Associations of dietary intake and nutrient status with micronutrient and lipid composition in breast milk of donor women

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. 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