Associations between Maternal Body Composition and Appetite Hormones and Macronutrients in ...

18 Pages·2017·0.98 MB·English

nutrients
Article
Associations between Maternal Body Composition
and Appetite Hormones and Macronutrients in
Human Milk
SambaviKugananthan1,2,†,ZoyaGridneva2,*,†,ChingT.Lai2,AnnaR.Hepworth2,
PeterJ.Mark1,FoteiniKakulas2andDonnaT.Geddes2
1 SchoolofHumanSciences,TheUniversityofWesternAustralia,CrawleyWA6009,Australia;
[email protected](S.K.);[email protected](P.J.M.)
2 SchoolofMolecularSciences,TheUniversityofWesternAustralia,CrawleyWA6009,Australia;
[email protected](C.T.L.);[email protected](A.R.H.);[email protected](F.K.);
[email protected](D.T.G.)
* Correspondence:[email protected];Tel.:[phone]
† Theseauthorscontributeequallytothiswork.
Received:25January2017;Accepted:7March2017;Published:9March2017
Abstract:Humanmilk(HM)appetitehormonesandmacronutrientsmaymediatesatietyinbreastfed
infants. This study investigated associations between maternal adiposity and concentrations of
HMleptin,adiponectin,proteinandlactose,andwhethertheseconcentrationsandtherelationship
betweenbodymassindexandpercentagefatmass(%FM)inabreastfeedingpopulationchangeover
thefirstyearoflactation. Lactatingwomen(n=59)providedmilksamples(n=283)atthe2nd,5th,
9thand/or12thmonthoflactation. Concentrationsofleptin,adiponectin,totalproteinandlactose
weremeasured. Maternal%FMwasmeasuredusingbioimpedancespectroscopy. Highermaternal
%FMwasassociatedwithhigherleptinconcentrationsinbothwhole(0.006±0.002ng/mL,p=0.008)
andskimHM(0.005±0.002ng/mL,p=0.007),andprotein(0.16±0.07g/L,p=0.028)concentrations.
Adiponectinandlactoseconcentrationswerenotassociatedwith%FM(0.01±0.06ng/mL,p=0.81;
0.08±0.11g/L,p=0.48,respectively). Wholemilkconcentrationsofadiponectinandleptindidnot
differsignificantlyoverthefirstyearoflactation. Thesefindingssuggestthatthelevelofmaternal
adiposityduringlactationmayinfluencetheearlyappetiteprogrammingofbreastfedinfantsby
modulatingconcentrationsofHMcomponents.
Keywords: leptin;adiponectin;maternalbodycomposition;percentagefatmass;lactation;human
milk;breastfeeding;appetitehormones;macronutrients;protein;lactose
1. Introduction
Humanmilk(HM)istheoptimalnutritionforterminfantsasitcontainsauniquelybalanced
profileofmacronutrientsalongwithmicronutrients,hormones,antibodies,bioactivemolecules[1]and
cells[2,3],whichadequatelysupportthenutritionalneeds,appropriategrowth,immunoprotectionand
physiologicaldevelopmentoftheinfant[4,5]. Itiswelldocumentedthatprolongedbreastfeedingis
associatedwithdecreasedprevalenceofoverweightandobesityacrossthelifecourse[6,7]. Enhanced
appetitecontrolinadultswhohavebeenbreastfedasinfantshasbeenpartlyattributedtoregulatory
appetitehormonespresentinHM[8–10],whichincludeleptinandadiponectin[8,11]. Inratpups,
acuteorchronicadministrationofleptinbyintraperitonealinjectionhasbeenshownnottoreduce
foodconsumptionbuttomodulatetheexpressionofneuropeptidesandreceptorsinvolvedinthe
regulationoffeedingbehaviour(neuropeptideY(NPY)andproopiomelanocortin(POMC))[12],while
inmicepupsleptindeficiencycausedprofounddisruptionsinthedevelopmentoftheprojectionsof
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thearcuatenucleusofthehypothalamus[13]. Thesefindingsindicatethatleptinplaysaneurotrophic
roleandcontributestothedevelopmentalprogrammingofthehypothalamicappetitecircuitryduring
theneonatalperiod, precedingleptin’sacuteregulationoffoodintakeinadults. Adiponectinhas
anti-inflammatorypropertiesandimprovesbothfattyacidmetabolismandsensitivitytoinsulin[14].
Inmice,adiponectininhibitstension-sensitivegastricvagalafferentmechanosensitivity,modulating
satietysignalsinbothleanandobeseanimals,whilesimultaneouslyincreasingthemechanosensitivity
ofmucosalgastricvagalafferentinanobesity-inducedmodel[15].
Additionally,theconcentrationofmacronutrientsinHM,namelyprotein,fatandlactose,may
alsobeinvolvedinregulationoftheinfantappetitecontrol[9,16]. Differencesintheconcentrations
ofthesefactorsmaypartlyexplainthevariabilityinbreastfeedingpatternsobservedininfantswho
feedondemand[17]. Assuch,anunderstandingofthefactorsthataffectconcentrationsofappetite
hormonesandmacronutrientsinHMiscritical,asitpresentsauniqueopportunityfortheprevention
ofunfavourableearlydevelopmentalprogrammingandsubsequentobesity.
Leptin is secreted into the maternal circulation by white adipocytes and is subsequently
transferred into the mammary ductal system via diffusion or a receptor-mediated transport
mechanism [18]. Leptin is also contributed by lactocytes [19,20]. Previous studies have identified
a positive correlation between maternal body mass index (BMI) and both maternal serum [21,22]
andskimHMleptin[21–24],despitedifferingmethodologiesinleptinmeasurement[25]. However,
noconsistentrelationshipbetweenmaternaladiposityandHMleptinconcentrationshasbeenshown
acrosslactation,withMirallesetal. (2006)reportingonlymoderatecorrelationsbetweenmaternalBMI
andHMleptinduringthefirst6monthsoflactation(R=0.387;p<0.01)[8],whileBronskyetal. (2011)
sawnoconsistentrelationshipoverthefirst12monthsoflactation[11], despiteutilizingasimilar
leptinmeasurementmethodology.
Thereareanumberoffactorsthatmaycontributetotheseconflictingfindings. BMIisapoor
measure of adiposity, as it fails to adequately differentiate between adipose tissue and lean body
mass[26,27]. InvestigationsbetweentherelationshipofmaternalBMIandleptinhavebeenconducted
predominantlyinskimHM,whichexcludesthefatandcellularcomponentsofHM[21,24]. Further,
leptinconcentrationshavebeenshowntobehigherinwholeHMcomparedtoskimHM[20,23,28].
In contrast, maternal serum concentrations of adiponectin are lower if weight and BMI
are higher [29,30]. Results associating maternal BMI and adiponectin concentrations in HM
are conflicting [25], with several studies showing no associations [11,31,32] and two studies
counter-intuitivelyreportingapositiveassociation[33,34]. Aswithleptin,useofBMIasameasure
of maternal adiposity may contribute to these conflicting findings. Also, both increasing [11,35]
and decreasing [30,33,36] trends in adiponectin concentrations across the lactation period have
beenreported.
Similartoitseffectsonappetitehormones, itispostulatedthatmaternaladiposityinfluences
macronutrientconcentrationsinHM.Again,resultsareconflicting.Excessiveadiposetissuestoragehas
beenshowntoimpairaminoacidandmonosaccharidemetabolismandtransport[37,38],yetincreased
serumaminoacidlevelswerefoundinmotherswithmoreadiposetissue[39]. Inlactatingwomen,
higher concentrations of HM protein were associated with lower BMI in one study [40], yet with
higherBMI[41]andhigheradiposity[42]inothers. BMIalsowasfoundtoassociatepositivelywith
concentrationofHMgalactose[41]. Thereisapossibilitythattheeffectofmaternalbodycomposition
(BC)onlybecomesevidentinlatebutnotearlylactation,whenthefataccumulatedduringpregnancy
isdepleted[42]. Moreprecisemeasurementsofmaternaladiposityacrossthelactationperiodare
neededtoelucidateeffectsonHMcomposition.
ThisstudyinvestigatedrelationshipsbetweenmaternaladiposityandHMleptin,adiponectin,
totalproteinandlactose. Further,itinvestigatedtherelationshipbetweenpercentagefatmass(%FM)
and BMI, and the change in maternal adiposity and component concentrations over the first year
oflactation.
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2. MaterialsandMethods
2.1. StudyParticipants
Fifty-ninepredominantlyCaucasian,English-speaking,breastfeedingmotherswererecruitedvia
theAustralianBreastfeedingAssociation(ABA)andthroughexternalnetworking. Inclusioncriteria
were: healthysingletons,gestationalage≥37weeks,fullybreastfedat2and/or5months[43]and
maternal intention to breastfeed until 12 months. The exclusion criterion was: maternal smoking.
Participantswererecruitedduringtheir2nd,5th,9thand12thmonthoflactationandinvitedtocome
backatanysubsequenttimepoints. Twenty-oneparticipantscontributedsamplesattwoormoretime
points.Allparticipantsprovidedinformedwrittenconsentandansweredasecureonlinequestionnaire
thatwasadministeredandsecurelystoredattheuniversity. ThisstudywasapprovedbytheHuman
ResearchEthicsCommitteeofTheUniversityofWesternAustralia(RA/4/1/4253)andregisteredwith
theAustralianNewZealandClinicalTrialsRegistry(ACTRN12616000368437).
2.2. HumanMilkSampleCollection
HMsampleswerecollectedonsiteatourresearchlaboratoryatKingEdwardMemorialHospital
for Women (Subiaco, Perth, Australia). Pre-feed and post-feed milk samples (~5 mL each) were
obtainedfromthebreast(s)theinfantfedfrombyhandexpressionorwithabreastpumpandwere
analysedseparately.Sampleswerecollectedbetween9:30and11:30a.m.tominimisepossiblecircadian
influencesonthemilkcomposition. Sampleswerestoredat−20◦Cforlaterbiochemicalanalysis.
2.3. AnthropometryandBodyComposition
Maternalweightwasmeasuredusinganelectronicscale(±0.1kg;Seca,Chino,CA,USA).Height
wasself-reportedbyparticipantsormeasuredagainstacalibratedmarkedwall(accuracy±0.1cm).
BMIwascalculatedaskg/m2.
Percentagefatmass(%FM)wasmeasuredwithwholebodybioimpedance(wristtoankle)using
anImpediMedSFB7tetra-polarbioelectricalimpedanceanalyser(Impedimed,Brisbane,Australia)
withtheparticipantinasupinepositiononanon-conductivesurfaceaccordingtothemanufacturer’s
instructions. Beforeeachsession,theexternalcalibrationofthebioelectricalimpendenceanalyserwas
testedwithacalibrationTestCell(ImpediMed,Brisbane,Australia). Tenconsecutivemeasurementsof
%FMweretakenwithin1–2minandaveraged. Within-participantcoefficientofvariationformaternal
%FMwas0.21%. Allmeasurementsweremadeafterthebreastfeedingsession.
2.4. LeptinandAdiponectinMeasurements
LeptinconcentrationsinwholeandskimHMweremeasuredusingtheDuoSetHumanLeptin
enzyme-linkedimmunosorbentassay(ELISA)(R&DSystems,Minneapolis,MN,USA)asdescribed
previously[28]. Thedetectionlimitwas0.05ng/mLwitharecoveryof96.3%±1.2%(n=10)forskim
milkand97.1%±9.1%(n=10)forwholemilkleptinandaninter-assaycoefficientofvariation(CV)
of<7.2%.
AdiponectinwasmeasuredinwholeHMusingtheBiovendorHumanAdiponectinSandwich
ELISAkit,(LifeTechnologies,Asheville,NC,USA).Thedetectionlimitwas1ng/mL,witharecovery
of96.2%±3.2%(n=10)andaninter-assayCVof<2.5%.
2.5. ProteinandLactoseMeasurements
Protein content was measured using the Bradford assay according to the methods of
Mitoulas et al. [44]. The detection limit was 1.03 g/L, with a recovery of 97.2% ± 1.4% (n = 10)
andaninter-assayCVof<1.9%.
Lactoseconcentrationwasmeasuredusingtheenzymatic–spectrophotometricmethodoutlined
byKuhnetal.[45]accordingtothemethodsofMitoulasetal.[44]. Thedetectionlimitwas30mM,
witharecoveryof98.2%±4.1%(n=10)andaninter-assayCVof<3.5%.
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2.6. StatisticalAnalyses
StatisticalanalyseswereperformedusingR2.15.1forWindows[46]. Thepackagesnlme[47]and
lattice[48],andRColorBrewer[49]wereusedforlinearmixedeffectsmodelinganddatarepresentation
respectively. Descriptivestatisticsarereportedasmean±standarddeviation(SD)andrangeunless
otherwisestated;modelparametersarepresentedasestimate±standarderror(SE).
Inordertocollectsystematicinformationovertimeandatfixedmomentsintimeandtomake
betteruseofthecollecteddata,acombineddataapproachthatconsidersindividual-levelrandom
effectstoaccountforparticipantsmeasuredattwoormorestudysessionswasadopted. Wefurther
contrastedtheresultsfromthecombineddataandfromthelongitudinalsubsettoconfirmourfindings.
During this study, infants were measured at least at one of the four time points (2, 5, 9 and
12 months postpartum). An approximate sample size was calculated using the ‘Linear multiple
regression: fixedmodel: r2increase’optioninG*Power[50]asifthiswasacross-sectionalstudywith
equalnumbersateachtime. Allowingfourpredictors(onemaineffect,threegroupcontrasts),α=0.05
and22participantsateachtimepoint(88samplepoints=22participants×4timepoints)gavethe
studypowerof0.80todetectaneffectsizeof0.15. Thisapproachwasselectedasthereisnoclosed
form expression suitable for the calculation of sample sizes for this research design [51], with the
considerationthatlongitudinalstudydesignismorepowerful. Tomaintainpredictedpowerandto
addressissuesrelatingtomissedvisits,suchasinabilitytoattendduetoillnessandunwillingnessof
mothersapproachedat2months(n=8)tocommittoastudythatrequiresbreastfeedingto12months,
therecruitmentofparticipantscontinuedpast22, resultingin111sessionsfor59(21longitudinal,
38cross-sectional)participants.
BCdataat9monthsoflactationismissingfortwolongitudinalparticipants. Missingdataalso
occurredforallmilkcomponentsduetoinsufficientmilksamplevolumes. Missingdatawasdealt
with using complete case (regression models) or available case (descriptive statistics) approaches.
Milksampleswerenotpooledforbiochemicalanalysis;thus,measureswerenotaveraged. Sample
sizesarepresentedinTable1.
Table1.Samplesizesusedinstatisticalanalyses.
MonthofLactation 2 5 9 12 Total
Participants*
Cross-sectional - - - - 38
Longitudinal - - - - 21
Total - - - - 59
Sessions
Cross-sectional 8 8 13 9 38
Longitudinal 15 21 19 18 73
Total 23 29 32 27 111
Samples(completecases)
Cross-sectional 19 21 33 27 100
Longitudinal 41 55 47 40 183
Total 60 76 80 67 283
Samples(availablecases)**
Wholemilkadiponectin 66 79 86 72 303
Wholemilkleptin 66 79 86 72 303
Skimmilkleptin 62 77 85 71 295
Totalprotein 64 78 87 69 298
Lactose 65 78 86 67 296
*Thenumberofparticipantsateachtimepointisthesameasthenumberofsessions,thusnotspecifiedinthetable;
**Thenumberofsamples(includingpre-feedandpost-feed)analysed;thisdiffersbycomponent.
LinearmodelswereusedtoinvestigateassociationsbetweenmaternalBMIor%FM(predictors)
andeachofthecompositionvariables(responses),withandwithoutcontrollingformonthoflactation
Nutrients2017,9,252 5of18
(four-level factor or linear predictor). Associations with month of lactation were assessed using
omnibus F-tests and specific post-hoc tests comparing each of the subsequent time points with
2 months. Appropriate random effects were selected by comparing four models for each analysis
usingalikelihoodratiotest. Modelswere(a)linearregression,andlinearmixedeffectsmodelswith
randomeffectsofoneof:(b)effectofgeneralinter-individualvariationpresentinthestudypopulation;
(c)effectofthemonthoflactationsampleswerecollectedat,inadditiontointer-individualvariation;
and(d)theeffectofpre-andpost-feedsamplesalongwithinter-individualvariation. Whetherthe
overalleffectofmaternaladiposityonHMcomponentconcentrationsdiffersbymonthoflactationwas
alsoinvestigatedbyincludinginteractionsbetweenBMI/%FMandthemonthoflactation(factoronly).
Toallowforrealisticinterpretationoftheinterceptvaluesinthemodeloutputs,maternalBMIand
%FMhavebeencentredattheupperboundsofthe‘healthy’range(25kg/m2forBMI,and33%for
%FM)[52,53]. Wheresignificantoutliervalueswereidentifiedfromakerneldensityplot,modelswere
runwithandwithoutthesevaluestodeterminehowtheymightbeinfluencingthefindings.
Anintercept-onlylinearmixedeffectsmodelwasusedtocalculatethecoefficientofvariationfor
maternal%FMmeasurements(n=10,10measurementseach).
3. Results
3.1. Participants
ParticipantadipositymeasuresandHMcomponents’concentrationsareshowninTable2. Mean
maternalagewas33.4±4.2yearsandparitywas1.8±0.8atthestartofthestudy. Atthefirstsession
attendedateither2,5,9or12monthspostpartumparticipantswereclassifiedasbeingunderweight
(BMI<18.5,5%,n=3;%FM<21,7%,n=4),ofnormalweight(BMI18.5–24.9,54%,n=32;%FM21–32.9,
50%,n=29),overweight(BMI25–29.9,24%,n=14;%FM33–38.9,28%,n=16)orobese(BMI>30,
17%,n=10;%FM>39,15%,n=9)[53]. Infantmale/femaleratiowas33/26.
Table2. Maternaladiposityandhumanmilkcomponentsconcentrationspresentedatthemonths
after birth for combined subset (n = 59) expressed as mean ± standard deviation (SD, range).
Someparticipants(n=21)contributedmilksamplesatmultipletimepoints.
2 5 9 12 Total
MonthofLactation
(n=23) (n=29) (n=32) (n=27) (n=111)
27.0±7.3 23.5±4.5 23.9±5.2 24.4±5.5 24.6±5.7
MaternalBMIa
(20.1–51.3) (17.0–35.2) (16.9–37.2) (18.2–37.2) (16.9–51.3)
34.9±6.4 32.5±6.0 30.9±7.9 31.3±7.2 32.3±7.0
Maternalfatmass(%)
(19.6–49.3) (23.2–47.2) (16.7–47.9) (19.4–45.3) (16.7–49.3)
0.55±0.29 0.50±0.17 0.53±0.15 0.54±0.13 0.53±0.19
Wholemilkleptin(ng/mL)
(0.21–2.24) (0.20–0.85) (0.21–0.99) (0.24–0.89) (0.20–2.24)
0.34±0.21 0.27±0.07 0.26±0.09 0.26±0.08 0.28±0.12
Skimmilkleptin(ng/mL)
(0.19–1.46) (0.20–0.48) (0.19–0.76) (0.19–0.54) (0.19–1.46)
11.12±4.39 9.30±3.94 8.46±2.26 11.07±7.88 9.88±5.05
Adiponectin(ng/mL)
(5.62–25.62) (5.17–29.67) (4.56–20.29) (4.74–54.92) (4.56–54.92)
12.94±6.15 11.7±5.70 10.83±4.63 12.83±6.74 11.96±5.82
Totalprotein(g/L)
(6.54–31.51) (7.00–34.76) (3.32–29.69) (6.60–36.89) (3.32–36.89)
67.54±9.05 68.07±8.10 68.37±8.83 69.70±9.11 68.41±8.75
Lactose(g/L)
(50.35–89.06) (50.92–110.07) (51.81–100.05) (51.00–98.36) (50.35–110.07)
Dataaremean±SDandranges. Concentrationsofcomponentsaremeasuredinbothpre-andpost-feedmilk
samples.aBMI—bodymassindex.
Nutrients2017,9,252 6of18
3.2. ChangesinComponents’ConcentrationwithFeeding(Pre-andPost-Feed)
HM component concentrations did not differ between pre-feed and post-feed samples in
univariatemodelsorafteraccountingforthemonthoflactationasalineareffectmodelorasafactor
(Table3).
Table3.Relativeconcentrationsofpre-feedhumanmilksamplescomparedtopost-feedsampleswith
andwithoutaccountingforpossiblemonthoflactationeffects.
AccountingforMonth AccountingforMonth
Univariatea
Predictor ofLactation(Linear)b ofLactation(Factor)b
PE±SE p PE±SE p PE±SE p
Adiponectin(ng/mL) 0.45±0.42 0.29 0.45±0.42 0.30 0.45±0.42 0.29c
Leptin(ng/mL)
Wholemilk −0.008±0.016 0.65 −0.008±0.017 0.65 0.007±0.016 0.66
Skimmilk −0.002±0.009 0.84 −0.002±0.009 0.86c −0.001±0.009 0.87c
Protein(g/L) −0.14±0.32 0.68 −0.14±0.32 0.68 −0.14±0.32 0.68
Lactose(g/L) −0.36±0.91 0.69 −0.38±0.88 0.66 −0.39±0.88 0.66
Dataareparameterestimate±SE.Analyseswererunonpre-andpost-feedsamplesusingcompletecaseapproach.
aEffectsofpredictorstakenfromunivariatelinearmixedeffectsmodels;bEffectsofpredictorstakenfromlinear
mixedeffectsmodelsthataccountedforthemonthoflactationaslinearmaineffectorasafactor;c Monthof
lactationissignificant(p<0.036).PE—parameterestimate;SE—standarderror.
3.3. AssociationsinCombinedSubset
3.3.1. DifferencesinConcentrationsofHumanMilkComponentsatDifferentMonthsofLactation
Table4presentsthechangesinHMcomponents’concentrationsinthecombinedsubset(n=57)
atfourtimepointsduringfirst12monthsoflactation.
While component concentrations differed by the month of lactation within participants for
all components (lactose: p = 0.031; adiponectin, whole and skim milk leptin, protein: p<0.001),
noconsistentmonthoflactation-relatedpatternswereseenforwholemilkleptin(p>0.47),protein
(p>0.37)andlactose(p>0.26).
Skim milk leptin decreased non-linearly over the months of lactation (univariate: p=0.024).
Post-hoctestsshowedthatadiponectinconcentrationat9monthswas−2.27±0.88ng/mLlower
(p=0.013)thanthatat2monthsoflactation(univariate: p=0.042)(Table4).
Table4. Associationsbetweenhumanmilkappetitehormonesandmaternaladiposity. Valuesare
parameterestimates±standarderror(n=57).Someparticipants(n=21)contributedmilksamplesat
multipletimepoints.
Adiponectin(ng/mL) WholeMilkLeptin(ng/mL) SkimMilkLeptin(ng/mL)
Predictor
PE±SE p PE±SE p PE±SE p
Univariatemodelsb
BMI 0.10±0.07 0.17 0.01±0.003 <0.001 0.008±0.002 <0.001a
%FM 0.01±0.06 0.81 0.006±0.002 0.008 0.005±0.002 0.007
Monthd - 0.042 - 0.52 - 0.024
Intercept 10.58±0.71 - 0.55±0.03 - 0.34±0.02 -
5e −1.39±0.87 0.12 −0.06±0.05 0.18 −0.07±0.03 0.038
9e −2.27±0.88 0.013 −0.01±0.05 0.75 −0.08±0.03 0.011
12e −0.26±0.93 0.78 −0.02±0.05 0.72 −0.10±0.03 0.005
Adjustedmodelfor%FM(monthoflactationaslinearmaineffect)c
Intercept 9.76±0.61 - 0.52±0.03 - 0.32±0.02 -
%FM 0.008±0.06 0.90 0.006±0.002 0.007 0.004±0.002 0.021
Monthd −0.05±0.09 0.57 0.003±0.004 0.47 −0.008±0.003 0.012
Nutrients2017,9,252 7of18
Table4.Cont.
Adiponectin(ng/mL) WholeMilkLeptin(ng/mL) SkimMilkLeptin(ng/mL)
Predictor
PE±SE p PE±SE p PE±SE p
Adjustedmodelfor%FM(monthoflactationasafactor)c
Intercept 10.59±0.72 - 0.54±0.03 - 0.34±0.02 -
%FM −0.01±0.06 0.86 0.006±0.002 0.009 0.004±0.002 0.028
Monthd - 0.044 - 0.51 - 0.063
5e −1.40±0.88 0.12 −0.04±0.04 0.34 −0.06±0.03 0.070
9e −2.30±0.90 0.014 0.01±0.05 0.76 −0.07±0.03 0.031
12e −0.29±0.95 0.77 0.007±0.05 0.88 −0.09±0.03 0.011
AdjustedmodelforBMI(monthoflactationaslinearmaineffect)c
Intercept 9.76±0.60 - 0.52±0.03 - 0.33±0.02 -
BMI 0.09±0.07 0.19 0.01±0.003 <0.001 0.008±0.002 <0.001
Monthd −0.04±0.09 0.62 0.002±0.004 0.59 −0.008±0.003 0.005
AdjustedmodelforBMI(monthoflactationasafactor)c
Intercept 10.51±0.71 - 0.54±0.003 - 0.34±0.02 -
BMI 0.07±0.07 0.33 0.01±0.003 <0.001 0.007±0.002 0.001
Monthd - 0.063 - 0.74 - 0.039
5e −1.23±0.89 0.17 −0.03±0.04 0.50 −0.05±0.03 0.13
9e −2.13±0.89 0.021 0.01±0.04 0.78 −0.07±0.03 0.025
12e −0.16±0.94 0.87 0.004±0.05 0.92 −0.09±0.03 0.007
Dataareparameterestimate±SE.Analyseswererunonpre-andpost-feedsamplesusingcompletecaseapproach.
aSignificantp-valuesareinboldfont;bEffectsofpredictorstakenfromunivariatelinearmixedeffectsmodels;
cEffectsofpredictorstakenfromlinearmixedeffectsmodelsthataccountedforthemonthoflactationaslinear
maineffectorasafactor;dOmnibusF-test;ePost-hoctestwithreference2months.Abbreviations:BMI—body
massindex;%FM—percentagefatmass;PE—parameterestimate;SE—standarderror.
3.3.2. AssociationsbetweenMaternalAdiposityandHMLeptin
Table4presentsassociationsbetweenadiposityandHMcomponents’concentrationsseeninthe
combinedsubset(n=57)atfourtimepointsduringfirst12monthsoflactation.
AssociationsbetweenMaternalAdiposityandHMLeptin
Higher%FMandBMIwereassociatedwithhigherconcentrationsofbothwhole(Figure1a,b)
andskimmilk(Figure2a,b)leptin(Table4). Accountingforthemonthoflactationasamainlinear
effect or as a factor did not change the associations with %FM and BMI for both whole and skim
milk leptin. Significant negative interactions were seen between %FM and the month of lactation
for whole milk leptin, (2 m: reference; 5 m: −0.02 ± 0.01, p = 0.023; 9 m: −0.02 ± 0.01, p = 0.003;
12 m: −0.03 ± 0.01, p < 0.001; month of lactation as a factor: p = 0.008), and for skim milk leptin
(2 m: reference; 5 m: −0.02 ± 0.01, p < 0.001; 9 m: −0.02 ± 0.01, p < 0.001; 12 m: −0.02 ± 0.01,
p=0.002;monthoflactationasafactor: p<0.001);andalsobetweenBMIandthemonthoflactation
for whole milk leptin, (2 m: reference; 5 m: −0.02 ± 0.01, p = 0.026; 9 m: −0.03 ± 0.01, p < 0.001;
12m: −0.03±0.01,p<0.001;monthoflactationasafactor: p<0.001),andforskimmilkleptin(2m:
reference;5m: −0.02±0.01,p<0.001;9m: −0.02±0.01,p<0.001;12m: −0.01±0.01,p=0.005;
monthoflactationasafactor: p=0.001),indicatingthattheassociationbetweenadiposityandleptin
weakensoverthefirst12monthsoflactation.
Removing statistically significant outliers resulted in either weakening or an absence of the
associationbetweeneither%FMorBMIandwholeandskimmilkleptinintheunivariatemodels
(%FM: 0.004 ± 0.002 ng/mL, p = 0.066; 0.003 ± 0.001 ng/mL, p = 0.043, respectively; BMI:
0.004±0.002ng/mL,p=0.065;0.004±0.002ng/mL,p=0.066,respectively)andafteraccounting
for the month of lactation as a linear effect (%FM: 0.004 ± 0.002 ng/mL, p = 0.039, age: p = 0.17;
0.002 ± 0.001 ng/mL, p = 0.12, age: p = 0.030, respectively; BMI: 0.005 ± 0.002 ng/mL, p = 0.053,
age: p = 0.25; 0.004 ± 0.002 ng/mL, p = 0.039, age: p = 0.17, respectively) or as a factor (%FM:
Nutrients2017,9,252 8of18
0.004±0.002ng/mL,p=0.044,age: p=0.37;0.002±0.001ng/mL,p=0.14,age: p=0.15,respectively;
BMI: 0.004 ± 0.002 ng/mL, p = 0.072, age: p = 0.53; 0.004 ± 0.002 ng/mL, p = 0.044, age: p = 0.37,
Nutrients 2017, 9, 252  8 of 18 
respectively). Nointeractionbetweeneither%FMorBMIandthemonthoflactationasafactorwas
Nutrients 2017, 9, 252  8 of 18 
seen(%FM:wholemilkleptin: p=0.37;skimmilkleptin: p=0.13;BMI:wholemilkleptin: p=0.24;
%FM or BMI and the month of lactation as a factor was seen (%FM: whole milk leptin: p = 0.37; skim 
skimm%FimlMki ll keoprl teBipnM:t ipIn  a=:n p0d.=1 t3h0;e .B1 mM8)o.I:n wthh oofl ela mctialtki olenp atsin a:  pfa =c t0o.r2 4w; assk ismee mn i(l%k FleMp:t iwn:h po l=e  0m.1i8lk).  leptin: p = 0.37; skim 
milk leptin: p = 0.13; BMI: whole milk leptin: p = 0.24; skim milk leptin: p = 0.18). 
a b
a b
2.0 2.0
2.0 2.0
mL) mL)
Whole milk leptin (ng/Whole milk leptin (ng/mL) 1111....0505 Whole milk leptin (ng/Whole milk leptin (ng/mL) 1111....0505
00..55 00..55
00..55 00..55
0.0 0.0
0.0 2200 30 40 50 0.0 2200 30 40 50
2200 F3a0t mass (%) 40 50 2200 Body3 0mass index (kg/m402) 50  
Fat mass (%) Body mass index (kg/m2)  
Figure 1. Associations between whole human milk (HM) leptin and (a) maternal percentage fat mass; 
Figure1.Associationsbetweenwholehumanmilk(HM)leptinand(a)maternalpercentagefatmass;
aFnigdu (rbe) 1 m. Aastesornciaal tBioMnsI .b Cetowmebeinn wedh osuleb hseutm daanta m piolkin (tHs M(m) eleapsutirne dan idn  (par) em‐ aatnedrn paol spte‐freceedn tsaagme pfalte sm) aasrse;  
and(b)maternalBMI.Combinedsubsetdatapoints(measuredinpre-andpost-feedsamples)are
sahnodw (nb ) ams actreornssa‐ls eBcMtioI.n Calo m(pbailnee db lsuueb) saent dd altoan pgoitiundtsi n(aml e(adsaurrke db ilnu ep).r eL‐ iannesd  aproes tf‐ifxeeedd  seafmfecptlse sf)r oamre  
shownascross-sectional(paleblue)andlongitudinal(darkblue).Linesarefixedeffectsfromunivariate
ushnoivwanri aatse  lcirnoesasr‐ smecitxioedna el ff(epcatl em boldueel)s : apnadle  lbolnugei tdudotitneadl  l(idnaer—k cbolmueb)i.n eLdin ceosh aorret  (fTixaebdle  e4f)f;e cmtse dfiruomm  
linearmixedeffectmodels:palebluedottedline—combinedcohort(Table4);mediumbluedashed
bulnuiev ardiaasteh eldin elairn em—ixceodm ebfifneecdt  mcoodheolrst:  pwailteh  blouuet ldieortst edre lminoev—edco m(Sbeicnteiodn  co3h.3o.r2t) ;( Tsaobllied  4d); amrke dbiulume  
line—combinedcohortwithoutliersremoved(Section3.3.2);soliddarkblueline—longitudinalcohort
lbinluee—  ldoansghietudd ilninael —cochoomrtb (iTnaebdl e cSo1h).o rt  with  outliers  removed  (Section  3.3.2);  solid  dark  blue 
(TablilneeS—1)l.ongitudinal cohort (Table S1). 
a b
a b
1.5 1.5
1.5 1.5
mL) mL)
Skim milk leptin (ng/m milk leptin (ng/mL) 11..00 Skim milk leptin (ng/m milk leptin (ng/mL) 11..00
Ski 00..55 Ski 00..55
00..55 00..55
0.0 0.0
0.0 2200 30 40 50 0.0 2200 30 40 50
2200 F3a0t mass (%) 40 50 2200 Body3 0mass index (kg/m402) 50  
Fat mass (%) Body mass index (kg/m2)  
Figure 2. Associations between skim human milk (HM) leptin and (a) maternal percentage fat mass; 
FiguaFnirgedu 2(rb.e)A 2 m.s sAaotscesrioanctaiialo tBnioMsnbIs.e  bCtweotwmeeebneinnse ksdikm ismuhb husuemtm adanant mam piillokki n((HHts MMar))e ll eesphptotiiwnn naan nadsd  (ca(ra)o )msmsa‐staeetcretnriaonlna pallep r(ecpreacnleetna pgtaueg rfpealtef am) taamnssda;  ss;
andlaon(nbdg) i(mtbu)ad mtienarantle ar(dlnBaarlM kB IMp.uCIr. opCmloe)mb. iLbniienndeedss  uasrubebs esfietxtde ddaa tetaaffp epocotiisnn tftsrso aamrree  ussnhhioovwwarnnia aatses  clcirnrooessass‐rs- smeecitcxitoeiondan ela f(lfpe(acptlae m lpeoupdrupelrlsep:) l peaa)nladen  d
longploiuntrugpdilteiun ddaiolnt(atdel da( drlkianrpek—u prcuporlmpe)lbe.i)nL. eiLndine cseosah aroerretfi  (fxTixeaedbdlee e f4fffe)e;c cmttsse fdfrriooummm  uupnnuiirvvpaalreri iadattaees lhliinendeea larin rmem—ixixecodemd efbefifenfceetcd mt cmoodhoeodlrset: l wsp:aitplhea  le
puroppuuletrlpideloers td treoedtmteloidnv leeid—n e(c—Soecmcotibmoinbn i3en.d3e.dc2 o)c;h osohorolitrdt(  Td(Taabarbklel pe4 u4)r;);pm mlee elddiniiuuem—m lppounugrrppitllueed ddinaasashlh ecedodh liolnirnet e—(—Tcaocbmolemb Sib1ni)en.d e dcochoohrto wrtiwthi th
outliers removed (Section 3.3.2); solid dark purple line—longitudinal cohort (Table S1). 
outliersremoved(Section3.3.2);soliddarkpurpleline—longitudinalcohort(TableS1).
3.3.3. Associations between Maternal Adiposity and HM Adiponectin 
3.3.3. Associations between Maternal Adiposity and HM Adiponectin 
HM adiponectin was not significantly associated with either %FM or BMI in the univariate 
HM adiponectin was not significantly associated with either %FM or BMI in the univariate 
models (Table 4) or after accounting for the month of lactation. No interactions were seen between 
models (Table 4) or after accounting for the month of lactation. No interactions were seen between 
the month of lactation as a factor and either %FM (p = 0.51) or BMI (p = 0.62).  
the month of lactation as a factor and either %FM (p = 0.51) or BMI (p = 0.62).
Nutrients2017,9,252 9of18
3.3.3. AssociationsbetweenMaternalAdiposityandHMAdiponectin
HMadiponectinwasnotsignificantlyassociatedwitheither%FMorBMIintheunivariatemodels
(Table4)orafteraccountingforthemonthoflactation. Nointeractionswereseenbetweenthemonth
oflactationasafactorandeither%FM(p=0.51)orBMI(p=0.62).
Removingastatisticallysignificantoutlierdidnotchangetheconclusion(%FM:p≥0.50;BMI:
p≥0.083) and no interaction between the month of lactation and either %FM (p = 0.54) or BMI
(p=0.081)wasseen.
3.3.4. AssociationsbetweenMaternalAdiposityandHMProtein
Higher%FMwasassociatedwithhigherconcentrationsofproteininHMinunivariatemodel
(Table5;Figure3a). Accountingforthemonthoflactationmadetheassociationbetween%FMand
proteinconcentrationsweakerbutstillsignificant. BMIwasnotassociatedwithconcentrationsof
protein in HM in the univariate model (Figure 3b) or after accounting for the month of lactation.
No interaction with the month of lactation as a factor was seen for both %FM (p = 0.21) and BMI
(p=0.16).
Table 5. Associations between human milk macronutrients and maternal adiposity. Values are
parameterestimates±standarderror(n=57).Someparticipants(n=21)contributedmilksamplesat
multipletimepoints.
Predictor Lactose(g/L) Protein(g/L)
PE±SE p PE±SE p
Univariatemodelsb
BMI 0.06±0.14 0.66 0.14±0.09 0.14
%FM 0.08±0.11 0.48 0.16±0.07 0.028a
Monthd - 0.65 - 0.37
Intercept 67.32±1.43 - 12.90±1.11 -
5e 0.56±1.89 0.77 −1.39±1.48 0.35
9e 1.31±1.88 0.49 −2.47±1.47 0.10
12e 2.35±1.97 0.24 −0.63±1.55 0.69
Adjustedmodelfor%FM(monthoflactationaslinearmaineffect)c
Intercept 68.89±1.13 - 11.93±0.87 -
%FM 0.09±0.11 0.43 0.16±0.07 0.036
Monthd 0.08±0.15 0.59 −0.03±0.14 0.84
Adjustedmodelfor%FM(monthoflactationasafactor)c
Intercept 67.58±1.44 - 12.54±1.11 -
%FM 0.06±0.11 0.55 0.15±0.07 0.054
Monthd - 0.67 - 0.56
5e 0.60±1.85 0.75 −0.94±1.47 0.53
9e 1.37±1.87 0.50 −1.78±1.49 0.24
12e 2.27±1.96 0.25 −0.02±1.56 0.99
AdjustedmodelforBMI(monthoflactationaslinearmaineffect)c
Intercept 67.71±2.92 - 12.10±0.88 -
BMI 0.06±0.13 0.63 0.13±0.09 0.16
Monthd 0.20±0.17 0.26 −0.07±0.14 0.61
AdjustedmodelforBMI(monthoflactationasafactor)c
Intercept 67.55±1.44 - 12.69±1.12 -
BMI 0.06±0.13 0.66 0.12±0.09 0.21
Monthd - 0.69 - 0.48
5e 0.63±1.88 0.74 −1.01±1.50 0.50
9e 1.29±1.87 0.49 −2.14±1.49 0.16
12e 2.22±1.95 0.26 −0.36±1.56 0.82
Dataareparameterestimate±SE.Analyseswererunonpre-andpost-feedsamplesusingcompletecaseapproach.
aSignificantp-valuesareinboldfont;bEffectsofpredictorstakenfromunivariatelinearmixedeffectsmodels;
cEffectsofpredictorstakenfromlinearmixedeffectsmodelsthataccountedforthemonthoflactationaslinear
maineffectorasafactor;dOmnibusF-test;ePost-hoctestwithreference2months.Abbreviations:BMI—body
massindex;%FM—percentagefatmass;PE—parameterestimate;SE—standarderror.
Nutrients2017,9,252 10of18
Nutrients 2017, 9, 252  10 of 18 
a b
30 30
Protein (g/L) 20 Protein (g/L) 20
10 10
00 00
2200 30 40 50 2200 30 40 50
Fat mass (%) Body mass index (kg/m2)  
FigFuirgeur3e. 3. AAssssoocciiaattiioonnss bebtewtweeene pnroptreointe cionncceonntcraetniotrna atinodn (aa)n mdat(ear)naml apteerrcneanltapgee rfcaet nmtaagsse; afnadt (mb)a ss;
maternal BMI. Combined subset data points are shown as cross‐sectional (pale orange) and 
and(b)maternalBMI.Combinedsubsetdatapointsareshownascross-sectional(paleorange)and
longitudinal (dark orange). Lines are fixed effects from univariate linear mixed effect models: pale 
longitudinal(darkorange). Linesarefixedeffectsfromunivariatelinearmixedeffectmodels: pale
orange dotted line—combined cohort (Table 5); solid dark red line—longitudinal cohort (Table S1). 
orangedottedline—combinedcohort(Table5);soliddarkredline—longitudinalcohort(TableS1).
3.3.5. Associations between Maternal Adiposity and HM Lactose 
3.3.5. AssociationsbetweenMaternalAdiposityandHMLactose
Neither %FM or BMI were associated with concentrations of lactose in HM in univariate model 
Neither%FMorBMIwereassociatedwithconcentrationsoflactoseinHMinunivariatemodel
(Table 5) or after accounting for the month of lactation. No interaction with the month of lactation as 
(Taba lfeac5t)oor rwaafste sreeancc foour nBtMinIg (pf o=r 0t.h19e),m bount tah siogfnliafcictaantito pno.sNitiovein intetrearacctitoionnw wiaths steheenm beotnwtheeonf %laFcMta tainodn as
afatchteo rmwonatshs oeef nlacfotartiBoMn (I2( pm=: r0e.f1e9r)e,nbcue;t 5a msi:g 0n.1ifi2 c±a 0n.t33p,o ps i=t i0v.7e1i;n 9t emra: c−t0io.1n4 w± 0a.s30s,e pe n= b0.e6t3w; 1e2e nm%: 0F.5M8 ±a nd
the0m.31o,n pt h= 0o.f06la8c; tmatoinotnh (o2f mlac:traetifoenre ansc ae ;fa5ctmor:: 0p. =1 20.±0290).,3 i3n,dpic=at0in.7g1 t;h9atm as:s−oc0ia.1t4ion± b0e.t3w0e,epn= %0F.M63 ;an12d m:
0.58la±cto0s.e3 1st,rpen=gt0h.0e6n8s ;omveorn tthhe ofifrslta 1ct2a mtioonntahss aoff laaccttoarti:opn.=  0.029),indicatingthatassociationbetween
%FMandlactosestrengthensoverthefirst12monthsoflactation.
3.4. Associations in the Longitudinal Subset 
3.4. AssociationsintheLongitudinalSubset
3.4.1. Participants 
3.4.1. Participants
Longitudinal (n = 21) participants’ characteristics and HM components’ concentrations are 
shLoownng iitnu dTianbalel (Sn2=. P2a1r)tipcaiprtainctips ainn tlso’ncghiaturadcitnearli sstuicbsseatn wdeHreM gceonmerpalolyn elenatsn’ecro tnhcaenn itnra tthioen csoamrebisnheodw  n
inTsaubblseetS, 2b.uPt anrotnicei pwaenrets uinndleornwgeiitguhdt.i nAatl tshueb fsirestt wseesrseiogne tnheerya wllyerlee calnaessriftiheadn asin: ntohremcaolm wbeiinghedt (BsuMbIs et,
but1n8o.5n–e24w.9e,r 6e7u%n, dne =r w14e;i %ghFtM. A 2t1t–h3e2.fi9,r s5t7%se,s nsi =o n12t)h, eoyvewrwereeigchlats (sBifiMeId 2a5–s:29n.o9r, m19a%l,w ne =ig 4h; t%(FBMM I331–83.58–.92,4 .9,
67%2,9n%=, n1 4=; 6%) FoMr o2b1e–se3 2(.B9M,5I7 >% 3,0n, =141%2), ,no =v e3r;w %eFigMh t>( B39M,1I42%5,– n2 9=. 93,)1 [95%3],. nIn=fa4n;t% mFaMle:3fe3m–3a8le.9 ,ra2t9io% w,nas= 6)
10:11. 
orobese(BMI>30,14%,n=3;%FM>39,14%,n=3)[53]. Infantmale:femaleratiowas10:11.
3.4.32..4L.2o. nLgonitguidtuindainlaCl hCahnagnegseisn inC Coonncecenntrtraattioionnss ooff HHuummaann MMiillkk CCoommppoonneenntst s
Table S1 presents the changes in HM component concentrations in the longitudinal subset (n = 
TableS1presentsthechangesinHMcomponentconcentrationsinthelongitudinalsubset(n=21
21 participants, 73 sessions). While component concentrations differed by the month of lactation 
participants,73sessions). Whilecomponentconcentrationsdifferedbythemonthoflactationwithin
within participants for all components (lactose: p = 0.020; adiponectin, whole and skim milk leptin, 
participantsforallcomponents(lactose: p=0.020;adiponectin,wholeandskimmilkleptin,protein:
protein: p < 0.001), no consistent month of lactation‐related patterns were seen for adiponectin (p > 
p<0.001),noconsistentmonthoflactation-relatedpatternswereseenforadiponectin(p>0.32),whole
0.32), whole milk leptin (p > 0.11) or lactose (p > 0.46). Although the overall pattern for protein was 
milkleptin(p>0.11)orlactose(p>0.46). Althoughtheoverallpatternforproteinwasnotsignificant
not significant (p > 0.10), post‐hoc tests showed that protein concentration at 9 months was 3.80 ± 1.65 
(p>0.10),post-hoctestsshowedthatproteinconcentrationat9monthswas3.80±1.65g/Llower
g/L lower than that at 2 months of lactation (univariate, p = 0.027). Skim milk leptin decreased 
thanthatat2monthsoflactation(univariate,p=0.027). Skimmilkleptindecreasednon-linearlyover
non‐linearly over the months of lactation (univariate: p = 0.007). 
the monthsoflactation(univariate : p=0.007).
3.4.3. AssociationsbetweenMaternalBodyMassIndexandPercentageFatMass
A strong relationship (p < 0.001) was observed between maternal BMI and %FM in the
lon gitudinal subset, with a one-unit increase in BMI associated with a 1.07% ± 0.17% increase in