Growth, Tblamir, Status, Erytbrocyte Fatty Add Composition, and VisualAcuity in FulltermInfants Fed Breastmilk,Formula,or Evaporated Milk
AThesis presentedto the DepartmentcfBlochemistry
of
MemorialUniversityofNewfoundland
by
Ursula McCloy
lnpartialfulfillmentofthe requirementsforthedegree of
MastersofScience
May,1996
lCU. McCloy1996
1...1
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ABSTRACT
The effectsof feedingbreast-nijk(BM>, commercialformulas(F), or evaporated milkformula(EM) on growth,thiaminstatus,erythrocytephosphatidylethnnolarnine (PE) fattyacid compositionand visualacuity in 100healthyfull terminfants were determined.
Growth,thiaminstatus,and erythrocytePE fattyacid compositionwas determinedat birth, 3,and 6 monthsof age.Thiaminstatuswas assessedby determiningthe erythrocyte transketolaseactivity(TKA)and the fattyacid compositionofmilkand blood wasdeterminedby gas-liquid-chromatogr.aphy.Preferentiallooking acuitywas assessed at 3 and 6 months of age usingthe acuity card procedure.
Then:were no significantdifferencesinweight,length,or head circumference betweenany feedinggroups. However,dailyweightgainbetweenthree and six months was significantlylowerfor the BM group (weightgain:~±SO,BM"15.5±4.0 g,F=
20.4:I::5.8g, EM=20.2:t 6.8 g, II<:0.05).As well,breastfedinfants had slightlylower headetreumfereneegrowthvelocitybetweenthreeand six monthsof age.
Therewere no differencesinthe thiaminpyrophosphate effect betweengroups, however,8M infantshad significantlylowertranskctolaseactivitythan the F group whichcorrelatedwithenergybut not thiamin Intake.
The breastmilkobtainedfrom mothersin the study containedon average(%total fattyacids by weight),12.1%linoleicacid (18:2(n-6»),2.1% linolenic(18:3(n-3)),and 0.2%docosahexaenoic acid (22:6(n-3» .Infantsinthe F groupconsumedeither Similac or Enfalac.Similaccontained30.5%18:2(n-6),and 4.9%18:3(n-3). Enfalac contained
17.1%18:2(n~6)and 1.8%18:3(n~3J.EM contained(%totalfattyacids)2.1% 18:2(n-6), and 0.8%18:3(n-3).Onlybreaatmilkcontained22:6(n.3).
Inthe circulation,Ffedand EM fedinfantshadlower arachidonicacid (20:4(n-6»
at3 months{pc0.05)than8Mfed infants(_::I:SO,20.3:2.8,8M.18.3::1: 2.7,F,18.7±
2.7,EM,%totalfattyacids)butnot at 6 months,andEMfed infants had loweradrenic acid (22:4(0.6»atboth 3 and6months. 22:6(0-3)wasthe highestin the8M groupat both3(6.0 ±I.7,DM, 3.1 ::1:0.8, F,4.1±0.9,EM,%total fatty acids)and 6 months of age (5.3 ::1:1.6.BM. 2.9:i0.8. F. and 4.2±1.3.EM.%total fattyacids)followedbyEMthen F.Visualacuitywas higherin the DM groupthan EM {3.86±0.29 cycles/degree vs 3.29
±0041 ,3 mos,9.03±0.29vs754±0.25.6 mos,p<0.05)with intennediate valuesin the F group(NScompared to EM and DM).Differencesseeninvisual acuitymay be due to me low 18:3(n-3) inEM of 0.3%ofenergy and are nol reflectiveof22:6(n~3)in circulation.
From the resultsof the presentstudyit appearsthatEM fonnulasmay not meet theessentialfattyacid requirementsfor optimalvisualacuity,however.itmay be adequate for thiaminandoptimumgrowth. The differences seen inthe 8Mgroup in growth and TKAcan berelatedto lower energyintakes and donot indicateany deficiencies.
iii
ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr.James K.Frie' for hiscontinuedhelp, guidance,and encouragementthroughoutthisresearch project.Iwould alsoliketo thank Dr.G. Herzbergand Dr.W.Andrews who werenot only membersofthegraduate supervisal)' commitee,but also wereactivelyengagedintheproject.Dr G.Herzbergand hisresearchassistant CraigSkinnermustalsobeacknowledgedfor theirallowanceand assistancein conductingthefatty acidanalysesin their laboratory.Aspecialthankyouas wellforthe cooperation of Dr.M.Courage of the Departmentof Pyschologyand her researchassistantNatalie Finlayfor theirperfonnance ofvisual acuity testing.
A veryspecial thank you goes towards AllisonMcDonaldand Claude Mercerwho were integralto thisresearch project.
Technicalassistancefrom SoniaNolan and SeanFryeris also greatly Appreciated.
I alsoacknowledgethelaboratorytechnologistsat the Grace General Hospital, theJanewayChild HealthCentre,and theCarbonearGeneral Hospital as wellas the hospitalsthemselvesfor theircooperationandclinicspace.
Mostimportantly,I wouldlike to thank the parentsand theirinfantswho participatedinthis study,for withoutthem the studywouldnot have been possible.
Thisresearch was fundedbyHealth Canada(NationalHealthResearchand DevelopmentProgram,NHRDP).
iv
TABLEOF CONTENTS
ABSTRACT .
ACKNOWLEDGEMENTS CHAPTER1.0 INTRODUCTION.
1.1 History or infant feeding.
1.1.1 Advancements
...(ii)
.. (iv)
. 1 ...I ...2
1.1.2 Evaporatedmilk introduction... . .4
1.1.3 Prevalenceof feedingpractices 5 1.2Growth and developmentininfancy .. 1.3Thiamin . 1.3.1 Absorption and Transport.. 1.3.2 Deficiency... 1.4 Fattyacids . 7 ..9
...10
...10
.•...12
1.4.1 Fatty acid nomenclatureand defInitions... .12
1.4.2 Metabolism andsupply.... . 13
1.4.2.1Endogenoussupply... . 13
1.4.2.2Dietarysupply. ...16
1.4.2.3Regulationofpolyunsaturated fatty acidsynthesis.... ..17
1.4.3 N·6 Fatty acids.... ...19
1.4.4 N· 3 Fattyacids... . 21
CHAPTER2.0 PROBLEM STATEMENT 26
3.7Fattyacidanalyses ..
CHAPTER 3.0 METHODS 3.1Subjects 3.2Protocol 3.3Dietaryintakes.
3.4 Anthropometry 3.5Visual acuity ..
3.6Sample collection . 3.6. 1 Milk 3.6.2Blood...
....29
...29
... ...30
. 30 ..., 32
...32
... 33
... ...33
. 33 .. 34
...•...51
. 53 ...34
...37
...37
...41
...43
..."" ...43
... ...43
...45
..51
...51
4.4.1 Dietary intakes " ..
4.4.2 Transketolase activityand TPPe . 4.5 Fatty acids... .. ..
3,7,1 Erythrocytelipid"
3.7.2 Milk ..
3.8Thiaminassay . 3,9 Statistical analyses.
4.4 Thiamin..
CHAPTER 4.0 RESULTS 4.1Subjects...
4.2 Dietaryintakes...
4.3 Growth,
vi
4.5.1 f\.tilk .53
4.5.2 Erythrocytephospholipid _ 59
4.5.2.1Saturates...•....••...•..•.M 59 4.5.2.2Mcncunsaturatedfaay acids _ 62 4.5.2.3N-6Fatty acids
4.5.2.4N·)Fatty acids .
4.6Visual acuity ..
62 ...64
.. 64
CHAPTER 5.0 DiSCUSSiON . ...10
5.1Growth ..
5.2Thiamin ..
...10
. 13
5.3Fatty acids 75
5.3.1 Milk composition 77
5.3.1.1Hwnanmilk 77
5.3.1.2 Conunercialformula M 18
5.3.1.3Evaporatedmilk 19
5.3.2 Clinicaldeficiencysigns &0
5.3.3 81000... . 80 5.3.3.1Circulatingfatty acid composition
is afunctionofbolhfattyacidratios and absolute intakes... ...80
5.3.3.2 N·9Fattyacids , .. ...81
5.3.3.3N·6Fattyacids " 83
S.3.3.3.1linolc:icacid(18:2(n-6» 83
vii
5.3.3.3.2Arachidonicacid(20:4(n-6»...•.•..84
5.3.3.3.3 Adrenic acid (22:4(n-6) 85
5.3.3.4N-3 Fattyacids...•...•..~...•....•••...86 5.3.3.4.1a-Linolenicacid(l8:3(n-3» _ 86 S.3.3.4. 2 Eicosapentaenoicacid(20:5(n-3» 86 5.3.3.4.3Docosapentaenoicacid(22:5(n-3+n-6 » 87 5.3.3.4.4Docosahexaenoicacid(22:6(n-3» 89
CHAPUR6.0 CONCLUSIONS 95
REFEREN CES 98
APPEND IX A 108
APPEND IX B 110
APPENDIX
c
117APPENDIX D...•...•...•...•...•••••.••••...•••...••....•...•...•.119
viii
Figu re 1.1 1.2
1.3 4.1 4.2 4.3 4.4 4.5 4.6
Listof Fieuru
P. gt
Structure oCThiarnin. 10
Str'UCtW'eSof theMajorFatty Acids _._ 14
MajorPathways ofF_ttyAddBiosynthesis 15
Erythroc:yIe Transkelolase Activity 55
ThiaminPyrophosphate Effect. 57
N·6 FattyAcids... . 63
N.3FattyAcids... .. 65
Visual Acuity... .. 68
FrequencyDistributionof VisualAcuity Scores 69
ix
Table
ListofTables
Page 3.1 Incub ation Chart forDetenninationofErythrocyte
Transk etolase ActivityandtheThiaminPyrophosphateEffect ....39 3.2 DeterminationofPentose UtilizedinTran sketolaseAssay... ..40 4. 1 Characteristicsof SubjectGroups .. ...44
4.2 MacronutrientIntakes Per Day .. .. .46
4.3 Weight and Weight Gain Velocity... ...47
4.4 LengthandLengthGainVelocity... .48
4.5 HeadCircumferenceand HeadCircumference Growth Velocity .49 4.6 ZScores... . 50 4.7
4.8
ThiaminIntakes ..
TransketolaseActivity , .
....52
...54 4.9 ThiaminPyrophosphateEffect (1'PPe)...
4,10 FattyAcid CompositionofBreast Milk, Formula,and Evaporated Milk ..
4.11 ErythrocytePE Fatty Acid Composition ..
4.12 VisualAcuity .
....55
..58 ...•...60
.. 67
CHAPT E R 1.0 INTRODUCTION
1.1 Histo ry of infant feeding
Successfullactationhas beena requirementfor survivalamongallmammals.
However.recently,hwnans have beenan exception.Whilealternate[cedinghas occurred ona large scalein the past 60 yearswithapparentsuccess,thisis an extremelyshorttime whencomparedwiththe overallhistoryofhwnan lactation,As such.itshouldbe recognizedas a relativelyshort term biologicalexperiment.with wtknownlongterm consequences(Nevilleetal,1983).
Althoughitis assumedthatalternatefeeding to breastmilk is a recent development.feedingbettleshavebeen foundinexcavationsas farback as 2000B.C.in France,and in the Nile Basindatedaround 500 B.C.A perforatedcow'shomwas first mentionedinthethirdcentury in the TalmudandbecamecommonintheMiddleagesin thepeasant classes.In1565,the feedingof goat'sor cow'smilk throughahom afterthe thirdmonth oflife was recommended(Wickes,1953).
The onlyalternatives to breastmilkbefore the nineteenthcenturywereeither animalmilkora formof starchygruel.Thetintdehydratedmilkswere madein1855.
witha patentfor a processof slowlyconcentratingsweetenedcow's milk.Nestlefirst producedcondensed milkintinbottlesin1866.followedby several other companies.
By1883 there were27 brands of patentinfant food (Wickes, 1953).
Withthis increaseinartificial infantfood, the mortalityratesalso increased.The mortality rateinParisin 1777was 80%forinfants artificially fed,and 59-82%in Berlin
versusonly9%forthose breastfed in thenineteenthcentury (Cone, 1976) A.lthough othermodesofinfantfeeding wereattempted, breastfeedingwasstill themost common mode weJlinto thetwentieth centuryaccompanied bylate weaning ages.In a surveyof UScitiesbetween1911-1916,58percent stillbreastfedat the ageof oneyear [Fomon. 1974).
1.1.1 Advancemen ts
Inthe40yearperiodbetween 1880 and1920.several major advancesoccurred which madebottlefeeding botheconomicallyfeasibleandreasonablysafe forinfants.
Theseadvancesweremainly (Fomon,1974):
I)saferwatersupplies andofsanitarystandards formilk storage and handling;
2)development of easily cleansed andsterilized bottles andnipples;and 3)alteration of curd tension ofmilk.
I) Safety of waterand milk.Withthe chlorinationofwaterandgarbagedisposal improvements,watersafetywasmuch improvedinthelate1800's,Aswell.general sanitationwasimprovedwith theidentification of the colon-bacillusand the determinationthattheorganisms causingbloodydiarrhea wereofthedysentery group.
Somecontrolled.;leatingr;,fmilk was introducedinGenn anyandtheUS.but pasteurizationwasnotintroduceduntilmuchlater.Bacterialcontamination was therefore a problemand itwasnotuntilthe early part ofthe twentieth century,withtheadvent of thekitchen icebox,that the storageofmilkbecame feasible.
In1920. the reduction of bacterialcontentbythe acidificationofmilkwas
attempted.Originally,thebenefitsofthisprocesswasthereductionofitsbuffetin g capacitywhichwasthoughttopromotebacterialgrowth, decrease the flowof'pancreatic juiceandbile,andinhi bit gastric secretion. However, itsmost importantbenefitwas actua llyadecreaseincurd tension.
Inthe early 1900's condensingof milk was patentedwith sugar added to increase keepingqualities.This wasused widely ininfant feeding.However,it wasfound to be unsatisfactoryas aninfant food probably due10thehigh caloriccontent. Thesanitary open top can allowedclean fillingsothatevaporated milkcould be marketedin cans.
However,evaporated milk wasnotwidelyusedininfant feedinguntilthe 1920's.
2)Feedingdevices.The feedingdevices consistingofspoutedpots made of pott ery,pewter,orsilve r used until thelateeighteenth century were difficultto clean and cumbersome.Glassbottlesenabledmuch more thorough cleaning.Nippleswere formerlymadeofa tannedheifer'steal, cork,wood,ordecalcifiedivory.In1864,a patentfor a rubbernippleplaced at the end of aflexible feeding tube was obtained.
Rubbernipplesdirectlyattach edto narrowmouthed glassbottles wereinuse bythe beginning of the twentiethcentury.
3)Curdlension.Whencow'smilkenters thestomachthehydrochloric acid presentcauses the precipitation(coagulation) of thecaseinand Calcium ,whic his caned thecurd.Theremain ing watery portion containsmost of the lactose and wheyproteins.
When coagulated,cow's milkhas avery high curdtensionand causes gastrointe stinal disturbances.Fresh human milk.however,contains little caseinand fonns a soft floccul ent curd.The processingof cow's milkby acidilicalion,dilution,boiling,
modificationof mineralcomposition.andtreatment withenzymes,alldecreasecurd tension,makingthe milk moredigestible forthe infant. Theseadvancementsinthe processingof cow's milk led thewayforthe wide acceptanceof evaporatedmilkand the declineof breastfeeding.
1.1.2 Evapor atedmilk intreduetlcn
The introductionof evaporatedmilk in thelate1920's washeraldedby leading pediatriciansfor its digestibility.Marriott andSchoenthal.prominent pediatriciansat the time, reportedthe followingin a studyin1929:
...evaporated milk mixtureswereunifonnilywelldigested .. There were no cases in whichitwas foundnecessaryto substitutesome otherfonn of milk fortheevaporatedmilkbecauseofuntowardsymptomsor failure todo well.
Theresultsofevaporated feedingsofnewbominfantsappearto us toindicate that this form ofmilk isreadily digestibleand well utilizedby veryyoung infants (Marriot,etai,1929).
Evenmuch later, in1966, Dr.BenjaminSpeck.in hisinfant feedingbook promotes breastfeedingas a firstchoicebut if it is not convenient,he writes;
There isnothing mysteriousaboutaformula. Itis usuallya comblnarioaof evaporated or pasteurized.fresh milk,waterand some added auger to make it a littlecloserto breastmilkIn its composition.. doctors have been able,
graduallyto testout simplerfonnulas...whichhave worked wellandhave madeinfant feedingmore fun forbabiesandmothers...Evaporatedmilkhas become increasinglypopularbecauseitis inexpensive.convenient to keep, safe. easily digesledbymostbabies(Speck.1966).
Theacceptance ofthe medical establishmentoffonnula feedingandthelack of breastfeedingpromotion allowed breastfetding10decline.thusaffecting boththe nutritionalstatusofinfants andcausingprobable economichardshipfor families.
1.1.3 Pre valence o((ec dingpraetiees
Theprevalence ofvarious feedingpracticesdifferswith bothregional variation and incomelevel. Damcollectedduringthe Nutrition Canadasurvey of 1970-1972.
reportedthat 24% ofthe 895infantssurveyed consumedevaporatedmilk.There was showntobea markedrelalionshipbetweenincome levelandtypeofmilk consumed.Of thoseinfants fedevaporatedmilk. 52%\\''CTeinthelowest incomegroup,22% inthelow income group.and only80/.inthe"other"group(Myeres.1979).
Therewas alsoaregional variation.with fhe Atlanticprovinces and Quebec havinga muchhigherrate of bonle feedingthan the Pacificregion.Evaporatedmilk formulas werefed morefrequentlyin theAtlanticprovincesthan in the rest of Canada.
InAtlanticCanada.41%ofinfants were fedEM at oneweekof age (Myeres,1979).In the USin1972,fewerthan S% werefed evaporatedmilkand about 70%were fed co mmercial formulas(Martinez.1976), a drop froman estimated 800/.in1960 (Cone.
1981).By 1979,in a studydone in Montrealand Toronto,71%nfinfantswere being breastfedatImonthof age.Themeanduration ofbreastfeedingwas3.5months.The rest consumedformulawithwhole cow'smilkaddedto the dietsgraduallyafterone month.Evaporated milk wasnot a factor.ItWITSconsumedbyonly 2%of infantsat 12 monthswith no apparent usage beforethis time(Yeung,1981).
InNewfoundland,the use ofevapo rated milkininfantfeedingdid notbecome widespreaduntilafterconfederationin1949due,inpart,to theincreasedflowof governmentmoney.BeforeConfederation,breastfeeding wascomm on,but by1960.
almostall infantfeedingwasevaporatedmilkWith thisrapidincrease in the use of evapo rated milkcame a sharpriseininfant ilescurvyinNewfoundland, with 77cases reportedin1959,Of the 40ofthosc 77who answereda survey,nonehadbreastfedtheir infants,and all hadfedevaporatedmilk.Thefigure forbreastfeedingwas considered to beaslowas 10%.Part of thedecline was associatedwiththeadvice bydoctorsto stop breastfeedingwhentheincidence of tuberculosiswashigh,thuscreat ing insome women'smindsa relationshipbetweenbreastfeeding and tuberculosis.As well, breastfeeding wasconsidereda signofpov ertyheldover from thedepression years (Seve rs et al,1961).
Altho ughitwas knownby doctorsthatbreastfeeding prevented infantilescurvy, educationwas consideredtoo slow a processwith segmentsofthepopulation too hard to reach. Thereforeit was recommendedto supplementevaporatedmilkwith vitaminC instead ofpromotingbreastfeeding(Severset ai,1961).InMarch, 1964, the Federal Food and Drug Regulationswerechangedto allowvitamin C tobeadded to evaporated
milk,andthe incidence of scurvydeclined(Severs,1964).Withthis supp lementation, evaporated milk came to be seen as aprotection againstscurvy.even forinfants.This may havefurther encourageditsuse in infant feeding.
Whiletheprevalenceof evaporated milk feedingdeclinedinthe U.S.andCanada.
itremained commoninNewfoundland.InNewfoundland in1978,Mackey andOrr foundthai.inhospital, 17%ofinfants werebreastfed, 60% receivedform ula,and 220/, receivedevaporatedmilkfonnula.Aloneweekof age, 4]% of the metropolitan. 47%of theurban. and 72%ofthe ruralinfantswere consumingevaporatedmilk(Mackey and OrT,1978).
By1992,Matthewsetal(1992), foundthatevaporatedmilk feeding,although having droppedsubstantially,remainedconunoninNewfoundland.This wasparticularly truein infants of lowerincome,less educatedmothers.By onemonth of age, 32%,55%.
and14%were fedbreastmilk, commercialformulas,and evaporatedmilk.respect ively.
At fourmonthsof age.evaporatedmilkfeedingrose to21~Gof infantsandbreastfeeding dropped102] %.This breastfeeding Tateis about halfthe nationalaverage.Itis therefore evidentthai thereare culturaland traditional factorsinvolved,as wellaseconomica l, in Newfoundlandfeedingpractices.With a poor economy,itis doubtful thatthe useof evaporatedmilkwill decline.
1.1 Growth and development in infancy
Therate of gaininbothweightandlengthisfaster during thefirstyear oflifethan atany otherage. As seenby growth curves.the earlymonthsshow veryrapid growth
followedbyagradualdeceleration towardsthe endoftheyear.By 12 monthstheinfant's we ightwillbetripled,and lengt h increa sed by 50%.Bodyrat increase sto 25%of body weight,withbody water decre asing. Ph ysiologica l and metabolicfunctions. such as renal function, stomachcapacity, anddigestiv eability,stabilize.Neurom uscular,social and psy chologic aldevelop ment,also occurrapidlyat thistime (Beal,1980).
Anthr opometricmeasure ments areusedwidelyin the assess mentof nutrition al sta tus.Nutr itional anthropometrybasbeen defm ed as:
...measureme nts ofthevariation s ofthe physicaldimension s andtheglOSS com position of thehumanbod yatdifferentagelevelsand degre esof nutrition.(Jelliffe,1966)
The semeasurementsarcpartic u larly importantwhenthere is achro n ic imba lance betweenintakes ofprotein andenergy.Anthropo metric indicesare drawnfromeithera sing le measurement,such aswei ghtfor age, or from acombination,such asweight and height.Referencedatacanbeobtainedfromeitherlocalor internation al sour ces. Local referencedata iscom piledfrom a localelitegroupof healthy, well- n ourished individu als, whe reas internationaldataiscompiledfroma cross-sectio nalsamp le, usingwell stan dardized procedur es,withthemajority of thesample populationobtainin gits full growthpotential(Gibson. 1990).
The WorldHealthOrgan izationrecommend stheNationalCent refor Health Statistics (NCHS)referencegro wthdataasan international standardsinceit meetsthe criteria ment ionedabove.The valuesl'Jrweight,length,and weighttorlength from birth to 36 months aretakenfrom longitudinal datacollectedby theFels ResearchInstitute
betwe en1960and 1975.Themeas urement s were takenfrom720 wh it emiddleclass infan tsandch ild ren,wit hthe maj o rityfed proprieta ry formu la-based pr o ducts (Gibson, 1990).
However,theap propriate nessofthese chartsas aninte rnational standard has been questi oned. Recent ev idencefrom Britain,Canada, Australia, Finland, andthe US has shown thatbre astfed ba b ies matchthegrowth chartsuntilthree monthsof age.However, this is followe dby reducedgrowth velocitywith breas tfedinfants fallin g below the medianat6-9 months(Whitehead etal,1981,Chan dra,1982,Hitchcock etal, 1982, Duncan et ai,1984,Salmo npera etal,1985 , White headet ai,1984,De we yer el,1992).
Thelower growth ratesand energyintakes found inbreastfed infantsis notassociated withdetriment al conseq uencessuc has red ucedact iv ityleve l,increase dmorbid ity,or differingbehav iouraldevelopme nt (Dewey , et ai, 19 91).Thes eresults suggest that NCHSgrowthchartsmaybeinapp ropriatefor breastfed infantsand nee dtobe revised.
1.3 Thiamin
Thiaminisa wate rsolublevit aminwithext ensive bio logicalrol e s(Figu re1.1), As a componen tonhecoenzymethiaminpyrophosphate,itfunctionsasa coenzymein bioche m ical re a ctionsrelatedto carbo hydra te metabo lismlea d ing tothe formatio nof carbondioxide(Yeung,19 83).This includesthe oxidativede ca rboxylation of a- ketoaci d s and pyruvate, transketol a se reactionsofth e penrose phosphate:pathway , as we ll as actingas a coenzyme infatty acidsynthe s is.Itisalso involv ed inthe decarbo x ylation ofbranc hed ch a inammo acids. Thia min may alsohave a noncoenzyme ro leinneur onal
membraneexcitability(Tsang,et al,1988).
Figure 1.1 Str uctureofTblamin.
1.3.1 Absorptio n and tra nsport
Thiamin absorptioncanbeboth active or passive.dependingon the concentration.
Atlow concentrations.itis active,occurring through a Nat-dependent,carrier-mediated process primarily in thejejunum. At high intakesabsorptionis mainlypassive. Therate of absorptionis quitehigh. exceptin the cases of excess ethanol consumptionorfolate deficiency.Thiaminis convertedto its activeform,the phosphateesterthiamin diphosphate(TOP)(commonlyknown as thiaminpyrophosphate(TPP)) in the mucosal cells, and is thenreleasedinto the plasma.Inthe brainTOPcan alsobeconverted to thiamintriphosphate(TTP) (Huntetal,1990).
1.3.2 Defidency
The primaryresultofthiamindeficiencyis impairedcarbohydratemetabolism, manifesting itselfinberi-beri.Infantile beri-berimay occurbetweenone and fourmonths
10
ofage (Tsangetal.,1988),and is characterizedbydiminished urinarythiamin excretion, progressiveedemaandacutecardiacfailure.oftenresultingindeath.Severethia min deficiency indevelopedcountriesis rare,withonlytwo casesreported in Canada andthe US since195&. The causeineach of thesecases wasa changeto unsupplemente dsoy milkformula,which containedalmostnothiamin(Davis,et al, 1958,Cochrane,etel, 1961),Beri-beri hasalso beenfoundin breastfedinfants of thiamindeficient mothersin Asian countrieswherepolishedriceis the staplefood(Thangangkul, 1966).However, theincidenceof moderate deficiency hasnotbeenstudiedmuchininfants andyoung children.Thisgroupmaybemost atriskforlongtermeffectsofthiamin deficien cy becausesuch defic iencymayimpairmyelinat ioninthe rapidlydevelopingbrain(Haas, 1988).
The measurementof transketolaseeIU:)11le activityinerythrocytesis currentlythe most reliableindexofthiamin status as it givesanindicationof theadequacyofbody stores of thiamin(Gibson,1990).Measureme nt ofthe activityoflhisenzymein erythrocytesis most frequentlyusedsincethe erythrocytesare amongthe firsttissuesto be affected bythiamindepletion(Brio,1967).Inthiamin deficency,the basalacit ivityof transketolaseis low,andtheadditionofTPPwillproducean enhancementofenzyme activity.The percentincreaseinactivityis known asthe "TPPeffect".A TPPeffect of
<15% is considerednormal,and>25% is considered deficient(Brinet ai,1965).
Theurinary excretionofthiamin isanother commonlyuseddeterminantof thiamin status.Itis notconsideredas reliable:as theTPP effectas itis onlya reflec tionof immediateintake andmaynotbeareliable indexoftissue stores,distributionoractual
11
biochemicalfunctioning.The excretionor thiamindecreasesproportionatelytointake untilacrit icalpointafterwhichonlyvariable andminorchanges ofexcret ionoccur.
Thiaminintake,tranketolase activity,andurinaryexcretionofthiaminare known tobe relatedatintakes below30!J.g!kcal(Sauberlic h ,1979).Therequirementfor allage groups,therefore,witha safety factorincluded,is40!J.g/keal(Nutrition Reconunendations, 1990).
1.4 Fattyadds
Fat isacritical componentin aninfant'sdiet,comprising50%ofenergyinta ke. Besidesbeing amajorenergysource,itisan almostunlimitedfonnorenergystorage, and actsas avehicle forabsorptionand transportoffat solublevitamins.Italsosupplies fattyacids ofthe0-3and0-6 serieswhichcannot besynthesizedinmammaliancell s.
Dietarylinolenic (18:3(n-3» andlinoleic(18:2(n-6» acidsfulfilltheserequirements and can bedesaturated and elongatedto the morebioactiveproducts .Theserattyacids are requiredfor nonnalgrowth,cellmembranecomposition andfunction,andas precu rsors for importanthormonal substances suchasprostaglandins andleukotrienes.Howe ver,it is importan t tonote thatquantitatively(76%inrats),theirmajormetabolicrouteisp- oxidationtoCO~(Cunnaneetel.1995).The central ne rvoussystemis 50%lipid,second only10adiposetissue,with most ofitstructural, composedmainl y ofthe deseterated and elongated productsoftheessentialfattyacids.Thispredictsan essentialfunctionalrole inthe neural development of thegrowinginfan t(Moya, 1993).
12
1.4 .1 Flttyaeid nomelltlature and deflaldcns
Thenomenclaturefor fattyacidsusedwillbethe numberof carbonatoms followedbyacolonand thenumberofdoublebonds.Thefattyacid familyisidentified bythedistnnceofthe firstdoublebondfromIhemethyl end(n-scarbens)of thefatty acid carbonchain.The positionwheredesaturario noccurs andtherespectivedesaturas e enzymeisdesignated bythe carbonnumberfrom thecarboxylend(6.).Thetermlong chain polyunsaturatedfatty acids(LCPUFA)is used todescribefatty acidscontaining20 Dr22carbons, andgreaterthan2doublebonds,Thestructuresandnamesofthemajor fattyacidsare infigure1.2,
1.4.2 Metabolisman dsupply
1.4.2 .1 Endogenoussu p p\y
Fattyacids can originatefromva rioussour ces.Theycanbeproducedde novo from acetylCoA,whic hisproducedinmitochond riafrom carbohydrate(viapyruvate oxidation), amin oaciddegradation,or fatty aciddegradatio n.Thisbiosynthe sisoccursin manymammali ancellsinvolvingthesequential additionof two carbon units,usually culminatinginpalmiticacid(16:0).Themajorpathwaysof LCPUFA biosynthesisare shown infigur e1.3,Palmiticacid canbe furtherelongatedto stearicacid(18:0)ineither mitoc hondriaorinmicrosomes(Ral'm.19 89).Altemae ly, inlesscommonroutes,it can bedesaturated andelongated orbesimplyelongatedtolongchainsaturated20·26carbon fattyacids(Cook,1991 ). Desaturationandelongati ontakesplaceon theendo plasmic
13
HaC ~COOH
a -Unolen icICId(C1B:3,N-3)
H.C ~COOH
Elcosapentaenolcacld (C20: 5 ,n-3)
H .C ~COOH
Docosahexaen olcacid(C22:6. n-3)
Arachidonicadd(C20:4,,..s)
H.C ~ COOH
Un o lelelcid(C1B:2. n-6) Figure1.2 Strue auresoftheMajor FairyAcid"
14
DeI OVOordlf'f:
Fromdiet:
16:O--+11 ~20~2 2:O--t 24:o-26 :0
1
N·huies N·JSCM N-hrnn
18:2{a-6) 18;3(n-J) 18;I(I·91---t lO:I(n -9 }-f22;1(n-9)-+24;1(n-91
1
11:3(n -6 )
1
se rieJ 2eicosanoids- 20:3(n-6 )
1 1
18:4(n·])
1
20:4(n·)
1 1
18:2(n·9)
1
20:2(n·9)
1
tt.6 dcsaNratiOll
Elongation
tt.5desatur ation series2 eico sanoids.--10:4(D-6) 20:5(n·3) 20:3(n-9)
series2leu kotrienes 1- scriesJeicosanoids.serin5leukotrienes
1
U:4(o-41
1
24:4(n-6)
!
24:S{n-6)
!
22:5(n·6)
1
Elongation22;5(11-3)
J
EIonp!ion24;5(~J)
1
tt.6 desalUraliOll24:6f.·])
1
p-oxldalion22:6(D-3)
Figu re1.3 MajorPathwaysoffat tyAdd Biosynthesis.
{AdaptedfromCook.1991.Innis,1992,andSp recher,1992)
15
reticulum membranes ofmany tissueswhichcontainelongatingenzymes, as well as 6.9, 6.6,and 6.5 desaturases.Theseoxygen-dependent,ironcontaining desaturesesare key amphipathicenzymes assembledin an electrontransportsystemthat contains the flavoenzymeNADH-cytochromebsreductase,andcytochromebs[Rawn,1989).
The6.9 desaturaseisthegatewayforproductionofthe n-9family of fatty acids, the onlyendogenouslyproduced polyunsatwatedfattyacids.Stearicacidcan be desaturated bythisenzymeto oleicacid(18:l(n-9»and eitherfurther elongatedand desaturated toeicosatriencicacid (20:3(n-9»or just elongatedto variousminor monounsaturatedproducts.20:3(n-9)can partiallyreplacefor some physicalfunctionsof the n-6 and n-3serieswithin membranes.howeveritisnot a precurserofprostaglandins and cannot alleviatesignsof essentialfatty aciddeficiency(Cook. 1991).
1.4.2.2 Dietary sup ply
Many of the abovementionedendogenously producedfatty acids can alsobe suppliedin the diet.However.mammalian cellscannot endogenouslyproducefatty acids ofthen-3and n-6 series.This is due to theabsenceof the 6.12 and 6.15 desaturases requiredto producethe0· 3 and 0-6 seriesof fatty acids.Onlyplants and insects havethis capacity.Fattyacidsoflheseseriesprovide muchof the fluid coreof cel!membranesand arctheprecursorsfor eicosanoidandprostaglandinbiosynthesis (Cook. 1991).
Therefore,dietary sourcesare essentialand must beprovidedinthe dietprimarilyas the precursorslinoleicacid (18:2(n-6» andlinolenicacid(18:3(0-3»ortheirlonger chain derivatives.Alternately,the fatty acids.16:2 (n-6)and16:3(0-3).found in small amounts
16
ingreenvegetab les.canbeelongated to18:2(n-6)and 18:3(0-3)respectively (Sprecher, 1968,Cunnane etai, 1995)
For infants, a varyin g supplyof18:2 (n-6) and 18:3(n-3)fany acidsisprovidedin themilksupply.Formulas, whichare vegetable oil based. containonly the 18 carbon essentialfattyacids.However,animal based milk, suchashumanmilk,else cont ain the longer chainderivatives (20and22carbonchain).After absorpt ionthe fattyacidsare incorpo rated intochylorrucronsin the intestineandreleased into the circulation.The fany acids are takenup bytheliver where theymaybeelongatedand desaturatedinto other products. Repackaging then occurs intolipoproteins,andthey are released into
plasma (Bazanet al, 1993 ).Organs such asthe brain,retina.and liver selectively uptakerequiredLCPUFA from the circulation.although theydo have limitedcapacityfor synthes isof thesefatty acidsfrom theappropriateprecursor.Redblood cells, often used as amarker to determinetheavailabil ity offattyacids forincorpora tion into memb ranes.
donot contain theenzyme snecessaryfor elongation and desaturationof fattyacids.
However , they incorporat e significant amounts of LCPUFA's through theturnover of phospho lipidfattyacids.This occurs through deacylationandreacylatio nofthe phospho lipid fatty acidsfromplasma. as we ll asthemuchslowerexchange of intact phospho lipids(Innis,1992 ).
1.4.2.3 Regulat ion ofpo)yunssturatedfatty acid synthesis
Since 0·6,0-9.and n-3fatty acidsal\rely on the same enzymesinthe microsomal electro ntransport system ,thereis competition among potential substrates foragiven
17
desaturaseenzyme(lnnis,199 1).As seeninfigure 1.3, the threefamili es offatty acids all undergosimilarreactions,alte rnatingbetweenelongation (the additionof a two carbon unit) and desaturation.18:1(n-9),fonned endogenously,18:2(n-6), and 18:3(0-3), obtained fromthediet,all comp eteforthe.6.6 desaturase,whichacts at carbon 6-7.
Preferential desaturationoccursinthe order18:3(n-3)>18:2(n-6)>18: l(n-9) (Brenner,1974).Theserelative affinities explain the rise in 20:3(n-9)seen inconcurrent 18:3(n-3) and 18:2(n-6) deficienc yand its nearabsenceunder conditionsof adequate essential fatty acidstatus. As aresult of this competitionfor the same enzymere latively higheramountsof tissue 0-3 derivatives are produced withalow dietaryn-3/0-6ratio, suggesting thatthebalanceas wellas absoluteamountsofeachseriesaffects the ability for synthes isof longer chainderivatives. The rateof .6.6 desatura tionis also regul ated by the concentrationof the reactionsubstrateand product.High amounts of substrate inhibit enzyme activity,whea reas low amounts of productinduce enzyme activity(Brenneret al, 1966,1969).
Dietary intakeof mineralsalso isa factorin LCPUFA biosynthesis.Lowzinc intakeimpairs A6desaturatio n,reducing 20:4(n-6) as well as20:3(n-9)production {Cunnane etal, 1995).As well,high copper intake increasesthe fonnation of20:3(n-9) evenwilhadequate18:2(n-6)intake (Cunnane,1985).Thereforea diet which is both low in zincand coppermay resultill impaired20:3(n -9)production .
The liSdesaturaseacts atcarbon 5-6,produc ing elcosapentaenolcacid (EPA-20:5 (n.3)andarachidonicacid (AA-20:4 (0-6 )), immediate precursors tothe prostaglandins.
Ithas beendemonstratedthat .6.5desaturationof therespective fatty acidpathwaysis
18
inhibitedby 18:2(n-6)and18:3(n·3) (Brenner, 1974). Thus it appearsthat thereis a controlmechanism to ensure that excessiveamounts ofn-3or n-6LCPUFA'sarenot formedinthe presence of exceptionallyhighamountsof18carbonprecursor.
IIwasoriginallyassumedthatdosatutationatthe .6.4positionoccurredthrougha .0.4desaturase. However, itwasrecently determinedthattwoelongations from 20 to 22 carbons and 22 and24 carbonproductsoccurs, followedby.6.6desaturaucnand~
oxidation inmitochondriaand/or peroxisomesto 22:6 (n-3)and22:5 (n-6)(Sprecher, 1992).
1.4.3 N-6racty acids
Theessentiality of linoleicacid was firstestablishedin 1929byBurr andBurr in experiments in whichyoung ratswerefedfor severalmonthsonafatfreediet.Growth retardation.scalydermatitus,increasedtransepidermalwaterloss andreproductivefailure were fOW1d (Burr,et at.1929). Laterthis essentialitywasestablished in human infantsby Hansenand others.Infants were maintainedforup to severalmonths on dietsbasedon skim milk containing<0.04 to0.07%of caloriesfrom linoleicacid.Theyquickly developedlargestools,and withinweeksthe skin becamedry, thickenedand later desquamationwithoozing occurred inthe intertriginousfolds. Dietaryadditionsof linoleicacid orfoodcontaining linoleic acidrelieved or preventedthe condition. Diets based oncow's milk.evaporated whole cow's milkformula,or skim milkwith added butterfatdid notproduce ihe clinicalconditions outed above and actuallyacted as therapeuticdietsfor recovery(Hansen et al, 1963).These studies. however, did not
19
involveintakesinthe crucialrangeof 0.07and1.3%kcatas linoleicacid,thereforea moreprecise cut off of when clinical signsensueisunknown.
Biochemicalindiceshave alsobeen usedto determinethe presence ofEFA deficiency.Dueto thepreferential desaturation ofn-Jand0· 6fatty acids,rarelydo fatty acidsof the0·9 series accumulate.However,duringn-jor 0-6deficiency,the accumulationof primarily20:) (n·9)occurs,accompaniedby a reducedarachidonicacid (20:4(n-6»level. Studiesdeterminingthefattyacidcompositionin erythrocytes,plasma and hearts of rats haveshownthat at intakesfallingbelowabout I%ofenergy aslinoleic acid,the triene:tetraeneratiorapidly increases(Holman, 1960).However,18:3(n-3) and 18:I(n-9)were alsooften missing fromthe dietsin thesestudies,sothe specific requirementfor 18:2(n-6)is difficultto judge.
Minimalrequirements of linoleicacid havealsobeendeterminedfrom the amount needed tomaintaintissue levelsof arachidonic acid in variousratorgans.Bour-net al (1990)conducted a study of21 day oldratsin whichthe levelof linolenicacid waskept at 0.4%of calorieswhilethe linoleicacid amountwas increased.It was foundthat the liver,lung andkidneyhad thehighest requirementforlinoleicacid at 2.4% of energy after which point 20:4(n-6)didnotincreasefurther.However.a levelof 2.4%as linoleic acid was requiredto producemaximalaccumulationof 22:4(n-6)inthe brain.
Theimportanceof an adequatesupplyofn-6 fattyacids in the diet is demonstratedinautopsyresultsof infants. Thesehavedemonstratedthatthe0-6 fatty acidscontinuetobedepositedintheforebrainafterbirth,with arachidonicand adrenic acids the mostabundant(Martinez,1992).A dietarysupplyof20:4(0-6)or22:4(n-6)
20
doesnot appeartobearequirement.however, as dietswithabundant 18:2(n· 6) withouta preformed supplyproduceaccumulationintheeNSsimilarto infantsfedbreestmilk, whichdoes containpreformed sources(Makrides,1994,Farquharson.1992).Autopsy analyseshavenotbeenperformed oninfants fedreduced amountsof linoleicacid;
thereforeminimalrequirementsformaximumaccretion of n·6 LCPUFAareunknown.
The degreeto whichthese fatty acidsdecreasewithinorgansduring a deficiencyand any possiblefunctionaleffects are unknown.
Boththe amountsfound in breastmilkand triene:tetraene ratios have helped establishthe requirementsfor 18:2(n~6)to bein the rangeof1-4.5%kcal (Nutrition Recomendations.1990).The1990 CanadianRNIfor infants04 monthsofageis4.5% total energy.basedprimarilyonthe averageamounts foundin breastmilk(Nutrition Recommendations.1990).Inthelate 70'sanargumentwasmade forvaluesaslow as 0.6% keel,due to the absenceof anyreported cases ofEFA deficiency even when evaporatedcow'smilk was prevalent(about0.8%kcal as18:2(n.6»(Cuthbertson,1976).
Themuch higheramountsseeninmother's milk,incombinationwithwhatisknown aboutamountsrequired for optimumstructurallipidlevelsof linoleicor arachidonic acids, membrane associatedfunctions,andeicosanoidmetabolism,have indicatedthat linoleicacid deficiencymaybesilent,withovert clinicalsigns onlyin extremecases (lnnis,1991).
1.4.4 N-)fattyacids
The establishmentof a-linolenicacid asessentialforhumans has takenmuch
21
longerthan linoleicacid.due inpart to the lack of easilyrecognizabledeficiency symptoms.lack of increaseof20:3(n-9).the resistanceof tissues to n-3depletion.and thelow dietaryrequirementforn-J fatty acids (Innis.1991).a-Linolenic acid deficiency is mainlyof importanceduringdevelopmentsince,afterthis time.casesof deficiency have occurredonlyduringverylong periodsof intravenousnutritional supportat extremelylowlevels of 18:3 (n-3) (Carlson,1991).
A specificrolefor18:3 (n-3) has not been found.however.its elongationand desaturationproducts,primarilyeicosapentaenoic acid (EPA,20:5(n-3»and docosahexaenoicacid (DHA.22:6(n-3»performmanyessentialroles.EPA is a direct pret:uBorto eicosanoidswhichhelpregulate bloodclotformation,immuneresponse.and theinflammationresponseto injwy and infection (Whitney et al,1993).DHA isfoundin very highlevelsincentralnervoussystem(CNS) membranes,such as thevisualelements of theretinaand synaptictenninal membranes(Clandininetal,1980).
Dietslow in n-3fattyacidsasseen inWesterndietsis associatedwith increased risk of heart andinflammatorydiseasesas a resultofthe increased production of themore potent 20:4(n-6)derivedeicosanoids(Sinclair.1991).However thesignificanceof low n-Jfattyacidsoneicosanoidmetabolismin infantsisunknown.
DHA(22:6n-3)continuesto increase inthe brainuntilatleast two years of age.
Anadequatesupply,therefore, offatty acids fromthe n-J fattyacid seriesis criticalfor brain developmentevenafter birth (Martinez, 1992).The veryhigh amounts(as % total fatty acids)of22:6(n-3),inparticular,present in gray matleroflhebrain (30%).retina, especially the rod outersegment(38%). andtestes. acrossmanyspecies indicatestheir
22
importance (Salem, 1985).To determineminimal requirements,the amountof 18:3(0-3) that supports madmal accumulation of n-]fatty acidderivativesinvarious organshas been used.Inrats,0.3to 0.7%kcal8S 18:3(n-3) has producedmaximal concentrations of22:6(n-3)in wholebrainlipid, synaptic membranes.retlna, and myelin (Bcurreet 81.1989).Inpiglets,2%ofenergy as (I_linolenic acidproduces adequateaccretionof 22:6\n-3)for synaptic membranesand the retina,howeverformulas with0.4% of energy aslinolenicaciddo nOI.ascomparedtothosefedsow milk.As well,piglets fedformulas with no 22:6(n-3)containing 1.5%versus 0.75%ofenergyas 18:3(n-] )hadhigher 22:6(n-3}in the brainandliver(Innis.1992.ArbuckleetaI,1992.Hrboticky et al, 1990).
Inhuman infants.autopsyanalysisofCNStissue has shown thaI dietswitha-linolenic acidin therangeof0.5-0.8%energyhavelowercerebral cortex22:6(n-3)thaninfants brrastfed,howeverthey have similar retinal composition (Makrides etal,1994, Farquharson elal.1992).
Functional lS'iCumentinrats,primatesandbothprrtenn andfullterminfantsof dietaryn-J adequacyhas been done primarilythroughmcaurements of visualsystem development.Alink betweenvision andn-Jfattyacid deficiency was firstobserved when ayoungpatientontotal parenteralnutrition (TPN)experiencedblurred vision (Holmanet ai,1982). The relatlcnshlpnfdietaryintakeof 0-3Cany acids, organ accretion.and visualdevelopmentwas then establishedin inCantrhesusmonkeysby Ncuringeret al (1984.1986).Infantrhesus monkeyson dietsveryhigh in linoleicacid (75%totalCatty acids),hut low in a-linolenicacid(0.3% total fatty acids),had decreased accretion ratesof n·3derivatives.alteredelcctroretinographic responses,andreduced
23
visual acuity.
These functionaleffect, are explainedbyDHA',criticalrole in the visual process.
The highestlevels0(22;6(n-3)inthebody(SOmol%)arefoundinthediskmembranes oftheoutersegment'of photorcccptorcells.Thesecell, are thesiteofv ision initiation, andarcpacked withvisual pigment.Therodscontainthepigment rhodopsinwhich appearstorequire specificallytho;presence of2 2:6(n.3)for optical activity.as phospholipidscontaining22:6(n-3)associate with rhodopsinstrongly(Neuringer,1993. Deese et al,1981).
Methods commonly used to measure visualfunctionarevisualacuity or electroretinograms.Visualacuity measures corticalfunctionand electroretinography measuresretinaleffects.Electroretinogramsmeasurethe electricalsignal thatthe photoreceptorcellsemitwhenstimulated by light. Themost commonlyusedcsethcdsof visual acuityarevisual-evoked potentialacuity (YEP)and preferential looking acuity
"(pL).YEP testinginvolvesthe responseof the infant measuredby electrodesto changes
in visual panems on avideo screen.PL.mostcommonly wing the Telleracuity card procedure.involves showing an infanta set of cards withstripesofvaryingwidthon one side and a blankscreen ontheother.By observing the looking behaviourofthe infant.
theexaminerdetermineswhichside thestripesarcon(Teller.1988).Thesevisual acuity tesls arc basedon the inherenttendencyforinfantstogazeat adiscriminable pattern ratherthanablank screen,and when their.(. ;;,t can nolongerdiscerna pattern, itis consideredtobetheiracuity score(Neuringer,1993).
Studies onpreterm infantsconswningdietslacking 22:6(n.3)butwith reasonable
24
amountsofIS:3(n-3}haveconsistently demonstrated a decreasein visual function as compared withinfants fedmarine-oilbased fonnulas or breastmilkwhich contain preformed22:6{n-3}(Uauy et ai, 1992,Carlsonetal,1993).It thus appearsthat 22:6(0- 3) isessentialin pretenn infants. Pretenn infants, however,in contrastto those born fullterm,areparticularlyat risk due10 Iherapid accretionof22:6(n-3)inthe retina and eNSduring the last trimesterof pregnancy(Martinez, 1992).Infullterm infants, whether preformed22:6(0-3)is requiredinthe dietto produceoptimalvisual functionas wellor if adequateamountsof its precursorl8:3(n-3}can fulfill lherequirementshas produced mixed results, dependingon the levelof I8:3(n-3}as well as thetypeof visual testing being performed.(Makrides,1993, Innis, 1994, Birch, 1992}. Researchhas shown that full term infants fed fonnulas withoutpreformed 22:6 (n-3)containing 18:3(n-3)as 1%of energy couldmatchthevisual functionof breastfed infants(Innis,1994),however,0.4%
(Birch. 1992),or 0.5-0.8%(Makrides,1993) of energy as 18:3(0-3)does not.Therefore, moreresearch mustbedone10 clarify whether adequate 18:3 (n-3) is enoughto fulfillthe eNSrequirementsfor n-J LCPUFA's.
25
CHAPfER2.0 PRO BLEMSTATEMENT
From the1930's to the late 60's thefeedingofevaporated milk formulasinNorth America was prevalent(Cone,1981).Although evaporated milk usagehasdropped worldwide,it remainscommoninNewfoundland.Canada.due to bothtraditionaland economic reasons(Matthewsetal,1992).Evaporatedmilkformula,although adequate forenergy,contains excessproteinand lessfat than fonnulaorbreastmilk.Evaporated milkalsois low in iron,copper,zinc,selenium,vitaminsBI •K,and A and bothessential fany acids,linoleicanda-linolenic(Nestle,1991,Litov, 1991,Sanders et ai,1979).
Thiamin,inpanicular,indilutedevaporatedmilk (1:2 dilution)containsonly0.30 mgl lOOO kcalcomparedtothe recommended intakeof 0.40mgllOOOkcal forinfants (Nestle.1991).Breastmilk,on average, contains only 0.22mgllOOOkcal (Health and Welfare Canada. 1987).As there are very few studies on thiamin status in infancythis recommendationis based more on adultdatathan infant.Studies wbichhave been done did not reportdietary intakes of thiamin ortypeof feeding.Thiamin'sroleinenergy metabolism and neurologicaldevelopmentemphasizes the imponancein detennining its adequacyinvarious diets.
There has been a resurgenceof interestin recent yearsofthe fany acid requirements of infanIs whichhasledto areexaminationof the adequacy of thefatty acid composition invariousfonnulas.This has been led by the discoverythaithe circulating fatty acidcompositioninerythrocytes and plasma isknown10differin infants either breastfedorfedformulasofdiffering ratty acidcomposition(Innis,1991). Aswell,
26
visionhasbeen known to beaffectedininfants fedinadequatequantities ofessential fatty acids(Makrides et ai,1994, Uauyet el,1992). Breastmilkcontains both theessential fatty acidsand their long chain polyunsaturatedproducts (LCPUFA's), commercialformulas contain essentialfattyacidsbutdo not contain LCPUFA's (Innis,1991).LCPUFA'sare found in vel)'lowamounts in evaporatedmilk (Sanders et ai, 1979» . The central nervous system (CNS)accreteshigh amountsof LCPUFA'sduringthebraingrowthspurt which continuesuntil 18monthsofage,indicatingthatanadequatesupplyininfantsis critical (Clandinin etal.1980).
The presentstudy,therefore.soughtto compare the effects offe eding breastmilk, commercialfonnulas,orevaporated milk on growth,thiaminstatus. and fattyacid status inhealthy,fullterm infants.
The objectives ofthe presentstudywere:
I) toobserveandcompare thegrowthpatternsofallgroups bydetennininglength, weight,and head circumference
2) 10determinethe adequacyof thiaminintakes ofallgroups by:
a) comparisonofactualintakes with recommendedintakes
b) measuringthiamin status througherythrocyte transketolase activityandthe thiaminpyrophosphate effect, a functionalassay ofthiamin adequacy J) to determine theadequacyofthefatty acidcontentfor infantsby:
a) comparingthefattyacid composition of all milk and fonnulaconsumed b) comparingthefattyacidcompositioninthephosphatidylethanolamine
fractionoferythrocytes
27
c) comparingthe performanceon a functionaltest of fany acid adequacy (visualacuity).
28
CHAPTERJ.O METHODS
3.1 Subject!
Healthy.fullterminfantswererecruited from eithertheGrace Generalhospitalin St.John'sorthrough publiehealthnursesinsurroundingareas.All infants wereeligible if theyweighedbetween2500-4500grams at birth.were of3 8-42weeks gestation.had nohealthproblemsoranomalies.andinformed parental consentwas obtained (appendix A).BetweenJanuaryandOctobe r of1993. mothers ofeligible infants wereapproached beforedischarge6'01\"1hospital by the researchnurse .Theplannedmethodof feeding was thendeterminedwizhno recomme ndations oradvicegiven.Thosewith an intention to eitherfeed human milk (8 M),the commerci al fonnula'sSimilac· orEnfalac'(F).or an evaporated miikform ula (EM) exclusively for the dura lion ofthestudy(sixmonths) were asked to take part.Forattendanceal eachofthethreeandsixmonthclinics. the nlothers received aS40stipend tohelp offset costs suchas travelor childcare.
Subj ectswere recruitedinconsecutiveorder~'ntilapproximalely35 infantswere recruitedineach group.Duetoswitching of feed groups.moving. or traveldifficulties.
somesubj ects were losl fromfollow-up.Atthree months theenrollment was 8M(0'"'35).
F(0-34).and EM(0-31).withafinalenrollmentof~Oineachgroupatsixmonths.As this study is one part ofalargerstudy on the overall nutritional status of theseinfants.an adequateamoun tof blood could not alwaysbeobtained foralltests, thereforethere were incompletedatasets forsome infants.
29
3.2 Protocol
Atbaseline «3 days of age) a blood samplewas obtainedvia a heel prick before dischargeat the GraceGeneral Hospital and weight,length.and headcircumferences were obtainedfrom hospitalrecords.At threeandsix months ofage(±14 days)the subjectsattended a researchclinic atthe Janeway Child HealthCentrein St.John'sor the CarbonearGeneralHosp italwherea bloodsamplewastaken viavenipuncture.Weight.
length,and headcircumferencewere measured,visualacuity testin gwas performed,and the dietaryrecordwasreview ed.Amilk samplewas obtainedfromallmothersstill breastfeedingat the three andsixmonth sclinics and a formulasample from the othersat the three month clinic.As well.a questionnai reregardingparentalinformationsuchas maternal age, education. andsocioeconomicstatus was complet edat the threemonth clinic(appendix B).Duringthe study mothers weretelephoned atbiweekly intervalsby theresearch nurseto determin ecompliance.As well,beforeeach of thethreeand six monthclinics,the mothers wereprovided informationin writing abouttheclinic appointment.the milksample.and howtofill out the dietaryreco rd(appendixC).All blood sampleswere takenin cooperationwithstaff laboratorytechnologists at the respectivehospitalsand visualacuitytesting wasperformedin cooperationwith Dr. Mary Co urage (Departmentof Psychology).Theprotocol and procedureswere approved by all hospitaland university Ethics Committees.
3.3 Dieta ry intakes
Dietaryintakewasobtained fromthreeday dietary records. Thesewere mailed to
30
the motherand containedinstructionson howtorecordfoodintake,withcolumnsfor amountconsumed,brandnames offoods,andtimeoffeeding.Theywere askedto fill these out in the lastthreedaysbefore the clinic visit. At the clinic.thediet retard was brieflyreviewedand anyinconsistencies wereclarified. Thetimingof solid foodor supplementary milk introductionwasalsodetermined.Ifthefood record was not completed,a 24 hour recallwasperformed.The main feed, anyvitaminormineral supplements,solid foodsorsupplemental formulawererecorded.All intake was converted tograms, assigned afoodcode,andenteredontothe computerdatabase (Friel et ai,1985). Thedatabase wasupdatedwith nutrientinformationobtainedfromthe variousformulaand food companies. Asthe volume ofbreastmilkintake wasnot determined,intakewasbasedon anestimationof milk consumptionof750 mVdayat three months.At six months ofage,basedondata from partiallyandexclusively breastfedinfants, exclusivelybreastfed infants(>90%estimatedenergyfrom breastmilk) were estimatedtoconsumeon average 77kcallkg,and those who supplementedwere estimatedtoconsume 81kcallkg(Hienig etal,1994).Energyintaxewascalculated ona per kilogrambasisand energyfromsolidfood or formulaswere subtracted fromthe total, with theremainderof intakeattributedto breastmilk. Theenergy intake from breastmilk wasconvertedto the equivalentgramsandwas enteredontothecomputersothat an estimateof contibutionof breastmilkfornutrientintakecouldbedetermined.Thefatty acid composition wasdeterminedfrom analysingmilk samplesobtainedfrom each subjectin allgroupsat thethree monthclinic as well asthose still breastfeeding atthe six monthclinic.
3\
3.4 Anthropometry
All anthropometricmeasurementsweredonein triplicate hy the same two examiners. Recumbent length was measuredusinganin-house infantometer (Memorial University TechnicalServices) to thenearest millimeter.Weight was detenninedusing a pediatricspring scale with apan.A towel was placedon thescale and the weight was taken to the nearest gram when the infantwaslying quietly.Calibration was done at the start of each clinic using a 5 kg weight. Head circumference was measuredusing non- stretch tape (Ross Laboratories, Columbus, Ohio).Zscores for length for age, weight for age, and weight for length werecalculated using CASP (Anthropometric Software Program, version3.0,1987, Division of Nutrition, Center for Health Promotion and Education, Centersfor Disease Control, Atlanta,Ga).The calculation is as follows:
(Actual anthropometricvalue - median reference value)+Standard deviation(S.D.).
Reference values were those obtained from the National Center for Health Statistics growth data (Hamill et ai, 1979).
3.5 Visual acuity
Binocularpreferentiallooking (visual)acuity wastested using the Teller acuity card procedure.This test is based on the inherent tendencyfor infants to gaze at a discriminable pattern rather than a blank screen and has been describedindetail elsewhere (Teller et ai, 1988).The Teller Acuity Cards (vlstecb Inc.) consist of a set of 16 rectangular gray cards, 15 ofwhich contain a black and white square grating (stripes) embedded in a luminance-matched gray background, which is to the left or ,;Jltof a
32
small centralpeephole.Atrainedobserver , positioned55em fromthe infant,showedthe subjecttheseriesof cardsbeginningwith thegratingof lowestspatialfrequency(i.e..the widest stripes)of0.3cycles/degreeandadvancingin half-octave stepsfromcoarse10fine gratings.Thetester,whowasunaware of whichsidecontainedthe gratings, determined it byobservingtheinfant's looking behaviourthroughthe peephole.The finestgrating resolveddeterminedthe visualacuiryscore.
3.6 Samplecollection
3.6.1 Milk
Formulaor breastm ilksamples wereobtainedfromallmothers at thethreemonth clinicand fromthe motherswhowerecontinuingto breastfeedat the sixmonthclinic.A 15ml polypropylene screw-top vial(ComingCentrifuge)wasmailed withthediet record forallbreestfeedingmothers.Themothers wereaskedtohand express a sample, keep it intherefrigerator without freezing,andbringittothe clinic.Samples of milkfrom the remaininginfants waspoured offdirectly fromthe current bottlethe infant hadbeenfed.
Forallsamples approximatelythree mlswasplacedin screwtop glass vials and placedon ice.nitrogengas was blown over it.andthe samples were frozenat -70°Cuntil analysis.
3.6.2 Blood
At baseline.500·800 IIIofblood wastakenvia a heelprick and collectedin mtcrctalners(Beclon Dickinson).At three and six monthcollections,approxim atelytwo
33
to three millilitre (ml) ofblood was takenfrom the infant' sann by venipunctur eand drawn intovacutainers(BectonDickinso n)containing EDTAas anticoagulant.Baseline bloodsamp les were placed onice and transportedimmediatelyto theuniversity laboratoryforseparation, washin g,and aliquoting. However,bloodsamplesobtainedat thethreeandsilt monthclinicswerehandledin4°C cold roomson site.Bloodsamples werecentrifuged(5415 Eppendorfcentri fuge. Brinkman)at4°C at3-4000rpm. plasma wasremoved.andthe red cells werewashedthree to four times in ice coldsalineat 4°C.
Theblood was then immediatelyaliquotedfor various tes ts.For the thiamin assay.200- 400piof erythrocytes were dilutedwithequal amounts of distilledwaterand storedin plasticeppendorf tubes. For the fatty acidassay.100to 300pioferythrocyt es were placedin4mlglassscrew-topvialsandblown over with nitrogen gas.Allsamples were storedat-70°C.
3.7 Fattyaddanalyses
3.7.1 Ery tbrocyte lipid
Comparativeanalysesof the fatty acid compositionofred bloodcell andplasma glycerophosphol ipidsamong infants fed various fonnulas andbreastm ilk havebeenused asa measureoftheadequacyof essential fattyacids (Innis. 1991).Inthepresent study, the fatty acid compositionof thephosph at idyleth anolamine (PE) fraction of erythrocytes wasdetermin ed The PE fractionwas used as it is concentratedprimarilyontheinner part ofthe redcell membraneand shouldthereforebelessproneto dietary fluctuations
34
andlIleoretiCtlllyprovide abetterindexof org anfatty acid composition.
Thestepsinthe analysesbrie fly invo l ve:ertraction oflipidfromwashed erythrocytes,separationofphospholipids bythinlayerchromatograpby.methylat ionof the fattyacids.andsubseq uentquantificationandidentificationofrelative ameuntsof Canyacidsbygas·liquickhromatogra phy.Thefatty acid compositionofthe vario us formulasand breastmilkwas alsodeterm ined.similar to erythrocytes,however.totallipid wasused for ana lyses.
The stepsinerythro cytefattyacid analysesare asfollow s:
i)Euraction.Red blood cell lipidswere extrac ted bythemethod cf'Rcse and Oklande r(Rose eral,1965).Anequalvolumeof distilledwaterwasadde dto thered cells.vortexed, andallowedtostand forISminutes.Isopropano l(HPLegrade)as eleven timestheredcellvolume(I I:I) was addedslowly with occasionalvor texing over an hour.Chlorofonn(HPLCgrade) as seventimesthe originalredcellvolumewa s added.the samplewasvcn exee, andallowedtostandforone hour.Thesamplewas then centrifuged for fiveminutesat highspeed andtheextrectwaspouredoff into 100 ml cylinders.The extractwaswashed sequentiallywilhtwentytim esthevolume as2:I chloroform:methano l and0.2thevolumeas 0.31%Kel.follow edby0.2 thevolumeIS methanol/water(I:I. vlv) (Felch,19 5 7).Eac h time thecylinderwas inverted andthen allo...ed to stand untillayerseparationoceured.Theto p aqueous layerwas thenremoved by aspira tion.The lipid ext ractwasdriedunder nitrogen gasand redissolvedinSOIII of chloroform:methanol (9:I).
ii)ThinLayer Chromatography.Indiv idualphospholipidclasses were res o lved
35
bythethinlayerchromalogra phy methodofShipskyetai,(1964)(chlorofonn,methanol, acetkecid,andwater, 2S:IS:4:2byvo lume)onsilieaGclGplatesandspotswere identifiedbycomparisonwithCOI11JtIercialpho spholipid standards(SigmaChemica lCo.).
The bandswerevisua lizedbyiodinevapour.and!hephosphatidylethanolarnine(PE) bandwasscrapedonto waxpaperandtnnsferndintotestr obes.
iii)£JUlion.The phospholipidwaselutedfromthesilicaby adding2 ml of 9:1 ch lorofonnl methanol.vonex ing.and centrifugingfor10 minutesandpouringoffthe supernate ntintotran smethylationvials(Supelco).Thiswasrepeatedthree timesinorder torecoveralltheph ospholipid.
iv)Trammethylation.Thepho spholipidextract wasevaporated to drynessand the fattyectdsweremethylate d bytheadditionofl.5mlof94:6methanol:HCLand placedina 6SoC oven forIShrwithhydroquin oneISanantioxidant.
v)Recowryofll'ttlhylesun ,Therattyaddmethylesters(FAME's)were re co veredfromthe mcthanollHCLsolutionbyadding l.Smlefhexane,anddrawingoff theupper(organic)layer.This wasperformedthreetim estoensuremaximumsamp le recovery.W. lcr(I.Sml)wasthenaddedand the hcxane layercontainingthe FAME's wasagaindrawnoff.Thisstepwasrepealedtwice andW35followedbypla cingthe sa mples ina_20°C freezer,tofreezeanyremainingwate r.The sam plewaspouredoff andevapo rated10dryness.
vi)Gas·liquid-chromat ography .Themethylesters weredissolved inCS1•and placedininsertvia ls.Fattyacid methylesters weresepar atedby gas-liquid- chromatogra phyusingISupelcowax30mcapillarycolumni'laHewlettPac kard5890
36
Series II GC. Oventemperaturewas 190°C,ramped to220°Cat IS minata rate of 5°C/m in for12min and the injecti onportandflameionizationdetectortemperatures were230°C.Identificationsoffattyacids weredeterminedbycomparingretent ion times with authenticstandards andquantifiedbytotalweightpercent (Nu-Chek-Preplnc., Supelcotnc.).Integrationofpeakareaswas donebyHewlettPackard 3365Series11 Chemstationsoftware.Peaksthat couldnotbeidentifiedwere discountedfromthearea percent.Therefore,the weightpercent calculatedwas oftotal identifiedfattyacids.
3.7.2 Milk
Beforelipidextraction, breastmilksampleswereheattreatedina SO°Cwaterbath {or90seconds to inactivat elipase activity(Silman etaI,1984).Lipidsfromformulas andbrea nmilk wereextracted with chlorofonn:methanol(2:I)bysuction filtrationto removeprecipitate.Fattyacidmethylesters werethenformed directlyfromthe lipid extract andthe remainderof thepreparationwasas forerythrocytes,mentionedabove.
As>98% ofthe peaks cou ldbeidentifiedas knownrany acids, therelativeweight percents were notrecalculated.Breastmilksampleswereanalyzedfrom 29/35mothers.
Representativesamplesof theother formulaswerealsoanalyzed(EM.0=6.Similac", 0=5,Bnfalac",n"3).
3.8 Thiamin3S58Y
Erythrocytetransketolaseactivityandthethiaminpyrophosphateeffect(TPPe) wasmeas uredbythemethodofBrin etai, (1965)to serveas a functionalevaluationof
37
thiaminadequacy.Transketolase,anenzyme ofthepenrosephosphatepathway,is dependenton the activeformofthiamin,thiaminpyrophosphate (TPP).Inthered blood cells,as thiamin reservesbecomedepleted,transketolase activityis reduced,and is recoveredonly through theprovisionof TPP. 1he test is based onthe followingreaction which requirestransketolaseboundto thiamin pyrophosphate;
""-
Xylclose-s-phosphate+ribose-S-phosphate- - rsedobepnnose-r-phosphate -t-glyceratdehyde-Scphosphate Thedisappearanceof the added substrate,nbc se-s-phosphete (transkelolase substrate in pentosephosphatepathway), at pH 7.4 and 37°Cgivesa measureof transketolaseactivityinugpentoseutilizedlml hemolysatelhour.Thepercentincrease in activityofredcells saturatedwithadded TPPcomparedtothosewithonly endogenous IPPgivesanindication of thiaminadequacy inthetis sues, called theTPP effect(Brinet al,1965).
Initially,thehemolysate was preincubatedin abuffered mediumwithorwithout added TPP for30 min,in orderforthe coenzyme(IPP) toattachto transketolase.Itwas then incubatedwithanexcess ofsubstrate{ribose-c-phosphate}for 60 minat37°C,with trichloroacetic acid(TCA)usedto stopthereactionanddenature theprotein (incubation chart,table3,I).Thesamples werethencentrifugedand theprotein-freefiltratewas used to determinethe amountofpenroseutilized,The pentosesremaining,whichconsistedof an equilibriummixtureof ribose-S-pbosphate.ribulose-s-phcsphate,andxylulose-S- phosphate,were determined bya colorimetricassay (table3.2).Inthisassay,these
38
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pentoseswereconve rtedto furfuralandfurfura l derivatives byconcentratedHClandthe co ndensatio nofapolyhydric phenol (orcinol)inthepresenceof metalions(FE"")tofonn co lourcom plexes.Thesecompoundshaveanabsorption maxim a at670nmandthe absorbance swere read against ablank setalzeroinaMiltonRoy Spectron ic601 spe ctrophotometer.
Thestandardsdeterm inetheopticaldens ityper microgram ofpentoseandfro m this the amoun t ofpenroseinthe other tubes canbecalcu lated. Thecalculationin appendixD determinesthemicrograms peetose utilizedper milliterper hour (transketola se activity) ,Bycomparing the relati veactivi tyoftransketclasewithor withoutthe additionof TPP,theTPP effectcan bedetermined.Rangesforthiamin ade quacyusedwere thoseesta blished byBrinet al(1965):
Thiamin cond ition TPPEffe ct
Norm al <15%
Marginal 15-25%
Defic ient >25%
3.9 Statistic al lnalym
Thediet effec t sateach timewere analyzcd byoneway analysis of va riance (ANOYA)withthe least signifi cantdiffe rencetest (LSD) as thepost-hoctest .Thefatty acid compositi ondata wasarcs inetransformedbeforeANaYAto correctforthenon- nonnal distributi onof percentage data(Zar,1984).Theeffectoftim e wasdeterminedbya series ofpaire d t-e sts,using aBonferroniccrrectt o n forthe numberofcomparisons.
41
Regressionanalysiswasusedto determinecorrelat io nsbetweenvariables.All bioche micaland growthdatawas analysedusing SPSSlt.Visualacuity wasanalysed usingrepealedmeasuresanalysesofvariancewithNeuman-Keulsas the post-hoctest.
Statistical significanceforallanalyseswas assigned to p c0.05,
42
CHAPTER4.0 RESULTS
4.1 Subjects
Subjectgrou pcharacteristicsare presente dintable4.1.The mate rnalageof these fe edingEMwassign ificantlyyoungerthaneither othergroup.Aswell,breastfeeding mothersav er agedthehighestineducation and socioecon omicindices,followedby formulafee d ingmoth ers.thenmothersfeedingEM.Therewasnodifferencesin number ofchildren betweenfeedgroups.
4.2 Dietaryinlakes
Ofthebreastfedinfan ts,at threemonths.29 ofthe 35 infan tswereexclusively breastfed.Five infan tswerereceiving suppleme ntalfonn ularangingfrom51030%
en ergy.Twoinfantswere rec eivingbo th cereal
«
1"0ener gy)andsupplem entalformula.On einfantwas reciev ing <I% of energyascerea landno supplem entalformula.IntheF group,at threemonthsof age,29infan ts werefedSimilac~.and fiveinfant s werefed Enfalac".Allwerefed formu lafrombirth.Twelveinfants were receivingsolidfood s co m prisingup t04%ofenergy.primarilyascereal .IntheEMgrou p, 16infantswere consuming solidfoods,rangingfrom<I%to19%of energy,prim arilyascustardand cereal.ThreeinfantsinrheEMgroup consumedcommerc ialformula forthe firsttwoto threeweeks.before sw itching exclusively toEM .
Alsi x months.inthe BMgroup.90f30infantshadbeenweanedbetween 31/2 and5112months. withfour infants exc lusivelybreastfed.Eleveninfantsconsumed
4J
T.ble4.1 Cb....eterbtlesoflubJeetgroups.
Baselin e- 3 montbs 6montb s
30 30 30
14 17 21
16 13 9
Sub jrc:1 Num ber(D) Su M
F
8M 35 18 17
34
17 17
EM 31
22 9
BM EM
Matem al age 29.5:1::4.2" 27.7±4.7" 24.5±S.3~
(yn)
Soelnecoe omlc 41.3±18.6" 33.8±16.8~ 22.2±7.9' Index'
Maternal 6.2±1.7" 5.1±1.9b 3.6±1.3' [dueatlonl
/ICbildren 2.3±1.9 1.9 ±0.9 2.2±1.2
Values wirhdifferent superscripts inanyrow aresignificantly different(. :I::SO)(p<
0.05).
I,]SeeappendixBforexplanation.
