www.jped.com.br
ORIGINAL
ARTICLE
Effect
of
maternal
vitamin
A
supplementation
on
retinol
concentration
in
colostrum
夽
,
夽夽
Evellyn
C.
Grilo
a,∗,
Mayara
S.R.
Lima
a,
Lahyana
R.F.
Cunha
b,
Cristiane
S.S.
Gurgel
a,
Heleni
A.
Clemente
a,
Roberto
Dimenstein
aaUniversidadeFederaldoRioGrandedoNorte(UFRN),Natal,RN,Brazil
bUniversidadeFederaldeCampinaGrande(UFCG),CampinaGrande,PB,Brazil
Received7February2014;accepted14May2014 Availableonline2September2014
KEYWORDS
Colostrum; Fasting;
Postprandialperiod;
Supplementary feeding;
VitaminA
Abstract
Objective: ToinvestigatetheeffectofvitaminAsupplementationontheretinolconcentration incolostrumunderfastingandpostprandialconditions.
Methods: Thiswasaquasi-experimentalstudy,withbeforeandafterassessments,conducted with 33patients treatedatapublicmaternity hospital.Bloodandcolostrum sampleswere collectedunderfastingconditionsintheimmediatepostpartumperiod.Asecondcolostrum col-lectionoccurredtwohoursafterthefirstmealoftheday,atwhichtimeamegadoseof200,000 IUofretinyl palmitatewasadministered.Onthefollowingday,thecolostrumwas collected againunderfastingandpostprandialconditions.Serumandcolostrumretinolconcentrations weredeterminedbyhighperformanceliquidchromatography.
Results: The serum retinol concentration was 37.3 (16.8-62.2) g/dL, indicating adequate nutritionalstatus.Thecolostrumretinolconcentrationbeforesupplementationwas46.8 (29.7-158.9)g/dLinfastingand67.3(31.1-148.7)g/dLinpostprandialcondition(p<0.05),showing anincreaseof43.8%.Aftersupplementation,thevalueswere89.5(32.9-264.2)g/dLand102.7 (37.3-378.3)g/dLinfastingandpostprandialconditions,respectively(p<0.05),representing anincreaseof14.7%.
Conclusions: Thisstudydemonstratedthatmaternalsupplementationwithhighdosesof vita-minAinpostpartumresultedinasignificantincreaseoftheretinolconcentrationincolostrum underfastingconditions,withanevengreaterincreaseafterameal.
©2014SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Allrightsreserved.
夽 Pleasecitethisarticleas:GriloEC,LimaMS,CunhaLR,GurgelCS,ClementeHA,DimensteinR.EffectofmaternalvitaminA
supple-mentationonretinolconcentrationincolostrum.JPediatr(RioJ).2015;91:81---6.
夽夽
StudyconductedatUniversidadeFederaldoRioGrandedoNorte(UFRN),Natal,RN,Brazil. ∗Correspondingauthor.
E-mail:evellyn-cg@hotmail.com(E.C.Grilo),mayara.lima@yahoo.com.br(M.S.R.Lima). http://dx.doi.org/10.1016/j.jped.2014.05.004
PALAVRAS-CHAVE
Colostro; Jejum;
Períodopós-prandial;
Suplementac¸ão
alimentar;
VitaminaA
Efeitodasuplementac¸ãomaternacomvitaminaAsobreaconcentrac¸ãoderetinolno colostro
Resumo
Objetivo: Investigaroefeitodasuplementac¸ãocomvitaminaAsobreaconcentrac¸ãoderetinol noleitecolostroemcondic¸õesdejejumepós-prandial.
Métodos: Estudoquase-experimental,dotipoantesedepois, realizadocom33parturientes atendidasem umamaternidadepública, dasquaisforamcoletadas,em jejum,amostrasde sangueeleitecolostro,nopós-partoimediato.Umasegundacoletadecolostroocorreuduas horasapós aprimeira refeic¸ão do dia, momentoem que uma megadose de 200.000UI de palmitatoderetinilafoiadministrada.Nodiaseguinte,umanovacoletadecolostrofoirealizada emcondic¸õesdejejumepós-prandial.Asconcentrac¸õesderetinolnosoroenocolostroforam determinadasporcromatografialíquidadealtaeficiência.
Resultados: Aconcentrac¸ãoderetinolséricofoide37,3(16,8-62,2)g/dL,evidenciandoum estadonutricionaladequado.Nocolostro,aconcentrac¸ãoderetinolantesdasuplementac¸ão foide46,8(29,7-158,9)g/dLemjejume67,3(31,1-148,7)g/dLemcondic¸õespós-prandiais (p<0,05),mostrandoumaumentode43,8%.Apósasuplementac¸ão,osvaloresforamde89,5 (32,9-264,2)g/dLe102,7(37,3-378,3)g/dLemjejumepós-prandial,respectivamente(p< 0,05),representandoumaumentode14,7%.
Conclusões: Estetrabalhodemonstrouqueasuplementac¸ãomaternacomaltasdosesde vitam-inaAnopós-partoresultouemumaumentosignificativodaconcentrac¸ãoderetinolnocolostro emcondic¸õesdejejum,sendoestevaloraindamaiorapósarefeic¸ão.
©2014SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Todososdireitos reservados.
Introduction
VitaminAisessentialforhumangrowthanddevelopment,
preserves vision, and contributes to the proper
function-ing of the immune system, defending the body against
infections.1
VitaminAdeficiency(VAD)canleadtodisorderssuchas xerophthalmiaandnightblindnessinchildhood,aswellas anemiaandlowresistancetoinfections,whichcanincrease the severity of infectious diseases and the risk of death. Childrenofpreschoolageandpregnantwomenare consid-eredthepopulationsatgreatestriskforVAD;itisestimated thatapproximatelyone-third of theworld’s population of preschoolersand15%ofpregnantwomenarebiochemically deficient,mainlyinAfricaandSoutheastAsia.2
StudiesindicatethatvitaminAdeficiency constitutesa publichealth problemin the Northand Northeastregions andsomepartsofSoutheasternBrazil.3In2006,theNational
DemographicandHealthSurvey(NDHS)indicatedthatthe prevalenceofVADinBrazilis17.4%inchildrenyoungerthan 5yearsand12.3%innon-pregnantwomenofreproductive age.4
Pregnant and lactating women have a higher require-mentofvitaminA,andtheriskofdeficiencyisaggravated by low nutrient intake and the emergence of infections inthese groups.1,5 The World HealthOrganization (WHO),
theUnitedNationsInternationalChildren’sEmergencyFund (UNICEF), and the International Vitamin A Consultative Group (IVACG) recommend the provision of high doses of vitaminA(200,000IU)untilthe60thdayafterdelivery to postpartumwomenfromregionswherethedeficiencyofthis nutrientisendemic.6
Breastmilkisa sourceof energyand nutrientsat ade-quate amounts for the infant’s nutrition, which includes proteins,lipids,carbohydrates,minerals,vitamins, lympho-cytes,immunoglobulins, andgrowthfactors.7 Colostrumis
the milk secretion of the first dayspostpartum, and sev-eralstudieshavedemonstratedtheprotectiveeffectagainst neonatalmortalityoffeeding thenewbornwiththismilk, especiallywhenofferedinthefirsthouroflife.8
According to Black et al., 9 the risk of a newborn to
haveitsreservesexhaustedisgreaterwhenthereis mater-nalmicronutrientdeficiency.Thus,thevitaminAcontentof breastmilkisthemaindeterminantofthenutritionalstatus ofvitaminAinthenewborn.
Rossetal.10affirmthatretinolistransferredtothemilk
intwoways:throughtheretinolbindingprotein(RBP)and throughchylomicrons.However,themechanismofvitamin Atransfertomilkisyettobefullyunderstoodinhumans, andisbeingstudiedinanimalmodels.11
Thus, the present study aimed to evaluate the effect ofmaternal vitaminAsupplementationonretinol concen-trationinhumancolostrumunderfastingandpost-prandial conditions,aimingtocontributetotheunderstandingofthe mechanisms of retinol transfer to the mammary gland in humans,asmostofthestudiesfoundintheliteraturewere performedinanimals.
Methods
Thiswasaquasi-experimentalinterventionstudy,assessed
beforeandafterthe intervention,carriedoutina
conve-niencesample.Thestudyincluded33voluntarypostpartum
departmentofapublicmaternityhospital.Thesamplesize
wascalculatedconsideringanalphaerrorof5%,studypower
of80%,andthesizeoftheeffectequalto0.60.Basedon
thesecalculations,aminimumsamplesizeof33participants
wasestimated.12
Womenwithmorbidities(diabetesmellitus,cancer,liver andgastrointestinaltractdiseases,heartdiseases,syphilis, andhumanimmunodeficiencyvirusinfection),adolescents, womenwhohadpretermbirthorwithmultipleormalformed fetuses,aswellasthosewhoreportedhavingused supple-mentscontainingvitaminAduringpregnancyorimmediate postpartumperiodwerenotconsidered eligiblefor partic-ipation inthe study.Participants whowerein accordance withtheselectioncriteriawereincludedinthestudyafter signingthe informedconsentformapprovedby theEthics CommitteeinResearchoftheUniversityHospital.
Dataandbiologicalmaterialcollection
Blood(5mL)and colostrummilk(2mL)samples were
col-lected from postpartum women under fasting condition,
before the first meal of the day. Blood was obtained by
venipuncture performed by a maternity nursing
profes-sional. The milk was collected by trained undergraduate
students, through manual expression of a single breast,
at the beginning and end of the breastfeeding. A second
collectionof colostrumoccurred twohours afterthe first
meal of the day (postprandial condition). Subsequently,
a capsule of 200,000 IU (60mg) of retinyl palmitate was
administered.On thefollowingday, anewcolostrummilk
collection was performed under fasting and postprandial
conditions. The samples were collected in polypropylene
tubes protected from light and kept under refrigeration
duringtransporttotheFoodBiochemistryandNutrition
Lab-oratory.Thebloodsampleswerecentrifugedfor10minutes
(500xg)forserumseparationandremoval,whichwasstored
at -20◦ C as well as 500L aliquots of colostrum milk,
until the analyses were performed. Maternal and
obstet-ricdatawerecollectedfrommedicalrecordsofpostpartum
women.
Quantificationofretinolinmaternalserumand colostrummilk
Retinolwasextractedfromcolostrummilksamples
accord-ingto theadapted method ofGiuliano etal.13 Extraction
ofserumretinolwasperformedaccordingtothemethodof Ortegaetal.14
Thedriedextractsobtainedfrommilkandserumsamples were dissolved in absolute ethanol and 20L were ana-lyzedinthehighperformanceliquidchromatography(HPLC) equipment.Themobilephaseusedwas100%methanolinan isocraticsystemwithaflowof1.0mL/min,andthe wave-lengthusedwas325nm.
The identification and quantification of retinol in the samples wasestablished by comparing the peak obtained in the chromatogram with the area of retinol standard (Sigma®, MO, United States). The concentration of the standardwasconfirmedbyspecificextinctioncoefficientfor retinol(E1%,1cm=1780to325nm)inabsoluteethanol.15
Method accuracywasevaluatedbyextraction recovery test.Themeanrecoveriesobtainedfortheretinolacetatein milkandserumsampleswere96%and95%,respectively, sug-gestingextractionefficiency.Acceptablerecoveryintervals aregenerallybetween80%and110%.16
The linearityof thecurvewasdeterminedbyassessing the proportionality between the detector response and differentretinol standard concentrations. The calibration curve was obtained by linear regression (peak area as a functionofstandardconcentration)andtheobtained corre-lationcoefficientwas0.990,allowingforthequantification ofretinolbytheexternalstandardmethod.
Statisticalanalysis
TheShapiro-Wilkstestwasusedfortheassessmentof
nor-mality of the retinol values. The study data consist of
paired samples and presented a non-normal distribution,
thusthenonparametricWilcoxontestforpairedsampleswas
used.Differenceswereconsideredsignificantwhenp<0.05.
Spearman’scorrelationtestwasusedtoevaluatethe
asso-ciationbetweenretinollevelsinserumandcolostrummilk.
StatisticalanalyseswereperformedusingtheMinitab15
Sta-tisticalsoftware(Minitab,StateCollegePA,UnitedStates)
andRDevelopmentCoreTeamsoftware(Lucent
Technolo-gies,NJ,UnitedStates).
Results
The median concentration of retinol in the serum of 33
women was 37.3 (16.8 to 62.2) g/dL, considered
ade-quateaccordingtothereferencevalues(>20g/dL).17 In
colostrum milk, the median retinol concentration before supplementationwas46.8(29.7to158.9)g/dLunder fas-ting and 67.3 (31.1 to 148.7) g/dL under postprandial condition(p<0.05),showinganincreaseof43.8%afterthe meal.Aftersupplementation,thevalueswere89.5(32.9to 264.2)g/dL and 102.7 (37.3 to378.3) g/dL under fas-ting and postprandial conditions, respectively (p < 0.05), equivalenttoanincreaseof14.7%(Table1).Furthermore, comparisonofretinollevelsincolostrummilkunderfasting conditionbeforesupplementationwithpostprandialretinol levelsaftersupplementationshowedanincreaseof119,4%. Retinol levelsin thecolostrum milkof fastingmothers beforesupplementation showed no correlation with their serumretinolvalues.Thecorrelationsbetweenretinol lev-elsofcolostrumsamples,collectedatdifferenttimes,are showninTable2.
Discussion
Untiltheendofpregnancy,abalanceddietisimportantto
guaranteethetransferofnutrientstothefetus,inorderto
prepareitforbirthandlactation.VitaminAplaysan
impor-tantroleduringtheseperiods,asitisinvolvedincellgrowth
andproliferationprocesses,suchasthosethatoccurduring
pregnancy,lactation,andearlychildhood.5
Table1 Retinolconcentration(g/dL)incolostrummilkandserumofpregnantwomeninapublicmaternityhospital.
Median IQI25-75 LL UL
Milk(BS,fasting) 46.8 33.9-102.9 29.7 158.9
Milk(BS,postprandial) 67.3 44.1-105.5 31.1 148.7
Milk(AS,fasting) 89.5 63.1-137.7 32.9 264.2
Milk(AS,postprandial) 102.7 76.6-150.5 37.3 378.3 Maternalserum(BS,fasting) 37.3 31.2-50.3 16.8 62.2
IQI25-75,interquartileinterval;LL,lowerlimit;UL,upperlimit;BS,beforesupplementation;AS,aftersupplementation.
thisintervention resultsin an increase inretinol levelsin
breastmilk.18,19
Serum retinol levels observed in the present study (37.3g/dL) were lower than those found in women from Germany (49.3g/dL)20 and Southeastern Brazil
(48.1g/dL),21andsimilartothoseobservedinwomenfrom
NortheasternBrazil(40.1g/dL).22
Colostrum milk retinol levels in fasting conditions before supplementation were lower than those found in women from Northeastern Brazil (100.3g/dL and 92.9±50mg/dL)22,23.Thesedifferencesmaybeduetothe
largersample size in the aforementioned studies andthe factthatretinollevelsinmilkwereexpressedasmeanand standarddeviation,astheyshowednormaldistribution.
In thepresent study,when retinollevels inmilk under fastingandpostprandialconditionswerecompared,a sig-nificantincreasewasobservedinthelatteronthetwodays ofcollection.Inthestatisticalanalysis,thevaluesofr indi-catedastrong correlation(r=0.65)between theselevels, bothbeforeandaftersupplementation(Table2).
AnimalstudieshavedemonstratedthatvitaminAis trans-ferred to breast milk from two sources: retinol-binding protein(RBP)andretinylesterstransportedbylipoproteins (chylomicrons).24
Under fasting conditions, almost all circulating retinol is associated with RBP, and levels of this protein do not vary with the ingestion of large amounts of vitamin A. However, retinyl esters transported by chylomicrons vary accordingtothecontentofvitaminAinthediet,andthese lipoproteins contribute to the transport of retinol to the mammarygland.10,11,25---27
In this study,the increase in colostrumretinol after a mealsuggeststhatinhumans,inadditiontothemechanism ofretinoltransfertothemammaryglandviaRBP,thereisan additionalwaythroughwhichthisnutrientistransferred.As thisincreasewasobservedinpostprandialconditions,itis
believedthatthisadditionalmechanismoccursvia chylomi-crons,asdemonstratedinanimals.Thus,itissuggestedthat vitamin A supplementation should be administered close to mealtimes to achieve active participation of chylomi-crons andconsequently, better utilization of thesupplied vitamin.
AccordingtoBlaneretal.,27 approximately60%of
vita-minAistransferredtothemammaryglandbychylomicrons andrelies onlipoprotein lipase(LPL) bindingsites andon the hydrolysis of retinyl esters of chylomicrons through the action of this enzyme, whose activity increases dur-inglactation.AhigherpercentageofingestedvitaminAis preferentiallytargetedtothemammary glandinlactating women,incontrasttowhatoccursintheabsenceof lacta-tionstates.10,25,26 Itisalsolikelythatagreatertransferof
vitaminAtothemammaryglandoccursduetoanincreasein RBP-retinolbreastreceptorstoformcolostrummilk.28This
evidence corroboratestheresults obtainedin thepresent study,which showedan increase inthe amount of retinol afteramealandsupplementation.
The vitaminA releasedduring the hydrolysisof retinyl estersmaybere-esterifiedforhumanbreastmilksecretion orstoredinepithelialcells,whichexplainsthemaintenance ofhighlevelsofvitaminAinhumanmilkhoursafter supple-mentation,asobservedin thisstudy.Thisoccurswiththe participation of the enzyme that esterifies retinol and is present in breast tissue, called acyl-coenzyme A: retinol acyltransferase (ARAT).25,26,29,30 In addition,liverstores of
vitaminAarealsoincreasedaftersupplementationand con-tributetohigherlevelsofretinolinbreastmilk.
Thus, it is suggested that vitamin A supplementation during postpartum increases the percentage of retinol in colostrum milk, which, in postprandial conditions, occurs throughthecontributionofchylomicronretinylesters pro-ducedasaconsequenceofthemeal.Chylomicronremnants areclearedmainlybytheliver,butextrahepaticuptakeof
Table2 Correlationbetweenretinolconcentrationsinthemilkofmothersunderfastingandpostprandialconditions,before andaftervitaminAsupplementation.
Correlation(r)a BS,fasting BS,postprandial AS,fasting AS,postprandial
BS,fasting 1.00 0.65 0.50 0.28
BS,postprandial 0.65 1.00 0.32 0.26
AS,fasting 0.50 0.32 1.00 0.65
AS,postprandial 0.28 0.26 0.65 1.00
Spearman’scorrelation.
BS,beforesupplementation;AS,aftersupplementation.
theremnantsareimportantinthedeliveryofvitaminAto mammarytissue,beingtransportedbyRBP.
As shown in Table 2,31 the statistical analysis
demon-strated a strong correlation between thelevelsof retinol incolostrummilkofwomenbeforeandafterthemealon the first day, i.e., beforematernal supplementation with vitamin A.However, thelevels of retinol under postpran-dialconditiononthefollowingdayaftertheadministration of vitamin A supplement showed a weak correlation with fastingretinollevelsonthefirstday.
One explanation for the above-mentioned weak corre-lationis thatthereappears tobea limitfor theincrease in the levelsof micronutrients in breastmilk, which may occur due tosaturation of theproteins involved in trans-ferpathwaysofretinol fromblood tothemammary gland (lipoproteinsandRBP),aswellastheenzymesinvolvedin re-esterification and secretion of vitamin A in colostrum. Suchsituationwouldpreventanexcessiveincreaseofretinol inmilk,indicatingtheexistenceofanadaptivemechanism topreventthepassageof excessiveamountsofretinol to breastmilk,thusprotectingthebabyagainstthetoxicityof thismicronutrient.
It is knownthat theconcentration of retinol inhuman serum is greatly controlled by liver homeostasis mecha-nisms. On the other hand, this study showed that the biochemicalstateofretinolinbreastmilkcanbemodified accordingtodietarychanges.Thus,forthediagnosisofthe nutritionalstatusofvitaminA,itisnecessarythatthemilk collectionisperformedunderfastingconditions.
Thisstudyobservedanincreaseinretinollevelsinhuman colostrumunderpostprandialconditionsandaftermaternal supplementationwithretinylpalmitate.Thisindicatesthat the mechanism of retinol transfer tothe mammary gland via chylomicrons,alreadyknowninanimals,alsoprobably existsinhumans.Furthermore,itwaspossibletoverifythe effectivenessofmaternalsupplementation,indicatingthat thesupplyofhighdosesofvitaminAtowomeninthe imme-diatepostpartumperiod,andespeciallyafterameal,isan effectivestrategytoimprovethenutritionalstatusofretinol inlactatingwomenandtheirinfants.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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