Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Endocrinology & Metabolism
journal homepage: www.elsevier.com/locate/beem

Lipids in human milk

Hans Demmelmair, Scientist *, Berthold Koletzko, Pediatrician
Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children's Hospital, Univ. of Munich Medical
Centre, Ludwig-Maximilians-Universita €t München, Lindwurmstrasse 4, 80337, München, Germany

a r t i c l e i n f o
Fat is the main energy providing component in human milk and
Article history: comprising a complex mixture of different lipid species, with
Available online 14 November 2017 quantitative dominance of triglycerides. After elucidating the fatty
acid composition, more recent research has looked at influencing
Keywords: factors and the importance of specific lipids. Here we review
milk fat quantitative aspects of maternal metabolism which contribute to
long-chain polyunsaturated fatty acid the milk fatty acid composition, especially considering essential
milk-fat-globule membrane fatty acids and their long chain polyunsaturated derivatives. In this
palmitic acid context studies with stable isotopes have indicated the importance
stable isotope tracer
of maternal body pools for mediating the effects of diet on milk
composition.
Furthermore, the importance of positioning of palmitic acid at
the glycerol backbone of triglycerides is discussed, and the phos-
pholipids of the milk fat globule membrane are described and
examples for their potential importance for infant development
are presented.
© 2017 Elsevier Ltd. All rights reserved.

Introduction

Many studies have shown the benefits of human milk for infants in relation to gastrointestinal
function, risk of infectious diseases, growth, neurological development and development of the
immune system [1]. Milk fat is the major source of energy provided with human milk, but it is also

Abbreviations: LA, linoleic acid; ALA, a-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; MFGM, milk fat globule
membrane; TG, triacylglycerol; LC-PUFA, long chain polyunsaturated fatty acid; PUFA, polyunsaturated fatty acid.
* Corresponding author.
E-mail addresses: Hans.Demmelmair@med.uni-muenchen.de (H. Demmelmair), Berthold.Koletzko@med.uni-muenchen.de
(B. Koletzko).

https://doi.org/10.1016/j.beem.2017.11.002
1521-690X/© 2017 Elsevier Ltd. All rights reserved.
58 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

important for the provision of essential fatty acids, lipid soluble vitamins and bioactivities of specific
components [2].
Human milk fat provides almost 50% of the energy intake of young infants, corresponding to intakes
around 25 g/day for the age up to 6 months [3,4]. While the lipid content of mature human milk does
not seem to change much with stage of lactation, there is a huge inter-individual and intra-individual
variation [5,6]. While concentrations of protein and lactose are relatively constant during the day, fat
contents tend to be higher during the day and lower during the night (Fig. 1). Most striking is the
increase of the fat content during each breast feeding. Saarela et al. reported for foremilk fat contents
only slightly above 2% and hindmilk fat contents of almost 6% during the first six months, which agrees
with observations of other studies [7,8] (see Fig. 2).
Milk fat content and breast fullness are related and about 70% of the variance of fat content can be
explained by the amount of milk in the breast before and after feeding [9,10]. A long inter-feeding
interval leads to a full breast with low fat content, corresponding to a low foremilk fat content. If
the baby drinks a huge volume of milk, the breast will be widely empty with a low remaining volume,
corresponding to a high hindmilk fat content. As both these factors cannot be standardized in studies a
reliable estimate of the fat content of the milk consumed by the baby cannot be obtained from hind- or
foremilk samples only. Analyzing both hind- and foremilk enables a better estimate of the fat content,
but cannot easily provide the true fat content, as the increase of the fat content during sucking is not
linear [9,11]. Thus, reliable estimates of the milk fat content can only be obtained by pumping a full
breast not used by the infant and analyzing a representative sample, but this does not necessarily
reflect the fat content of the milk consumed by the baby, if it consumes less and only empties the breast
e.g. by half. So far the reasons for the increase of the fat content during breast feeding have not been
fully elucidated. One possibility suggested is that fat globules attach to the alveolar surface and during
breastfeed the morphology of the lactocyte and the shape of the alveolus change with a decrease in the
available surface. Together with the greater shear forces generated towards the end of the feeding this
leads to the excretion of more fat globules with milk towards the end of the feed [12]. In agreement
with this hypothesized mechanism is the observation that diameters of milk fat globules determined
by laser light scattering in fore- and hindmilk of 13 women only showed a non-significant trend to a
higher diameter in hindmilk compared to foremilk (4.6 ± 2.1 vs. 4.2 ± 1.0 mm), indicating that the
higher fat content in hindmilk is mainly due to a higher number of globules than in the foremilk rather
than to larger globules [13]. Although the amount of fat per volume is strikingly different between fore-
and hindmilk, the fatty acid composition of total milk fat is not different [14e16]. The fatty acid

1.2
relative difference from mean

0.8

0.6

0.4

0.2

0
morning daytime evening night
fat lactose protein
Fig. 1. Changes of the human milk macronutrients expressed relative to the corresponding average content (set to 1) of protein,
lactose and fat according to data of Saarela [97].
 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68 59

Fig. 2. Fat content (M ± SD) of foremilk and hindmilk secreted by mothers of term infants at the indicated time points of lactation.
Figure drawn based on data by [97].

composition is primarily determined by the milk triglycerides forming the core of the milk fat globules.
It remains to be investigated whether phospholipids forming the surface of the globules are also similar
in fore- and hindmilk.
Milk triacylglycerols (TG) are formed in the endoplasmic reticulum from fatty acids either taken up
from the circulation or synthesized de novo in the mammary epithelial cells from glucose. The first and
rate-limiting step in fatty acid synthesis is conversion of acetyl-CoA to malonyl-CoA, and then fatty acid
synthase catalyzes the sequence of reactions, of which each adds a two-carbon unit to the growing
fatty acyl chain. While in most tissues the synthesis is terminated after building a 16 carbon chain, the
cytosol of mammary epithelial cells contains an acyl thioester-hydrolase (thioesterase II) that termi-
nates fatty acid synthesis already once a carbon chain length of 8e14 carbons is achieved, resulting in
the de novo synthesis of medium-chain and intermediate chain fatty acids and explaining the high
content of these fatty acids in milk [17]. Long-chain fatty acids are imported from the plasma, where
they are either released from TG in chylomicrons or very low density lipoproteins by lipoprotein lipase
or circulate as non-esterified fatty acids. Once in the cell, fatty acids may be combined with CoA and
bound to acyl-CoAebinding protein or they can be bound to a fatty acidebinding protein likely
responsible for maintaining a readily available fatty acid pool for TG synthesis. Fatty acids synthesized
de novo or imported from the circulation are joined with glycerol-3-phosphate by transacylases located
in the endoplasmic reticulum to form TGs for incorporation into microlipid droplets. These coalesce
into larger cytoplasmic lipid droplets, which progress toward the apical membrane of the cell, become
enveloped in plasma membrane, and pinch off from the cell, bearing a continuous coat of specialized
plasma membrane called the milk fat globule membrane (MFGM). While many details of the process
from forming microlipid droplets to the exocytosis of milk fat globules with average volume adjusted
diameters around 4 mm and the characteristic tri-layered membrane are not yet fully understood, it
can be assumed that for this process some of the MFGM proteins are important [18,19]. Not fully
understood is the regulation of milk fat synthesis, which probably depends on the availability of
substrates and some hormones including prolactin, growth hormone and insulin [17].
Neutral lipids including TG, diacylglycerols, monoacylglycerols, sterolesters contribute 98% (wt/wt)
or more to the total fat in milk, and about 95% of these are contributed by fatty acids [20]. Therefore, it is
their fatty acid composition which largely determines the nutritional and physicochemical properties
60 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

of human milk fat. The sources of milk fatty acids and potential determinants of the relative contri-
bution are of interest. Well established are effects of the carbohydrate to fat ratio in the maternal diet
on medium and intermediate chain fatty acid content which increase with more energy contributed by
carbohydrates, as indicated by interventional [21] and observational studies [22] and demonstrated in
a tracer study [23].

Polyunsaturated fatty acids in milk

The longer chain fatty acids are either directly derived from the diet, i.e. via chylomicrons, or they
had been incorporated into fatty acid storage pools, e.g. adipose tissue or hepatic lipids, before
incorporation into milk. Both of these routes are in agreement with the well documented influence
of dietary n-6 and n-3 fatty acids on milk polyunsaturated fatty acid composition [24,25]. This cor-
responds to a short term influence of diet (direct transfer) and a long term effect of dietary habits
(incorporation into a slow turnover body pool before milk incorporation) on the milk fatty acid
composition. Correspondingly fatty acids synthesized, elongated or desaturated in the liver or
peripheral tissues can be expected to be incorporated into milk fat. Specific emphasis in this context
has been put on essential fatty acids and long chain polyunsaturated fatty acids (LC-PUFA), i.e. the
elongated and further desaturated derivatives of the essential fatty acids linoleic acid (C18:2n-6, LA)
and a-linolenic acid (C18:3n-3, ALA). The quantitatively major LC-PUFA in human milk on the n-6 side
are dihomo-g-linolenic acid (C20:3n-6) and arachidonic acid (C20:4n-6, ARA) and correspondingly on
the n-3 side eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3, DHA) [20]. In a
review including 65 studies a low variation of ARA compared to DHA between different populations
with different dietary habits has been described [26].
The perinatal period is a period of intensive growth of the brain, which contains high amounts of
ARA and DHA, suggesting high requirements and importance of LC-PUFA for the development of
neurological and cognitive functions. Furthermore, LC-PUFA are involved in inflammatory and
immunological processes, suggesting importance of a balanced LC-PUFA supply for optimal maturation
of the infantile immune system and LC-PUFA might influence adipocyte differentiation and as
consequence obesity risk [27].
Although a recent Cochrane review did not find the available results from randomized clinical trials
fully conclusive [28], their importance is reflected in recommendation for maternal DHA intake and
for the DHA content of infant formulas [29]. An intensive discussion about the importance of ARA
provision with infant formulas is ongoing [30e32]. Maternal intake of fatty fish or supplements shall
ensure adequate n-3 LC-PUFA supply for the fetus during pregnancy and for the infant during the
breastfeeding period [33] Thus, maternal metabolic disposition of fatty acids has to be considered for
the association between diet and milk fatty acids, including LC-PUFA.

Sources of polyunsaturated fatty acids in milk

The essential fatty acids in human milk are derived from the maternal diet, either directly or after
intermediate incorporation into a body storage pool. Diet and endogenous synthesis are the sources of
milk LC-PUFA, and for both steps incorporation into storage pools is a possibility. Oral application of
stable isotope labeled fatty acids and subsequent frequent measurement of isotopic enrichment and
concentration of milk fatty acids in combination with determination of milk volume produced was
used in several studies to quantify the transfer of fatty acids into milk. For palmitic and oleic acids two
studies have shown that about 10% of the applied tracers are transferred into milk within 48e72 h after
tracer intake [34,35]. The metabolic disposal of ingested LA including its conversion into LC-PUFA was
studied in detail in German and Mexican women. In the German women 1 mg 13C labeled LA per kg
body weight was applied in lactation weeks 2, 6 and 12. Before tracer intake and three times per day
during the following 5 day period breast milk samples were collected, the volume of milk produced
was recorded and dietary intake was calculated from weighed protocols [36]. LA tracer was rapidly
transferred into milk with maximal enrichment already 12 h after intake. The proportion of the
ingested 13C-LA transferred into milk as LA did not differ significantly with duration of lactation (2nd
week: 12.7 ± 1.4%, 6th week: 13.1 ± 2.5%, 12th week: 11.7 ± 2.7%, M ± SE). At all three time points about
 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68 61

0.2% of the tracer appeared in milk as dihomo-g-linolenic acid and 0.02% as ARA. By extrapolation to
the whole diet one could estimate that about 30% of milk LA is directly transferred from the diet,
whereas about 11% of milk dihomo-g-linolenic acid and 1.2% of milk ARA originate from direct
endogenous conversion of dietary LA. The findings in the Mexican women were similar with about 16%
of the tracer transferred into milk, again corresponding to 30% of milk LA, and also the results for
endogenously synthesized ARA were confirmed [37]. Assuming a similar transfer of dietary ARA as
observed for LA it could be estimated from the quantitative data on dietary intake and milk output, that
in the Mexican women the dietary intake of ARA contributed about 10% to milk ARA. Thus, neither
endogenous conversion nor current ARA intake could be identified as a major source of milk ARA. The
assumption of the similar transfer of ARA as measured for LA is justified by the study of Fidler et al. [35],
who had provided palmitic acid, oleic acid, and DHA labeled with 13C and observed a median cumu-
lative DHA recovery of 8% (7.0e9.6, Q1eQ3) in milk, similar to the above mentioned transfer of palmitic
and oleic acids. Thus, it can be assumed that for both major LC-PUFA in human milk, ARA and DHA,
about 10% are directly derived from dietary intake. One has to be aware that this is a very rough
estimate, as a two week supplementation with 200 mg DHA per day did not change the tracer indicated
transfer rate of 8%, and in combination with the increased intake (250 vs. 90 mg/day) diet derived milk
DHA of 20 and 7 mg per day can be estimated from the reported transfer rate [35]. Although the DHA
supplementation led to a significant increase of the percentage of DHA in milk fat, the secreted amount
of DHA per day (milk volume [l/day] * fat content [g/l] * DHA contribution [g/g]) only increased from 57
to 92 mg/d, and the corresponding contribution of direct transfer would be 12% or 21%, respectively.
Thus, contribution of direct transfer seems to be variable and to depend on many influencing factors
including acute intake.
The complexity of the process is increased by the observation that ALA, the precursor of the n-3
series LC-PUFA, seems to behave quiet differently from LA. In an experiment following the design of the
previously mentioned studies 10 women ingested 0.6 mg 13C-ALA/kg during the 5th week of lactation
and milk samples were collected for a five-day period, while dietary intakes were assessed. There were
7.3 ± 1.1% (M ± SE) of the tracer directly transferred as ALA into milk and about 0.25% of the tracer were
found in milk LC-PUFA with ALA originating tracer hardly detectable in DHA [38]. Combining intake
and milk data it was estimated that about 65% of milk ALA was directly derived from maternal diet.
Thus, in contrast to LA the major portion of milk ALA is directly derived from the diet. In the ALA study
and in the LA studies in addition to milk excretion also conversion of the tracer fatty acids to CO2 and
exhalation had been quantified. The finding was that in the German mothers about 20% of the orally
ingested tracer LA were converted to CO2, and in the Mexican women, depending on maternal BMI, 8 or
22% were oxidized [36,39]. The corresponding percentage in the ALA study was 35 ± 2.7% [38]. For both
n-6 and n-3 series fatty acids hardly any direct conversion of the dietary essential fatty acids into milk
LC-PUFA was found, but the contribution of ALA from body stores to milk ALA seems to be much lower
than the contribution of stored LA to milk LA. This leads to a relatively low ALA content in milk, but at
the same time a relatively high direct contribution of dietary intake of ALA to milk ALA. Direct transfer
from diet was identified as a major source of the fatty acid in milk only for ALA, while for palmitic acid,
oleic acid, LA and DHA direct transfer from diet seems not to be a major route. This applies to diet
derived LC-PUFA and to LC-PUFA endogenously produced from the essential fatty acids. The tracer
studies could not measure the relative importance of endogenous synthesis and uptake of preformed
dietary LC-PUFA for milk LC-PUFA. This indicates that a huge portion of these milk fatty acids passes
through large pools with slow transfer, such as adipose tissue, which leads to tracer concentrations
below the detection limits. It is highly plausible that slow turnover pools are involved (Fig. 3), but with
the applied study designs it was not possible to decide whether these pools store essential fatty acids
(precursors) before conversion, whether they store LC-PUFA (product), or whether both pass slow
turnover pools before appearance in milk. In any case such pools are suggested by the small and long
lasting enrichment observed in milk ARA and DHA [36e38].
The relevance of endogenous synthesis for milk LC-PUFA is also well demonstrated by several
studies, which showed that similar to plasma, red blood cells and adipose tissue the genotype of the
desaturases and elongases involved in the conversion of essential fatty acids into LC-PUFA is associated
with the corresponding fatty acid percentages in milk [40e43]. The studies agree in significant asso-
ciations of genotype and n-6 fatty acids, but not all studies found significant associations with DHA.
62 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

Fig. 3. Schematic presentation of the pathways of long chain polyunsaturated fatty acid (LC-PUFA) derived by endogenous
conversion from dietary polyunsaturated fatty acids (PUFA) into milk. Although not individually quantified direct transfer (red),
conversion and transfer after PUFA storage (blue), conversion into LC-PUFA followed by storage and transfer (green) and PUFA
storage before conversion and LC-PUFA storage after conversion followed by transfer (green broken line) could be identified as
potential routes.

This is in line with findings from ALA supplementation studies where either no effects on DHA with
daily ALA doses of 1.4e10.7 g given during the lactation period [44,45] were reported, or a DHA increase
from 0.3 to 0.8% DHA with an ALA dosage of 10.1 g/day starting from pregnancy month 6 and being
associated with 3% ALA in milk compared with 1% on habitual diet [46] was found. There are several
observational studies, which showed closer associations of maternal DHA intake with milk DHA levels
than of maternal ARA intake with milk ARA levels [24,47]. A study comparing Swedish and Chinese
mothers showed that in both populations there were significant associations for DHA dietary intake
and milk contents (Sweden r ¼ 0.54, China r ¼ 0.71) although due to different life styles fatty acid
composition of diet and milk were clearly different between countries [48]. In several randomized
clinical trials it was shown that supplementation of pregnant and lactating women with fish oil or
other n-3 LC-PUFA sources led to a significant increase of DHA in human milk [35,49e51]. Based on
these findings a linear dose response relationship is assumed, such that a supplementation with 1.1 g of
DHA per day is needed to achieve a DHA percentage of 1% in milk fat [52]. There are only a few studies
which tested the effects of n-3 supplementation on milk fat composition in combination with ARA
supplementation. In a Dutch study 88 breastfeeding women received either DHA (220 mg/day),
DHA þ ARA (220 mg/day each) or a placebo from gestation week 16 until postnatal week 12, with milk
samples collected in the second and 12th week of lactation [53]. In both supplemented groups DHA-%
were significantly higher than in the placebo group and the addition of ARA led to higher median
ARA-% in milk at both time points (week 2: 0.67% vs. 0.59% [placebo group] and 0.55% [DHA group],
week 12: 0.48% vs. 0.39% and 0.39%). As the supplementation had started already mid pregnancy this is
fully in agreement with the findings of the tracer studies, as the increased ARA excretion with milk
could be the result of increased direct transfer from the diet and increased ARA contribution to the flux
of fatty acids into milk passing maternal storage pools. In a similar study, Weseler et al. supplemented
breastfeeding women with DHA þ eicosapentaenoic acid (400 mg/day) alone or in combination with
different dosages of ARA (200 or 400 mg/day) from postnatal week 3 onwards and collected milk
samples 2 and 8 weeks later [54]. The ARA supplementation increased milk ARA-% in a dose dependent
fashion already within two weeks, and the higher dose attenuated the supplementation effect of DHA.
This rapid change of ARA in milk questions the assumption of Del Prado [37] that only 10% of milk ARA
is directly derived from the diet. Nevertheless, based on the information about average fatty acid
 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68 63

percentages provided by Weseler et al. [54] and assuming a daily milk production of 788 ml with a fat
content of 4.1% [7] it can be estimated that the amount of additional ARA excretion required for the
observed ARA-% increase is 8e12% of the supplement dose (Table 1). The calculated transfer agrees well
with the 10% direct transfer estimated previously [37]. Overall, the findings from the tracer studies
build a basis to understand how milk fatty acid composition can be improved with maternal
supplementation and where additional direct supplementation of the infant is required.

Palmitic acid in human milk

A milk fatty acid which starts to receive increased attention is palmitic acid, which contributes
around 25% to human milk fatty acids [55]. Of interest is the positioning of the palmitic acid at the
glycerol backbone. In contrast to most TG synthesized by mammalians palmitic acid in human milk TG
is mainly positioned at sn2, the central carbon atom of the glycerol backbone [4]. In human milk fat
more than 50% of the fatty acids esterified at sn2 can be palmitic acid, while oleic acid, usually the
major milk fatty acid, contributes only 12% to the sn2 fatty acids [4]. In contrast, in plant oils that are
widely used as the fat source for infant formulae only very little palmitic acid is located at sn2, but it
mainly occupies the outer carbons of the glycerol [4]. Infantile digestive lipases primarily cleave off the
fatty acids at sn1,3 and free fatty acids originate from these in the intestinal lumen, while the sn2 fatty
acid is left as monoglyceride which facilitates solubility and absorption [56,57]. The advantages of
palmitic acid at the sn2 position in human milk for fat and mineral absorption, intestinal comfort, at
least temporarily for bone density and crying behavior of the infants have recently been summarized in
systematic and narrative reviews [58e60]. There are indications that the palmitic acid position may
also be of importance for lipoprotein and non-esterified fatty acid metabolism [61,62]. A rodent study
suggests that it is not the amount, but the position of palmitic acid in the dietary fat which influences
N-acylenthanolamides, including anandamide levels [63]. Considering the analgesic effects of
anandamide this might contribute to the explanation of the observed association between the position
of palmitic acid in the dietary TG and crying behavior [58]. Based on the known interactions of
N-acylethanolamides and nuclear receptors this also suggests potential contributions to programming
effects.

Milk fat globule membrane

Although the MFGM is only some nanometers thick, while the diameters of the milk fat globules
are in the micrometer range, MFGM could be very important for the physiological properties of
human milk. MFGM might contribute to the differences in outcome between breastfed and formula
fed infants, which continue to exist even though the difference has become smaller with improved
formula qualities [64]. The lipid component of infant formulae is usually plant oil based and thus some
specific components of MFGM are absent [65]. This relates to MFGM proteins, but also to MFGM lipids,
which contribute about 30% to the dry weight of MFGM [66]. As expected from the cellular origin of

Table 1
Simplified model calculation relating the ARA-% in human milk observed by Weseler et al. to the supplement dosages using data
on milk production and fat content form the literature [7] estimating the percentage of dietary arachidonic acid (ARA) directly
transferred into milk.

Group Control DHA þ EPA DHA þ EPA þ low ARA DHA þ EPA þ high ARA

ARA intake (mg/day) 0 0 200 400

week 2 ARA-% 0.45 0.43 0.53 0.55
week 2 ARA excretion (mg) 145 139 171 178
week 2 excretion increase above control (mg) 0 6 26 32
week 2 ratio excretion increase/intake (%) e e 13 8
week 8 ARA-% 0.41 0.4 0.49 0.56
week 8 ARA excretion (mg) 132 129 158 181
week 8 excretion increase above control (mg) 0 3 26 48
week 8 ratio excretion increase/intake (%) e e 13 12
64 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

the MFGM, the major lipids are phosphatidylcholines, phosphatidylethanolamines and sphingo-
myelins, which each contributes between 20 and 40% to total MFGM phospholipids, while phos-
phatidylinositol and phosphatidylserine contribute 5e10% each [67e69]. Similar to the total fat
content, a wide range of total phospholipid concentrations from 140 to 410 mg/l has been reported
[67] with different observations in relation to change with advancing lactation period [67,70,71].
Comparing the fatty acid composition of milk total lipids and phospholipids reveals some general
tendencies although there are huge differences in the fatty acid composition between the individual
phospholipid classes. The oleic acid and, except for phosphatidylcholine, the palmitic acid contents
are lower in milk phospholipids than in total lipids, while stearic acid tends to be higher in phos-
pholipids [20,67,72,73]. Regarding LC-PUFA the content is generally higher than in total lipids. ARA
contributes up to 12% to phosphatidylethanolamine or phosphatidylinositol, but the percentage in
sphingomyelin is about 0.4% and thus similar to total lipids, while DHA contributes up to 5% and 3% in
phosphatidylethanolamine and phosphatidylserine, respectively [67]. Phospholipids contribute only
1e2% to total lipids [20]. Therefore, these numbers agree with the estimation of Harzer et al. that
about 85% of the milk LC-PUFA are contributed by TG and only 15% are phospholipid bound [74].
Although there are no good indications in humans that bioavailability of TG and phospholipid bound
LC-PUFA is different [75], the metabolic disposition including the incorporation into brain might be
affected [76]. Thus, the nutritional importance of the MFGM lipids is not primarily based on their LC-
PUFA content, but they provide a variety of specific lipids and some bioactivities in the gastro-
intestinal tract could be important.
Sphingolipids are based on a sphingoid backbone. In MFGM the dominating sphingolipid is
sphingomyelin (phosphocholine as head group) and in much smaller amounts glucosylceramides
(glucose), lactosylceramides (lactose), and with more complex glycosyl residues gangliosides
(monosaccharides, N-acetylgalactoseamine, sialic acid and others) occur [77]. Intact sphingomyelin is
not absorbed, but seemed to accelerate maturation of the intestine in rat pups [78]. By the activity of
alkaline sphingomyelinase and ceramidase, which are expressed on the apical membranes of intestinal
epithelial cells, absorbable sphingosine is derived from sphingomyelin, which can be converted to
spingosine-1-phosphate [77]. Sphingosine has been found to induce cell cycle arrest and apoptosis and
to inhibit protein kinase c [79]. In the intestine sphingosine-1-phosphate may be involved in the
regulation of immune functions [80]. Further rat studies suggest systemic effects of dietary sphingo-
myelin, including the increase of myelination in the nervous system in a deficit model [81,82]. In
relation to neurodevelopment, the gangliosides in the MFGM might be important, considering the
ganglioside content in nervous tissue, the high requirement for the rapid brain growth in the perinatal
period, and the demonstrated uptake of dietary gangliosides [83]. Gangliosides are exclusively located
in the MFGM and act as decoy receptors for pathogens, which may prevent infections of infants, and
they can modulate the behavior of immune cells [84].
MFGM phosphatidylcholine and sphingomyelin contribute about 10% to the total choline intake
of infants [85,86]. Thus, in quantitative terms water soluble choline in milk is more important, but
there are good indications that metabolization of free and esterified dietary choline is different and
that it may have specific effects on plasma cholesterol levels and even brain development [87].
Furthermore MFGM is a source of cholesterol [66] and it contributes sialic acid via glycosylated
proteins and gangliosides [84,88] to the dietary intake of breastfed infants. There are good
indications from in vitro studies, animal studies and observational studies that several MFGM
compounds are important for infant development, including the development of cognitive func-
tions [89e91].

Clinical trials testing milk fat globule membrane components

Several randomized and double-blinded trials have studied the effects of adding cow milk derived
MFGM to infant diets on health and cognitive function. In 6e12 month old infants in Peru, supple-
mentation with MFGM enriched protein significantly decreased the duration of diarrhea episodes and
the incidence of bloody diarrhea by almost 50% considering confounding factors [92].
In a Swedish study, exclusively formula-fed term infants were randomized before the age of 2
months to a formula supplemented with a protein-rich MFGM preparation (4% of total protein) or a
 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68 65

standard formula with the aim to study effects of MFGM supplementation. Primary outcomes were
growth and neurodevelopment of the infants, and secondary outcomes included disease symptoms
[93,94]. The formulae were fed until the age of 6 months and the infants were followed until the age of
12 months, along with a breast-fed reference group. At age 12 months follow up data were collected
from 73 infants in the MFGM group, 68 infants in the standard formula group and 72 breast-fed infants.
The higher dietary cholesterol availability from the supplemented formula was reflected in higher
average serum cholesterol in MFGM infants compared to infants fed standard formula at 4 and 6
months [95]. During the study period the supplemented group showed significantly less otitis media
than the standard formula group (1% vs 9%), as well as a lower incidence (25% vs. 43%) and a shorter
duration of antipyretic use [93]. Neurodevelopment of the infants was assessed at the age 12 months
using Bayley Scales of Infant and Toddler Development. Scores of the motor and verbal domains were
not different between the randomized groups, but the MFGM group obtained significantly higher
scores in the cognitive domain compared to the standard formula group (105.8 ± 9.2 vs. 101.8 ± 8.0,
M ± SD), which did not differ significantly from the breast-fed group (106.4 ± 9.5). This confirms
findings in Indonesia, where infants received standard formula or the same formula supplemented
with complex milk lipids from 2 to 24 weeks of age [96]. After the intervention period, serum
ganglioside concentrations were significantly higher in supplemented infants, who showed signifi-
cantly better performance in the Griffiths Mental Development Scale [96].

Conclusions

Randomized controlled clinical trials in infants are required to define infant needs and to develop
guidance on preferable feeding strategies. However, interventions tested in randomized clinical trials
are developed based on physiological and mechanistic studies that improve the knowledge about
relationship between maternal diet and milk composition, the effects of metabolism and genotype, and
biological functions. Model experiments, detailed chemical analyses and clinical studies highlighted
the importance of quantitatively minor milk components and details for infant development. Further
progress in understanding and development of interventions and the implementation of improved
infant feeding strategies may be expected in the years to come.

Practice points

 Milk fat content increases about 2e3 fold during each breast feeding. Preferential feeding of
pumped hindmilk, with a high fat and energy content, to poorly growing preterm infants can
help to enhance weight gain.
 The polyunsaturated fatty acid content in human milk and hence the supply to the breastfed
infant is modulated by the maternal dietary polyunsaturated fatty acid supply. Regular
consumption of sea fish including oily fish or the use of fish oil supplements enhance the milk
content of omega-3 DHA.
 Human milk contains complex lipids and milk fat globule membranes which are not provided
with conventional infant formulae based on vegetable oils as the lipid source. Recent
randomized infant trials indicate that the provision of milk fat globule membranes may
reduce infections and enhance neurodevelopmental outcomes.

Acknowledgements

This work has benefitted from financial support by the Commission of the European Community,
the 7th Framework Programme Early Nutrition (FP7-289346), the Horizon 2020 Research and Inno-
vation Programme DYNAHEALTH (No 633595), and the European Research Council Advanced Grant
META-GROWTH (ERC-2012-AdG, no. 322605). This article does not necessarily reflect the views of the
Commission and in no way anticipates the future policy.
66 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

References

[1] Prell C, Koletzko B. Breastfeeding and complementary feeding. Dtsch Arztebl Int 2016;113:435e44.
[2] Delplanque B, Gibson R, Koletzko B, et al. Lipid quality in infant nutrition: current knowledge and future opportunities.
J Pediatr Gastroenterol Nutr 2015;61:8e17.
[3] Grote V, Verduci E, Scaglioni S, et al. Breast milk composition and infant nutrient intakes during the first 12 months of life.
Eur J Clin Nutr 2016;70:250e6.
[4] Innis SM. Dietary triacylglycerol structure and its role in infant nutrition. Adv Nutr 2011;2:275e83.
[5] Koletzko B, Rodriguez-Palmero M, Demmelmair H, et al. Physiological aspects of human milk lipids. Early Hum Dev 2001;
65(Suppl:S3eS18).
*[6] Gidrewicz DA, Fenton TR. A systematic review and meta-analysis of the nutrient content of preterm and term breast milk.
BMC Pediatr 2014;14:216.
[7] Kent JC, Mitoulas LR, Cregan MD, et al. Volume and frequency of breastfeedings and fat content of breast milk throughout
the day. Pediatrics 2006;117:e387e95.
[8] Marangoni F, Agostoni C, Lammardo AM, et al. Polyunsaturated fatty acid concentrations in human hindmilk are stable
throughout 12-months of lactation and provide a sustained intake to the infant during exclusive breastfeeding: an Italian
study. Br J Nutr 2000;84:103e9.
*[9] Daly SE, Di Rosso A, Owens RA, et al. Degree of breast emptying explains changes in the fat content, but not fatty acid
composition, of human milk. Exp Physiol 1993;78:741e55.
[10] Khan S, Hepworth AR, Prime DK, et al. Variation in fat, lactose, and protein composition in breast milk over 24 hours:
associations with infant feeding patterns. J Hum Lact 2013;29:81e9.
[11] Woodward DR, Rees B, Boon JA. Human milk fat content: within-feed variation. Early Hum Dev 1989;19:39e46.
[12] Atwood CS, Hartmann PE. Collection of fore and hind milk from the sow and the changes in milk composition during
suckling. J Dairy Res 1992;59:287e98.
[13] Mizuno K, Nishida Y, Taki M, et al. Is increased fat content of hindmilk due to the size or the number of milk fat globules?
Int Breastfeed J 2009;4:7.
[14] Hall B. Uniformity of human milk. Am J Clin Nutr 1979;32:304e12.
[15] Gibson RA, Kneebone GM. Effect of sampling on fatty acid composition of human colostrum. J Nutr 1980;110:1671e5.
[16] Koletzko B. Zufuhr, Stoffwechsel und biologische Wirkungen trans-isomerer Fetts„uren bei S„uglingen. Die Nahr 1991;35:
229e83.
[17] Neville MC, Picciano MF. Regulation of milk lipid secretion and composition. Annu Rev Nutr 1997;17:159e84.
[18] Smoczynski M. Role of phospholipid flux during milk secretion in the mammary gland. J Mammary Gland Biol Neoplasia
2017;22:117e29.
[19] McManaman JL. Lipid transport in the lactating mammary gland. J Mammary Gland Biol Neoplasia 2014;19:35e42.
[20] Jensen RG. Lipids in human milk. Lipids 1999;34:1243e71.
[21] Nasser R, Stephen AM, Goh YK, et al. The effect of a controlled manipulation of maternal dietary fat intake on medium
and long chain fatty acids in human breast milk in Saskatoon. Can Int Breastfeed J 2010;5:3.
[22] Koletzko B, Thiel I, Abiodun PO. The fatty acid composition of human milk in Europe and Africa. J Pediatr 1992;120:
S62e70.
[23] Mohammad MA, Sunehag AL, Haymond MW. De novo synthesis of milk triglycerides in humans. Am J Physiol Endocrinol
Metab 2014;306:E838e47.
[24] Kim H, Kang S, Jung BM, et al. Breast milk fatty acid composition and fatty acid intake of lactating mothers in South Korea.
Br J Nutr 2017;117:556e61.
*[25] Yuhas R, Pramuk K, Lien EL. Human milk fatty acid composition from nine countries varies most in DHA. Lipids 2006;41:
851e8.
*[26] Brenna JT, Varamini B, Jensen RG, et al. Docosahexaenoic and arachidonic acid concentrations in human breast milk
worldwide. Am J Clin Nutr 2007;85:1457e64.
[27] Demmelmair H, Koletzko B. Importance of fatty acids in the perinatal period. World Rev Nutr Diet 2015;112:31e47.
[28] Jasani B, Simmer K, Patole SK, et al. Long chain polyunsaturated fatty acid supplementation in infants born at term.
Cochrane Database Syst Rev 2017;3, CD000376.
[29] Koletzko B, Boey CC, Campoy C, et al. Current information and Asian perspectives on long-chain polyunsaturated fatty
acids in pregnancy, lactation, and infancy: systematic review and practice recommendations from an early nutrition
academy workshop. Ann Nutr Metab 2014;65:49e80.
[30] Brenna JT. Arachidonic acid needed in infant formula when docosahexaenoic acid is present. Nutr Rev 2016;74:329e36.
[31] Hadley KB, Ryan AS, Forsyth S, et al. The essentiality of arachidonic acid in infant development. Nutrients 2016;8:216.
[32] Koletzko B. Human milk lipids. Ann Nutr Metab 2016;69(Suppl 2):28e40.
[33] Koletzko B, Cetin I, Brenna JT, et al. Dietary fat intakes for pregnant and lactating women. Br J Nutr 2007;98:873e7.
[34] Hachey DL, Thomas MR, Emken EA, et al. Human lactation: maternal transfer of dietary triglycerides labeled with stable
isotopes. J Lipid Res 1987;28:1185e92.
[35] Fidler N, Sauerwald T, Pohl A, et al. Docosahexaenoic acid transfer into human milk after dietary supplementation: a
randomized clinical trial. J Lipid Res 2000;41:1376e83.
[36] Demmelmair H, Baumheuer M, Koletzko B, et al. Metabolism of U13C-labeled linoleic acid in lactating women. J Lipid Res
1998;39:1389e96.
[37] Del Prado M, Villalpando S, Elizondo A, et al. Contribution of dietary and newly formed arachidonic acid to human milk
lipids in women eating a low-fat diet. Am J Clin Nutr 2001;74:242e7.
*[38] Demmelmair H, Kuhn A, Dokoupil K, et al. Human lactation: oxidation and maternal transfer of dietary (13)C-labelled
alpha-linolenic acid into human milk. Isot Environ Health Stud 2016;52:270e80.
[39] Villalpando S, Del Prado M, Lance A, et al. [13C]linoleic acid oxidation and transfer into milk in stunted lactating women
with contrasting body mass indexes. Am J Clin Nutr 2001;74:827e32.
 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68 67

*[40] Lattka E, Rzehak P, Szabo E, et al. Genetic variants in the FADS gene cluster are associated with arachidonic acid
concentrations of human breast milk at 1.5 and 6 mo postpartum and influence the course of milk dodecanoic, tetra-
cosenoic, and trans-9-octadecenoic acid concentrations over the duration of lactation. Am J Clin Nutr 2011;93:382e91.
[41] Morales E, Bustamante M, Gonzalez JR, et al. Genetic variants of the FADS gene cluster and ELOVL gene family, colostrums
LC-PUFA levels, breastfeeding, and child cognition. Plos One 2011;6, e17181.
[42] Molto-Puigmarti C, Plat J, Mensink RP, et al. FADS1 FADS2 gene variants modify the association between fish intake and
the docosahexaenoic acid proportions in human milk. Am J Clin Nutr 2010;91:1368e76.
[43] Yeates AJ, Love TM, Engstrom K, et al. Genetic variation in FADS genes is associated with maternal long-chain PUFA status
but not with cognitive development of infants in a high fish-eating observational study. Prostagl Leukot Essent Fat Acids
2015;102e103:13e20.
[44] Francois CA, Connor SL, Bolewicz LC, et al. Supplementing lactating women with flaxseed oil does not increase doco-
sahexaenoic acid in their milk. Am J Clin Nutr 2003;77:226e33.
[45] Mazurier E, Rigourd V, Perez P, et al. Effects of maternal supplementation with Omega-3 precursors on human milk
composition. J Hum Lact 2017;33:319e28.
[46] Valenzuela R, Bascunan K, Chamorro R, et al. Modification of docosahexaenoic acid composition of milk from nursing
women who received alpha linolenic acid from chia oil during gestation and nursing. Nutrients 2015;7:6405e24.
[47] Kresic G, Dujamovic M, Mandic ML, et al. Relationship beteween Mediterranean diet and brest milk fatty acid profile: a
study in breastfeeding women in Croatia. Dairy Sci Technol 2013;93:287e301.
[48] Xiang M, Harbige LS, Zetterstrom R. Long-chain polyunsaturated fatty acids in Chinese and Swedish mothers: diet, breast
milk and infant growth. Acta Paediatr 2005;94:1543e9.
[49] Sherry CL, Oliver JS, Marriage BJ. Docosahexaenoic acid supplementation in lactating women increases breast milk and
plasma docosahexaenoic acid concentrations and alters infant omega 6:3 fatty acid ratio. Prostagl Leukot Essent Fat Acids
2015;95:63e9.
[50] Helland IB, Saarem K, Saugstad OD, et al. Fatty acid composition in maternal milk and plasma during supplementation
with cod liver oil. Eur J Clin Nutr 1998;52:839e45.
[51] Makrides M, Neumann MA, Gibson RA. Effect of maternal docosahexaenoic acid (DHA) supplementation on breast milk
composition. Eur J Clin Nutr 1996;50:352e7.
[52] Brenna JT, Lapillonne A. Background paper on fat and fatty acid requirements during pregnancy and lactation. Ann Nutr
Metab 2009;55:97e122.
[53] van Goor SA, Dijck-Brouwer DA, Hadders-Algra M, et al. Human milk arachidonic acid and docosahexaenoic acid contents
increase following supplementation during pregnancy and lactation. Prostagl Leukot Essent Fat Acids 2009;80:65e9.
*[54] Weseler AR, Dirix CE, Bruins MJ, et al. Dietary arachidonic acid dose-dependently increases the arachidonic acid
concentration in human milk. J Nutr 2008;138:2190e7.
[55] Jensen RG. Handbook of milk composition. NewYork: Academic Press; 1995.
[56] Bar-Yoseph F, Lifshitz Y, Cohen T, et al. SN2-Palmitate reduces fatty acid excretion in Chinese formula-fed infants. J Pediatr
Gastroenterol Nutr 2016;62:341e7.
[57] Carnielli VP, Luijendijk IHT, Van Goudoever JB, et al. Structural Position and amount of palmitic acid in infant formulas:
effect on fat, fatty acid, and mineral balance. J Pediatr Gastroenterol Nutr 1996;23:553e60.
[58] Bar-Yoseph F, Lifshitz Y, Cohen T. Review of sn-2 palmitate oil implications for infant health. Prostagl Leukot Essent Fat
Acids 2013;89:139e43.
[59] Miles EA, Calder PC. The influence of the position of palmitate in infant formula triacylglycerols on health outcomes. Nutr
Res 2017;44:1e8.
[60] Petit V, Sandoz L, Garcia-Rodenas CL. Importance of the regiospecific distribution of long-chain saturated fatty acids on
gut comfort, fat and calcium absorption in infants. Prostagl Leukot Essent Fat Acids 2017;121:40e51.
[61] Nelson CM, Innis SM. Plasma lipoprotein fatty acids are altered by the positional distribution of fatty acids in infant
formula triacylglycerol and human milk. Am J Clin Nutr 1999;70:62e9.
*[62] Innis SM, Nelson CM. Dietary triacyglycerols rich in sn-2 palmitate alter post-prandial lipoprotein and unesterified fatty
acids in term infants. Prostagl Leukot Essent Fat Acids 2013;89:145e51.
[63] Carta G, Murru E, Lisai S, et al. Dietary triacylglycerols with palmitic acid in the sn-2 position modulate levels of
N-acylethanolamides in rat tissues. Plos One 2015;10, e0120424.
[64] Koletzko BV, Shamir R. Infant formula: does one size fit all? Curr Opin Clin Nutr Metab Care 2016;19:205e7.
[65] Berger A, Fleith M, Crozier G. Nutritional implications of replacing bovine milk fat with vegetable oil in infant formulas.
J Pediatr Gastroenterol Nutr 2000;30:115e30.
[66] Dewettinck K, Rombaut R, Thienpont N, et al. Nutritional and technological aspects of milk fat globule membrane ma-
terial. Int Dairy J 2008;18:436e57.
[67] Cilla A, Diego Quintaes K, Barbera R, et al. Phospholipids in human milk and infant formulas: benefits and needs for
correct infant nutrition. Crit Rev Food Sci Nutr 2016;56:1880e92.
[68] Claumarchirant L, Cilla A, Matencio E, et al. Addition of milk fat globule membrane as an ingredient of infant formulas for
resembling the polar lipids of human milk. Int Dairy J 2016;61:228e38.
[69] Thakkar SK, Giuffrida F, Cristina CH, et al. Dynamics of human milk nutrient composition of women from Singapore with
a special focus on lipids. Am J Hum Biol 2013;25:770e9.
[70] Zou XQ, Guo Z, Huang JH, et al. Human milk fat globules from different stages of lactation: a lipid composition analysis
and microstructure characterization. J Agric Food Chem 2012;60:7158e67.
[71] Giuffrida F, Cruz-Hernandez C, Bertschy E, et al. Temporal changes of human breast milk lipids of Chinese mothers.
Nutrients 2016;8.
[72] Smit EN, Martini IA, Mulder H, et al. Estimated biological variation of the mature human milk fatty acid composition.
Prostagl Leukot Essent Fat Acids 2002;66:549e55.
[73] Wang L, Shimizu Y, Kaneko S, et al. Comparison of the fatty acid composition of total lipids and phospholipids in breast
milk from Japanese women. Pediatr Int 2000;42:14e20.
68 H. Demmelmair, B. Koletzko / Best Practice & Research Clinical Endocrinology & Metabolism 32 (2018) 57e68

[74] Harzer G, Haug M, Dieterich I, et al. Changing patterns of human milk lipids in the course of the lactation and during the
day. Am J Clin Nutr 1983;37:612e21.
[75] Sala-Vila A, Castellote AI, Campoy C, et al. The source of long-chain PUFA in formula supplements does not affect the fatty
acid composition of plasma lipids in full-term infants. J Nutr 2004;134:868e73.
[76] Wijendran V, Huang MC, Diau GY, et al. Efficacy of dietary arachidonic acid provided as triglyceride or phospholipid as
substrates for brain arachidonic acid accretion in baboon neonates. Pediatr Res 2002;51:265e72.
[77] Nilsson A. Role of sphingolipids in infant gut health and immunity. J Pediatr 2016;173(Suppl:S53e9).
[78] Motouri M, Matsuyama H, Yamamura J, et al. Milk sphingomyelin accelerates enzymatic and morphological maturation of
the intestine in artificially reared rats. J Pediatr Gastroenterol Nutr 2003;36:241e7.
[79] Duan RD, Nilsson A. Metabolism of sphingolipids in the gut and its relation to inflammation and cancer development.
Prog Lipid Res 2009;48:62e72.
[80] Kunisawa J, Kiyono H. Immunological function of sphingosine 1-phosphate in the intestine. Nutrients 2012;4:154e66.
[81] Haruta-Ono Y, Setoguchi S, Ueno HM, et al. Orally administered sphingomyelin in bovine milk is incorporated into skin
sphingolipids and is involved in the water-holding capacity of hairless mice. J Dermatol Sci 2012;68:56e62.
[82] Oshida K, Shimizu T, Takase M, et al. Effects of dietary sphingomyelin on central nervous system myelination in
developing rats. Pediatr Res 2003;53:589e93.
[83] Palmano K, Rowan A, Guillermo R, et al. The role of gangliosides in neurodevelopment. Nutrients 2015;7:3891e913.
[84] Rueda R. The role of dietary gangliosides on immunity and the prevention of infection. Br J Nutr 2007;98(Suppl 1):
S68e73.
[85] Maas C, Franz AR, Shunova A, et al. Choline and polyunsaturated fatty acids in preterm infants' maternal milk. Eur J Nutr
2017;56:1733e42.
[86] Zeisel SH, Char D, Sheard NF. Choline, phosphatidylcholine and sphingomyelin in human and bovine milk and infant
formulas. J Nutr 1986;116:50e8.
[87] Lewis ED, Fields CJ, Jacobs RL. Should the forms of dietary choline also be considered when estimating dietary intake and
the implications for health? Lipid Technol 2015;27:227e30.
[88] O'Riordan N, Kane M, Joshi L, et al. Structural and functional characteristics of bovine milk protein glycosylation.
Glycobiology 2014;24:220e36.
[89] Wang B. Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. Adv
Nutr 2012;3. 465Se72S.
[90] Carlson SE. Early determinants of development: a lipid perspective. Am J Clin Nutr 2009;89. 1523Se9S.
[91] Leermakers ET, Moreira EM, Kiefte-de Jong JC, et al. Effects of choline on health across the life course: a systematic review.
Nutr Rev 2015;73:500e22.
[92] Zavaleta N, Kvistgaard AS, Graverholt G, et al. Efficacy of an MFGM-enriched complementary food in diarrhea, anemia,
and micronutrient status in infants. J Pediatr Gastroenterol Nutr 2011;53:561e8.
[93] Timby N, Hernell O, Vaarala O, et al. Infections in infants fed formula supplemented with bovine milk fat globule
membranes. J Pediatr Gastroenterol Nutr 2015;60:384e9.
*[94] Timby N, Domellof E, Hernell O, et al. Neurodevelopment, nutrition, and growth until 12 mo of age in infants fed a
low-energy, low-protein formula supplemented with bovine milk fat globule membranes: a randomized controlled trial.
Am J Clin Nutr 2014;99:860e8.
[95] Timby N, Lonnerdal B, Hernell O, et al. Cardiovascular risk markers until 12 mo of age in infants fed a formula supple-
mented with bovine milk fat globule membranes. Pediatr Res 2014;76:394e400.
[96] Gurnida DA, Rowan AM, Idjradinata P, et al. Association of complex lipids containing gangliosides with cognitive
development of 6-month-old infants. Early Hum Dev 2012;88:595e601.
*[97] Saarela T, Kokkonen J, Koivisto M. Macronutrient and energy contents of human milk fractions during the first six months
of lactation. Acta Paediatr 2005;94:1176e81.