalues,particularlyatthe femoralneck,pointingoutthecatabolicroleofPTHon corti-calbones.15,20_These_studies_have_also_shown_that_decreased

levelsofserumcalciumareassociatedwithdefectsin min-eralizationand consequentlylowBMD.13 _In_line_with_these

studies,Hosseinpanahetal.reportedanegativecorrelation

betweenPTHlevelsandBMDvaluesatfemoralneckinthe

absenceofsimilarcorrelationsbetweenserum25(OH)Dand

BMDofothersites.21_Sadat_Ali_et_al.22_showed_that_vitamin_D

levelssignificantlyinfluenceBMDreadingamongSaudi indi-viduals,pointingoutasignificantpositivecorrelationbetween 25OHDlevelandBMDandsignificantnegativecorrelationwith parathyroidhormoneinthestudiedgroups.Thepresentstudy, onthecontrary,reportedPTHtobeinverselycorrelatedwith BMDvalues atall sites rather than spine. Thecorrelation, however,wasreportedtobesignificant onlyatthefemoral trochanter.

The present study was conducted on healthy

individ-ualsbasedontheirself-reportedhistory;apotentialbiasof undiagnosedunderlyingdiseases,therefore,isprobable. Addi-tionally, the cross-sectional nature ofthe present study is animportantlimitation ofthis study. Moreover,this study

only measured fivebiochemical markers, namely PTH and

25(OH)D,whilemorerecentstudieshavelinkedmarkerssuch asintactosteocalcin(OC)toBMDvaluesandfracturerisk.23,24

Inaddition,25(OH)Dmeasurementswereperformedinwinter, when its levelsare believed tobeatthe lowestlevel com-paredtoothermonthsoftheyear.Itshouldbenotedthatthe presentstudywasanobservationalstudyinwhichtheeffectof importantfactorssuchaspopulationdifferences(gender,age, ethnic,sex,extentofsunexposure,andvitaminDintake)was notassessed.Largeprospectivestudies,therefore,areneeded tobetterevaluatethecorrelationbetweenbiochemical mark-ersandBMDvaluesindifferentpopulations.

Conflict

of

interest

Dr.Dimaireportsreceivingpaymentsforboardmembership, consultancy, expert’s testimony, travels, meetings and lec-turesforhisworkonosteoporosis(Amgen,Daiichi-Sankyo, EliLilly,Kyphon,Medtronic,MerckSharp&Dohme,Novartis, Nycomed,Sanofi-Aventis,Servier).Hisinstitution,the Med-icalUniversity ofGraz, alsoreportsreceivinggrantsforDr.

Dimai’s work on osteoporosis (Amgen, Daiichi-Sankyo, Eli

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Acknowledgement

Theauthorsofthisstudywouldliketoacknowledgethe per-sonneloftheEndocrinologyandMetabolismResearchCenter, thelaboratorystaffand allthosewho kindlycooperatedin conductingthisstudy.

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