Ci a ion: U e a-Velasco, N.;
Mon ealeg e-Poma , A.; Kelle , K.;
Escude -Vieco, D.; Fon echa, J.; Cal o,
M.V.; Megino-Tello, J.; Se ano, J.C.E.;
Ga cía-La a, N.R.; Pallás-Alonso, C.R.
Associa ions o Die a y In ake and
Nu ien S a us wi h Mic onu ien
and Lipid Composi ion in B eas
Milk o Dono Women. Nu ien s
2023,15, 3486. h ps://doi.o g/
10.3390/nu15153486
Academic Edi o : Tania Siahanidou
Recei ed: 20 June 2023
Re ised: 1 Augus 2023
Accep ed: 5 Augus 2023
Published: 7 Augus 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
nu ien s
A icle
Associa ions o Die a y In ake and Nu ien S a us
wi h Mic onu ien and Lipid Composi ion in B eas Milk
o Dono Women
Noelia U e a-Velasco 1,2,3,* , Ad iana Mon ealeg e-Poma 4, K is in Kelle 2,5, Diana Escude -Vieco 2,5,
Ja ie Fon echa 6, Ma ía V. Cal o 6, Ja ie Megino-Tello 6, JoséC. E. Se ano 7, Nadia Raquel Ga cía-La a 1,2,5,†
and Ca men R. Pallás-Alonso 1,2,3,†
1Depa men o Neona ology, 12 de Oc ub e Uni e si y Hospi al, 28041 Mad id, Spain;
[email p o ec ed] (N.R.G.-L.); [email p o ec ed] (C.R.P.-A.)
2Resea ch Ins i u e i+12, 12 de Oc ub e Uni e si y Hospi al, 28041 Mad id, Spain;
[email p o ec ed] (K.K.); [email p o ec ed] (D.E.-V.)
3Facul y o Medicine, Complu ense Uni e si y o Mad id, 28040 Mad id, Spain
4Epidemiología Clínica, Pon i icia Uni e sidad Ja e iana, Hospi al Uni e si a io San Ignacio,
Bogo á110231, Colombia; [email p o ec ed]
5“Aladina-MGU”—Regional Human Milk Bank, 12 de Oc ub e Uni e si y Hospi al, 28041 Mad id, Spain
6Food Lipid Bioma ke s and Heal h G oup, Ins i u e o Food Science Resea ch (CIAL), CSIC-UAM,
28049 Mad id, Spain; [email p o ec ed] (J.F.); m [email p o ec ed] (M.V.C.); [email p o ec ed] (J.M.-T.)
7Depa men o Expe imen al Medicine, Facul y o Medicine, Uni e si y o Lleida, 25008 Lleida, Spain;
[email p o ec ed]
*Co espondence: noelia.u [email p o ec ed]; Tel.: +34-913-901-929
†These au ho s con ibu ed equally o his wo k.
Abs ac :
The in luence o he die and nu i ional s a us o milk dono s on he nu i ional composi ion
o dono human milk (DHM) is unknown. The p esen s udy aimed o de e mine he nu i ional
p o ile o DHM and he associa ions be ween dono s’ die a y in ake and nu i ional s a us and
he mic onu ien and lipid composi ion in DHM. Fo his pu pose, 113 dono s comple ed a ood
equency ques ionnai e, p o ided a i e-day weighed die a y eco d, and collec ed milk o i e
consecu i e days. Nu ien de e mina ions in dono s’ e y h ocy es, plasma, u ine, and milk we e
pe o med. Mul iple linea eg essions we e conduc ed o he e alua ion o he associa ions. We
highligh he ollowing esul s: DHM docosahexaenoic acid (DHA) was posi i ely associa ed wi h
dono s’ plasma DHA con en and dono s’ DHA in ake (R
2
0.45, p< 0.001). Fo e e y 1 g/day DHA
in ake, an inc ease o 0.38% in DHA con en and 0.78% in o al omega-3 con en was obse ed in DHM
(R
2
0.29, p< 0.001). DHM sa u a ed a y acids we e posi i ely associa ed wi h e y h ocy e dime hyl
ace als, plasma s ea ic acid, ans a y acids in ake, and b eas eeding du a ion and nega i ely
associa ed wi h e y h ocy e ma ga oleic acid (R
2
0.34, p< 0.01). DHM cholecalci e ol was associa ed
wi h plasma cholecalci e ol le els and dai y in ake (R
2
0.57, p< 0.01). O he weake associa ions
we e ound o ee hiamin, ee ibo la in, py idoxal, dehyd oasco bic acid, and he lipid p o ile
in DHM. In conclusion, he die and nu i ional s a us o dono s in luence he a y acid p o ile and
mic onu ien con en o DHM.
Keywo ds:
b eas milk; human milk bank; associa ions; dono s; die ; nu i ional s a us; lipid p o ile;
i amins; mine als; docosahexaenoic acid
1. In oduc ion
Dono human milk (DHM) is conside ed he bes eeding op ion o p e e m in an s
when hei mo he ’s own milk is no a ailable. I s use is inc easing in neona al uni s, p i-
ma ily o in an s a high isk o de eloping nec o izing en e ocoli is o eeding in ole ance,
such as hose weighing <1500 g and/o hose a less han 32 weeks o ges a ional age (GA),
Nu ien s 2023,15, 3486. h ps://doi.o g/10.3390/nu15153486 h ps://www.mdpi.com/jou nal/nu ien s
Nu ien s 2023,15, 3486 2 o 24
in an s wi h congeni al hea disease, o hose wi h se e e in es inal diso de s [
1
–
5
]. These
in an s a e in a si ua ion o “nu i ional eme gency”; he e o e, knowledge o he essen ial
nu ien s in DHM and esea ch in o he ac o s con ibu ing o i s composi ion should be
a p io i y.
Acco ding o s udies ca ied ou in di e en geog aphical a eas o he wo ld, he e is
e idence ha he con en o a y acids (FAs) and some mic onu ien s in human milk (HM)
depends on he ma e nal deposi s o each nu ien a he end o ges a ion and ma e nal
in ake du ing he lac a ion pe iod [
6
–
9
]. Howe e , mos s udies ha e assessed he FA
p o ile and in o ma ion on mic onu ien s is much mo e limi ed.
In his con ex , mic onu ien s can be classi ied in a simpli ied way in o wo g oups
wi h espec o hei sec e ion pa e ns in milk in ela ion o ma e nal in ake and s a us
and he esponse o supplemen a ion. In gene al, wa e -soluble i amins (excep ola e),
a -soluble i amins, iodine, and selenium a e conside ed o belong o a g oup o mi-
c onu ien s o which sec e ion in o HM is dependen on ma e nal in ake o s a us, and
ma e nal supplemen a ion may inc ease hei concen a ions in milk [
8
–
11
]. Acco dingly,
iodine, i amin A, hiamin, ibo la in, i amin B6, and cobalamin ha e been es ablished as
p io i y nu ien s o b eas eeding women [
12
]. The beha io o i amin E is somewha
di e en , as nei he plasma no se um concen a ions no he usual ma e nal die a ec he
i amin E concen a ion in HM, al hough i amin E supplemen a ion has been shown o
inc ease colos um alpha- ocophe ol le els [
7
,
13
]. On he o he hand, he concen a ions o
ola e and o he mine als and ace elemen s in HM emain ai ly cons an ega dless o
ma e nal in ake o s a us, al hough ma e nal s o es may be a ec ed i he in ake o hese
nu ien s is insu icien ; ma e nal supplemen a ion wi h he nu ien s o his second g oup
is pa icula ly bene icial o he mo he a he han o he in an [8–12].
Ne e heless, he esul s o he di e en su eys ha e no always been conco dan [
7
],
and he au ho s o wo sys ema ic e iews on his opic [
8
,
9
] highligh ed he small sample
sizes and he la ge he e ogenei y in he s udies. In addi ion, women’s die s a y o e ime
and acco ding o he coun y in which hey li e and hei cul u al en i onmen [
14
–
17
].
Fo his eason, i is essen ial o ha e upda ed e e ences in each coun y on he nu ien s
in HM.
Mo eo e , HM is a e y complex and dynamic luid, and i s nu i ional composi ion
depends no only on ma e nal o in an ac o s bu also on i s ch onobiology (i.e., i changes
wi h he du a ion o lac a ion, he ime o day, he leng h o ime ha elapses be ween
eedings, he du a ion o eeding, and he ci cadian hy hm) [
7
,
18
–
21
]. All hese ac o s can
ac as con ounde s when s udying changes in HM con en in ela ion o die o ma e nal
nu i ional s a us and in e e e wi h he in e p e a ion o he da a.
Fu he mo e, DHM equi es i s own s udies as i is no compa able o b eas milk in
some espec s [
22
]. Fi s ly, HM dono s cons i u e a pa icula popula ion o se e al easons.
Milk dono s comp ise a he e ogeneous popula ion o b eas eeding mo he s in e ms o
he du a ion o p egnancy and s age o lac a ion. S udies ca ied ou in Spain and o he
coun ies ha e shown how he sociodemog aphic cha ac e is ics o dono women di e
om hose o he gene al popula ion [
23
,
24
]. Also, in addi ion o b eas eeding hei own
child, dono s exp ess su plus milk o dona ion, which migh lead o inc eased ma e nal
nu i ional needs and a di e en nu i ional milk p o ile. Secondly, DHM is subjec ed o p o-
cedu es ha may al e i s nu i ional p o ile, such as collec ion, mixing, mul iple con aine
changes, eeze– haw cycles, s o age, and pas eu iza ion [
22
,
25
]. Fo example, a dec ease in
i amins C and B6 in b eas milk due o exposu e o ligh and eezing has been obse ed,
as well as a dec ease in i amins C and B6 and ola e due o pas eu iza ion
[26–30]
. A ecen
s udy ound ha , du ing long- e m s o age o human milk, e ige a ed o ozen, he mem-
b ane phospholipids o milk a globules we e deg aded and iacylglyce ols we e eleased
om he co e, wi h sphingomyelin being he glyce ophospholipid showing he highes a e
o lipolysis [
31
]. Typically, om ex ac ion un il i is adminis e ed o he ecipien in an s,
DHM is exposed o ligh and unde goes wo eeze– haw cycles, as well as p ocessing,
usually in ol ing Holde pas eu iza ion [
26
,
30
]. O e all, da a indica e ha Holde pas eu -
Nu ien s 2023,15, 3486 3 o 24
iza ion a ec s se e al componen s o milk bu mainly bioac i e and immunomodula o y,
a he han nu i ional, componen s [
26
]. Al hough clinical p ac ices show ha bene icial
p ope ies o DHM pe sis a e Holde pas eu iza ion, new pas eu iza ion echniques a e
being in es iga ed o imp o e he biological and nu i ional quali y o DHM [
32
,
33
]. Las ly,
in 2020, Pe in published a sys ema ic e iew on he nu i ional composi ion o DHM,
highligh ing he need o u u e s udies on he mic onu ien con en especially, as da a on
he i amin and mine al composi ion o DHM we e sca ce [22].
The e o e, because o he g ea impo ance o nu i ion in he de elopmen o e y
p e e m o c i ically ill newbo ns, i is essen ial o ha e in o ma ion on he nu i ional
composi ion o DHM and he ac o s ha migh impac i , such as die , in o de o imp o e
ou ecommenda ions o dono s. To he bes o ou knowledge, he only s udy in which
a die a y assessmen o milk dono s was pe o med was a andomized, con olled ial
abou he e ec o dono s’ supplemen a ion wi h docosahexaenoic acid (DHA) on he DHA
con en in DHM conduc ed in he Uni ed S a es [34].
Hence, he aim o his s udy was o de e mine, in a la ge popula ion o dono s om
a milk bank in Mad id (Spain), he mac onu ien composi ion, lipid p o ile ( a y acid
p o ile, lipid classes, ela i e composi ion o phospholipids, and molecula species o ia-
cyclglyce ols), and mic onu ien composi ion ( ee hiamine, ee ibo la in, nico inamide,
py idoxal, pan o henic acid, olic acid, cobalamin, asco bic acid, dehyd oasco bic acid,
e inol,
α
- ocophe ol,
γ
- ocophe ol, cholecalci e ol, 25(OH)D
3
, iodine, calcium, phospho-
ous, and selenium) o aw DHM and hei associa ions wi h dono s’ die a y in ake and
nu ien s a us.
2. Ma e ials and Me hods
2.1. S udy Design and Pa icipan s
This s udy was pa o a la ge esea ch p ojec , a c oss-sec ional obse a ional s udy
conduc ed a he Aladina MGU Regional Human Milk Bank (RHMB) a he “12 de Oc ub e”
Uni e si y Hospi al in Mad id, Spain. Two p e ious a icles based on his esea ch p ojec
ha e ecen ly been published [35,36].
The main aim o he p esen wo k was o s udy he co ela ions be ween he die ,
nu i ional s a us, and nu i ional milk composi ion (mac onu ien s, lipids, wa e -soluble
and a -soluble i amins, calcium, phospho ous, and selenium) o milk dono s. Fo his
pu pose, we ec ui ed dono s by oppo uni y a he RHMB be ween Augus 2017 and
Feb ua y 2020. The inclusion c i e ion was o be an ac i e dono in ou RHMB (i.e., o
ha e dona ed a leas once in he las wo mon hs) wi hou communica ion ba ie s. In ou
RHMB, he equi emen s o be a milk dono a e ha po en ial dono s mus ha e a heal hy
li es yle, no be su e ing om illnesses o aking any medica ions ha a e incompa ible
wi h he dona ion, be success ully b eas eeding hei own child, and wan o dona e
hei su plus milk olun a ily and al uis ically. No mally, milk dono s a e accep ed om
3 weeks pos pa um o ensu e ha b eas eeding is well es ablished. The e a e no exclusion
c i e ia ega ding he age o he dono o he ges a ional age o he child, no is he e an
uppe limi on he leng h o b eas eeding. Women who wish o dona e milk a e he dea h
o hei baby, ege a ians o egans wi h no mal plasma cobalamin alues commi ed
o aking he ecommended i amin B12 supplemen s, and women wi h well-con olled
hypo hy oidism a e accep ed as dono s. The s a is ical calcula ion o he sample size
was unde aken in G*Powe 3.1 [
37
]; o an e ec size o 0.15 (mode a e), an alpha o 0.05,
powe o 0.80, and 8–10 p edic o s a mos , assuming losses o 5%, he sample size was
115 milk dono s.
The s udy was ca ied ou in ag eemen wi h Decla a ion o Helsinki and was ap-
p o ed by he Clinical Resea ch E hics Commi ee o he Hospi al Uni e si a io “12 de
Oc ub e” (p o ocol code 15/269). All pa icipan s ga e w i en in o med consen .
Nu ien s 2023,15, 3486 4 o 24
2.2. S udy P o ocol
The ull s udy p o ocol and labo a o y s udies ha e been p e iously desc ibed in
de ail in he abo emen ioned s udies [35,36].
The s udy p o ocol is shown in Figu e 1.
Nu ien s 2023, 15, x FOR PEER REVIEW 4 o 26
The s udy was ca ied ou in ag eemen wi h Decla a ion o Helsinki and was ap-
p o ed by he Clinical Resea ch E hics Commi ee o he Hospi al Uni e si a io “12 de
Oc ub e” (p o ocol code 15/269). All pa icipan s ga e w i en in o med consen .
2.2. S udy P o ocol
The ull s udy p o ocol and labo a o y s udies ha e been p e iously desc ibed in
de ail in he abo emen ioned s udies [35,36].
The s udy p o ocol is shown in Figu e 1.
Figu e 1. S udy p o ocol lowcha . * P o ided o he RHMB du ing he i s 15 days a e he end o
he s udy. Abb e ia ions: S-D, sociodemog aphic; FFQ, ood equency ques ionnai e.
In summa y, he s udy o pa icipa ing dono s comp ised a heal h and sociodemo-
g aphic su ey; soma ome ic measu emen s; blood and u ine de e mina ions o s udy
hei nu i ional s a us; a die a y s udy including a ood equency ques ionnai e (FFQ)
and a i e-day weighed die a y eco d, aking in o accoun he in ake o pha macological
nu ien supplemen s; and a de ailed s udy o he nu i ional composi ion o hei milk.
A he i s isi o he RHMB (day 0), as ing u ine and blood samples we e col-
lec ed. Plasma and ed blood cells we e ob ained om he blood. All biological samples
we e ozen a −80 °C un il analysis o he biochemical indica o s o nu i ion. In addi ion,
milk dono s p o ided hei in o med consen , unde wen a soma ome ic s udy (weigh ,
heigh , and body mass index calcula ion), illed in he heal h and sociodemog aphic
su ey, and comple ed a ood consump ion equency ques ionnai e. Dono s we e
ained in how o comple e he i e-day weighed die a y eco d and collec and keep
ozen milk samples a home. They also we e supplied wi h he equi ed ma e ial.
Wi hin he ollowing 15 days a e he isi o he RHMB, he i e-day die a y e-
co ding and he collec ion o he i e-day milk samples we e ca ied ou almos concom-
i an ly o 6 days in a ow, wi h milk collec ion s a ing 1 day a e he s a o he die a y
eco ding and ending 1 day a e he eco ding o he die had been comple ed. We en-
su ed ha he DHM we analyzed me he same condi ions and unde wen he same
p ocesses as he aw milk dona ed o ou RHMB. The e o e, in o de o eplica e he
dono s’ milk exp ession ou ine, pa icipan s we e no asked o exp ess milk a any pa -
icula ime, he only equi emen being comple e emp ying o one o bo h b eas s, and
he milk was collec ed in he same s e ile anspa en bo les ha he dono s use unde
no mal dona ion condi ions. F om days 2 o 5, dono s collec ed a 25 mL milk sample
Figu e 1.
S udy p o ocol lowcha . * P o ided o he RHMB du ing he i s 15 days a e he end o
he s udy. Abb e ia ions: S-D, sociodemog aphic; FFQ, ood equency ques ionnai e.
In summa y, he s udy o pa icipa ing dono s comp ised a heal h and sociodemo-
g aphic su ey; soma ome ic measu emen s; blood and u ine de e mina ions o s udy
hei nu i ional s a us; a die a y s udy including a ood equency ques ionnai e (FFQ)
and a i e-day weighed die a y eco d, aking in o accoun he in ake o pha macological
nu ien supplemen s; and a de ailed s udy o he nu i ional composi ion o hei milk.
A he i s isi o he RHMB (day 0), as ing u ine and blood samples we e collec ed.
Plasma and ed blood cells we e ob ained om he blood. All biological samples we e
ozen a
−
80
◦
C un il analysis o he biochemical indica o s o nu i ion. In addi ion, milk
dono s p o ided hei in o med consen , unde wen a soma ome ic s udy (weigh , heigh ,
and body mass index calcula ion), illed in he heal h and sociodemog aphic su ey, and
comple ed a ood consump ion equency ques ionnai e. Dono s we e ained in how o
comple e he i e-day weighed die a y eco d and collec and keep ozen milk samples a
home. They also we e supplied wi h he equi ed ma e ial.
Wi hin he ollowing 15 days a e he isi o he RHMB, he i e-day die a y eco ding
and he collec ion o he i e-day milk samples we e ca ied ou almos concomi an ly o
6 days in a ow, wi h milk collec ion s a ing 1 day a e he s a o he die a y eco ding and
ending 1 day a e he eco ding o he die had been comple ed. We ensu ed ha he DHM
we analyzed me he same condi ions and unde wen he same p ocesses as he aw milk
dona ed o ou RHMB. The e o e, in o de o eplica e he dono s’ milk exp ession ou ine,
pa icipan s we e no asked o exp ess milk a any pa icula ime, he only equi emen
being comple e emp ying o one o bo h b eas s, and he milk was collec ed in he same
s e ile anspa en bo les ha he dono s use unde no mal dona ion condi ions. F om
days 2 o 5, dono s collec ed a 25 mL milk sample om each exp ession (a leas one pe
day) and oze i a
−
20
◦
C. On day 6, hey collec ed and oze one comple e milk ex ac ion.
Dono s we e asked o deli e hei die a y eco ds and hei ozen milk samples o he
RHMB du ing he i s 15 days ollowing he end o he s udy. Dono s anspo ed he
milk samples in iso he mal con aine s wi h cold packs, and he milk emained ozen a
he RHMB a −20 ◦C un il hawing o aliquo ing.
Nu ien s 2023,15, 3486 5 o 24
In he RHMB, di e en milk samples om he same day we e mixed and aliquo ed,
bu he milk om di e en days was no mixed, as he composi ion o he milk om each
day was analyzed indi idually.
The e o e, he milk samples ha we e analyzed wen h ough a p ocess o ex ac ion,
eezing a he dono s’ homes, anspo o he RHMB, and hawing o collec he aliquo s,
simila o he p ocess ha DHM usually unde goes a ou RHMB. The milk samples we e
subsequen ly ozen un il analysis in he espec i e labo a o ies a
−
80
º
C ins ead o
−
20
◦
C,
as he eezing ime was much longe han ha o he DHM a he RHMB. Be o e analysis
in he labo a o y, he milk was hawed again. Thus, he s udied milk unde wen a o al o
wo eeze– haw cycles, jus like he DHM in ou RHMB, he only di e ence being ha i
was no pas eu ized.
The milk samples collec ed om days 2 o 5 we e used o he s udy o i amins and
mine als, and he comple e milk collec ion sample om day 6 was used o he in eg al
cha ac e iza ion o he lipid ac ion. The da a on he in ake o ood, be e ages, and
pha macological nu ien supplemen s ob ained om he i e-day die a y eco d we e
analyzed using DIAL
®
so wa e (DIAL.EXE Ve sion 3, Feb ua y 2014, Alce Ingenie ía,
Mad id, Spain) o calcula e daily nu ien in akes [
38
]. Nu ien in akes we e compa ed
wi h he Recommended Die a y Allowances/Adequa e In akes p o ided by he Ame ican
Ins i u e o Medicine [
39
–
44
] and he Popula ion Re e ence In akes/Adequa e In akes
p o ided by he Eu opean Food Sa e y Au ho i y [45] o lac a ing women.
2.3. Nu ien Analysis
Lipid analyses we e conduc ed by he Food Lipid Bioma ke s and Heal h G oup a
he Ins i u e o Food Science Resea ch (CIAL, CSIC-UAM). Fa y acid me hyl es e s we e
analyzed by GC-MS in e y h ocy es, plasma, and milk. A e a ex ac ion om human
milk samples om 20 andomly selec ed dono s, he sepa a ion and quan i ica ion o lipid
classes we e pe o med using an HPLC e apo a i e ligh sca e ing de ec o (ELSD), and
he de e mina ion o TAG molecula species was unde aken using GC-FID.
Vi amin and mine al analyses we e conduc ed by he NUTREN-Nu igenomics G oup
o he Depa men o Expe imen al Medicine a he Uni e si y o Lleida, Spain. Mic onu i-
en de e mina ions we e ca ied ou using ch oma og aphic analyses, mass spec ome y,
and immunoassays. F ee hiamin, ee ibo la in, nico inamide, py idoxal, pan o henic
acid, olic acid, cobalamin, asco bic acid, dehyd oasco bic acid, e inol,
α
- ocophe ol,
γ
-
ocophe ol, cholecalci e ol, 25(OH)D
3
, iodine, calcium, phospho ous, and selenium we e
de e mined in milk. The e y h ocy e glu a hione educ ase ac i i y coe icien (EGRAC),
ibo la in, nico inamide, pan o henic acid, py idoxamine, and hemoglobin we e de e -
mined in e y h ocy es. The EGRAC, which assesses e y h ocy e glu a hione educ ase
ac i i y in e ms o an excess o la ine adenine dinucleo ide compa ed o baseline ac i -
i y, is a unc ional p ope y used o de e mine he nu i ional ibo la in s a us. Thiamin,
ibo la in, nico inamide, pan o henic acid, py idoxine, py idoxamine, olic acid, cobal-
amin, holo anscobalamin, homocys eine, asco bic acid, e inol, 25(OH)D
3
, 1,25(OH)
2
D
3
,
α
- ocophe ol,
γ
- ocophe ol, iacylglyce ols, o al choles e ol, HDL-choles e ol, and LDL-
choles e ol we e de e mined in plasma. C ea inine, me hylmalonic acid, iodine, sodium,
calcium, and phospho ous we e de e mined in u ine.
De e mina ions o mac onu ien s ( o al a , p o eins, and ca bohyd a es) in DHM
we e ca ied ou a he RHMB using Fou ie - ans o m mid-in a ed (FT-MID) spec oscopy
in a milk analyze (MilkoScan FT2, FOSS S.A., Ba celona, Spain).
A ull desc ip ion o he analy ic echniques employed o each o he nu ien s is
a ailable in a p e ious pape [36], as men ioned abo e.
2.4. S a is ics
S a is ical analysis was pe o med wi h he STATA 14 p og am.
Fo desc ip i e analysis, quali a i e a iables we e p esen ed as absolu e and ela i e
equencies and quan i a i e a iables as medians and he in e qua ile ange o as means

Nu ien s 2023,15, 3486 6 o 24
and he s anda d de ia ion depending on hei dis ibu ion, which was es ablished by
pe o ming he Shapi o–Wilk no mali y es .
Fo he mul i a ia e analysis o associa ions, mul iple linea eg essions (MLRs) we e
pe o med using each o he lipids, mac o- and mic onu ien s in DHM as dependen
a iables, excep o iodine, which had been p e iously s udied [
35
]. Fo i amins and
mine als, he a e age o he ou -day milk con en o each pa icipan was used. The
independen a iables we e as ollows: he clinical and sociodemog aphic cha ac e is ics;
he soma ome ic s udy esul s; he le els o nu ien s in dono s’ plasma, e y h ocy es, and
u ine; he esul s o in ake om he FFQ; and he a e age in ake o nu ien s eco ded by
dono s in he i e-day die a y eco d. The independen a iables wi h signi ican associa-
ions (p< 0.05) wi h milk con en in he bi a ia e analysis we e en e ed in o he models.
Once he independen a iables and hei p obable in e ac ions we e in oduced in each
o he models, he a iables wi h p> 0.05 we e emo ed, and con ounding a iables we e
e alua ed un il he mos pa simonious model was ob ained. Each model was diagnosed,
and some ou lie s we e emo ed o imp o e he i . Fo each model, eg ession diagnosis
was pe o med wi h he esiduals; he assump ions o linea i y wi h each independen
a iable we e assessed wi h sca e plo s and, in addi ion, homoscedas ici y o a iance
was assessed wi h he B eusch–Pagan es and no mal dis ibu ion wi h p–p plo s. The
independence o obse a ions was also assessed wi h i and omi ed a iable bias was
es ed wi h o es . The ollowing diagnos ic plo s we e used o he in luen ial poin s:
esidual e sus i ed alues ( plo ), augmen ed componen -plus- esidual plo s (acp
plo s), le e age agains no malized squa ed esiduals (l 2plo ), and Cooks’ dis ance. In
he models’ ables, he in luencing poin s iden i ied and emo ed o each model a e
indi idual analyses we e eco ded.
3. Resul s
3.1. Popula ion S udied
A o al o 114 milk dono s we e ec ui ed: 112 we e omni o es (93 wi h ull- e m
in an s, 16 wi h p e e m in an s who we e no hospi alized, and 3 wi h p e e m in an s
<32 weeks
o ges a ional age and/o wi h <1500 g bi hweigh hospi alized in he neona-
ology se ice) and 2 we e o o-lac o ege a ians (1 wi h a p e e m in an 36
+4
weeks o
ges a ional age and he o he wi h a ull- e m in an ). One o he omni o e dono s le he
s udy ea ly; hus, comple e da a we e ob ained om 113 dono women.
Tables 1–4show he esul s o he sociodemog aphic and heal h su ey, including he
cha ac e is ics o he dono s’ b eas ed in an s and b eas eeding habi s.
The median age o dono s was 35.6 yea s and hey had a median body mass index
(BMI) o 22.9 kg/m
2
, simila o hei p e-p egnancy BMI (22.1 kg/m
2
). BMI a he ime
o he s udy was classi ied as no mal o 71% o he sample (Table 1). In e ms o die a y
habi s, 29% epo ed ha ing changed ea ing habi s in he las wo yea s owa ds a heal hie
die o due o changes in dai y consump ion. The e we e 18 dono s wi h some ype o
ood es ic ion: dai y (n= 6), ish (n= 3), mea (n= 3), eggs (n= 2), nu s (n= 1), and
o he s (
n= 3
). In o al, 87 o he 109 dono s who speci ied he ype o sal consumed (79.8%)
epo ed aking iodized sal .
Fi y- i e pe cen o dono s epo ed ha ing no p e ious child en. Fou dono s had
win p egnancies, among whom wo los a e us (one o o-lac o ege a ian dono wi h a
e m in an and one omni o e dono wi h a p e e m in an 24
+4
weeks o ges a ional age)
and one los one o he wins a 27
+0
weeks o ges a ional age du ing he i s week o li e.
Ano he dono had one e al dea h a 22
+6
weeks o ges a ional age 2 mon hs p e iously
(Table 2).
Fi e pe cen o dono s we e b eas eeding mo e han one child ( andem o win
b eas eeding) a he ime o he s udy. The median ges a ional age a bi h was 39
+4
weeks, and 47% o child en we e male. Rega ding he 19 dono s wi h p e e m deli e ies,
he pos -mens ual/co ec ed age o he in an s anged om 30
+6
weeks o 17.5 mon hs.
Se en y- wo pe cen o mo he s employed simple elec ical b eas pumps (Tables 3and 4).
Nu ien s 2023,15, 3486 7 o 24
Rega ding nu ien supplemen a ion, 112 o 113 dono s (99.1%) epo ed consuming
pha macological nu i ional supplemen s du ing p egnancy and 102 o 113 dono s (90.3%)
du ing lac a ion. Mos pa icipan s ook supplemen s o olic acid, i amin B12, and iodine
du ing p egnancy. The numbe o dono s who ook supplemen s o each nu ien , bo h
du ing ges a ion and lac a ion, and he median daily dose ha hey consumed a e a ailable
in Table S1 in he Supplemen a y Ma e ial.
Table 1. Dono cha ac e is ics (n= 114).
Cha ac e is ic
Age (yea s) 35.6 (32.9, 38.7)
Weigh (kg) 60.5 (55.2, 70.6)
Heigh (cm) 164.1 (6.5)
P e-p egnancy BMI (kg/m2)22.1 (20.6, 24.8)
P e-p egnancy BMI (kg/m2) ca ego y
Unde weigh (<18.5) 4 (3.5%)
No mal (18.5–24.9) 84 (73.7%)
O e weigh (25–29.9) 15 (13.2%)
Obese (≥30) 11 (9.6%)
Cu en BMI (kg/m2)22.9 (21.1, 25.1)
Cu en BMI (kg/m2) ca ego y
Unde weigh (<18.5) 4 (3.5%)
No mal (18.5–24.9) 81 (71.1%)
O e weigh (25–29.9) 16 (14.0%)
Obese (≥30) 13 (11.4%)
Ges a ional weigh gain (kg) 11.3 (9.0, 14.0)
Pos pa um weigh e en ion (kg) 1.0 (−0.6, 2.5)
Numbe o li ing child en
0a–1 63 (55.3%)
2 39 (34.2%)
≥3 12 (10.5%)
Coun y o o igin: Spain 102 (89.5%)
Educa ion le el
Seconda y s udies 2 (1.8%)
Technical s udies 14 (12.3%)
Uni e si y s udies 98 (86.0%)
Cu en ly wo king 50 (43.9%)
Physical ac i i y
Seden a y 26 (22.8%)
Low ac i i y 60 (52.6%)
Ac i e/ e y ac i e 28 (24.6%)
Tobacco consump ion
P e iously 28 (24.6%)
Cu en ly 1 (0.9%)
Passi e smoking 24 (21.1%)
Ac i e smoking 1 (0.9%)
Alcohol consump ion
P io o p egnancy 55 (48.2%)
Du ing p egnancy 1 (0.9%)
Cu en ly 4 (3.5%)
Season du ing he s udy
Sp ing 30 (26.3%)
Summe 16 (14.0%)
Au umn 42 (36.8%)
Win e 26 (22.8%)
Quan i a i e a iables a e exp essed as means (s anda d de ia ions) when hey we e dis ibu ed pa ame ically
and as medians (25 h and 75 h pe cen iles) when hey we e dis ibu ed non-pa ame ically. Quali a i e a iables
a e p esen ed as he absolu e and ela i e equencies (%).
a
A e al dea h a 22
+6
weeks o ges a ional age and
450 g bi hweigh . Abb e ia ions: BMI, body mass index.
Nu ien s 2023,15, 3486 8 o 24
Table 2.
Diseases, medica ion in ake, and cha ac e is ics o he las p egnancy eco ded o he dono s
(n= 114).
n(%)
Diseases 141 (36.0%)
Endoc inological and me abolic diseases * 10 (8.8%)
Ca dio ascula diseases (hype ension) 1 (0.9%)
Respi a o y diseases (as hma) 6 (5.3%)
Immune diseases (alle gy, pso iasis, a opy) 7 (6.1%)
Spinal/medulla y pa hology 6 (5.3%)
Miscellaneous ** 13 (11.4%)
Medica ion in ake 112 (10.5%)
O al con acep i es 4 (3.5%)
Thy oid ho mone eplacemen he apy 4 (3.5%)
O he medicines *** 5 (4.4%)
Twin p egnancy 4 (3.5%)
P oblems in he las p egnancy
136 (31.6%)
Thy oid diso de s 17 (14.9%)
P eeclampsia 2 (1.8%)
Ges a ional diabe es 2 (1.8%)
In au e ine e al g ow h es ic ion 6 (5.3%)
In au e ine e al dea h 3 (2.6%)
O he p oblems **** 11 (9.6%)
1
The ca ego ies do no exclude each o he . * Endoc inological and me abolic diseases included ou cases o
well-con olled hypo hy oidism, ou cases o subclinical hy oid diso de s, and wo cases o hype choles e olemia.
** Miscellaneous included congeni al amylase–suc ase de iciency, a y li e , esophagi is, an iphospholipid syn-
d ome, u ei is, mig aines, polycys ic o a y synd ome, choles ea oma, enous insu iciency, ecu en u ina y
ac in ec ions, human papilloma i us in ec ion, and dep ession. *** O he medicines included an ihis amines,
an idep essan s, p o on-pump inhibi o s, an i e iginous medica ions, and o al b onchodila o s. **** O he p ob-
lems included he h ea o abo ion, h ea o p ema u e bi h, e o- e al ans usion synd ome, cho ioamnioni is,
oligohyd amnios, in ahepa ic choles asis, cy omegalo i us in ec ion, b onchi is, and u ina y ac in ec ions.
Table 3. Cha ac e is ics o in an s (n= 116).
Cha ac e is ic n *
Ges a ional age (weeks) 114 39+4 (38+2, 40+2); 22+6–42+3
Boy 116 55 (47.4%)
Bi h weigh (g ams) 116 3195.0 (2795.0, 3472.5); 450.0–4640.0
Bi h weigh pe cen ile 1
≤25 32 (27.6%)
25–75 116 73 (62.9%)
≥75 11 (9.5%)
Age o in an (mon hs)
114
0–6 45 (39.5%)
6–12 43 (37.7%)
12–50 26 (22.8%)
Pos mens ual age o p e e m in an s (weeks)
19 50.3 (38.6, 78.2)
Weigh pe cen ile o b eas ed child 2
113
≤15 17 (15.0%)
15–85 77 (68.1%)
≥85 19 (16.8%)
Quan i a i e a iables a e exp essed as medians (25 h and 75 h pe cen iles) because hey we e dis ibu ed non-
pa ame ically. Ranges a e shown a e he semicolon. Quali a i e a iables a e p esen ed as he absolu e and
ela i e equencies (%). * n= 116 includes a e al dea h a 22
+6
weeks o ges a ional age and wo pai s o wins;
n= 114
is due o he numbe o ges a ions; n= 113 excludes he e al dea h, a win baby who died in he i s week
o li e, and one child wi h missing da a.
1
Based on Olsen in au e ine g ow h cu es [
46
].
2
Based on he Wo ld
Heal h O ganiza ion (WHO)’s child g ow h s anda ds [47].
Nu ien s 2023,15, 3486 9 o 24
Table 4. B eas eeding cha ac e is ics (n= 114).
Cha ac e is ic n n (%)
Dono p e iously 114 20 (17.5%)
Du a ion o lac a ion o he p e ious child (mon hs)
51 a
0 1(2.0%)
3–6 2 (3.9%)
6–12 10 (19.6%)
12–24 22 (43.1%)
≥24 16 (31.4%)
Cu en lac a ion s age (mon hs) 114 7.0 (4.8, 12.0); 1.8–50.0
Type o lac a ion
113 b
Exclusi e 52 (46.0%)
Pa ial 61 (54.0%)
Sum o b eas eeding imes plus daily milk pumping
sessions
114
<5 12 (10.5%)
5–10 66 (57.9%)
>10 33 (28.9%)
Missing da a 3 (2.6%)
Tandem b eas eeding 114 5 (4.4%)
B eas eeding wins 114 1 (0.9%)
Type o milk ex ac ion *
114
Manual 7 (6.1%)
Mechanical b eas pump 12 (10.5%)
Simple elec ic b eas pump 82 (71.9%)
Double elec ic b eas pump 15 (13.2%)
Quan i a i e a iables a e exp essed as medians (25 h and 75 h pe cen iles) because hey we e dis ibu ed non-
pa ame ically. Ranges a e shown a e he semicolon. Quali a i e a iables a e p esen ed as he absolu e and
ela i e equencies (%).
a
Numbe o dono s wi h p e ious child en.
b
Due o one e al dea h. * The ca ego ies do
no exclude each o he .
3.2. Die Su ey and Nu i ional S a us
The esul s o he i e-day die a y eco d, he FFQ, and he analysis o nu ien s in he
e y h ocy es, plasma, and u ine o he dono s o he assessmen o hei nu i ional s a us
a e displayed as supplemen a y da a (Tables S2–S7). Re e ences [
36
,
48
–
72
] a e ci ed in he
Supplemen a y Ma e ials.
3.3. Composi ion o Dono Human Milk
Da a on he nu i ional composi ion o he DHM a e p esen ed in Tables 5–7. Table 5
p esen s he mac onu ien composi ion, he lipid classes’ p o ile, he ela i e composi ion
o phospholipids, and he molecula species o iacylglyce ols in he DHM. Table 6p esen s
he concen a ions o 30 FAs, and Table 7shows he concen a ions o 15 mic onu ien s in
he DHM.
Table 5.
Mac onu ien composi ion (g/100 mL milk), lipid classes’ p o ile (g/100 g a ), ela i e
composi ion o phospholipids (g/100 g pola lipids), and molecula species o iacylglyce ols con en
(g/100 g a ).
Nu ien Dono s
nMean (SE)
Mac onu ien s (g/100 mL milk)
Lipids
103
3.13 (0.17)
Ca bohyd a es 7.73 (0.03)
P o eins 1.17 (0.03)
Nu ien s 2023,15, 3486 16 o 24
3.4.3. Mac onu ien s Associa ions in DHM
No associa ions we e ound be ween ca bohyd a e and p o ein con en in DHM and
dono s’ clinical and soma ome ic cha ac e is ics, nu i ional biochemical de e mina ions,
o die a y in ake.
4. Discussion
This c oss-sec ional s udy in es iga ed he associa ions be ween mac onu ien s, he
a y acid p o ile, lipid classes, he ela i e composi ion o phospholipids, molecula species
o iacyclglyce ols, and 14 mic onu ien s ( ee hiamine; ee ibo la in; nico inamide;
py idoxal; pan o henic acid; olic acid; cobalamin; i amins C, A, D, and E; calcium;
phospho ous; and selenium) in aw DHM and he die , as well as he nu i ional s a us o
113 milk dono s om he Regional Human Milk Bank in Mad id, Spain. To he bes o ou
knowledge, his is he i s s udy in which hese associa ions ha e been e alua ed in dono
human milk. Fo his pu pose, dono s comple ed an FFQ, p o ided a i e-day weighed
die a y eco d, and collec ed milk o 5 consecu i e days. In addi ion, soma ome ic
measu emen s and nu ien de e mina ions in dono s’ e y h ocy es, plasma, u ine, and
milk we e pe o med.
One o he mos impo an indings o he p esen s udy was he co ela ion be ween
dono s’ DHA in ake and DHA plasma le els and he DHA con en o he aw DHM. This
esul is impo an because, in a ecen s udy [
91
], he DHA le els in DHM we e lowe
han in he mo he ’s own milk o e y p e e m in an s, indica ing ha DHM p o ides
an insu icien supply o DHA o hese pa ien s, who comp ise an al eady DHA-de icien
popula ion [
92
]. Ou esul s a e consis en wi h hose o he only s udy ha has de e mined
he DHA con en o DHM in ela ion o he DHA in ake o milk dono s. In ha andomized,
con olled ial conduc ed in Ohio and published in 2013 [34], milk dono s supplemen ed
wi h an algal-de i ed p oduc p o iding a DHA dose o 1 g/day had signi ican ly highe
DHA con en in hei milk han baseline samples, and DHA con en was o e ou imes
highe han he placebo g oup a e 14 days o supplemen a ion. Howe e , he sample size
was e y small, and he DHA in ake a baseline was only 23 mg/day. In ou obse a ional
s udy, he median dono DHA in ake was 310 mg/day, and o e e y 1 g/day o DHA
in ake, he adjus ed DHA con en in DHM inc eased by 0.38 g/100 g a . This inc ease was
somewha smalle han ha epo ed by Mak ides in 1996 [
93
] in a andomized s udy o
ma e nal supplemen a ion (lac a ing non-dono mo he s) wi h p e o med algae-de i ed
DHA a di e en doses, whe e he DHA con en o b eas milk inc eased by app oxima ely
0.1 g/100 g a o each 0.1 g o DHA in ake. In his sense, ou app oach adds in o ma ion
wi h espec o he eal ope a ing condi ions o a milk bank wi hou modi ying he die a y
and supplemen a ion habi s o he dono s. As pas eu iza ion has no been shown o
signi ican ly impai he DHA con en o DHM [
26
,
94
,
95
], i could be in e ed ha inc easing
dono s’ DHA in ake is a easible s a egy o achie e a highe DHA con en in pas eu ized
DHM. In addi ion, we ound a posi i e associa ion be ween DHA in ake and he pe cen age
o omega-3 in he DHM. On he o he hand, we ound no associa ion be ween he con en
o DHA in DHM and he in ake o i s p ecu so s, such as
α
-linolenic acid. In a p e ious
s udy, supplemen ing mo he s wi h laxseed oil, which is e y ich in linolenic acid, did no
enhance he DHA con en o hei milk [
96
]. Enhancemen o dono s’ DHA in ake can be
achie ed by bo h inc easing oily ish in ake and/o pha macological supplemen a ion [
97
].
In ou p e ious s udy compa ing he nu i ional milk composi ion o ege a ian o egan
lac a ing women s. omni o e human milk dono s, he milk DHA con en was lowe by
hal in he ege a ian/ egan women g oup as a esul o hei lowe in ake o DHA [
36
].
The a ailabili y o DHA supplemen s om algal oil o e s ege a ian o egan dono s he
possibili y o inc easing hei DHA in ake om a plan -based sou ce. Supplemen a ion
wi h 250 mg o algae-de i ed DHA daily has been shown o inc ease plasma DHA con en
in omni o es, ege a ians, and egans [
98
], and he use o a DHA/EPA supplemen was
posi i ely associa ed wi h he DHA con en in milk om lac a ing women ollowing egan,
ege a ian, and omni o e die s [
99
]. Thus, wi h he e idence a ailable a p esen , i could be

Nu ien s 2023,15, 3486 17 o 24
deduced ha one me hod o inc easing he DHA con en o DHM om ege a ian/ egan
mo he s is he in ake o algae-de i ed DHA supplemen s; howe e , o he bes o ou
knowledge, no andomized con olled s udy has been ca ied ou o con i m his.
We also ound a posi i e co ela ion be ween ma e nal PUFA in ake and he PUFA
con en in DHM. This posi i e co ela ion was ound in a p e ious wo k ha s udied he
PUFA con en in b eas milk o non-dono lac a ing mo he s du ing he i s mon h o
lac a ion [100].
I is ema kable ha o he co ela ions ound in his s udy ega ding lipid con en ha e
no been iden i ied be o e. The DHM linoleic acid con en inc eased wi h he e y h ocy e
linoleic acid con en , as well as, in e es ingly, wi h combined in ake o mea , ish, and
eggs. The p opo ion o DHM MUFAs inc eased wi h plasma MUFA le els and e y h ocy e
ma ga oleic acid le els. The p opo ion o DHM SFAs inc eased wi h inc eased e y h ocy e
DMA con en , plasma s ea ic acid, TFA in ake, and b eas eeding ime, bu dec eased wi h
inc eased e y h ocy e ma ga oleic acid con en .
In e ms o mic onu ien s, a ious associa ions we e ound be ween he dono s’
die /nu i ional s a us and he DHM composi ion.
Rega ding B-g oup i amins, we ound associa ions wi h he con en o ee hiamin,
ee ibo la in, and py idoxal in milk. We we e no able o demons a e posi i e associa-
ions be ween he milk con en o o he B-g oup i amins and ma e nal in ake o e y h o-
cy e/plasma le els, al hough all o hem (excep ola e) a e conside ed o be dependen on
die and ma e nal s o es [11].
F ee hiamin in DHM was posi i ely co ela ed wi h plasma hiamin le els and nega-
i ely associa ed wi h b eas eeding ime and he daily in ake o dai y. F ee ibo la in in
DHM was posi i ely associa ed wi h ibo la in in ake and i amin B2 supplemen a ion
du ing lac a ion. Py idoxal in DHM was posi i ely associa ed wi h i amin B6 supple-
men a ion du ing p egnancy and nega i ely associa ed wi h b eas eeding ime. In he
li e a u e, he concen a ions o hese h ee i amins in human milk a e s ongly associa ed
wi h ma e nal in ake [
7
–
9
,
101
]. Howe e , he in luence o ma e nal s o es on milk con en
depends on whe he he mo he has adequa e o poo s a us in he case o hiamin, while
i is con o e sial in he case o ibo la in and posi i e in he case o i amin B6 [
7
]. Ou
dono popula ion showed low plasma hiamin le els and 28% showed ibo la in de iciency
acco ding o he e y h ocy e glu a hione educ ase ac i i y coe icien s udy, bu , ne e he-
less, he popula ion showed adequa e le els o plasma ibo la in. We we e unable o assess
he i amin B6 nu i ional s a us o dono s because we did no de e mine plasma py idoxal
phospha e. In e ms o die , he median in akes o i amins B1, B2, and B6 we e abo e
he ecommended alues, and he p e alence o inadequa e in ake was 6.2%, 15.9%, and
1.8%, espec i ely. The con en o hese h ee i amins in he DHM can be conside ed low
compa ed o p e iously published da a [
75
,
76
,
79
] and conside ing ha ee hiamin should
comp ise abou 30% o he o al hiamin con en in HM [
7
], ee ibo la in should comp ise
abou 39% o he o al ibo la in con en , and py idoxal is he p edominan o m o i amin
B6 in b eas milk [
10
]. In addi ion, bo h plasma nico inamide le els and milk le els in ou
dono popula ion we e low [
77
], despi e adequa e niacin in ake in all he dono s s udied. I
emains unce ain whe he he low con en o ee hiamin, ee ibo la in, nico inamide,
and py idoxal in DHM was due o de icien ma e nal s a us; p olonged a e age b eas eed-
ing ime; o he deple ion o i amins exposed o pho odeg ada ion, eezing, and s o age.
On he o he hand, he le els o cobalamin in DHM we e adequa e wi h espec o he
e e ence alues o HM [
80
], and dono s’ in ake and plasma le els o his mic onu ien
we e also adequa e. A ailable e idence indica es a posi i e co ela ion be ween ma e nal
cobalamin in ake and milk i amin B12 concen a ion in women wi h de icien B12 in ake o
deple ed B12 s o es [
7
,
9
,
102
]. I is possible ha , in a popula ion such as ou s wi h adequa e
cobalamin in ake and s o es, he usual die does no a ec he concen a ion o cobalamin
in milk.
Dono s’ ui consump ion was associa ed wi h dehyd oasco bic acid con en in DHM.
One o he mos consis en indings o he wo sys ema ic e iews conduc ed on he e ec
Nu ien s 2023,15, 3486 18 o 24
o ma e nal die on he nu i ional composi ion o b eas milk [
8
,
9
] was he in luence o
ma e nal i amin C in ake on i s con en in milk, al hough he o al numbe o s udies
in which his esul was ound was only h ee [
101
,
103
,
104
]. In e es ingly, he s udy
published by Hoppu in 2005 [
104
] epo ed ha he i amin C concen a ion in he milk
o mo he s wi h a opic disease was associa ed wi h hei i amin C in ake bu no wi h
i amin C supplemen a ion. We we e also unable o de ec an associa ion be ween i amin
C supplemen a ion and i amin C con en in DHM. I should be no ed ha bo h ou
s udy and Hoppu’s we e obse a ional s udies in which he a es o he use o i amin
C-con aining supplemen s we e 43.5% and 50% and he median daily doses o i amin C
supplemen a ion we e 80 mg and 75 mg, espec i ely.
Fo a -soluble i amins, associa ions we e ound only wi h i amin D. The i amin
D con en o he DHM was simila o he usual alues o i amin D in b eas milk,
widely known o be low o he needs o he in an [
83
]. I was no ewo hy ha he dono
popula ion s udied showed a high pe cen age o i amin D de iciency (87.7%). The con en
o cholecalci e ol in DHM was posi i ely co ela ed wi h he plasma cholecalci e ol le els
(bu no wi h plasma 25(OH)D
3
le els) and he daily in ake o dai y. Fu he mo e, he
con en o 25(OH)D
3
in DHM inc eased wi h he use o i amin D supplemen a ion du ing
b eas eeding. These indings a e in line wi h obse a ions om p e ious s udies [
7
,
9
].
Nei he e inol no i amin E in DHM we e associa ed wi h dono s’ in ake o s a us. In he
case o i amin A, dono s’ s o es we e adequa e, and i has been epo ed ha bo h ma e nal
in ake and ma e nal s a us in luence milk con en when ma e nal s o es a e deple ed [
7
].
The
α
- ocophe ol con en o DHM was adequa e in ela ion o he e e ence alues o
HM [
84
], al hough he s a us o he dono s was de icien . In gene al, nei he ma e nal
s o es no die a y in ake o i amin E in luence HM i amin E concen a ions, al hough
i amin E supplemen a ion appea s o inc ease colos um alpha- ocophe ol le els [7,13].
Finally, we ound no associa ion be ween he ca bohyd a es, p o eins, lipid classes,
phospholipids, molecula species o iacylglyce ols, calcium, phospho us, and selenium
con en in he DHM and he independen a iables s udied. The selenium con en in
DHM was no associa ed wi h die a y in ake in he p esen s udy, al hough i is known
ha he die in luences selenium HM con en [
105
]. The calcium and selenium con en in
DHM was low and phospho us con en was adequa e acco ding o he e e ence alues o
HM [79,87–89].
In summa y, we s udied he ac o s ha migh in luence he nu i ional con en o
DHM, and we highligh he ollowing as s eng hs: he de ailed s udy o he supple-
men a ion habi s o dono s since ges a ion, he i e-day weighed die a y eco d, and he
de e mina ion o nu ien s in dono s’ e y h ocy es, plasma, and u ine. In addi ion, he di-
e a y s udy was ca ied ou concomi an ly wi h he collec ion o milk o e i e consecu i e
days. Milk om di e en dono s o di e en days was no pooled o allow us o de e mine
associa ions be ween he con en o each nu ien in DHM and dono s’ in ake, s a us, and
cha ac e is ics. Howe e , ou s udy has some limi a ions. I is impo an o poin ou ha
he s udied DHM comp ised aw milk, and al hough he impac o pas eu iza ion on he
nu ien con en in DHM has been p e iously s udied [
26
,
33
], we do no know i all he
associa ions obse ed would emain a e he pas eu iza ion o milk. Holde pas eu iza ion
is he mos widely used hea ea men and he mos s udied. Acco ding o a e iew on
he e ec o Holde pas eu iza ion [
26
], i amin C (asco bic acid + dehyd oasco bic acid)
and i amin B6 dec ease signi ican ly wi h Holde pas eu iza ion, while i amins D, B2, B3,
B5, and B12, as well as bio in, do no seem o be a ec ed. Vi amins A and E ha e shown
di e en esponses o Holde pas eu iza ion in di e en s udies. The o al lipid con en is
p ese ed a e Holde pas eu iza ion, as well as he a y acid composi ion. In pa icula ,
DHA is no a ec ed by Holde pas eu iza ion [
26
,
94
,
95
]. New ypes o p ocessing o DHM
a e being in es iga ed in o de o p ese e as many o i s p ope ies as possible, wi h di e -
en esul s in e ms o he deg ee o e en ion and loss o di e en nu ien s [
32
,
33
]. As a
nex s ep, i would be in e es ing o s udy he ela ionship be ween he die and nu i ional
s a us o dono s and he nu ien con en o he p ocessed milk as he inal p oduc o be
Nu ien s 2023,15, 3486 19 o 24
gi en o he ecipien in an s. Ano he limi a ion o ou s udy was ha , al hough we did
s anda dize milk collec ion in e ms o he ype o exp ession ( ull exp ession), we did
no s anda dize milk collec ion in e ms o he ime o milk exp ession o he ime ha
had elapsed since he p e ious eeding o exp ession, as ecommended [
106
]. We also
did no p o ec pho osensi i e i amins in milk om ligh . The g ea he e ogenei y in
he popula ion s udied in e ms o b eas eeding ime and du a ion o ges a ion was also
no ewo hy. I is known ha all hese ac o s in luence he nu ien s in milk. Howe e ,
we wan ed o ep oduce he eali y o ou human milk bank. On he o he hand, ou
esul s may no be gene alizable o con ex s o he han a human milk bank. Al hough we
con olled o an impo an numbe o co a ia es ha could ha e ac ed as con ounde s,
he la ge numbe o ac o s ha can modi y he nu i ional con en o human milk, as well
as he complexi y o nu ien me abolism—which is also in luenced by he gene ics o he
mo he –in an dyad i sel —makes i e y di icul o s udy in e ac ions be ween ma e nal
die and milk composi ion. The in e p e a ion o he esul s o biochemical nu i ional
indica o s in b eas eeding mo he s is also di icul due o he limi ed in o ma ion a ailable
o his popula ion and e en mo e so in he case o p olonged b eas eeding [
107
]. A longi-
udinal s udy design could ha e p o ided addi ional in o ma ion on changes in nu ien
s o es h oughou lac a ion and hei possible associa ion wi h he composi ion o DHM.
Also, gi en ha he beha io o some nu ien s in human milk wi h espec o he ma e nal
die may di e depending on he socioeconomic s a us o lac a ing mo he s, access o ood,
and ma e nal nu i ional s a us, he indings om ou popula ion o dono s, who exhibi ed
a high educa ional le el and li ed in a de eloped coun y, may no be gene alizable o
o he en i onmen s.
Howe e , we conside ha he esul s ob ained in his s udy a e ele an since we ha e
shown ha he e a e associa ions be ween he nu i ional composi ion in aw DHM and he
die , as well as he nu ien deposi s o dono s—despi e he he e ogenei y among he dono s
ega ding he du a ion o p egnancy and b eas eeding ime—and he p ocedu es o which
aw DHM is subjec ed, such as ex ac ion, eezing, mixing, and s o age in anspa en
glass con aine s.
5. Conclusions
In conclusion, he die and nu i ional s a us o dono s in luence he a y acid p o ile
and some o he mic onu ien s in he aw DHM. One o he mos impo an esul s was he
posi i e co ela ion ound be ween he DHA con en in he DHM and bo h he DHA in ake
and he plasma DHA le els o he dono s. In ou s udy, o e e y 1 g/day o DHA in ake,
an inc ease o 0.38% in DHA con en and 0.78% in o al omega-3 con en was obse ed in
DHM. DHM sa u a ed a y acids we e posi i ely associa ed wi h e y h ocy e dime hyl
ace als, plasma s ea ic acid, ans a y acids in ake, and b eas eeding du a ion and nega-
i ely associa ed wi h e y h ocy e ma ga oleic acid. DHM cholecalci e ol was associa ed
wi h plasma cholecalci e ol le els and dai y in ake. O he weake associa ions we e ound
o ee hiamine, ee ibo la in, py idoxal, dehyd oasco bic acid, 25(OH)D
3
, o al MUFAs,
o al PUFAs, and linoleic acid in DHM. We ound no associa ions be ween he ca bohy-
d a es, p o eins, lipid classes, phospholipids, and molecula species o iacyclglyce ols in
DHM and he clinical cha ac e is ics o he dono s o hei soma ome ic s udy, nu i ional
biochemical de e mina ions, o die a y in ake.
In es iga ion o he composi ion o DHM and he ac o s ha in luence i is essen ial.
In an s ecei ing DHM a e in a s a e o nu i ional eme gency, and i is he e o e desi able
ha he milk hey ecei e is in acco dance wi h hei needs. S udies such as he p esen one
may help o imp o e die e ic ecommenda ions o milk dono s o ensu e a mo e sui able
composi ion o DHM. Howe e , much emains o be lea ned and i is possible ha he
de elopmen o pe sonalized medicine will allow us o be e unde s and he a iabili y
in he composi ion o DHM and he di e en needs o p e e m in an s. Answe s o open
ques ions a e likely o be ound in his new ield.
Nu ien s 2023,15, 3486 20 o 24
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/nu15153486/s1, Table S1: Consump ion o he pha macological
supplemen s du ing p egnancy and he lac a ion o he human milk dono s (n= 113); Table S2: Die
su ey: i e-day die a y eco d. Daily nu ien in ake o he human milk dono s; Table S3: P e alence
o inadequa e in ake o speci ic nu ien s in human milk dono s; Table S4: Die su ey: i e-day
die a y dia ies. Numbe o ood se ings pe day consumed by he pa icipan s, heal hy ea ing
index (HEI), and eco ds o supplemen and iodized sal in ake among he human milk dono s;
Table S5: Die su ey: ood equency ques ionnai e (FFQ, he numbe o ood se ings pe day
o week consumed by he pa icipan s) esul s o he human milk dono s; Table S6: E y h ocy e
and plasma a y acid composi ions (g/100 g o o al FAs) o he human milk dono s; Table S7:
E y h ocy e, plasma, and u ine concen a ions o nu ien s and biochemical de e mina ions o he
human milk dono s.
Au ho Con ibu ions:
Concep ualiza ion, N.U.-V., D.E.-V., N.R.G.-L. and C.R.P.-A.; me hodology,
N.U.-V., D.E.-V., J.F., M.V.C., J.M.-T. and J.C.E.S.; o mal analysis, N.U.-V. and A.M.-P.; in es iga ion,
N.U.-V., K.K., D.E.-V., J.F., M.V.C., J.M.-T. and J.C.E.S.; w i ing—o iginal d a p epa a ion, N.U.-V.
and A.M.-P.; w i ing— e iew and edi ing, N.U.-V., A.M.-P., K.K., D.E.-V., J.F., M.V.C., J.M.-T., J.C.E.S.,
N.R.G.-L. and C.R.P.-A.; p ojec adminis a ion, N.U.-V., N.R.G.-L. and C.R.P.-A.; unding acquisi ion,
N.U.-V., D.E.-V., N.R.G.-L. and C.R.P.-A. All au ho s ha e ead and ag eed o he published e sion
o he manusc ip .
Funding:
This esea ch was unded by a g an om he Spanish Resea ch P ojec s in Heal h unded
by Ins i u o de Salud Ca los III (ISCIII)— he S a e Plan o Scien i ic and Technical Resea ch and
Inno a ion (g an FIS PI15/00995). Fu he mo e, his wo k ecei ed suppo om RETICS “Ma e nal
and Child Heal h and De elopmen Ne wo k” (SAMID Ne wo k) (RD16/0022/0015) and om he
Spanish Minis y o Science, Inno a ion, and Uni e si ies (p ojec PDI2020-114821RB-I00).
Ins i u ional Re iew Boa d S a emen :
The s udy was conduc ed in acco dance wi h he Decla a ion
o Helsinki and app o ed by he Hospi al 12 de Oc ub e’s Clinical Resea ch E hics Commi ee
(p o ocol code 15/269, 29 Sep embe 2015).
In o med Consen S a emen :
In o med consen was ob ained om all subjec s in ol ed in he
s udy.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho . The da a a e no publicly a ailable due o p i acy issues.
Acknowledgmen s:
We wish o hank ou dono s who ook he ime o pa icipa e in his s udy
and o kindly dona e hei human milk. Thank you o you gene osi y, which made his esea ch
possible. We also wan o acknowledge he con ibu ion o RETICS “Ma e nal and Child Heal h and
De elopmen Ne wo k” (SAMID Ne wo k).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
A slanoglu, S.; Co peleijn, W.; Mo o, G.; B aegge , C.; Campoy, C.; Colomb, V.; Decsi, T.; Domellö , M.; Few ell, M.; Hojsak, I.;
e al. Dono human milk o p e e m in an s: Cu en e idence and esea ch di ec ions. J. Pedia . Gas oen e ol. Nu .
2013
,57,
535–542. [C ossRe ] [PubMed]
2.
Mo o, G.E.; Billeaud, C.; Rachel, B.; Cal o, J.; Ca alla in, L.; Ch is en, L.; Escude -Vieco, D.; Gaya, A.; Lembo, D.; Wesolowska, A.;
e al. P ocessing o Dono Human Milk: Upda e and Recommenda ions F om he Eu opean Milk Bank Associa ion (EMBA).
F on . Pedia . 2019,7, 49. [C ossRe ] [PubMed]
3.
Wea e , G.; Be ino, E.; Gebaue , C.; G o slien, A.; Mileusnic-Mileno ic, R.; A slanoglu, S.; Ba ne , D.; Boquien, C.Y.; Bu in, R.;
Gaya, A.; e al. Recommenda ions o he es ablishmen and ope a ion o Human Milk Banks in Eu ope: A consensus s a emen
om he Eu opean Milk Bank Associa ion (EMBA). F on . Pedia . 2019,7, 53. [C ossRe ] [PubMed]
4.
Ab ams, S.A.; Lande s, S.; Noble, L.M.; Poindex e , B.B.; AAP Comi ee on Nu i ion; AAP Sec ion on B eas eeding; AAP
Comi ee on Fe us and Newbo n. Dono human milk o he high- isk in an : P epa a ion, sa e y, and usage op ions in he Uni ed
S a es. Pedia ics 2017,139, e20163440. [C ossRe ]
5.
Ma ini, S.; Beghe i, I.; Annunzia a, M.; Ace i, A.; Galle i, S.; Ragni, L.; Don i, A.; Co aglia, L. En e al Nu i ion in Te m In an s
wi h Congeni al Hea Disease: Knowledge Gaps and Fu u e Di ec ions o Imp o e Clinical P ac ice. Nu ien s
2021
,13, 932.
[C ossRe ]
Nu ien s 2023,15, 3486 21 o 24
6.
Na ional Academies o Sciences, Enginee ing and Medicine; Heal h and Medicine Di ision; Food and Nu i ion Boa d. Nu i ion
Du ing P egnancy and Lac a ion: Explo ing New E idence: P oceedings o a Wo kshop; The Na ional Academies P ess: Washing on,
DC, USA, 2020; ISBN 9780309679244.
7. D o , D.K.; Allen, L.H. O e iew o nu ien s in human milk. Ad . Nu . 2018,9, 278S–294S. [C ossRe ]
8.
B a i, F.; Wiens, F.; Deca li, A.; Dal Pon , A.; Agos oni, C.; Fe a oni, M. Impac o ma e nal nu i ion on b eas -milk composi ion:
A sys ema ic e iew. Am. J. Clin. Nu . 2016,104, 646–662. [C ossRe ]
9.
Keikha, M.; Bah eynian, M.; Saleki, M.; Kelishadi, R. Mac o- and Mic onu ien s o Human Milk Composi ion: A e They Rela ed
o Ma e nal Die ? A Comp ehensi e Sys ema ic Re iew. B eas eed. Med. 2017,12, 517–527. [C ossRe ]
10.
Allen, L.H. B Vi amins in B eas Milk: Rela i e Impo ance o Ma e nal S a us and In ake, and E ec s on In an S a us and
Func ion. Ad . Nu . An In . Re . J. 2012,3, 362–369. [C ossRe ]
11.
Smilowi z, J.T.; Allen, L.H.; Dallas, D.C.; McManaman, J.; Rai en, D.J.; Rozga, M.; Sela, D.A.; Seppo, A.; Williams, J.E.; Young,
B.E.; e al. Ecologies, syne gies, and biological sys ems shaping human milk composi ion—A epo om “B eas milk Ecology:
Genesis o In an Nu i ion (BEGIN)” Wo king G oup 2. Am. J. Clin. Nu . 2023,117, S28–S42. [C ossRe ]
12.
Allen, L.H. Mul iple mic onu ien s in p egnancy and lac a ion: An o e iew. Am. J. Clin. Nu .
2005
,81, 1206–1212. [C ossRe ]
[PubMed]
13.
Lima, M.S.R.; Dimens ein, R.; Ribei o, K.D.S. Vi amin e concen a ion in human milk and associa ed ac o s: A li e a u e e iew.
J. Pedia . 2014,90, 440–448. [C ossRe ] [PubMed]
14.
Ba e a, C.; Valenzuela, R.; Chamo o, R.; Bascuñán, K.; Sando al, J.; Sabag, N.; Valenzuela, F.; Valencia, M.P.; Puig edon, C.;
Valenzuela, A. The impac o ma e nal die du ing p egnancy and lac a ion on he a y acid composi ion o e y h ocy es and
b eas milk o chilean women. Nu ien s 2018,10, 839. [C ossRe ] [PubMed]
15.
Bu, T.; Tang, D.; Liu, Y.; Chen, D. T ends in Die a y Pa e ns and Die - ela ed Beha io s in China. Am. J. Heal h Beha .
2021
,45,
371–383. [C ossRe ] [PubMed]
16.
G ech, A.; Rangan, A.; Allman-Fa inelli, M. Mac onu ien Composi ion o he Aus alian Popula ion’s Die ; T ends om Th ee
Na ional Nu i ion Su eys 1983, 1995 and 2012. Nu ien s 2018,10, 1045. [C ossRe ] [PubMed]
17.
Fulgoni, K.; Fulgoni, V.L. T ends in o al, added, and na u al phospho us in ake in adul ame icans, nhanes 1988–1994 o nhanes
2015–2016. Nu ien s 2021,13, 2249. [C ossRe ]
18.
Ch is ian, P.; Smi h, E.R.; Lee, S.E.; Va gas, A.J.; B eme , A.A.; Rai en, D.J. The need o s udy human milk as a biological sys em.
Am. J. Clin. Nu . 2021,113, 1063–1072. [C ossRe ]
19.
Hampel, D.; Shahab-Fe dows, S.; Islam, M.M.; Pee son, J.M.; Allen, L.H. Vi amin Concen a ions in Human Milk Va y wi h Time
wi hin Feed, Ci cadian Rhy hm, and Single-Dose Supplemen a ion. J. Nu . 2017,147, 603–611. [C ossRe ]
20.
Dawodu, A.; Tsang, R.C. Ma e nal Vi amin D S a us: E ec on Milk Vi amin D Con en and Vi amin D S a us o B eas eeding
In an s. Ad . Nu . 2012,3, 353–361. [C ossRe ]
21.
O ega, R.M.; López-Sobale , A.M.; Ma ínez, R.M.; And és, P.; Elena Quin as, M. In luence o smoking on i amin E s a us du ing
he hi d imes e o p egnancy and on b eas -milk ocophe ol concen a ions in Spanish women. Am. J. Clin. Nu .
1998
,68,
662–667. [C ossRe ]
22.
Pe in, M.T.; Bel o , M.B.; Hagado n, J.I.; McG a h, J.M.; Taylo , S.N.; Tosi, L.M.; B ownell, E.A. The nu i ional composi ion and
ene gy con en o dono human milk: A sys ema ic e iew. Ad . Nu . 2020,11, 960–970. [C ossRe ]
23.
Sie a-Colomina, G.; Ga cía-La a, N.R.; Escude -Vieco, D.; Alonso-Díaz, C.; And és Es eban, E.M.; Pallás-Alonso, C.R. Dono
milk olume and cha ac e is ics o dono s and hei child en. Ea ly Hum. De . 2014,90, 209–212. [C ossRe ]
24. Osbaldis on, R.; Mingle, L.A. Cha ac e iza ion o human milk dono s. J. Hum. Lac . 2007,23, 350–357. [C ossRe ]
25.
Pa ke , M.G.; S ellwagen, L.M.; Noble, L.; Kim, J.H.; Poindex e , B.B.; Puopolo, K.M.; Sec ion on B eas eeding; Commi ee on
Nu i ion, Commi ee on Fe us and Newbo n. P omo ing Human Milk and B eas eeding o he Ve y Low Bi h Weigh In an .
Pedia ics 2021,148, e2021054272. [C ossRe ]
26.
Peila, C.; Mo o, G.E.; Be ino, E.; Ca alla in, L.; Gi ibaldi, M.; Giuliani, F.; C esi, F.; Coscia, A. The e ec o holde pas eu iza ion
on nu ien s and biologically-ac i e componen s in dono human milk: A e iew. Nu ien s 2016,8, 477. [C ossRe ]
27. Lea , A.; Lansdowne, Z. Vi amins—Con en ional uses and new insigh s. Wo ld Re . Nu . Die . 2014,110, 152–166. [C ossRe ]
28.
Van Zoe en-G obben, D.; Sch ij e , J.; Van den Be g, H.; Be ge , H.M. Human milk i amin con en a e pas eu isa ion, s o age,
o ube eeding. A ch. Dis. Child. 1987,62, 161–165. [C ossRe ]
29.
Romeu-Nadal, M.; Cas ello e, A.I.; López-Saba e , M.C. E ec o cold s o age on i amins C and E and a y acids in human milk.
Food Chem. 2008,106, 65–70. [C ossRe ]
30.
Nessel, I.; Khashu, M.; Dyall, S.C. The e ec s o s o age condi ions on long-chain polyunsa u a ed a y acids, lipid media o s,
and an ioxidan s in dono human milk—A e iew. P os aglandins Leuko . Essen . Fa . Acids 2019,149, 8–17. [C ossRe ]
31.
Wei, W.; Cheng, J.; Yang, J.; Chen, C.; Jin, Q.; Song, J.; Wang, X. Phospholipid composi ion and a globule s uc u e change du ing
low empe a u e s o age o human milk. LWT 2021,150, 112050. [C ossRe ]
32.
O’Conno , D.L.; Ewaschuk, J.B.; Unge , S. Human milk pas eu iza ion: Bene i s and isks. Cu . Opin. Clin. Nu . Me ab. Ca e
2015
,
18, 269–275. [C ossRe ] [PubMed]
33.
Escude -Vieco, D.; Rod íguez, J.M.; Espinosa-Ma os, I.; Co zo, N.; Mon illa, A.; Ga cía-Se ano, A.; Cal o, M.V.; Fon echa,
J.; Se ano, J.; Fe nández, L.; e al. High-Tempe a u e Sho -Time and Holde Pas eu iza ion o Dono Milk: Impac on Milk
Composi ion. Li e 2021,11, 114. [C ossRe ]

Nu ien s 2023,15, 3486 22 o 24
34.
Valen ine, C.J.; Mo ow, G.; Pennell, M.; Mo ow, A.L.; Hodge, A.; Haban-Ba z, A.; Collins, K.; Roge s, L.K. Randomized
Con olled T ial o Docosahexaenoic Acid Supplemen a ion in Midwes e n U.S. Human Milk Dono s. B eas eed. Med.
2013
,8,
86–91. [C ossRe ] [PubMed]
35.
U e a-Velasco, N.; Kelle , K.; Escude -Vieco, D.; Se ano, J.C.E.; Ga cía-La a, N.R.; Pallás-Alonso, C.R. Assessmen o Iodine
Concen a ion in Human Milk om Dono s: Implica ions o P e e m In an s. Nu ien s 2022,14, 4304. [C ossRe ] [PubMed]
36.
U e a-Velasco, N.; Kelle , K.; Escude -Vieco, D.; Fon echa, J.; Cal o, M.V.; Megino-Tello, J.; Se ano, J.C.E.; Rome o Fe ei o, C.;
Ga cía-La a, N.R.; Pallás-Alonso, C.R. Human Milk Composi ion and Nu i ional S a us o Omni o e Human Milk Dono s
Compa ed wi h Vege a ian/Vegan Lac a ing Mo he s. Nu ien s 2023,15, 1855. [C ossRe ]
37.
Faul, F.; E d elde , E.; Lang, A.G.; Buchne , A. G*Powe 3: A Flexible S a is ical Powe Analysis P og am o he Social, Beha io al,
and Biomedical Sciences. Beha . Res. Me hods 2007,39, 175–191. [C ossRe ]
38.
O ega, R.; López-Sobale , A.; And és, P.; Requejo, A.; Apa icio, A.; Moline o, L. DIAL So wa e o Assessing Die s and Food
Calcula ions; Depa men o Nu i ion (UCM) & Alceingenie ía, SA: Mad id, Spain, 2013.
39.
Ins i u e o Medicine. Die a y Re e ence In akes o Calcium, Phospho us, Magnesium, Vi amin D, and Fluo ide; Na ional Academies
P ess: Washing on, DC, USA, 1997; ISBN 9780309063500.
40.
Ins i u e o Medicine. Die a y Re e ence In akes o Thiamin, Ribo la in, Niacin, Vi amin B6, Fola e, Vi amin B12, Pan o henic Acid,
Bio in, and Choline; The Na ional Academies P ess: Washing on, DC, USA, 1998; ISBN 9780309064118.
41.
Ins i u e o Medicine. Die a y Re e ence In akes o Vi amin C, Vi amin E, Selenium, and Ca o enoids; Na ional Academies P ess:
Washing on, DC, USA, 2000; ISBN 9780309069496.
42.
Ins i u e o Medicine. Die a y Re e ence In akes o Vi amin A, Vi amin K, A senic, Bo on, Ch omium, Coppe , Iodine, I on, Manganese,
Molybdenum, Nickel, Silicon, Vanadium, and Zinc; The Na ional Academies P ess: Washing on, DC, USA, 2001; ISBN 9780309072908.
43.
Ins i u e o Medicine. Die a y Re e ence In akes o Ene gy, Ca bohyd a e, Fibe , Fa , Fa y Acids, Choles e ol, P o ein, and Amino Acids;
The Na ional Academies P ess: Washing on, DC, USA, 2005; ISBN 030908525X.
44.
Ins i u e o Medicine. Die a y Re e ence In akes o Calcium and Vi amin D; Ross, C.A., Taylo , C.L., Yak ine, A.L., Del Valle, H.B.,
Ins i u e o Medicine, Eds.; Na ional Academies P ess: Washing on, DC, USA, 2011; ISBN 978-0-309-16395-8.
45.
EFSA (Eu opean Food Sa e y Au ho i y). Die a y Re e ence Values o nu ien s. Summ. Rep. EFSA Suppo . Publ.
2017
,98,
e15121.
46.
Olsen, I.E.; G o eman, S.A.; Lawson, M.L.; Cla k, R.H.; Zemel, B.S. New in au e ine g ow h cu es based on Uni ed S a es da a.
Pedia ics 2010,125, 214–224. [C ossRe ]
47.
Wo ld Heal h O ganiza ion. WHO Child G ow h S anda ds: Leng h/Heigh - o -Age, Weigh - o -Age, Weigh - o -Leng h, Weigh - o -
Heigh and Body Mass Index- o -Age: Me hods and De elopmen ; Wo ld Heal h O ganiza ion: Gene a, Swi ze land, 2006; ISBN
92-4-154693-X.
48.
Allen, L.H.; Ca iqui y, A.L.; Mu phy, S.P. Pe spec i e: P oposed Ha monized Nu ien Re e ence Values o Popula ions. Ad
Nu 2020,11, 469–483. [C ossRe ]
49.
O ega An a, R.; Requejo Ma cos, A. Nu iguía. Manual de Nu ición Clínica; Edi o ial Médica Paname icana: Mad id, Spain, 2015;
ISBN 9788498358674.
50.
Kennedy, E.T.; Ohls, J.; Ca lson, S.; Fleming, K. The Heal hy Ea ing Index: Design and applica ions. J. Am. Die . Assoc.
1995
,95,
1103–1108. [C ossRe ]
51.
Dapcich, V.; Sal ado Cas ell, G.; Ribas Ba ba, L.; Pé ez Rod igo, C.; A ance a-Ba ina, J.; Se a Majem, L. Emba azo y lac ancia.
Necesidades especiales. In Guía de la Alimen ación Saludable; Sociedad Española de Nu ición Comuni a ia, Ed.; Sociedad Española
de Nu ición Comuni a ia: Mad id, Spain, 2004; p. 82. A ailable online: h ps://www.nu icioncomuni a ia.o g/es/o as-
publicaciones (accessed on 2 Feb ua y 2023).
52.
Giu ida, F.; Flei h, M.; Goye , A.; Samuel, T.M.; Elmelegy-Masse ey, I.; Fon annaz, P.; C uz-He nandez, C.; Thakka , S.K.;
Monna d, C.; De Cas o, C.A.; e al. Human milk a y acid composi ion and i s associa ion wi h ma e nal blood and adipose
issue a y acid con en in a coho o women om Eu ope. Eu . J. Nu . 2022,61, 2167–2182. [C ossRe ]
53.
G aham, J.; Pee son, J.; Haskell, M.; Sh es ha, R.; B own, K.; Allen, L. E y h ocy e ibo la in o he de ec ion o ibo la in
de iciency in p egnan Nepali women. Clin. Chem. 2005,51, 2162–2165. [C ossRe ]
54.
Be ge , M.M.; Shenkin, A.; Schweinlin, A.; Am ein, K.; Augsbu ge , M.; Biesalski, H.K.; Bischo , S.C.; Casae , M.P.; Gundogan, K.;
Lepp, H.L.; e al. ESPEN mic onu ien guideline. Clin. Nu . 2022,41, 1357–1424. [C ossRe ]
55.
Ehsanian, R.; Ande son, S.; Schneide , B.; Kennedy, D.; Mansou ian, V. P e alence o low plasma i amin B1 in he s oke
popula ion admi ed o acu e inpa ien ehabili a ion. Nu ien s 2020,12, 1034. [C ossRe ]
56.
Pe eys, B.J.; F ank, E.L. Rapid de e mina ion o i amin B2 ( ibo la in) in plasma by HPLC. Clin. Chim. Ac a
2011
,412, 38–43.
[C ossRe ]
57.
And aos, S.; Jones, B.; Wall, C.; Tho s ensen, E.; Kussmann, M.; Smi h, D.C.; Lange, K.; Cli o d, S.; Sa e y, R.; Bu gne , D.;
e al. Plasma B i ame s: Popula ion epidemiology and pa en -child conco dance in child en and adul s. Nu ien s
2021
,13, 821.
[C ossRe ]
58.
Allen, L.H.; Mille , J.W.; De G oo , L.; Rosenbe g, I.H.; Smi h, A.D.; Re sum, H.; Rai en, D.J. Bioma ke s o Nu i ion o
De elopmen (BOND): Vi amin B-12 Re iew. J. Nu . 2018,148, 1995S–2027S. [C ossRe ]
59.
Pawlak, R.; Pa o , S.J.; Raj, S.; Cullum-Dugan, D.; Lucus, D. How p e alen is i amin B12 de iciency among ege a ians? Nu .
Re . 2013,71, 110–117. [C ossRe ]
Nu ien s 2023,15, 3486 23 o 24
60.
Sobczy´nska-Male o a, A.; Ha ing on, D.J. Labo a o y assessmen o ola e (Vi amin B9) s a us. J. Clin. Pa hol.
2018
,71, 949–956.
[C ossRe ]
61.
Bailey, L.B.; S o e , P.J.; McNul y, H.; Fenech, M.F.; G ego y, J.F., III.; Mills, J.L.; P ei e , C.M.; Fazili, Z.; Zhang, M.; Ueland, P.M.;
e al. Bioma ke s o nu i ion o de elopmen -Fola e e iew. J. Nu . 2015,145, 1636S–1680S. [C ossRe ]
62.
De Pee, S.; Da y, O. Biochemical indica o s o i amin A de iciency: Se um e inol and se um e inol binding p o ein. J. Nu .
2002,132, 2895–2901. [C ossRe ] [PubMed]
63.
Wo ld Heal h O ganiza ion. Se um Re inol Concen a ions o De e mining he P e alence o Vi amin A De iciency in Popula ions; Wo ld
Heal h O ganiza ion: Gene a, Swi ze land, 2011. A ailable online: h p://www.who.in / mnis/indica o s/ e inol.pd (accessed
on 28 Feb ua y 2023).
64.
U.S. Cen e s o Disease Con ol and P e en ion. Second Na ional Repo on Biochemical Indica o s o Die and Nu i ion in he U.S.
Popula ion; U.S. Cen e s o Disease Con ol and P e en ion: A lan a, GA, USA, 2012; ISBN 1499234783.
65.
Die a y Guidelines Ad iso y Commi ee. Scien i ic Repo o he 2015 Die a y Guidelines Ad iso y Commi ee: Ad iso y Repo o he
Sec e a y o Heal h and Human Se ices and he Sec e a y o Ag icul u e; Die a y Guidelines Ad iso y Commi ee: Washing on, DC,
USA, 2015.
66. D o , D.K.; Allen, L.H. Vi amin E de iciency in de eloping coun ies. Food Nu . Bull. 2011,32, 124–143. [C ossRe ]
67.
Henjum, S.; Mange , M.; Hampel, D.; B an sæ e , A.L.; Shahab-Fe dows, S.; Bas ani, N.E.; S and, T.A.; Re sum, H.; Allen, L.H.
Vi amin B12 concen a ions in milk om No wegian women du ing he six i s mon hs o lac a ion. Eu . J. Clin. Nu .
2020
,74,
749–756. [C ossRe ] [PubMed]
68.
No man, E.J.; Mo ison, J.A. Sc eening elde ly popula ions o cobalamin ( i amin B12) de iciency using he u ina y me hyl-
malonic acid assay by gas ch oma og aphy mass spec ome y. Am. J. Med. 1993,94, 589–594. [C ossRe ]
69.
Wo ld Heal h O ganiza ion. U ina y Iodine Concen a ions o De e mining Iodine S a us De iciency in Popula ions. Vi amin and
Mine al Nu i ion In o ma ion Sys em. A ailable online: h ps://apps.who.in /i is/bi s eam/handle/10665/85972/WHO_NMH_
NHD_EPG_13.1_eng.pd (accessed on 8 Ma ch 2023).
70.
Ahn, J.; Lee, J.H.; Lee, J.; Baek, J.Y.; Song, E.; Oh, H.S.; Kim, M.; Pa k, S.; Jeon, M.J.; Kim, T.Y.; e al. Associa ion be ween u ina y
sodium le els and iodine s a us in Ko ea. Ko ean J. In e n. Med. 2020,35, 392–399. [C ossRe ]
71. Foley, K.F.; Boccuzzi, L. U ine calcium: Labo a o y measu emen and clinical u ili y. Lab. Med. 2010,41, 683–686. [C ossRe ]
72.
Fe nández-Ruiz, L.; Rodelo Haad, C.; Rod íguez-Po illo, M.; San ama ía-Olmo, R. Va ia ion o phospha u ia acco ding o
phospho us in ake. Ac ual. Medica 2020,105, 18–26. [C ossRe ]
73.
Zhang, Z.; Wang, Y.; Yang, X.; Cheng, Y.; Zhang, H.; Xu, X.; Zhou, J.; Chen, H.; Su, M.; Yang, Y.; e al. Human Milk Lipid P o iles
a ound he Wo ld: A Sys ema ic Re iew and Me a-Analysis. Ad . Nu . 2022,13, 2519–2536. [C ossRe ]
74.
Gibson, R.S.; Rahmannia, S.; Diana, A.; Leong, C.; Hasza d, J.J.; Hampel, D.; Reid, M.; E ha d , J.; Su yan o, A.H.; So iah, W.N.;
e al. Associa ion o ma e nal die , mic onu ien s a us, and milk olume wi h milk mic onu ien concen a ions in Indonesian
mo he s a 2 and 5 mon hs pos pa um. Am. J. Clin. Nu . 2020,112, 1039–1050. [C ossRe ]
75.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA); Tu ck, D.; B esson, J.L.; Bu lingame, B.; Dean, T.; Fai wea he -
Tai , S.; Heinonen, M.; Hi sch-E ns , K.I.; Mangelsdo , I.; McA dle, H.J.; e al. Die a y e e ence alues o hiamin. EFSA J.
2016
,
14, 4653. [C ossRe ]
76.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA); Tu ck, D.; B esson, J.-L.; Bu lingame, B.; Dean, T.; Fai wea he -
Tai , S.; Heinonen, M.; Hi sch-E ns , K.; Mangelsdo , I.; McA dle, H.; e al. Die a y Re e ence Values o ibo la in. EFSA J.
2017
,
15, 4919. [C ossRe ]
77.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o niacin. EFSA
J. 2014,12, 3759. [C ossRe ]
78.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o pan o henic
acid. EFSA J. 2014,12, 3581. [C ossRe ]
79.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on nu ien equi emen s and die a y in akes
o in an s and young child en in he Eu opean Union. EFSA J. 2013,11, 3408. [C ossRe ]
80.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o cobalamin
( i amin B12). EFSA J. 2015,13, 4150. [C ossRe ]
81.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o i amin C.
EFSA J. 2013,11, 3418. [C ossRe ]
82. EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o i amin A.
EFSA J. 2015,13, 4028. [C ossRe ]
83.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic opinion on die a y e e ence alues o i amin D.
EFSA J. 2016,14, 4547. [C ossRe ]
84.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o i amin E as
α- ocophe ol. EFSA J. 2015,13, 4149. [C ossRe ]
85. Semba, R.D.; Delange, F. Iodine in human milk: Pe spec i es o in an heal h. Nu . Re . 2001,59, 269–278. [C ossRe ]
86.
Ande sson, M.; B aegge , C.P. The ole o iodine o hy oid unc ion in lac a ing women and in an s. Endoc . Re .
2022
,43,
469–506. [C ossRe ] [PubMed]
Nu ien s 2023,15, 3486 24 o 24
87.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o calcium.
EFSA J. 2015,13, 4101. [C ossRe ]
88.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o phospho us.
EFSA J. 2015,13, 4185. [C ossRe ]
89.
EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies (NDA). Scien i ic Opinion on Die a y Re e ence Values o selenium.
EFSA J. 2014,12, 3846. [C ossRe ]
90.
LASER Analy ica. Comp ehensi e li e a u e sea ch and e iew o b eas milk composi ion as p epa a o y wo k o he se ing o
die a y e e ence alues o i amins and mine als. EFSA Suppo . Publ. 2014,11, 629E. [C ossRe ]
91.
Cas illo, F.; Cas illo-Fe e , F.J.; Co dobilla, B.; Domingo, J.C. Inadequa e con en o docosahexaenoic acid (DHA) o dono human
milk o eeding p e e m in an s: A compa ison wi h mo he ’s own milk a di e en s ages o lac a ion. Nu ien s
2021
,13, 1300.
[C ossRe ]
92.
Cas illo Salinas, F.; Mon ane Ramón, A.; Cas illo Fe e , F.-J.; Domingo-Ca nice, A.; Co dobilla, B.; Domingo, J.C. E y h ocy e
Memb ane Docosahexaenoic Acid (DHA) and Lipid P o ile in P e e m In an s a Bi h and O e he Fi s Mon h o Li e: A
Compa a i e S udy wi h In an s a Te m. Nu ien s 2022,14, 4956. [C ossRe ]
93.
Mak ides, M.; Neumann, M.; Gibson, R. E ec o ma e nal docosahexaenoic (DHA) supplemen a ion on b eas milk composi ion.
Eu . J. Clin. Nu . 1996,50, 352–357.
94.
Valen ine, C.J.; Mo ow, G.; Fe nandez, S.; Gula i, P.; Ba holomew, D.; Long, D.; Wel y, S.E.; Mo ow, A.L.; Roge s, L.K.
Docosahexaenoic acid and amino acid con en s in pas eu ized dono milk a e low o p e e m in an s. J. Pedia .
2010
,157,
906–910. [C ossRe ]
95.
Baack, M.L.; No is, A.W.; Yao, J.; Colaizy, T. Long Chain Polyunsa u a ed Fa y Acid Le els in U.S. Dono Human Milk: Mee ing
he Needs o P ema u e In an s? J. Pe ina ol. 2012,32, 598–603. [C ossRe ]
96.
F ancois, C.A.; Conno , S.L.; Bolewicz, L.C.; Conno , W.E. Supplemen ing lac a ing women wi h laxseed oil does no inc ease
docosahexaenoic acid in hei milk. Am. J. Clin. Nu . 2003,77, 226–233. [C ossRe ]
97.
Valen ine, C.J. Ma e nal Die a y DHA Supplemen a ion o Imp o e In lamma o y Ou comes in he P e e m In an . Ad . Nu .
2012,3, 370–376. [C ossRe ]
98.
Ga cía-Maldonado, E.; Alco a, A.; Zapa e a, B.; Vaque o, M.P. Changes in a y acid le els a e consump ion o a no el
docosahexaenoic supplemen om algae: A c osso e andomized con olled ial in omni o ous, lac o-o o ege a ians and
egans. Eu . J. Nu . 2023,62, 1691–1705. [C ossRe ]
99.
Pe in, M.T.; Pawlak, R.; Dean, L.L.; Ch is is, A.; F iend, L. A c oss-sec ional s udy o a y acids and b ain-de i ed neu o ophic
ac o (BDNF) in human milk om lac a ing women ollowing egan, ege a ian, and omni o e die s. Eu . J. Clin. Nu .
2019
,58,
2401–2410. [C ossRe ]
100. Scopesi, F.; Cianghe o i, S.; Lan ie i, P.B.; Risso, D.; Be ini, I.; Campone, F.; Ped o i, A.; Bonacci, W.; Se a, G. Ma e nal die a y
PUFAs in ake and human milk con en ela ionships du ing he i s mon h o lac a ion. Clin. Nu .
2001
,20, 393–397. [C ossRe ]
101.
Koden so a, V.M.; V zhesinskaya, O.A. E alua ion o he i amin s a us in nu sing women by i amin con en in b eas milk.
Bull. Exp. Biol. Med. 2006,141, 323–327. [C ossRe ]
102. D o , D.K.; Allen, L.H. Vi amin B-12 in humanmilk: A sys ema ic e iew. Ad . Nu . 2018,9, 358S–366S. [C ossRe ]
103.
Salmenpe ä, L. Vi amin C nu i ion du ing p olonged lac a ion: Op imal in in an s while ma ginal in some mo he s. Am. J. Clin.
Nu . 1984,40, 1050–1056. [C ossRe ]
104.
Hoppu, U.; Rinne, M.; Salo-Väänänen, P.; Lampi, A.-M.; Pii onen, V.; Isolau i, E. Vi amin C in b eas milk may educe he isk o
a opy in he in an . Eu . J. Clin. Nu . 2005,59, 123–128. [C ossRe ]
105. Do ea, J.G. Selenium and b eas - eeding. B . J. Nu . 2002,88, 443–461. [C ossRe ]
106.
Mille , E.M.; Aiello, M.O.; Fuji a, M.; Hinde, K.; Milligan, L.; Quinn, E.A. Field and labo a o y me hods in human milk esea ch.
Am. J. Hum. Biol. 2013,25, 1–11, E a um in Am. J. Hum. Biol. 2013,25, 442. [C ossRe ] [PubMed]
107.
Ins i u e o Medicine (US) Commi ee on Nu i ional S a us du ing P egnancy and Lac a ion. Nu i ion du ing Lac a ion; Subcom-
mi ee on Nu i ion du ing Lac a ion: Washing on, DC, USA; Commi ee on Nu i ional S a us du ing P egnancy and Lac a ion:
Washing on, DC, USA; Food and Nu i ion Boa d: Washing on, DC, USA; Ins i u e o Medicine: Washing on, DC, USA; Na ional
Academy o Sciences: Washing on, DC, USA, 1991; ISBN 0309043913.
Disclaime /Publishe ’s No e:
The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .