monitoring fortification of bread folic acid

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©CrownCopyright December 2011 Ministry of Agricultureand Forestry



Monitoringvoluntaryfortificationofbreadwithfolicacid

K.E.Bradbury1,C.M.Skeaff 1,I.Oey2,S.Williams3,J.Mann1.

1DepartmentofHumanNutrition,2DepartmentofFoodScience,3Departmentof

PreventiveandSocialMedicine



Summary

Consumptionoffolicacidbeforeconceptionandthroughtheearlystagesofpregnancy

reducestheriskofhavingafoetalneuraltubedefect.Thebakingindustry,inresponse

toarequestfromtheNewZealandgovernment,agreedtothevoluntaryadditionoffolic

acid(200microgramsfolicacidper100gramsofbread)toapproximatelyonethirdof

allbreads.TheprogrammehasbeeninplacesinceApril/May2010.

Thepurposeoftheresearchprojectwastoanalysethefolicacidcontentofthetopten

rankedfolicacidfortifiedbreads,bysalesvolume,inboththeNorthIslandandthe

SouthIsland.Anotherpurposeoftheresearchwastoassess,inarepresentativesample

of300women,aged18to44yearsinWellingtonandDunedin,theconsumptionoffolic

acidfortifiedbread,aswellaswomen’sserumandredbloodcellfolatestatus.

Seventeenfolicacidfortifiedbreads,sevenfromtheNorthIslandandtenfromthe

SouthIsland,werecollectedforanalysisoffolicacidcontent.Fivebreads,threefrom



voluntaryfolicacidfortificationofbreadprogramme,werehigherthaninwomen18-44

yearsfromthe2008/09NewZealandAdultNutritionSurveyandfromthe1999

DunedinANZFASurvey.

Thevoluntaryadditionbythebreadindustryoffolicacidtoarangeofbreadshas

coincidedwithincreasedserumandredbloodcellfolatestatusofwomen.



Contents

Summary...........................................................................................................................................ii

Contents...........................................................................................................................................iv

ListofTablesandFigures...........................................................................................................vi

Introduction.....................................................................................................................................1

Contractservicerequirements:................................................................................................2

Methods.............................................................................................................................................4

Analysisoffolicacidcontentinbread....... .................. .................. ................. .................. ................4

ProcedureforselectingandcollectingbrandsofbreadcollectioninWellingtonand

Dunedin.......................................................................................................................................................................4

Procedureforcollectingbreadusedtostudythestabilityoffolicacidfortifiedinbread

d i h lf lif 5



Statisticalanalysis................ ................. .................. .................. .................. ................. .................. .......14

Results.............................................................................................................................................15

Folicacidcontentoffolicacidfortifiedbreads...........................................................................15

Folicacidstabilityovershelf-lifeoffolicacidfortifiedbread...............................................15

Responseratetosurveyrecruitment.............................................................................................15

Characteristicsofsurveyparticipants...........................................................................................18

Consumptionoffolicacidfortifiedbread,breakfastcereal,andspreads.........................18

Serumandredbloodcellfolateconcentrationsofparticipants...........................................19

Therelationbetweenbreadandbreakfastcerealconsumptionandserumandred

bloodcellfolateconcentrations.......................................................................................................21

Comparisonwithserumandredbloodcellfolatestatuspre-voluntaryfolicacid

fortificationofbread............................................................................................................................21

PredictionofchangeinNeuralTubeDefectratefollowingtheintroductionofthe

t f li id f tifi ti f b d 22



ListofTablesandFigures

Figure1:Overviewoffolateextractionprocess............................................................................6

Figure2:Overviewofthefolateisolationandpurificationprocess.......................................7

Figure3:Flowofparticipantsthrough2011FolateandWomen'sHealthSurvey.........17

Table1:Informationaboutfolicacidfortifiedbreadsanalysedinthissurvey...............35

Table2:Folicacidcontentoffolicacidfortifiedbread............................................................36

Table3:Folicacidcontentofbreadfromstarttoendofshelf-life(bestbeforedate)..37

Table4:Characteristicsofparticipantsinthesurvey..............................................................38

Table5:Characteristicsofparticipantsandnon-participantsinthesurvey...................39

Table6:Prevalenceofbread,breakfastcereal,andspreadconsumptioninthe

previousweek........................................................................................................................................40

Table7:Frequencyofconsumptionofallbreadandbreakfastcerealsintheprevious



Introduction

Theresultsofrandomisedcontrolledtrialsconductedinthemid-1980sshowedthat

periconceptionalsupplementationwithfolicacidreducedtheriskofneuraltubedefect

(NTD)-affectedpregnancies1,2.Furthermore,resultsfromobservationalstudieshave

shownthatwomenwhohaveredbloodcellfolateconcentrationsabove905nanomoles

perlitre(nmol/L)orplasmafolateabove15.8nmol/LhaveariskofNTDsimilarto

womentakingadaily400micrograms(µg)folicacidsupplement3.Themajorityof

womendonottakeadaily400µgfolicacidsupplementbeforeconceptionandthrough

theearlystagesofpregnancy4,5.Forthisreason,in2006,theNewZealandand

Australiangovernmentsregulatedmandatoryfortificationofbreadwithfolicacid–to

takeeffectinSeptember20096.Thefoodstandardwouldrequirebreadmanufacturers

toproducebreadcontaining80to180ugoffolicacidper100grams(g)ofbread.

Predictionsbasedonmodellingbreadconsumption–asreportedinthe1997New

ZealandNationalNutritionSurvey–indicatedthatmandatoryfortificationofbread

woulddeliver,onaverage,140µgoffolicacidperdaytowomenofchildbearingagein

N Z l d 7



andForestryalsowantedthefolicacidcontentoffortifiedbreadtobemeasuredaspart

ofmonitoringthevoluntaryfortificationprogramme.

Resultsfromthe2008/09NewZealandAdultNutritionSurveywouldbetheprimary

andbestsourceofinformationaboutthebloodfolatestatusofwomenofchildbearing

agebeforevoluntaryfortificationofbread.Apopulation-basedsurveyconductedin

1999inDunedin8wouldalsoproviderelevantinformationaboutbloodfolatestatusof

womenofchildbearingagepre-voluntaryfortification.Themajorpurposeofthisreportistogatherinformationonbloodfolatestatusaftervoluntaryfortificationby

conductingasmallpopulation-basedsurveyofwomen.

ThereisgoodevidenceinNewZealandthatcarefullyconductedsmallsurveys

employingrepresentativepopulationsamplingtechniquescanprovideestimatesof

nutritionalstatussimilartothoseofmuchlargernationalsurveys.Forexample,the

iodinestatusof300school-childreninDunedinandWellington9wasvirtuallythesame

asthatof1796NewZealandschool-agechildrenwhoparticipatedinthenationwide

2002Children’sNutritionSurvey10.

I thi t t th lt f k i i d b MAF Th t



ZealandFoodStandardsAuthority(ANZFA)surveywiththis2011Folateand

Women’sHealthsurveytodetermineiftherehasbeenachangeovertimein

bloodfolatestatusofwomenofchildbearingage;

4. Determinethefrequencyofconsumptionoffolicacidfortifiedfoodsin

womenofchildbearingageanddetermine,withinthe2011Folateand

Women’sHealthSurvey,therelationbetweenbreadconsumption–totalor

fortified–andbloodfolatestatus;

5. Undertakecomparativeanalysisofresultswithdatafrompreviousworkon

thebloodfolatestatusofwomenofchildbearingageconductedpriorto

fortification.



Methods

Analysisoffolicacidcontentinbread

ProcedureforselectingandcollectingbreadsamplesinWellingtonand

Dunedin

Theselectionoffolicacidfortifiedbrandsofbreadforinclusioninthesurveywasbasedonatop-tenrankingoffolicacidfortifiedbreadsbysalesvolume.TheBakingIndustry

ResearchTrustgatheredconfidentialinformationfromthefourmainbreadcompanies

inNewZealandtogeneratetwolistsofthetop-tenfolicacidfortifiedbreads,onefor

NorthIslandandoneforSouthIslandbreads,rankedbysalesvolumeineachIsland.All

breadsonthetwolistswereanalysed,withoneexception;twoofthebreadsinthetop-tenlistfortheNorthIslandfromthesamebranddifferedonlyintheusage,onefor

“toast”andtheotherfor“sandwich”.Folicacidwasonlyanalysedinthe“toast”version.

Therefore,between27/10/2010and8/02/2011,ninebrandsofbreadwereselected

foranalysisintheNorthIslandandtenintheSouthIsland.



Foreachbrand,acombinedloafwascreatedfromthethreeloavesbychoosingevery

alternateslicefromadifferentloaf,i.e.crustfromloaf1,2ndslicefromloaf2,3rdslice

fromloaf3,4thslicefromloaf1andsoon.Eachcombinedloafwashomogenisedand

extracted.

Procedureforcollectingbreadusedtostudythestabilityoffolicacid

fortifiedinbreadduringshelf-life

ThetoprankedfolicacidfortifiedbreadintheSouthIsland–awhitebread–andthe

toprankedfolicacidfortifiedbreadintheNorthIsland–awholewheatbread–were

usedfortheshelf-lifestudy.FourloavesoftheSouthIslandbreadwerecollectedfroma

Dunedinoutletonthemorningofextraction.FourloavesoftheNorthIslandbreadwere

courieredtoDunedinimmediatelyaftertheycameoffthepackaginglineinthe

afternoonandreceivedthefollowingmorning(3/02/11)inDunedin.

TwocombinedloavesweremadefromthefourSouthIslandloaves;thesamewasdone

withthefourNorthIslandloaves.Thefirstcombinedloafwasmadebyselectingthe

crustfromloaf1,2ndslicefromloaf2,3rdslicefromloaf3,4thslicefromloaf4,5thslice

f l f 1 d f h O hi fi bi d l f d h d



Samplepreparation: Homogenise thecombinedloaf (Magimix,CompactAutomatic

3100Multicuve,France).

Folateextraction:Weigh4gofhomogenateintriplicateintothecentrifugetube.Add

upto80gofextractionbuffer(50mMCHES-50mMHEPESpH7.85containing2%

(wt/v)ascorbicacid,and0.07%(v/v)β-mercaptoethanol).Homogenisethemixture

(PolytronPT2100Kinematica,Switzerland)for30satspeed13.

Bi-enzyme extraction: Cool samples immediately in an ice bath. Homogenise the

mixture (Polytron PT2100 Kinematica, Switzerland) for 30 s at speed 13. Bring

samplestopH7withHCl(4M).Add2mLα-amylase(0.02g/mL)toeachsample.

Heatextraction:Placesamplesinboilingwaterbathfor10min.

Incubate samples for 3 hours at

37°C. Continuously agitate

samples at 200 rpmwith theuse

of an orbital mixer incubator

(R k B i Vi i



Elute phosphate buffer (0.1M, pH7) through folate binding protein

affinity chromatography columns.Afterwards, apply15mLofbread

extracttothecolumn.

Thaw samples at 4-6oC immediately prior to folate isolation and

purification.Centrifuge thawedsamples for30minsat27216 gand

4°C(JA-20Beckmancentrifuge,USA).Filterthesupernatantthrough

a0.45µmPTFEfilter.

Apply5mL ofphosphate buffer (0.025M, pH7 containing 1MNaCl)

followedby5mLofphosphatebuffer(0.025M,pH7).



High-performanceliquidchromatographicanalysisoffolicacidextractsfrom

bread

Thehigh-performanceliquidchromatography(HPLC)methodforfolicacidwas

conductedbasedonthemethoddescribedbyVerlindeetal.12.A1200seriesHPLC

(AgilentTechnologies,Germany)equippedwithaUV-DADdetectorandEZChromElite

3.3softwarewasusedtoidentifyandquantifytheconcentrationoffolicacidinthe

samples.Folicacididentificationwasdonebasedonpeakpurityofcommerciallyavailablepurifiedfolicacidstandard.APrevailRPC18column(250×4.6mm,5µm

particlesize;GraceDavisonDiscoverySciences,Deerfield,Illinois,USA)wasusedto

separatefolicacid.Theconditionsforelutionoffolicacidwereasfollows:flowrate,

1mL/min;columntemperature,25°C;UVdetection,283nm.AgradientofHPLCgrade

methanoland0.1%(v/v)formicacidwasusedasmobilephase.Theruntimewas39

minutes;thegradientstartedat88%0.1%(v/v)formicacid,12%methanol,andwas

maintainedisocraticallyfor4.2minutes,afterwhichthemethanolconcentrationwas

raisedlinearlyto56%within12.6minutes(thusuntil16.8min).Subsequently,the

conditionwith56%methanolwasmaintainedisocraticallyuntil29.6minutesbefore

ilib i h i i i l di i f 12% h l 30 6 i h i



workingstandardwasused.Theconcentrationoffolicacidinthebreadsampleswas

expressedper100gedibleportion.

Surveydesigntoassessfolatestatusofwomen

The2011FolateandWomen’sHealthSurveywasacross-sectionalsurveyofwomenof

childbearingage(18–44years)carriedoutfromApriltoAugust2011intwocity

centresofNewZealand–aSouthIslandcentre,Dunedin,andaNorthIslandcentre,

Wellington.Thesurveyusedastratifiedrandomsamplingtechniquewiththeelectoral

roleasthesamplingframe.TheUniversityofOtago’shumanethicscommitteeapproved

thesurvey,andallparticipantsgaveinformedwrittenconsent.Thesurveywas

registeredwiththeAustraliaNewZealandClinicalTrialsRegistry

(ACTRN12611000463976).

Samplesize

Asamplesizeof300participants(150ineachcitycentre)wouldprovide90%power

(alpha=0.05,twosided)todetecta64nmol/Lchangeinredbloodcellfolate

concentrations–aboutonequarterofastandarddeviation–andachangeinthe

i f i h d bl d ll f l i b 905 l/L f



exclusivelymale–‘Baron’,‘Brother’,‘Count’,‘Father’,‘Lord’,‘Master’,‘Monsignor’,‘Mr’,

and‘Sir’–wereexcludedfromthesample.Localauthorityboundarieswereusedto

restrictthesamplestoourSouthIslandandNorthIslandcentres.ForourSouthIsland

centre–Dunedin–thesamplewasrestrictedtothoselivingwithinthelocalauthority

boundaryofDunedincity.ForourNorthIslandcentre–Wellington–thesamplewas

restrictedtothoselivingwithintheboundariesofthreelocalauthorities:Wellington

city,PoriruacityandHuttcity.

Thoselistedwerecategorisedintothefollowingagegroups:18–19;20–24;25–29;30–

34;35–39;and40–44years.Foreachofthecitycentres,arandomnumberwas

generatedforpotentialparticipantsandwithineachagecategorytherandomnumber

wasusedtocreateanorderedlist.

Thecensus2006informationontheproportionofwomenineachagecategoryfrom

18–44yearswasusedtodeterminetheproportionofwomeninourfinalsamplein

eachagecategory.Forexample,fromthecensus2006data,17%ofwomenintheage

range18–44yearswereintheagecategory20to24,therefore17%ofoursampleof

300womenineachcentrewasselectedfromthisagerange.Thecensus2006datalists

i fi i W d h 40% f i h



participate,acolouredbrochure,aconsentform,ageneralquestionnaire,afreepost

returnenvelope,astudypen,andanoptoutformiftheydecidednottoparticipate.

Secondly,allwomeninthesampleweresent,seventoeightdaysafterthefirstmailout,

apostcardwhichservedasathank-youtothosethathadalreadyreturnedtheirconsent

formandgeneralquestionnaire,andaremindertothosethathadnot.Thirdly,16days

aftertheinitialmailout,asecondinvitationwithreplacementinformationpackwas

senttothenon-responders.Finally,28daysaftertheinitialmailout,thenon-

respondentstelephonenumberswereobtainedfromthewhitepagesandtheywere

phonedtoinvitethemtoparticipateonefinaltime.Iftheirnumberwasnotlisted,orif

theycouldnotbereached,afinalreminderpostcardwassent.Duetoalowerthan

anticipatedresponserateintheWellingtonsample,asecondsamplewasselectedfrom

Wellingtonandcontactedfollowingthesameproceduresdescribedabove.Onehundred

andfourteenwomenwereinvitedtoparticipateinthesecondsampleround,whichwas

basedontheresponserateofthefirstWellingtonsampleatthetimeofthedecisionto

recruitasecondsample(around33%),withtheaimtorecruit150womenintotalfrom

Wellington.

G l i i



possible,theseeaterieswerecontactedandquestionedaboutthebrandofbreadsthey

used,orthebrandofingredientsiftheymadetheirownbread,totrytoidentify

whetherornotthebreadconsumedbytheparticipantcontainedfolicacid.Ifthiswas

unabletobeidentified,thebreadwasspecifiedasunknown.Theywerealsoaskedhow

ofteninthepastweektheyhadconsumedmarmite,vegemiteorvegemitecheesybite,

andwhethertheytookanysupplements.Participantswereathomewheninterviewed

andwereaskedtoreadoutthebrandnameofthepacketsofbreakfastcerealsand

breads,ifavailable,thattheyhadconsumedinthepastweek.Thesamewasdonewith

dietarysupplements.Theingredientslistonpacketsofbreads,breakfastcereals,and

supplementsthatwerereportedbytheparticipantswerecheckedatalocal

supermarketforthepresenceofaddedfolicacidoraddedfolate.Weclassifiedbreadas

folicacidfortifiediftheingredientsliststatedthebreadcontainedfolicacid;wedidnot

alterthisclassificationaccordingtoouranalysisofthefolicacidcontentofbreads.The

phoneinterviewwastypicallycompletedoneweekbeforetheclinicvisit,participants

weretoldatthebeginningabouttheimportanceofbeinghonestandtryingto

rememberatbestastheycould,andattheendoftheinterviewtheywereremindedto

eatastheynormallywouldandthatanyquestionstheyhadaboutfolatewouldbe



serumsamplesweredispensedandstoredat-80°Cwithinthreehoursofblood

collection.

Measurementofserumandwholebloodfolateconcentrations

Serumandwholebloodfolatewasmeasuredbymicrobiologicalassaywiththeuseof

thetestorganismLactobacillusrhamnosus,asdescribedbyO’Broin14.Samplestobe

assayedwerethawedjustpriortouse.Oncethawed,wholebloodwasfirstdilutedone

intenin1%ascorbicacidandincubatedat37°Cfor30minutes,thenfurtherdiluted

onein40in0.5%sodiumascorbate.Serumwasdilutedonein20in0.5%sodium

ascorbate.Serumandwholebloodfromthesameparticipantwereassayedin

quadruplicateonthesameplate.Increasingamountsoffolicacid(200pg/ml)were

usedforthestandardcurve;onestandardcurvewasconstructedperday.Plateswere

incubatedfor42hoursat37°Candthenreadonmicroplatereaderwiththewavelength

setat590nm.Linearinterpolationwasusedtoquantifytheconcentrationoffolatein

thebloodsamples.

Redbloodcellfolateconcentrationswerecalculatedaccordingtothefollowing



folateconentrationof906nmol/Lorhigherwasusedtocategorisewomenatverylow

riskofhavingaNTD-affectedpregnancy.

ThereductionintheprevalenceofNTDswaspredictedasdescribedbyWaldetal.17

andwasbasedonthechangeinserumfolateconcentrationsbetweenthe2008/09New

ZealandAdultNutritionSurveyandthe2011FolateandWomen’sHealthSurvey.

Statisticalanalysis

AllstatisticalanalysiswerecarriedoutusingSTATA(version11.2,StataCorp,College

Station,TX).Differencesinthecharacteristicsofparticipantsbetweenthetwocities,or

ofparticipantsandnon-participantsofthe2011FolateandWomen’sHealthSurvey

wereexaminedusingsimpleregressionforcontinuousvariablesandchi-squaredtest

forcategoricalvariables.Weusedmultiplelinearregressiontoexaminetherelation

betweenbloodfolatestatusanddiet.Becauseoftheirpositiveskew,serumandred

bloodcellfolateconcentrationswerelog-transformedbeforestatisticalanalysisandthe

differencesbetweengroupswerepresentedasratios.AtwosidedStudent’st-testfor

unpairedsampleswasusedtotestsignificantdifferencesinbloodfolatestatusbetween

the2008/09NewZealandAdultNutritionSurveyandthe2011FolateandWomen’s



Results

Folicacidcontentoffolicacidfortifiedbreads

ThedetailsaboutthebreadsselectedforfolicacidanalysisarepresentedinTable1.In

theNorthIsland,fourofthesevenbreadstestedcontained100–300µgfolicacidper

100gedibleportion(Table2),theotherthreebreadscontainedlessthan50µgper

100gedibleportion.IntheSouthIsland,twoofthetenbreadstestedhadfolicacid

concentrationsthatexceeded400µgper100gedibleportion,sixbreadscontained

100–300µgfolicacidper100gedibleportion.Theothertwobreads(D8andD9)

containedlessthan50µgper100gedibleportion.Themean(SD)andmedian

(interquartilerange)forall17breadswas151µg(131)and144µg(41,189),

respectively.

Folicacidstabilityovershelf-lifeoffolicacidfortifiedbread

Thestabilityoffolicacidduringthefivetosixdayshelf-lifeoffortifiedwhiteandwhole-

wheatbreadisshowninTable3.Thefolicacidcontentwasmarginallylower,by1µg



phoneinterviewnoraclinicvisit.Ofthese108participants,onedidnotcompletea

telephoneinterviewandfourpeopledidnotcompleteaclinicvisit.Therewere44

participantswhodeclinedtotakepart,23non-deliveries,andonepersonwhowasnot

eligibletoparticipateasshedidnotunderstandEnglish.Therefore,outofthe300

womenselectedtherewere122womenwhodidnotrespondtotheinvitation.The

responserateforthefirstWellingtonsamplewas39%[participantswhocompletedat

leastoneofaphoneintervieworclinicvisit/(totalselected–(non-deliveries+

ineligible),108/(300-24)].

InthesecondWellingtonsample,114womenwereinvitedtoparticipateinthestudy.

Thirty-sevenconsentedtoparticipate,fourcompletedneitheraphoneinterviewnora

clinicvisit,leaving32includedintheanalysis.Ofthesetwoparticipants,onedidnot

completeaphoneinterview,andonedidnotcompleteaclinicvisit.Therewere15

participantswhodeclinedtotakepart,and11non-deliveries.Therefore,outofthe114

womenselectedtherewere52whodidnotrespondtotheinvitation.Theresponserate

forthesecondWellingtonsamplewas32%[33/(114-11)].Theoverallresponserate

fortheWellingtonsamplescombinedwas37%.



Dunedin WellingtonA WellingtonB

310invitations

sent300invitations

sent

114invitations

sent

149consentedto

participate37consentedto

participate

38declined

totakepart

23non-deliveries

44declined

totakepart

23non-deliveries

1didnot

understand

English

15declined

totakepart

11non-deliveries

110consentedto

participate



Characteristicsofsurveyparticipants

Themeanageofparticipantswas33years(Table4)andtheirmean(SD)bodymass

index(BMI)was27(6)kilogramspermetre-squared(kg/m2).Themostcommon

ethnicitywasNewZealandEuropeanandother(81%).Almosthalfofthewomenhad

sometertiaryeducationandmorethan80%saidtheywereingoodorexcellenthealth;

onequarterofthewomenusedoralcontraceptiveagentsandathirdusedprescription

medications;threequartersofthewomendrankalcoholandasimilarproportiondrank

coffee.Almostonequarterofwomenhadvisited,inthepastyear,acountrywith

mandatoryfolicacidfortificationoffood.

Ingeneral,theage,BMI,educational,health,andincomecharacteristicsofwomenliving

inWellingtondidnotdifferfromthoseofwomenlivinginDunedin;however,there

werestatisticaldifferencesinethnicity,useofprescriptionmedication,andhistoryof

pregnancybetweenwomenrecruitedfromthetwocities.Therewasaslightlyhigher

proportionofMaoriandPacificwomeninWellingtonthaninDunedin( p=0.001);more

womeninDunedinwereusingprescribedmedication(39%comparedwith25%,

p=0.01)andmorewomeninDunedinhadpreviouslybeenpregnant(65%compared



consumedoneormoreslicesofbreadofunknownbrand.Abouthalfofwomen

consumedafolicacidfortifiedspread.Therewasasignificantcorrelationbetweentotal

slicesofbreadconsumedinthepreviousweekandfrequencyofusingfolicacidfortified

spreads(r=0.28, p<0.001).Therewasaninverseassociationbetweensmoking(Y/N)

andfrequencyofconsumptionofbreakfastcerealsinthepastweek(r=-0.21, p<0.001).

Breakfastcerealwasconsumedby72%oftheparticipantsintheweekpriorto

completingthetelephonefoodfrequencyquestionnaire(Table6).Forty-onepercentof

womenconsumedatleastonebowloffolicacidfortifiedbreakfastcerealinthepast

weekand31%consumedbreakfastcerealthatwaseithernon-fortifiedorcouldnotbe

identifiedassuch;ofthislattergroup,15(5%)consumedoneormorebowlsof

breakfastcerealofunknownbrand.

Therewerenosignificantdifferences( p>0.05)betweenWellingtonandDunedininthe

proportionofwomenconsumingfolicacidfortifiedbread,folicacidfortifiedbreakfast

cereal,orfolicacidfortifiedspreads.Theproportionofwomenwhoateanybreador

anybreakfastcerealalsodidnotdiffersignificantlybetweencities(datanotshown).

Amongstallwomen,themean(SD)consumptionofbreadandbreakfastcereal,was12



folateof30nmol/L(95%CI:28,32nmol/L)andgeometricmeanforredbloodcell

folateof996nmol/L(95%CI:945,1049)nmol/L).Themedianserumfolate

concentrationwas29nmol/L(interquartilerange:20,47nmol/L)andthemedianred

bloodcellfolateconcentrationwas989nmol/L(interquartilerange:744,1316).The

prevalenceofwomenwithdeficientserumandredbloodcellfolateconcentrations,

definedaslessthan6.8nmol/Lforserumandlessthan317nmol/Lforredbloodcell,

was1%.Almost60%ofwomeninthesurveyhadaredbloodcellfolateconcentration

906nmol/Lorhigher,aconcentrationassociatedwithNTDrisksimilartowomen

takinga400µgdailyfolicacidsupplement.

Meanserumfolateandredbloodcellfolateconcentrationsweresignificantlyhigherin

DunedincomparedwithWellington;however,afteradjustmentforfolicacid

supplementuse(Y/N)andconsumptionoffolicacidfortifiedbreakfastcereals(Y/N)

thetrendforslightlyhigherconcentrationsinDunedinwasnotsignificant.Theadjusted

ratioofthegeometricmeansofserumfolateinDunedinrelativetoWellingtonwomen

was1.13(95%CI:1.00,1.29; p<0.068)andforredbloodcellfolateitwas1.11(95%CI:

1.00,1.22; p<0.052).



Therelationbetweenbreadandbreakfastcerealconsumptionandserumand

redbloodcellfolateconcentrations

Participantswhoreportedconsumingfolicacidfortifiedbreadhadasignificantlyhigher

meanserumfolateconcentrationcomparedwithparticipantswhodidnotconsume

folicacidfortifiedbread(Table11).Theratioofgeometricmeansafteradjustmentfor

useoffolicacidsupplements,consumptionoffolicacidfortifiedbreakfastcereal,and

cityofresidencewas1.24(95%CI:1.05,1.48; p=0.012).Thesametrendinserumfolate

concentrationwasapparentforconsumersoffortifiedbreakfastcerealrelativetonon-

consumerswithanadjustedratioof1.15(95%CI:1.00,1.31; p=0.042).Meanredblood

cellfolateconcentrationswerenotsignificantlyhigherinconsumersoffolicacid

fortifiedbread,however,therewasatrendforhigherconcentrationsamongst

consumers;theadjustedratiowas1.12(95%CI:0.99,1.28; p=0.079).Participantswho

atefolicacidfortifiedbreakfastcerealshadsignificantlyhigherredbloodcellfolate

concentrationscomparedwiththosewhodidnotconsumefolicacidfortifiedbreakfast

cereal;theadjustedratiowas1.13(95%CI:1.02,1.25; p=0.019).

Therewasnorelationbetweenfrequencyoftotalbreadconsumption(slicesperweek)



withredbloodcellfolateconcentrations906nmol/Lorhigherwas33%(95%CI:26,

40)highercomparedwithwomenofsimilarageinthe2008/09NewZealandAdult

NutritionSurvey.Theage,bodymassindex,ethnicity,andNewZealandDeprivation

2006(NZDep2006)characteristicsofparticipantsaged18-44yinthe2008/09New

ZealandAdultNutritionSurveyandthe2011FolateandWomen’sHealthSurveyare

showninTable14;informationfromthetwosurveysabouteducationarenotshownin

theTable14becausethequestionsusedtogathertheinformationinthe2008/09New

ZealandAdultNutritionSurveywerenotreadilycomparabletothe2011Folateand

Women’sHealthSurvey.Fifty-ninepercentofwomenaged18-44yinthe2008/09New

ZealandAdultNutritionSurveyhadaqualificationbeyondhighschool.Thosewomen

withincompletequalifications,orqualificationsthattooklessthanthreemonthto

finishwereexcludedfromthiscategory.Sixty-ninepercentofparticipantsinthe2011

FolateandWomen'sHealthSurveyhadacertificateordiploma,oratleastsometertiary

study.Therefore,althoughtheeducationcategoriesinthetwosurveysarenotdirectly

comparable,theparticipantsappeartohavesimilareducationlevels.

Apopulation-basedsurveyof212women,18-45ylivinginDunedinwasconductedin

1999andshowedthatmedian(interquartilerange)redbloodcellfolateconcentration



Discussion

Folicacidcontentoffolicacidfortifiedbread

Themoststrikingresultfromouranalysisofthefolicacidfortifiedbreadswasthatfive

outofthe17breadshadfolicacidcontentslessthan50µgper100gedibleportion;

threeofthebrandswithverylowfolicacidcontentswerefromtheNorthIslandand

twowerefromtheSouthIsland.Weareconfidentintheaccuracyandprecisionofthe

results.AllbreadsthatwerepurchasedinWellingtonwerebakedinNorthIsland

factories,andallbreadspurchasedinDunedinwerebakedinSouthIslandfactories.

Weassessedtheaccuracyoftheanalyticalassaybyspikingsamplesofunfortifiedbread

withfolicacid;recoveryoffolicacidfromspikedsamplesexceeded90%.Thestandard

curvewaslinearandgaveexcellentreproducibility,basedonlowstandarddeviations

(Table2).

Therewerefivebreadscontaininglessthan50µg/100gedibleportion,ninebreads

containing100–200µg/100gedibleportion,onecontaining200–300µg/100gedible



Itisimportanttonotethatbreadsforourstudywerecollectedfromsupermarketson

oneoccasion;therefore,ouranalysiscanonlyreflectthefolicacidcontentatthatpoint

intimeandcannotautomaticallyreflectfolicacidlevelsinbreadatalltimes.Thethree

NorthIslandbrandsofbreadthatwefoundhadverylowfolicacidcontentswerethe

topthreeNorthIslandfolicacidfortifiedbreadsrankedbrandsbysalesvolume

(rankingnotshown,topreserveconfidentiality);thetwoDunedinbrandswith

undetectedfolicacidwererankedfifthandsixthintheSouthIsland.Thus,ifour

analysisofbreadreflectstheusualfolicacidcontentofthesebrandduringthesixto

twelvemonthspriortocollectingbloodfromtheparticipantsinourstudy,onewould

expectthefolatestatusofwomenresidinginDunedintobeslightlyhighercompared

withwomenresidinginWellingtonbecausethefoodsupplyinDunedinduringthat

periodwouldhavemorefolicacidfrombread.Thetrendforhigherfolatestatusin

DunedincomparedwithWellingtonwomenisconsistentwiththisproposition.Though

theadjustedratioofmeanserumconcentrationsinthetwocities(1.13, p=0.068),as

wellasthatforredbloodcellfolate(1.11, p=0.052),didnotdifferstatisticallyfrom1,

thelowerlimitoftheconfidenceintervalwas1.00forserumandredbloodcellfolate

concentrations(Table10).



difficulttoknowwhethertheydidnotwanttoparticipate,orwhethertheynever

receivedtheinvitationtoparticipate.Becauseourparticipantsweremorelikelytobe

olderandlessdeprivedthanthesamplefromwhichtheyweredrawn,itispossiblethat

ourparticipantsreflectaslightlyhealthiergroupcomparedwiththatofallNewZealand

womenofchildbearingage.However,thefactthatageandNZDepscorewerenot

predictorsofserumofredbloodcellfolateconcentrationinthe2011Folateand

Women’sHealthSurveysuggeststhattheslightlyolderageandlessdeprivationindex

scoreoftheparticipantsisunlikelytobiastheresults.

Consumptionoffolicacidfortifiedbread,breakfastcerealandspread

Thevastmajority,93%,ofthesurveyparticipantsreportedconsumingoneormore

slicesofbreadinthelastweek.ArecentreportbytheAustralianInstituteofHealthand

Welfarereported,basedonresultscompiledbyFoodStandardsAustraliaNewZealand,

thatbetween2001and2008about80-85%ofNewZealandwomen,14yearsorolder,

consumedbread18.Eighteenpercentoftheparticipantsinthe2011Folateand

Women’sHealthSurveywereabletoconfirmhavingconsumedoneormoreslicesof

fortifiedbreadinthelastweek.Theactualproportionofwomenconsumingfolicacid



fortifiedbreakfastcereal,butprobablynotasmuchasthatforbreadbecauseonly5%

ofwomenwereunabletoidentifyatleastonebrandofbreakfastcerealconsumed.

Fifty-threepercentofwomeninoursurveyreportedconsumingfolicacidfortified

spreadinthepreviousweek.ThereportbytheAustralianInstituteofHealthand

Welfare18showedthatjustover50%ofNewZealandwomen14yearsorolder

consumedyeastcontainingspreadsin2007and2008.

Theproportionofwomenconsuminganybreadorbreakfastcereal,whetherfortifiedor

not,didnotdifferbetweenWellingtonandDunedin.Furthermore,thefrequencyof

consumption(i.e.slicesperweekorbowlsperweek)ofbreadandbreakfastcereals

amongstallconsumers,consumersofbreadorbreakfastcereals,orconsumersof

fortifiedbreadorbreakfastcerealsdidnotdifferbetweenthecities.Thissuggeststhat

thedifferencesinmeanbloodfolatestatusofwomenresidinginWellingtoncompared

withthoseresidinginDunedindidnotresultfromadifferentprevalenceorfrequency

ofbreadandbreakfastcerealconsumptionbetweenthetwocities.

Therelationbetweenconsumptionoffolicacidfortifiedbreadorbreakfast

cerealandbloodfolatestatus



correspondingtypeofbread;thisseemsunlikely.Consumptionoffolicacidfortified

breakfastcerealwasassociatedwitha5nmol/Lhighermeanserumfolicacid

concentrationsthannon-consumersafteradjustmentforfolicacidfortifiedbread

consumption,folicacidsupplementuse,andcity.

Meanredbloodcellfolateconcentrationwashigherinconsumerscomparedwithnon-

consumeroffolicacidfortifiedbreakfastcereals;theadjustedratioofthegeometric

mean(1.13)wassimilartothatforserumfolate(1.15).However,consumptionoffolic

acidfortifiedbreadwasnotassociatedwithsignificantlyhighermeanredbloodcell

folateconcentration.Thereasonforthisisnotreadilyapparent.Itmaybeassimpleas

thenumberofwomenclassifiedasconsumersoffolicacidfortifiedbreadbeingtoo

smallagrouptogivetheanalysisadequatestatisticalpower.Furthermore,someofthe

breadsweclassifiedasfolicacidfortified–onthebasisoftheingredientlist–mayhave

containedlittlefolicacid.Attenuationbiasofmisclassifyingfolicacidfortifiedbread

consumermighthaveplayedarole,butonewouldexpectthistoalsoapplyforthe

serumfolateresults.Theupperleveloftheconfidenceintervalfortheadjusted

differenceinredbloodcellfolateconcentrationbetweenfolicacidfortifiedandnon-

fortifiedbreadconsumerswas1.28,avaluesufficientlylargetomakeitdifficultto



folicacidcontentoffortifiedbreakfastcerealstobesomewhathigherthanthat

reportedonthelabel–aphenomenonreferredtoas‘overages’20.

Theaccuracy,precision,andvalidityofthetelephonefoodfrequencyquestionnairewas

nottestedpriortothesurvey,therefore,itisimpossibletoknowtowhatextent

measurementerrormayhaveinfluencedtheresults.Ingeneral,dietarymeasurement

errorwouldtendtoincreasemisclassificationofparticipantsintocategoriesoffortified

ornon-fortifiedfoodconsumption,thus,ourresultsarelikelytobeanunderestimation

ofthetrueassociationbetweenconsumptionoffolicacidfortifiedbreadsandbreakfast

cereals.

Comparisonoffolatestatuspre-andpostthevoluntaryfolicacidfortification

ofbreadprogramme

Comparedtowomen18-44yearsofageinthe2008/09NewZealandAdultNutrition

Survey,thebloodfolateconcentrationsofparticipantsinthe2011FolateandWomen’s

HealthSurveyweresignificantlyhigher(Table13).Forredbloodcellfolatethe

differencebetweenthetwosurveyswas302nmol/L,andforserumfolate,the

differencewas7.9nmol/L.Theproportionofwomenwithredbloodcellfolate



introducedinApril/May2010,providedsomeoftheadditionaldietaryfolicacid;but

howmuch,isdifficulttoestablish.Oneargumentforlinkingthevoluntaryfortification

ofbreadwithimprovedfolatestatusofwomen,wasthenon-significanttrendforhigher

serumandredbloodcellfolateconcentrationsofwomeninDunedincomparedwith

Wellington;afteradjustmentforfolicacidsupplementuseandconsumptionoffolicacid

fortifiedbreakfastcereals.GiventhatthetopthreeNorthIslandranked(bysales

volume)folicacidfortifiedbreadswerefoundtohavelittlefolicacid,itisprobablethat

thefolicacidintakefrombreadwasslightlyhigherinwomenresidinginDunedinthan

inWellington.ThedemographiccharacteristicsofwomeninDunedindifferedslightly

fromwomeninWellington(Table4).InDunedin,therewasahigherproportionof

NewZealandEuropeanandOthersandlowerproportionofMaoriandPacific,ahigher

proportionofprescriptionmedicationusers,andhigherproportionofwomenwitha

pastpregnancy.However,inregressionanalysis,noneofthesefactorspredictedserum

orredbloodcellconcentrationsintheparticipants.Therefore,theslightdifferencesin

thedemographiccharacteristicsofwomeninthetwocitiesprobablydidnotcontribute

tothedifferencesinmeanserumandredbloodcellfolateconcentrations.Giventhat

womenconsumed,onaverage,1.7slicesofbreadperdayandthatasignificant



bloodcellfolateconcentrationsbetweenfastingandnon-fastingsamplescollectedfor

theNationalHealthandNutritionExaminationSurvey(NHANES)1999-2000.The

NationalInstitutesofStandardsandTechnology(NIST)certifiedreferencematerialfor

serumfolate(SRM1955)wasusedasqualitycontrolduringtheserumandredblood

cellfolateanalysisofthe2008/09NewZealandAdultNutritionSurveysamples,aswas

apooledplasmasample.Theanalysedvalues(n=19)ofthethreelevelNISTcertified

referencematerialfromthe2008/09NewZealandAdultNutritionSurveyfolate

analysiswere:6.2nmol/L(certifiedrange:4.9–6.3nmol/L),14.0nmol/L(certified

range:12–16nmol/L),and47.1nmol/L(certifiedrange:37–51nmol/L),with

CoefficientsofVariation(CVs)of9.8%,12.5%and8.9%,respectively.ThemeanandCV

forthe2008/09NewZealandAdultNutritionSurveypooledplasma(n=164)was19.2

nmol/Land16.5%,respectively.Theseanalyticalvalues,measuredduringthe2008/09

NewZealandAdultNutritionSurvey,fortheSRM1955aswellasthepooledplasmadid

notdiffersignificantlyfromthevaluesweobtained(seemethodssection,andAppendix

B)inthe2011FolateandWomen’sHealthStudyfortheexactlythesameSRM1955and

pooledplasmaqualitycontrols.Thecloseagreementshowsthattheaccuracyofthe

microbiologicalassaydidnotdifferbetweenthesurveysandthatanydifferenceinthe



Conclusions

Themedian(interquartilerange)folicacidcontentofthe17breadswetestedwas

detectedwas144(41,189)µgper100gofbread.Thevariationinfolicacidcontentof

thefolicacidfortifiedbreadswasconsiderable.Mostfolicacidfortifiedbreads

containedbetween100–200µgper100gedibleportion,however,fivebreads

containedlessthan50µgper100gedibleportion,andtwobreadscontained400–500

µgper100gedibleportion.

Folicacidaddedtowhiteandwhole-wheatbreadisstableovertheshelf-lifeofthe

bread.

Asmallproportion(18%)ofwomenwereabletorecallconsumingfolicacidfortified

bread.Breadwasconsumedbymorethan90%ofwomeninthesurveyandmean

consumptionwastwelveslicesinthepreviousweek.Therewasnodifferenceinthe

prevalenceorfrequencyofbreadconsumptionbetweenwomenresidinginWellington

orDunedin.



NutritionSurveyandthe1999DunedinANZFASurvey.Thissuggeststhatvoluntary

fortificationofbreadhascontributedtotheincreasedfolatestatusofwomen.



References

1. Anonymous.Preventionofneuraltubedefects:resultsoftheMedicalResearchCouncilVitaminStudy.MRCVitaminStudyResearchGroup.Lancet1991;338:131-7.

2. CzeizelAE,DudasI.Preventionofthefirstoccurrenceofneural-tubedefectsbypericonceptionalvitaminsupplementation.NewEnglJMed1992;327:1832-5.

3. DalyLE,KirkePN,MolloyA,WeirDG,ScottJM.Folatelevelsandneuraltube

defects.Implicationsforprevention.Jama1995;274:1698-702.

4. DobsonI,DevenishC,SkeaffCM,GreenTJ.Periconceptionalfolicaciduseamong

womengivingbirthatQueenMaryMaternityHospitalinDunedin.AustNZJObstetGynaecol2006;46:534-7.

5. SchaderI,CorwinP.HowmanypregnantwomeninChristchurchareusingfolicacidsupplementsinearlypregnancy?NZMedJ1999;112:463-5.

6. AnnetteKingMinisterforFoodSafety.NewZealand(MandatoryFortificationofBreadwithFolicAcid)FoodStandard2007.

7. FoodStandardsAustraliaNewZealand.ProposalP295:Considerationofmandatoryfortificationwithfolicacid.CanberraandWellington;2006.



16. SentiFR,PilchSM.AnalysisoffolatedatafromthesecondNationalHealthand

NutritionExaminationSurvey(NHANESII).JNutr1985;115:1398-402.

17. WaldNJ,LawMR,MorrisJK,WaldDS.Quantifyingtheeffectoffolicacid.Lancet2001;358:2069-73.

18. AustralianInstituteofHealthandWelfare.MandatoryfolicacidandiodinefortificationinAutraliaandNewZealand:supplenenttothebaselinereportfor

monitoring.Canberra:AIHW;2011.

19. QuinlivanEP,GregoryJF,3rd.Reassessingfolicacidconsumptionpatternsinthe

UnitedStates(19992004):potentialeffectonneuraltubedefectsandoverexposuretofolate.AmJClinNutr2007;86:1773-9.

20. RaderJI,WeaverCM,AngyalG.Totalfolateinenrichedcereal-grainproductsintheUnitedStatesfollowingfortification.FoodChem2000;70:275-89.

21. BrownRD,LangshawMR,UhrEJ,GibsonJN,JoshuaDE.TheimpactofmandatoryfortificationofflourwithfolicacidonthebloodfolatelevelsofanAustralianpopulation.

MedJAust2011;194:65-7.

22. PfeifferCM,CaudillSP,GunterEW,OsterlohJ,SampsonEJ.BiochemicalindicatorsofBvitaminstatusintheUSpopulationafterfolicacidfortification:resultsfromtheNationalHealthandNutritionExaminationSurvey1999-2000.AmJClinNutr

2005;82:442-50.



B r e a d C o d e N o .

1

Dateof

purchase

Cityof

purchase L o a f s a m p l e n o

Bestbefore

date B r e a d C o d e N o .

1

Dateof

purchase

Cityof

purchase L o a f s a m p l e n o

Bestbefore

date

NorthIsland SouthIsland

1 1

2 2

3 3

1 1

2 2

3 3

1 1

2 2

3 3

1 1

2 2

3 3

1 1

2 2

3 3

1 1

2 2

3 3

1 1

Table1Informationaboutfolicacidfortifiedbreadsanalysedinthissurvey

D6 28/10/10 Dunedin 1/11/10

D4 27/10/10 Dunedin 1/11/10

D5 27/10/10 Dunedin 1/11/10

D2 27/10/10 Dunedin 31/10/2010

D3 27/10/10 Dunedin 1/11/10

D1 27/10/10 Dunedin 31/10/2010

W5 17/01/11 Wellington 21/01/11

W6 17/01/11 Wellington 21/01/11

W3 9/11/10 Wellington 12/11/10

W4 9/11/10 Wellington 12/11/10

W1 9/11/10 Wellington 11/11/10

W2 17/01/11 Wellington 21/01/11



Table2Folicacidcontentoffolicacidfortifiedbread

BreadCodeNo.

Mean(SD)folicacidinbread

(µg/100gedibleportion)1

NorthIsland

W1 156(5)

W2 41(23)

W3 133(7)

W4 119(4)

W5 6(2)

W6 8(4)

W7 246(15)

SouthIsland

D1 189(25)

D2 158(19)

D3 452(39)

D4 420(21)

D5 105(4)

D6 144(6)

D7 190(9)

D8 4(1)

D9 4(1)

D10 184(8)1Based on triplicate measurement from three loaves



Breadcodeno. Typeofbread Saledate Shelf-lifedate

NI1 Brown 198(12) 180(15)

SI1 White 256(9) 255(30)

1Basedontriplicatemeasurementfromfourloaves

Mean(SD)folicacidinbread(µg/100gedibleportion)1

Table3Folicacidcontentofbreadfromstarttoendofshelf-life(bestbeforedate)



Table4Characteristicsofparticipantsinthesurvey

All Wellington Dunedin pvalue1

Number 288 141 147Age2 33(8) 33(8) 33(8) 0.635

BMI2 27(6) 26(6) 27(6) 0.493

Ethnicity 0.001

NZEO 233(81%) 101(73%) 132(90%)

Maori 26(9%) 16(12%) 10(7%)

Pacific 11(4%) 8(6%) 3(2%)

Asian 16(6%) 14(10%) 2(1%)Education 0.154

Lessthanhighschool 53(18%) 22(16%) 31(21%)

Highschoolgraduate 37(13%) 15(11%) 22(15%)

Certificateordiploma 16(6%) 5(4%) 11(8%)

Sometertiary 39(14%) 22(16%) 17(12%)

Tertiary 142(49%) 77(55%) 65(45%)

Gluten-freediet 9(3%) 5(4%) 4(3%) 0.687

Health 0.596Excellent 89(31%) 47(34%) 42(29%)

Good 159(55%) 74(53%) 85(58%)

Fair 38(13%) 18(13%) 20(14%)

Poor 1(0%) 1(1%) 0(0%)

Antibiotics3 41(14%) 21(15%) 20(14%) 0.754

Householdincome 0.106



Table5Characteristicsofparticipantsandnon-participantsinthesurvey

Participants Non-participants pvalue1

Number 288 725Agecategory

18-19y 19(7%) 36(8%)

20-24y 41(14%) 83(19%)

25-29y 32(11%) 86(20%)

30-34y 54(19%) 80(18%)

35-39y 67(23%) 78(18%)

40-44y 75(26%) 74(17%)Maoridescent 26(9%) 56(13%) 0.115

NZDep20062

1 54(19%) 63(14%)

2 26(9%) 58(13%)

3 41(14%) 32(7%)

4 31(11%) 31(7%)

5 25(9%) 31(7%)

6 33(12%) 33(8%)

7 19(7%) 39(9%)

8 16(6%) 52(12%)

9 22(8%) 42(10%)

10 21(7%) 56(13%)

Valuesarenumber(%)

<0.001

0.001



40

Fortified1

Non-fortified/

unidentified2 Any Fortified3

Non-fortified/

unidentified4 Any

All 288 18%(51) 75%(215) 9 3%(266) 41%(116) 31%(89) 72%(205) 53%(152)

Wellington 141 14%(20) 75%(106) 89%(126) 36%(50) 36%(50) 72%(100) 49%(69)

Dunedin 147 21%(31) 74%(109) 95%(140) 45%(66) 27%(39) 71%(105) 56%(83)

Valuesarepercentofparticipantsrecruited(n)

Percentagesmaynotmatchtototalrecruitedduetomissingdata

Table6Prevalenceofbread,breakfastcereal,andspreadconsumptioninthepreviousweek

4Participantswhodidnotconsumefolicacidfortifiedbreakfastcereal.Participantsinthiscategoryreportedeatingonlynon-fortifiedbrandsof

breakfastcereal,unidentifiedbrandsofbreakfastcerealoracombinationofboth.

1Participantswhoconsumedanyfolicacidfortifiedbread.Tobeincludedinthiscategoryparticipantsreportedeatingabrandofbread(atleast

oneslice)knowntobefortifiedwithfolicacid.

N o

. r e c r u i t e d Breadconsumers Breakfastcerealconsumers

Fortified

spread

consumers

2Participantswhodidnotconsumefolicacidfortifiedbread.Participantsinthiscategoryreportedeatingonlynon-fortifiedbrandsofbread,

unidentifiedbrandsofbreadoracombinationofboth.

3Participantswhoconsumedanyfolicacidfortifiedbreakfastcereal.Tobeincludedinthiscategoryparticipantsreportedeatingabrandof

breakfastcereal(atleastonebowl)knowntobefortifiedwithfolicacid.



Table7Frequencyofconsumption1ofallbreadandbreakfastcerealsinthepreviousweek

Place Bread(slicesperweek) pvalue2 Breakfastcereal(bowlsperweek) pvalue2

All 12(9) 3.4(2.8)

Wellington 12(9) 3.4(2.8)

Dunedin 13(8) 3.5(2.9)

1Mean(SD)consumptionamongstallparticipants

2 pvaluesareforthedifferencebetweenWellingtonandDunedinpartipants,testedusingsimplelinearregression

0.296 0.737



Place Bread(slicesperweek)2

pvalue3

Breakfastcereal(bowlsperweek)4

pvalue3

All 13(8) 4.8(2.2)


Dunedin 13(8) 4.9(2.2)

1Mean(SD)consumptionamongstconsumers


Table8Frequencyofconsumption1ofallbreadandbreakfastcerealsamongst

consumersinthepastweek

4Therewere205(72%),100(72%),and105(71%)consumersofbreakfastcerealsinAll,Wellington

andDunedin,respectively

2Therewere266(93%),126(91%),and140(95%)consumersofbreadinAll,Wellingtonand

Dunedin,respectively

0.636 0.528



Place Bread(slicesperweek)2

pvalue3

Breakfastcereal(bowlsperweek)4

pvalue3

All 13(8) 4.8(2.3)


Dunedin 13(8) 5.0(2.3)

1Mean(SD)consumptionofallbreadamongstfolicacidfortifiedproductconsumers


Table9Frequencyofconsumption1ofbreadandbreakfastcerealsinthepastweek

amongstconsumersoffolicacidfortifiedbreadorbreakfastcereal

2Therewere51(18%),20(14%),and31(21%)consumersofanyfortifiedbreadinAll,Wellingtonand

Dunedin,respectively

3Therewere116(41%),50(36%),and66(45%)consumersofanyfortifiedbreakfastcerealsinAll,

WellingtonandDunedin,respectively

0.887 0.179



44

Table10Serumandredbloodcellfolatestatusofparticipants

All Wellington Dunedin

Difference3

(95%CI)

Ratio4

(95%CI)

Adjustedratio5

(95%CI)

Serumfolate(nmol/L)

n 271 129 142

Mean(SEM) 36(1) 33(2) 38(2)

Geomean(95%CI) 30(28,32) 27(25,30) 32(29,35) 1.17(1.01,1.35)

p=0.033

1.13(1.00,1.29)

p=0.068

Median(IQR)1 29(20,47) 25(19,37) 33(20,49) 8(2,13)

p=0.004

<6.8nmol/L2 1%(0,2) 1%(0,2) 1%(0,2)

Erythrocytefolate(nmol/L)

n 271 129 142

Mean(SEM) 1096(30) 1034(44) 1152(42)

Geomean(95%CI) 996(945,1049) 935(866,1010) 1054(982,1131) 1.13(1.01,1.25)

p=0.025

1.11(1.00,1.22)

p=0.052

Median(IQR)1 989(744,1316) 897(675,1251) 1042(778,1381) 157(37,276)

p=0.011

<317nmol/L2 1%(0,2) 1%(0,2) 1%(0,2)

≤339nmol/L2 1%(0,2) 1%(0,2) 1%(0,2)

≥906nmol/L2 59%(53,65) 49%(40,58) 68%(61,76)

2Valuesare%(95%CI)3DifferencebetweenmediansofDunedinandWellinton

1Interquartilerange(IQR),1stand3rdquartile

4RatioofthegeometricmeansofDunedinrelativetoWellington

5RatioofthegeometricmeansofDunedinrelativetoWellingtonadjustedforuseoffolicacidcontainingsupplements(Y/N),and

breakfastcerealconsumption(Y/N)



45

Table11Serumandredbloodcellfolateconcentration(nmol/L)accordingtoconsumptionoffolicacidfortifiedbreadorbreakfastcereal

Measurement Fortified1

Non-fortified,

unidentified,or

nobread1

Adjustedratio

(95%CI)2

pvalue Fortified1

Non-fortified,

unidentified,or

nobread1

Adjustedratio

(95%CI)3

pvalue

Serum folate 37(32,44) 28(26,31) 1.24(1.05, 1.48) 0.012 33 (29, 36) 28 (25, 31) 1.15 (1.00, 1.31) 0.042

Redbloodcellfolate 1124(990,1276) 969(906,1026) 1.12(0.99,1.28) 0.079 1079(991,1176) 942(883,1006) 1.13(1.02,1.25) 0.0191Valuesaregeometricmean(95%CI)

2Ratioofthegeometricmeansoffolicacidfortifiedrelativetonon-fortified,unidentifiedornobreadadjustedforuseoffolicacidcontainingsupplements(Y/N),cityofresidence(Wellingtonor

Dunedin),andfortifiedbreakfastcerealconsumption(Y/N).Forexample,1.24canbeinterpretedasa24%highergeometricmeanserumfolateconcentrationinconsumersoffortifiedbread

comparedwithnon-fortifiedorunknownbread.3Ratioofthegeometricmeansoffolicacidfortifiedrelativetonon-fortified,unidentifiedornobreakfastcerealadjustedforuseoffolicacidcontainingsupplements(Y/N),cityofresidence

(WellingtonorDunedin),andfortifiedbreadconsumption(Y/N).

Bread Breakfastcereal



Measurement

Adjustedratioper

incrementofbread

(sliceperweek)1

pvalue

Adjustedratioperincrement

ofbreakfastcereal

(bowlperweek)2

pvalue

Serumfolate 1.005(0.998,1.013) 0.176 1.036(1.012,1.061) 0.003

Redbloodcellfolate 1.005(0.999,1.012) 0.082 1.031(1.013,1.050) 0.0011Valuesareadjustedratio(95%CI)perincrementaldifferenceinfrequencyofbreadconsumption;adjustedforuseoffolic

acidcontainingsupplements(Y/N),cityofresidence(WellingtonorDunedin),andbowlsofbreakfastcerealconsumption.

2Valuesareadjustedratio(95%CI)perincrementaldifferenceinfrequencyofbreakfastcerealconsumption;adjustedfor

useoffolicacidcontainingsupplements(Y/N),cityofresidence(WellingtonorDunedin),andslicesofbreadconsumption.

Forexample,1.036canbeinterpretedasa3.6%highergeometricmeanserumfolateconcentrationperincremental(one

morebowlperweek)differenceinbreakfastcerealconsumption.

Table12Therelationbetweenfrequencyofconsumptionofbreadorbreakfastcerealandserumor

redbloodcellfolate



47

2008/09NewZealand

AdultNutritionSurvey

2011Folateand

Women'sHealthSurvey

Meandifference

(95%CI)Ratio(95%CI) pvalue

Serumfolate(nmol/L)

Mean(SEM) 27.8(1.1) 35.6(1.4) 7.9(4.3,11.4) <0.001

Geo-mean(95%CI) 23(21,24) 30(28,32) 1.31(1.19,1.45) <0.001

Redbloodcellfolate(nmol/L)

Mean(SEM) 794(21) 1096(30) 302(229,374) <0.001

Geo-mean(95%CI) 720(686,755) 996(945,1049) 1.38(1.29,1.48) <0.001

≥906nmol/L(95%CI) 26%(22,31) 59%(53,65) 33%(26,40) <0.001

Table13Comparisonofthe2008/09NewZealandAdultNutritionSurveyandthe2011FolateandWomen'sHealthSurvey



2008/09NewZealand

AdultNutritionSurvey

2011Folateand

Women'sHealthSurvey

Number 663 288

BMI(kg/m2)

127(9) 27(6)

Age(y)1

32(11) 33(8)

Agecategory2

18-19y 7% 7%

20-24y 16% 14%

25-29y 17% 11%

30-34y 19% 19%

35-39y 19% 23%

40-44y 22% 26%Ethnicity

2

NZEO 78% 87%

Maori 16% 9%

Pacific 6% 4%

NZDep20063

1 5% 19%

Table14Characteristicsofparticipantsinthe2008/09New

ZealandAdultNutritionSurveyandthe2011FolateandWomen's

HealthSurvey



Appendix A

Mapshowing25kmradiusfromWellingtoncitycentre



Mapshowing25kmradiusfromDunedincitycentre



AppendixB

Folateconcentration(nmol/L)forexternalstandardreferencematerialandinternalpooledplasma.

NISTSRM19551

LevelI LevelII LevelIIIANSpooled

plasma

NISTSRM19551

"informationvalues"

mean±2SD

(±2SDrange)

5.6±0.7

(4.9to6.3)

14±2

(12to16)

44±7

(37to51)

2011Women'sFolate

andHealthSurveymean±SD(CV)

7.5±0.2(2.4)

n=4

15.6±0.5(3.0)

n=4

48.9±2.1(4.3)

n=4

19.2±2.4(12.3)

n=29

2008/09NewZealandAdultNutritionSurvey

mean±SD(CV) 6.2±0.6(9.8)n=19

14.0±1.8(12.5)n=19

47.1±4.2(8.9)n=19

19.2±3.2(16.5)n=164

1NationalInstituteofStandardsandTechnologyStandardReferenceMaterial1955:NISTSRM1955.TheNISTstandardreferencematerial1955

forfolateinhumanserumisprovidedasthreesampleseachwithadifferentconcentration(i.e.level).

monitoring fortification of bread folic acid

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