Follow-up of a randomized controlled trial (BeMIM study)

14 Pages·2017·1.71 MB·English

RESEARCHARTICLE
Association of infant formula composition
and anthropometry at 4 years: Follow-up of a
randomized controlled trial (BeMIM study)
ManjaFleddermann1,HansDemmelmair2,ChristianHellmuth2,VeitGrote2,BrankaTrisic3,
TatjanaNikolic4,BertholdKoletzko2*
1 HiPPGmbH&Co.VertriebKG,Pfaffenhofen,Germany,2 Dr.vonHaunerChildren’sHospital,University
ofMunichMedicalCentre,Ludwig-Maximilians-Universita¨tMunich,Munich,Germany,3 HiPPStudyCenter,
Belgrade,Serbia,4 InstituteforGynecologyandObstetrics,ClinicalCentreofSerbia,Belgrade,Serbia
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*[email protected]
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Abstract
Therelationshipsbetweennutrition,metabolicresponse,earlygrowthandlaterbodyweight
havebeeninvestigatedinhumanstudies.Theaimofthisfollow-upstudywastoassessthe
OPENACCESS long-termeffectofinfantfeedingongrowthandtostudywhethertheinfantmetabolomeat
theageof4monthsmightpredictanthropometryat4yearsofage.TheBelgrade-Munich
Citation:FleddermannM,DemmelmairH,
HellmuthC,GroteV,TrisicB,NikolicT,etal. infantmilktrial(BeMIM)wasarandomizedcontrolledtrialinwhichhealthyterminfants
(2018)Associationofinfantformulacomposition receivedeitheraprotein-reducedinfantformula(1.89gprotein/100kcal)containingalpha-
andanthropometryat4years:Follow-upofa
lactalbuminenrichedwheyandlong-chainpolyunsaturatedfattyacids(LC-PUFA),ora
randomizedcontrolledtrial(BeMIMstudy).PLoS
ONE13(7):e0199859.https://doi.org/10.1371/ standardformula(2.2gprotein/100kcal)withoutLC-PUFA,focusingonsafetyandsuitabil-
journal.pone.0199859 ity.Non-randomizedbreastfedinfantswereusedasareferencegroup.Ofthe259infants
Editor:JacobusP.vanWouwe,TNO, thatcompletedtheBeMIMstudyattheageof4months(anthropometryassessmentand
NETHERLANDS bloodsampling),187childrenparticipatedinafollow-upvisitat4yearsofage.Anthropome-
Received:November29,2017 tryincludingweight,standingheight,headcircumference,andpercentbodyfatwasdeter-
minedusingskinfolds(triceps,subscapular)andbioelectricalimpedanceanalysis.Plasma
Accepted:June10,2018
metaboliteconcentration,collectedinsamplesattheageof4months,wasmeasuredusing
Published:July5,2018
flow-injectiontandemmassspectrometry.Alinearregressionmodelwasappliedtoestimate
Copyright:©2018Fleddermannetal.Thisisan theassociationsbetweeneachmetaboliteandgrowthwithmetabolitesasanindependent
openaccessarticledistributedunderthetermsof
variable.At4yearsofage,therewerenosignificantgroupdifferencesinanthropometryand
theCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and bodycompositionbetweenformulagroups.Sixmetabolites(Asn,Lys,Met,Phe,Trp,Tyr)
reproductioninanymedium,providedtheoriginal measuredat4monthsofageweresignificantlyassociatedwithchangesinweight-for-age
authorandsourcearecredited.
z-scorebetween1to4monthsofageandBMI-for-agez-score(Tyronly),afteradjustment
DataAvailabilityStatement:Allrelevantdataare forfeedinggroup.Nocorrelationwasfoundbetweenmeasuredmetabolitesandlong-term
withinthepaperanditsSupportingInformation
growth(upto4yearsofage).Nolong-termeffectsofearlygrowthpatternswereshownon
files.
anthropometryat4yearsofage.Thecompositionofinfantformulainfluencesthemetabolic
Funding:Thestudyperformancewasfinancially
profileandearlygrowth,whilelong-termprogrammingeffectswerenotobservedinthis
supportedbyHiPPGmbH&Co.VertriebKG
study.
(Pfaffenhofen,Germany)whosuppliedthe
respectiveformulaeduringtheinterventionperiod
andprovidedsupportintheformofsalariesforthe
authors(BK,MF,HD,VG),butdidnothaveany
additionalroleinthedataanalysis,decisionto
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Formulaintakeduringinfancyandanthropometryat4years
publish,orpreparationofthemanuscript.MF Introduction
carriedoutanalyseswhileshewasemployedatthe
Associationsbetweeninfantfeeding,growthvelocitiesandlaterlifeoutcomesareknown
Dr.vonHaunerChildren’sHospitalMunich,Div.of
MetabolicandNutritionalMedicine.Sheisnowan [1,2,3,4].Rapidweightgainduringthefirsttwoyearsoflifeisassociatedwithanincreasedrisk
employeeofHiPPGmbH&Co.VertriebKG.BTis ofobesitylaterinlife,asdocumentedinseveralstudies[2,3,4].Therateofweightgaininthe
anemployeeofHiPPGmbH&Co.VertriebKG. firsttwoyearsoflifecaneffectivelybemodulatedbyfeedingchoicesduringinfancy[5].For
Furthermore,theauthors’workisfinancially
example,highproteinintakeduringinfancyislinkedtoanincreasedriskofrapidearlygrowth
supportedinpartbytheCommissionofthe
aswellaslaterobesity[1,6].
EuropeanCommunities,EarlyNutritionProjects
Resultsofamulticenter,double-blind,randomizedclinicaltrial(Europeanchildhoodobe-
(FP7-289346),DYNAHEALTH(H2020-633595)
andLIFECYCLE(H2020-SC1-2016-RTD),andthe sityproject),inwhichinfantsreceivedeitheraformulawithahigherproteincontentora
EuropeanResearchCouncilAdvancedGrant reducedproteincontentduringthefirstyearoflife[7],showthecorrelationbetweenhigh
META-GROWTH(ERC-2012-AdG322605).
earlyproteinintake,enhancedearlyandlong-termbodyweight,andassociatedmetabolicpat-
Competinginterests:Allauthorshavemade terns.Programmingeffectsofincreasedlevelsofaminoacidsinplasmaandtissue[7]andpos-
substantialcontributionstotheconceptionand siblecorrelationwithanincreasedadipogenicactivity[8]havebeenproposed.Asaturationof
designofthestudy,oracquisitionofdata,or
thebranched-chainaminoaciddegradationpathwaywasobservedwithahighproteinintake
analysisandinterpretationofdata,draftingthe
duringinfancy[9].TheEuropeanchildhoodobesityprojecthasindicatedthatlyso-phosphati-
articleorrevisingitcriticallyforimportant
dylcholineC14:0inserumof6montholdinfantsisassociatedwithweightgainduringinfancy
intellectualcontent.Eachauthorhasseenand
approvedthecontentsofthesubmitted andchildhood,buttheunderlyingmechanismsarenotfullyunderstood[10].
manuscript.Thisstudywasfinanciallysupportedin Whiletheinfluenceofproteinquantityduringearlylifeonlateroutcomesisreportedin
partbyHiPP&Co.VertriebKGwhosuppliedthe severalstudies,theinfluenceofproteinqualityonlongitudinalchangesinbodycompositionis
respectiveformulaeduringthemainstudyand
lessclear.
providedsupportintheformofsalariesforauthors
TheBelgrade-Munichinfantmilk(BeMIM)trialisarandomizedcontrolledtrialthat
(BK,MF,HD,VG).MFcarriedoutanalyseswhile
aimedtoassessthesuitabilityofaprotein-reducedinfantformulacontainingalpha-lactalbu-
shewasemployedattheDr.vonHaunerChildren’s
HospitalMunich,Div.ofMetabolicandNutritional minenrichedwheyandlong-chainpolyunsaturatedfattyacids(LC-PUFA).Theprimaryeval-
Medicine.SheisnowanemployeeatHiPPGmbH uation(safetyandsuitability)ofthetrialfocusedongrowthvelocities,adverseevents,markers
&CoVertriebKG.BTisanemployeeofHiPP&Co. offattyacidsandproteinstatusininfantsuptoanageof4months.Theaimofthefollow-up
VertriebKGandreceivedsalariesfordata
(FU)study,presentedhere,wastoexaminetheinfluenceoftheproteinintakebetweenthe
collection.Theseemploymentsdonotalterthe
agesof1–4monthsonanthropometryandbodycompositionattheageof4yearsandtoinves-
adherencetoPLOSONEpoliciesonsharingdata
andmaterials.Thespecificrolesoftheseauthors tigatetherelationshipbetweennutrition,earlygrowth,long-termgrowthandmetabolic
arearticulatedinthe‘authorcontributions’section. effects.Inaddition,itwasinvestigatedwhethertheinfantmetabolomeattheageof4months
mightpredictanthropometryat4yearsofage(long-termgrowth)ormayexplainshort-term
Abbreviations:BIA,bioelectricalimpedance
analysis;BMI,bodymassindex;CF,control growthuntil4monthsofage.
formula;FU,follow-up;IF,interventionformula;
LC-PUFA,long-chainpolyunsaturatedfattyacids.
Materialsandmethods
Studydesignandprotocol
ChildrenincludedintheBeMIMstudy,arandomized,double-blind,controlledinfantfeeding
trialwithparalleldesign[11],wereinvitedtoparticipateinthefollow-up(FU)visitat4years
ofage.IntheBeMIMtrial,healthyterminfantsreceivedeitheracontrolinfantformula(CF,
2.2gprotein/100kcal)oraninterventioninfantformulawithalowerproteincontent(IF,1.89
gprotein/100kcal),butahigherproportionalcontentofalpha-lactalbuminenrichedwhey,
andaddedLC-PUFA.Bothformulaehadidenticalwhey/caseinratiosof60/40andenergycon-
tentsof67kcal/100mLandwereprovidedtoinfantsuntiltheageof4months(intervention
period).
Of213formula-fedinfantsthatwereenrolledduringthefirstmonthoftheirlife,167
infants(IFn=85,CFn=82)completedtheinterventionstudyat4monthsandformedthe
intention-to-treatpopulation(perprotocol:IFn=46,CFn=35)[11].Non-randomized
breastfedinfants(BF,n=185)wereenrolledasareferencegroup.Atotalof92BFinfants
completedthestudyat4months(perprotocol:BFn=54).Children(n=259)whohad
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Formulaintakeduringinfancyandanthropometryat4years
completedthetrialwereinvitedtoparticipateintheFUstudyat4yearsofage.Thestudypro-
tocolwasapprovedbytheClinicalCenterSerbiaEthicalCommittee,andparentsprovided
theirinformedconsent.ThetrialwasregisteredatClinicalTrials.gov(NCT01094080)onFeb-
ruary1,2010.
Anthropometryincludingweight,height,headcircumferenceandskinfoldmeasurements
(triceps,subscapular)aswellasbioelectricalimpedanceanalysis(BIA)wereperformedby
trainedstudypersonnelat4yearsofage.Theinfants’medicalhistorybetween4monthsand4
yearsofagewasassessedbasedonhospitalandparentalreports.Weightwasdeterminedusing
aSeca711scale(Seca,Hamburg,Germany)equippedwithameasuringrod(Seca220)for
measuringheight.Headcircumferencewasmeasuredusingatapemeasure(Seca212).All
measurementswereperformedinduplicateanddocumentedwithanaccuracyof10gfor
weightand0.1cmforlengthandheadcircumference.Theequipmentwascheckedeverytwo
monthsusingacalibratedweightof5.00kgtoensureaccuracyofmeasurements.Therewas
neveradeviationofmorethan10g.Z-scoresforagewerecalculatedusinganthropometric
resultsrelativetoWHOgrowthstandards[12].
TricepsandsubscapularskinfoldsweremeasuredusingaHoltaincaliper(HoltainLtd,Cry-
mych,UK)attheleftbodyaxis.Allmeasurementswereperformedintriplicateanddocu-
mentedwithanaccuracyof0.2mm.Bodyfatpercentagewascalculatedviapredictiveskinfold
equationsaccordingtoSlaughteretal.1988[13].Bioelectricalimpedanceanalysiswasper-
formedinduplicateusingaBF906BodyFatAnalyzer(Maltron,Rayleigh,UnitedKingdom)
andrestinginasupinepositionbyplacing2electrodesatstandardizedpointsontheright
handand2electrodesontherightfoot.
Laboratoryanalyses
At4monthsofage,venousbloodsamplesweredrawn>2hrsafterthelastfeed.Plasmaali-
quotswerestoredat-80˚C,thentransportedondryicetotheDr.vonHaunerChildren’sHos-
pital(Munich,Germany)wheretheanalysesofphospholipidsandacyl-carnitinesusingflow-
injectionmassspectrometryaccordingtoUhletal.2016wereperformed[14].Plasmaamino
acidconcentrationshavebeenpresentedbefore[11,15].
Analyticalquality(intra-andinter-batchvariation)wasevaluatedbasedonthecoefficients
ofvariationobtainedfromrepeatedanalysisofaliquotsoftwostandardplasmasamples.
Statisticalanalysis
StatisticalanalyseswereperformedusingStata1/MP11.0(StataCorpLP,CollegeStation,TX,
USA).APearson’schi-squaretestwasusedforstatisticalcomparisonofcategoricaldata.
Analysesonanthropometryandbodycompositionat4yearsofage,aswellasgrowthfrom
birthuntil4yearsofage,arepresentedasmean±standarddeviation.Student’st-testwasused
fornormallydistributedcontinuousvariablestoestimatetheeffectofformulatypeonanthro-
pometricoutcome.Linearregressionmodelswereappliedtoestimatetheeffectofformula
typeonanthropometricoutcome,includingchildren’sage,therespectivebaselinevalueat30
daysofage(ifavailable),gender,maternalageandmaternalsmokingstatusasadjustedvari-
ables.Effectsizesarepresentedascoefficientand95%confidenceintervals.Significancewas
acceptedatp<0.05.
Metabolomicanalyses(carnitineandcarnitineesterconcentrations)at4monthsofageare
presentedasmediansandinterquartileranges(IQR,25thand75thpercentile).TheKruskal-
Wallisranktestwasusedforabnormallydistributedcontinuousvariables.Linearregression
modelswereappliedtoestimatetheassociationsbetweeneachmetaboliteandWHO-stan-
dardizedweight/length/weight-for-length/BMIbetweenbirthand4yearsofage,with
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Formulaintakeduringinfancyandanthropometryat4years
metabolitesasindependentvariables.Duetotheanalysisof129metabolites,Bonferroni-cor-
rectedp-values(0.05/129=0.00038)wereusedforsignificanceevaluation.Allanalyseswere
repeatedwithadjustmentforfeedinggroup.
Anthropometryoutcomesat4yearsofagewereanalyzedforintention-to-treatandper-proto-
colpopulations.Theintention-to-treatanalysisincludedallrandomizedsubjectsthatreceived
studyformuladuringtheinterventionperiod.Theper-protocolanalysisincludedonlydatafrom
subjectscomplyingwithpredefinedconditionsduringtheinterventionperiod:nointakeoffor-
mulaotherthanstudyformula,amaximumdailyintakeof50mlteaandlessthan3spoonsof
complementaryfoodperweek.Missingdataatindividualtimepointswerenotreplaced.
Results
Population
Atotalof259children(IF:85,CF:82,BF:92infants)thatcompletedtheBeMIMstudyat4
monthsofagewereinvitedtoparticipateintheFUvisit.Outofthisoriginalstudypopulation,60
childrencouldnotbelocated(IF:16,CF:20,BF:24infants),andtwelveparents(IF:4,CF:3,BF:
5infants)declinedtheinvitationtoparticipate(Fig1),resultinginadistributionof65childrenin
theIF,59intheCFand63intheBFgroupwhocompletedtheFUstudy.Atotalof42childrenin
theIF,25intheCFand40intheBFgroupwerepartoftheper-protocolpopulationintheFU
study.TheFUstudytookplacebetween21.01.2014and08.04.2015.Numbersofdrop-outsfrom
theoriginalstudypopulationandthereasonsfordropping-outdidnotdifferbetweengroups.
ThecharacteristicsofthestudypopulationareshowninS1Table.Therewerenodiffer-
encesbetweenformulagroupsat4yearsofage.Breastfedchildrendifferedfromformula-fed
childrenintermsofmaternaleducationalstatus,whichisconsistentwithobservationsatbase-
line(age1month).Themeanageatfollow-upwas3.95±0.03years,withnodifference
betweenrandomizedformulagroups.
Fig1.CONSORTchart:Numbersofparticipantsforrandomization,allocation,follow-upandanalysis.(cid:3)Ofwhich3infantswereexcludedfromanalyses.
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Formulaintakeduringinfancyandanthropometryat4years
Furthermore,thestudypopulationat4yearsofagedifferedslightlyfromtheoriginalstudy
populationwithrespecttoageofmothersatdeliveryandpercentageofmotherssmokingdur-
ingpregnancy.Theageofmothersatdeliverywassignificantlyhigherinfamiliesthatpartici-
patedintheFUstudythaninthosewhodidnot(31.6vs.29.4years,p=0.003).The
percentageofmotherswhosmokedduringpregnancywaslowerinFUparticipantsthanin
non-participants(21%vs.35%,p=0.03).Nodifferenceswereobservedwithrespecttomater-
naleducationalstatusorparentalbodymassindex(BMI).
Growth,z-scoresandbodycompositionofchildrenupto4yearsofage
Infantgrowthintheinterventionalstudyuptotheageof4monthswaspublishedinFledder-
mannetal.[11].Fig2presentsz-scores(weight-for-age,length-for-age,BMI-for-age,weight-
for-length)between1monthand4yearsofageofchildrenparticipatingintheFUvisit.The
increaseinweightandlengthz-scoresbetween1and4monthsofagewashigherforIFthan
forCFinfants(Table1),whiletheincreaseofBMIz-scoresdidnotdiffer.After4monthsof
ageasignificantlylowerincreaseinz-scores(forweightandlength)wasobservedintheIF
comparedtotheCFgroup.Theadjustmentforconfoundershadnoeffectongroupdifference
ofchangeofz-scoreduringthefirst4yearsoflife.
At4yearsofage,therewerenosignificantdifferencesinanthropometryandbodycompo-
sitionparametersbetweengroupsinintention-to-treat(Table2)andper-protocolpopulation
(S2Table).Adjustmentforgenderandconfoundersdidnotchangethisfinding.
BMIwaspositivelyrelatedtothepercentageofbodyfatestimatedbyBIAorskinfolds,
whiletherewasastrongercorrelationforskinfolds(R=0.62,p<0.001)thanforBIA(R=0.36,
p<0.001).Bodyfatpercentageestimatesobtainedbybothmethodscorrelatedwell(R=0.50,
p<0.001).
Seriousillnesses
Theincidenceofseriousillnesses(anyhospitalisationupto4yearsofage)wascomparable
betweenallgroups(p=0.25).NoseriousillnesswasobservedintheIFgroup,while3children
intheCFgroup(twochildrenwithinfections,onechildwithmentaldisorder)and2children
intheBFgroup(onechildwithinfection,onechildwithhypothyroidism)developedoneseri-
ousillness.Additionally,onechildintheCFgroupdevelopedtwoseriousillnesses(cryptor-
chidismandarteriapulmonalisstenosis).Wefoundnoindicationsforacausalroleofthe
studyformulabeingresponsibleforanyoftheseadverseevents.
Plasmaacyl-carnitinesandbranched-chainaminoacids
Atotalof250plasmasamplesofinfantsaged4months(IF:80,CF:79,BF:91)wereanalyzed
toinvestigate59differentcarnitineestersand425phospholipids.Fourteencarnitineesters
hadintra-batchandinter-batchcoefficientsvariationoflessthan30%andthuswereconsid-
eredforfurtherstatisticalanalysis.Absoluteconcentrationsofphospholipids,sphingomyelins
[14]andofplasmaaminoacids[11]havebeenpublishedpreviously.
Fouracyl-carnitinesshowedstatisticallysignificantgroupdifferences(Table3).Acyl-carni-
tineC8:1,C12:0andC14:0weresignificantlyhigherintheIFgroupcomparedtotheCF
group,whileacyl-carnitineC3:0wassignificantlylowerintheIFcomparedtoCFinfants.
Metabolitesinrelationtogrowth
Toidentifypredictorsoflateranthropometrymeasures,associationsbetweenmetabolitesand
absoluteanthropometricoutcomeat4monthsor4yearsofageorbetweenmetabolitesand
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Formulaintakeduringinfancyandanthropometryat4years
Fig2.Meanz-scoresforweight,length,BMIandweight-for-lengthintheintervention,controlandbreastfed
groupsfrom1monthto4yearsofageoftheintention-to-treatpopulation(follow-upchildrenonly).
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Formulaintakeduringinfancyandanthropometryat4years
Table1. Changeofz-scoresduringthefirst4years.
Formulagroup EstimateddifferencebetweenIFandCF1 Breastfedgroup
Intervention Control
n Mean±SD N Mean±SD n Mean±SD
Changeinz-scorefrom1monthto4months–allinfants
WAZ 82 0.51 ± 0.68 82 0.29 ± 0.77 0.21(-0.01,0.42);p=0.06 92 0.08 ± 0.75
LAZ 82 0.34 ± 0.72 82 0.05 ± 0.77 0.21(0.003,0.42);p=0.047 92 -0.07 ± 0.78
BMIZ 82 0.48 ± 0.94 82 0.38 ± 0.99 0.14(-0.13,0.41);p=0.32 92 0.17 ± 1.05
WLZ 82 0.66 ± 1.15 82 0.70 ± 1.07 0.11(-0.17,0.36);p=0.44 92 0.61 ± 1.17
Changeinz-scorefrom1monthto4months–follow-upchildrenonly
WAZ 65 0.50 ± 0.62 59 0.23 ± 0.76 0.29(0.06,0.52);p=0.01 63 0.05 ± 0.69
LAZ 65 0.36 ± 0.73 59 0.07 ± 0.81 0.25(0.01,0.48);p=0.04 63 -0.15 ± 0.76
BMIZ 65 0.45 ± 0.86 59 0.30 ± 0.98 0.23(-0.07,0.52);p=0.13 63 0.19 ± 0.99
WLZ 65 0.65 ± 1.06 59 0.58 ± 1.10 0.22(-0.09,0.53);p=0.16 63 0.65 ± 1.11
Changeinz-scorefrom4monthsto4years
WAZ 65 0.34 ± 0.84 59 0.72 ± 0.66 -0.37(-0.64,-0.10);p=0.007 63 0.63 ± 0.80
LAZ 65 0.40 ± 0.92 59 0.72 ± 0.85 -0.33(-0.65,-0.02);p=0.04 63 0.37 ± 0.85
BMIZ 65 0.08 ± 1.16 59 0.34 ± 0.82 -0.26(-0.62,0.11);p=0.17 63 0.51 ± 0.98
WLZ 65 0.08 ± 1.17 59 0.33 ± 0.84 -0.25(-0.62,0.12);p=0.18 63 0.53 ± 0.98
Changeinz-scorefrom1monthto4years
WAZ 65 0.84 ± 0.96 59 0.95 ± 1.05 -0.08(-0.40,0.25);p=0.64 63 0.68 ± 0.88
LAZ 65 0.76 ± 1.09 59 0.80 ± 1.07 -0.09(-0.42,0.25);p=0.61 63 0.22 ± 0.97
BMIZ 65 0.52 ± 1.10 59 0.64 ± 1.11 -0.03(-0.39,0.33);p=0.88 63 0.70 ± 1.22
WLZ 65 0.73 ± 1.19 59 0.91 ± 1.16 -0.03(-0.38,0.32);p=0.87 63 1.18 ± 1.35
WAZ,weight-for-agez-score;LAZ,length-for-agez-score;BMIZ,BMI-for-agez-score;WLZ,weight-for-lengthz-score;IF,interventionformula;CF,controlformula.
Datapresentedasmean±standarddeviation.Significantdifferences(Student’st-test,p<0.05).
1Derivedfromlinearregressionadjustedforbaselinevalueattheageof1month(ifapplicable)andgender,95%confidenceintervalinparentheses.
https://doi.org/10.1371/journal.pone.0199859.t001
growth(changeinz-scorebetween1monthand4monthsor4monthsand4years)wereper-
formedusinglinearregressionandBonferroni-correctedp-valuesofp<0.00038.
Theanalysesontheinfantmetabolomeattheageof4monthsandpredictionofanthro-
pometryupto4yearsofageshowednoassociationbetweenmetabolitesandlong-term
growth(absoluteanthropometricparameterat4yearsofageandchangeingrowthbetween4
monthsand4yearsofage).
Short-termgrowthupto4monthsofageindicatednoassociationbetweenmetabolitesand
absoluteanthropometricoutcomeat4monthsofage.Furthermore,nocorrelationwasfound
betweenmetabolitesandthechangeinlength-for-ageorweight-for-lengthz-scoresbetween1
monthand4monthsofage.However,acorrelationwasfoundbetweenmetabolitesand
changeinweight-for-ageandBMI-for-agez-scoreduringthesametimeperiod(Table4)A
totalof12metabolites(8aminoacids,freecarnitine,3phosphatidylcholines)showedsig-
nificantcorrelationwithchangeinweight-for-ageandBMI-for-agez-scorefrom1to4
monthsofage(Table4).Includingfeedinggroupasadjustedvariable,6metabolitesremained
significantlyassociatedwiththechangeinweight-for-agez-score(Asn,Lys,Met,Phe,Trp,
Tyr)andBMI-for-agez-score(Tyronly).
Onlytheassociationbetweenchangeinweightbetween1and4monthsofageandTrpor
Tyrindicatingasignificantp-valuemettheBonferroni-correctedp-valuethresholdwhen
additionaladjustmentsforbaselineweight-for-agez-score/BMI-for-agez-scoreandsmoking
statusduringpregnancyweredoneinmetabolitesidentifiedinTable4.
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Formulaintakeduringinfancyandanthropometryat4years
Table2. Anthropometryandbodycompositionat4yearsofage(intention-to-treatpopulation).
Formulagroup EstimateddifferencebetweenIFandCF2 Breastfedgroup
Intervention Control
n Mean±SD n Mean±SD n Mean±SD
Weight
(kg) 65 17.7 ± 2.45 59 17.9 ± 2.31 -0.15(-0.96,0.66);p=0.72 63 17.9 ± 2.29
(z-score) 65 0.57 ± 1.00 59 0.64 ± 0.94 -0.08(-0.41,0.25);p=0.65 63 0.65 ± 0.91
Height
(cm) 65 108 ± 4.45 59 108 ± 4.45 -0.31(-1.76,1.13);p=0.67 63 108 ± 1.05
(z-score) 65 1.11 ± 1.05 59 1.25 ± 1.05 -0.07(-0.42,0.27);p=0.67 63 1.05 ± 0.94
Head
(cm) 65 51.2 ± 1.61 59 51.2 ± 1.33 0.03(-0.41,0.47);p=0.89 63 51.3 ± 1.22
BMI
(kg/m2) 65 15.2 ± 1.66 59 15.2 ± 1.22 0.07(-0.46,0.60);p=0.80 63 15.4 ± 1.56
(z-score) 65 -0.14 ± 1.15 59 -0.16 ± 0.91 -0.04(-0.40,0.33);p=0.85 63 0.02 ± 1.12
Weight-for-height
(z-score) 65 -0.16 ± 1.12 59 -0.19 ± 0.87 -0.03(-0.39,0.33);p=0.86 63 0.01 ± 1.08
BIAmeasurement
Bodyfat(kg) 65 2.21 ± 1.33 59 2.07 ± 1.12 0.15(-0.29,0.58);p=0.51 62 2.26 ± 1.33
Leanmass(kg) 65 15.5 ± 2.19 59 15.8 ± 2.05 -0.23(-0.99,0.54);p=0.56 62 15.7 ± 1.82
BodyfatfromBIA(%) 65 12.3 ± 6.52 59 11.5 ± 5.56 0.84(-1.31,2.98);p=0.44 62 12.3 ± 5.77
Skinfolds
Triceps(mm) 64 9.51 ± 2.58 59 9.14 ± 2.25 0.35(-0.48,1.18);p=0.41 63 9.48 ± 2.46
Subscapular(mm) 64 6.16 ± 2.12 59 6.01 ± 1.75 0.15(-0.54,0.83);p=0.67 63 6.26 ± 2.19
Bodyfatfromskinfolds(%)1 64 15.0 ± 3.73 59 14.6 ± 3.19 0.43(-0.76,1.63);p=0.48 63 15.1 ± 3.64
IF,interventionformula;CF,controlformula;BIA,bioelectricalimpedanceanalysis;BMI,bodymassindex.Datapresentedasmean±standarddeviation.Significant
differences(Student’st-test,p<0.05)
1Slaughteretal.1988[13].
2Derivedfromlinearregressionadjustedforbaselinevalueattheageof1month(ifapplicable),gender,ageatvisit,maternalageandmaternalsmokingstatus,95%
confidenceintervalinparentheses.
https://doi.org/10.1371/journal.pone.0199859.t002
Discussion
Theshort-termevaluationofaninterventionformulawithalowerproteincontent,containing
alpha-lactalbuminenrichedwheyandLC-PUFA,showeddifferentialgrowtheffectscompared
toanormalproteincontrolformulauptotheageof4months[11].Incontrast,nogroupdif-
ferencewasdetectedattheageof4years.
Anthropometryat4yearsandcomparisontootherstudies
Ahighrateof72.2%ofchildrenfromthemainstudyparticipatedintheFUstudywithasig-
nificantlyhigherpercentageofchildrenoriginatingfromtheper-protocolthantheintention-
to-treatpopulation(p=0.02).Theobesityrateof6.95%iscomparabletothatreportedin
anotherSerbianstudy[16].TheidentifiedcorrelationbetweenBIAandskinfoldmeasurement
wassimilartoOrtaetal.(2014)inchildrenaged9.5years(R=0.74,p<0.001)[17].Thecur-
rentevidenceconcerningtheeffectofproteinintakeduringinfancyonlong-termbodycom-
positionoutcomeisnotclear[18].
ChildrenpreviouslyfedIFshowedadequategrowthupto4yearsofagethatwasnotdiffer-
entfromgrowthcomparedtotheotherstudygroup.Whiletheaveragegrowthrate(changein
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Formulaintakeduringinfancyandanthropometryat4years
Table3. Median(interquartilerange,IQR)carnitineandcarnitineesterconcentrations(μmol/L)ofinfantsintheintervention,controlandbreastfedgroupatthe
ageof4monthsoftheintention-to-treatpopulation.
FormulaGroup p(Interventionvs.Control) Breastfed
Intervention Control
n Median IQR n Median IQR n Median IQR
Freecarnitine 80 48.9 42.8 53.3 79 50.7 42.0 55.8 0.41 91 44.0 40.2 50.1
Totalacyl-carnitines 80 10.3 9.52 11.6 79 10.2 8.77 11.1 0.11 91 10.2 8.67 11.5
Short-chainacyl-carnitines(<C5) 80 9.04 8.11 10.2 79 8.93 7.86 9.78 0.35 91 9.18 7.69 10.2
Carn.a.C2.0 80 8.04 7.20 9.42 79 7.83 6.65 8.59 0.13 91 8.47 6.98 9.36
Carn.a.C3.0 80 0.68 0.59 0.80 79 0.79 0.69 0.90 0.0002 91 0.62 0.47 0.74
Carn.a.C4.0 80 0.25 0.20 0.28 79 0.25 0.22 0.32 0.42 91 0.19 0.14 0.24
Carn.a.C8.1 80 0.73 0.41 0.91 79 0.42 0.31 0.52 <0.0001 91 0.14 0.09 0.23
Long-chainacyl-carnitines((cid:21)C10) 80 0.78 0.63 0.98 79 0.77 0.68 0.93 0.89 91 0.78 0.69 0.99
Carn.a.C10.1 80 0.21 0.16 0.27 79 0.24 0.19 0.31 0.01 91 0.22 0.17 0.31
Carn.a.C12.0 80 0.11 0.08 0.16 79 0.09 0.06 0.11 0.0002 91 0.10 0.08 0.16
Carn.a.C14.0 80 0.03 0.02 0.04 79 0.02 0.01 0.03 0.0004 91 0.04 0.03 0.05
Carn.a.C14.1 80 0.12 0.10 0.14 79 0.12 0.10 0.15 0.85 91 0.12 0.10 0.14
Carn.a.C14.2 80 0.03 0.02 0.04 79 0.03 0.02 0.04 0.48 91 0.04 0.03 0.05
Carn.a.C16.0 80 0.10 0.08 0.12 79 0.09 0.08 0.10 0.05 91 0.09 0.07 0.11
Carn.a.C16.1 80 0.02 0.01 0.03 79 0.02 0.01 0.02 0.47 91 0.02 0.02 0.03
Carn.a.C18.0 80 0.03 0.03 0.04 79 0.03 0.02 0.04 0.15 91 0.04 0.03 0.05
Carn.a.C18.1 80 0.12 0.09 0.14 79 0.11 0.10 0.13 0.85 91 0.10 0.08 0.12
Carn.a.C2.0/totalacyl-carnitines 80 0.76 0.75 0.79 79 0.77 0.75 0.78 0.58 91 0.82 0.79 0.84
Long-chainacyl-carnitines/totalacyl-carnitine 80 0.08 0.06 0.09 79 0.08 0.07 0.09 0.38 91 0.08 0.07 0.10
Carn.a.C2.0/freecarnitine 80 0.16 0.14 0.19 79 0.15 0.13 0.19 0.05 91 0.18 0.16 0.22
Long-chainacyl-carnitines/freecarnitine 80 0.02 0.01 0.02 79 0.02 0.01 0.02 0.36 91 0.02 0.01 0.02
Dataarepresentedasmediansandinterquartileranges.P-valueswerecomputedwiththeuseoftheKruskal-Wallisranktest(Bonferroni-correctedp-valueof
p<0.00038).Acyl-carnitineswithachainlength(cid:21)C10aredefinedaslong-chainacyl-carnitines,andwithachainlength<C5asshort-chainacyl-carnitines.
https://doi.org/10.1371/journal.pone.0199859.t003
weight-for-age,length-for-agez-score)duringthefirst4months(interventionperiod)was
higherinIFcomparedtoCFinfants(discussionsee[11]),thegrowthofIFchildrenslowed
downcomparedtoCFchildrenbetween4monthsand4yearsofage(FUperiod)suggestinga
catch-downgrowtheffect.Furthermore,changeinBMIdidnotdifferduringtheintervention
andFUperiodindicatingaproportionallyhighergrowthofweightandlengthinIFinfants.
Interestingly,Putetetal.[19]comparedinfantsfedwithastandardformula(2.7gprotein/100
kcal)tothosereceivingalow-proteinformula(1.8gprotein/100kcal)andfoundnoimpacton
growthvelocitiesduringthefirstyearoflife,anthropometryat4yearsofage(expressedas
z-scores)andbodycompositionat3or5yearsofage[19].AlsotheIGF-Iconcentrations
(markerlinkingeffectsofnutritiontogrowthininfants)at4and9monthsofage,didnotdif-
ferbetweentheformulagroups[19].Putetetal.speculatedthatthedifferenceinproteincon-
tentsofstudyformulaemayhavebeentoosmalltofindadifferenceinplasmaIGF-I
concentrationandrelatedgrowth[19].
Acyl-carnitinesasdegradationproductsofbranched-chainaminoacids
Groupdifferencesinacyl-carnitineconcentrationsinourstudymaybeexplainedbyacompo-
sitionaldifferenceinthestudyformulae(S3Table).Thehigherplasmacontentofacyl-carni-
tineC3.0ininfantsreceivingthestandardformulamaybearesultofthehigheraminoacid
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Formulaintakeduringinfancyandanthropometryat4years
Table4. AssociationsbetweenmetabolitesandchangeinweightandBMIz-scorefrom1monthto4monthswithandwithoutadjustmentforfeedinggroup.
Withoutadjustmentforfeedinggroup Withadjustmentforfeedinggroup
Regressioncoefficient (95%CI) p-value Regressioncoefficient (95%CI) p-value
Metabolitesrelatedtochangeinweight
PCaec38:4 -0.044 (-0.065,-0.023) 0.0001 -0.045 (-0.071,-0.018) 0.001
PCaec40:4 -0.327 (-0.502,-0.152) 0.0003 -0.303 (-0.512,-0.094) 0.005
PCaec40:5 -0.196 (-0.293,-0.099) 0.0001 -0.193 (-0.312,-0.074) 0.002
Freecarnitine 0.021 (0.010,0.031) 0.0002 0.019 (0.008,0.029) 0.0009
Asn 0.017 (0.010,0.024) <0.0001 0.016 (0.008,0.024) 0.0001
Ile 0.009 (0.004,0.013) 0.0001 0.008 (0.004,0.013) 0.0005
Leu 0.005 (0.002,0.007) 0.0001 0.004 (0.002,0.007) 0.0007
Lys 0.004 (0.003,0.006) <0.0001 0.004 (0.002,0.006) <0.0001
Met 0.023 (0.012,0.033) <0.0001 0.022 (0.010,0.033) 0.0002
Phe 0.015 (0.009,0.021) <0.0001 0.014 (0.008,0.020) <0.0001
Trp 0.017 (0.010,0.024) <0.0001 0.017 (0.010,0.024) <0.0001
Tyr 0.010 (0.007,0.014) <0.0001 0.010 (0.006,0.014) <0.0001
MetabolitesrelatedtochangeinBMI
Lys 0.005 (0.002,0.007) 0.0003 0.005 (0.002,0.007) 0.0006
Phe 0.015 (0.007,0.023) 0.0003 0.014 (0.006,0.023) 0.0009
Tyr 0.010 (0.005,0.015) 0.0002 0.010 (0.005,0.015) 0.0003
Onlymetabolitessignificantlyrelatedtoweight-for-age/BMI-for-agez-scorechangeresultingfromtheunadjustedlinearregressionmodelarelisted.
https://doi.org/10.1371/journal.pone.0199859.t004
contentinthisformula.Furthermore,thehighercontentsofacyl-carnitineC8.1,C12.0and
14.0ininfantsreceivingtheIFmightberelatedtoitshighercontentofmedium-chainfatty
acids.SimilarobservationswerereportedinthestudybyBeneetal.,inwhichplasmaconcen-
trationsofmedium-chainacyl-carnitineswererelatedtotheformulacontentofmedium-
chaintriglycerides[20].
Branched-chainaminoacidsarepreferentiallyoxidizedinmuscle[21].Ininfantsaged6
months,thecatabolismofbranched-chainaminoacidstoitsrespectivedegradationproducts
followssaturationkinetics,whichatveryhighplasmabranched-chainaminoacidconcentra-
tionmightresultinadverseregulatoryeffects[9].Inhibitingeffectsonthebeta-oxidationof
fattyacidsmightincreasebodyfatstorage[9].However,theaminoacidandcarnitineester
concentrationsfoundintheBeMIMinfantsdidnotshowindicationsofsaturationkinetics.
OnlyfourinfantshadIleconcentrationsabovethepreviouslyreportedbreakpointconcentra-
tionof136μmol/L,andonlytwoinfantshadLeuconcentrationsabovethepreviouslyreported
breakpointconcentrationof234μmol/L[9].Thisislikelyduetothefactthattheproteincon-
tentoftheBeMIMcontrolformulawaslower(2.2g/100kcal)thanthehighproteinformula
studiedbyKirchbergetal.[9](infantformula2.9g/100kcal,follow-onformula4.4g/100
kcal).
Metabolicparametersassociatedwithearlygrowth
IntheBeMIMstudy,plasmametaboliteconcentrationsat4monthsofagedidnotshowany
relationtoanthropometryat4monthsor4yearsofage.Nolong-termeffectsofmeasured
metaboliteconcentrationsonanthropometryat4yearsofagewerefound.Thisisincontrast
toRzehaketal.[10],whoidentifiedlysophosphatidylcholineLPCaC14:0at6monthsofageas
beingasignificantlyrelatedriskofoverweightorobesityat6yearsofage.
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