The lipid components of human milk provide the infant with
energy and essential micronutrients, and they also serve specific
roles to support gastrointestinal function, lipid and lipoprotein
metabolism, neurodevelopment, and immunity. There
have been significant advances both in food technology, which
enables the supply of new lipid preparations, and in lipidomic
analyses, which offer insight into the biological effects of complex
lipids in infancy. These will pave the way for improvements
in the feeding of infants who cannot be breastfed.
Human milk lipids provide a major portion of the total energy
intake in infants (approximately half of the energy supply). The
concentration of milk lipids varies greatly between individuals,
during the day, and throughout the course of breastfeeding.
The hindmilk contains a higher fat composition and a correspondingly
larger mean size of the milk fat globule. The outer
layer of the milk fat globule membrane (MFGM) consists of a
bilayer of amphipathic lipids, primarily phosphatidylcholine,
sphingomyelin, and cholesterol, as well as cerebrosides, gangliosides,
and others. These components are highly bioactive.
The biological importance of MFGM is gaining increased attention
after several clinical trials reported benefits of adding components
of MFGM to infant formula. Current evidence supports
the provision of omega-3 docosahexaenoic acid along with
omega-6 arachidonic acid with infant formula. The recent revision
of the European legislation that was implemented in 2016
stipulates that all infant and follow-on formula must contain
between 20 and 50 mg omega-3 docosahexaenoic acid per 100
kcal without a minimum requirement of arachidonic acid. This
is a novel concept never clinically tested for suitability and safety
of healthy infants from birth, and indications of possible adverse
effects exist. Therefore, we recommend not to use such formula
until conclusive data on their safety might become available in
Grote V, Verduci E, Scaglioni S, Vecchi F, Contarini G, Giovannini
M, et al: Breast milk composition and infant nutrient intakes
during the first 12 months of life. Eur J Clin Nutr 2016;70:250–
Human milk lipids provide a major portion of the
energy supply to breastfed infants as well as essential
vitamins, polyunsaturated fatty acids, complex lipids,
and bioactive components.
Recent data evaluating the addition of preparations of
complex lipids with or without milk fat globule
membranes to vegetable oil-based infant formula
show promising indications for potential
improvements of infant development and reduction
of infection risk.
Analyses of gene-diet interaction following the concept
of Mendelian randomization add to the evidence that
the supply of long-chain polyunsaturated fatty acids in
infancy is causally related to improving cognitive
development and to reducing asthma risk at school
age. Current evidence supports the provision of
omega-3 docosahexaenoic acid along with omega-6
arachidonic acid with infant formula.
Significant methodological progress both in food
technology enabling the provision of new lipid
preparations and in lipidomic analyses offers major
opportunities to explore the biological effects of the
supply of complex human milk lipids.
Human milk lipids provide the infant with energy and essential
vitamins, polyunsaturated fatty acids, and bioactive
components. Adding complex lipids and milk fat globule
membranes to vegetable oil-based infant formula has the
potential to enhance infant development and reduce infections.
Cholesterol provision with breastfeeding modulates
infant sterol metabolism and may induce long-term benefits.
Some 98–99% of milk lipids are comprised by triacylglycerols,
whose properties depend on incorporated fatty
acids. Attention has been devoted to the roles of the longchain
polyunsaturated fatty acids docosahexaenoic (DHA)
and arachidonic (ARA) acids. Recent studies on gene-diet
interaction (Mendelian randomization) show that breastfeeding
providing DHA and ARA improves cognitive development
and reduces asthma risk at school age particularly
in those children with a genetically determined lower activity
of DHA and ARA synthesis. It appears prudent to follow
the biological model of human milk in the design of infant
formula as far as feasible, unless conclusive evidence for the
suitability and safety of other choices is available. The recent
European Union legislative stipulation of a high formula
DHA content without required ARA deviates from this concept,
and such a novel formula composition has not been
adequately evaluated. Great future opportunities arise with
significant methodological progress for example in lipidomic
analyses and their bioinformatic evaluation, which
should enhance understanding of the biology of human
milk lipids. Such knowledge might lead to improved dietary
advice to lactating mothers as well as to further opportunities
to enhance infant formula composition.
Lipids are a major source of energy provided with human
milk to the infant [1, 2], but they also provide essential
nutrients such as polyunsaturated fatty acids (PUFA)
and lipid soluble vitamins. Many studies have demonstrated
important biological effects of the milk lipids
provided to the recipient infant, for example on gastrointestinal
function, lipid and lipoprotein metabolism,
membrane composition and function, infant growth,
neurodevelopment, and immune function .
Human milk lipids provide a major portion of the total
energy intake in young infants, with a mean 44% of the
energy supply  (Fig. 1). The average intake of human
milk lipids in fully breastfed infants amounts to 21.42 g/
day between birth and 6 months of age . This results in
an impressive 3.9 kg of human lipid supplied during the
first half year of life to fully breastfed infants, equivalent
to some 35,000 kcal provided by human milk lipids alone
during the first 6 months of life. While the mean lipid
content in human milk is relatively stable during the
course of the first months of lactation, there is very wide
interindividual and intraindividual variation of milk fat
concentrations (Table 1) [4–6]. In fact, among the macronutrients
in milk, fat shows the most variable concentration.
For example, in mature milk samples collected at
the infant age of 2 months, we find a coefficient of variation
of 37.3% for milk fat but only of 14.4% for lactose and
12.9% for protein . Milk fat content tends to increase
with longer duration of breastfeeding and varies during
the course of a day [1, 6]. Milk fat concentration increases
with an increasing time interval to the preceding milk
expression from the same breast, and it increases with
maternal fat deposition in pregnancy indicated by the degree
of gestational weight gain . Milk fat increases during
the course of each breastfeeding meal, with markedly
higher milk fat contents in hindmilk (at the end of feeding)
than in foremilk (at the beginning of the feed) (Fig. 2)
. This may be of biological benefit in that infants will
initially get milk rich in the essential water-soluble substrates,
whereas those who are hungrier and drink more
milk obtain milk with an increasing fat and energy content
to satisfy their caloric needs. Of interest, the increase
of milk fat content during the meal is accompanied with
a marked increase in the mean size of milk fat globule.
Thereby, hindmilk has a higher ratio of triglycerides in
the core of the milk fat globule (providing energy) to the
surface membranes (rich in phospholipids, complex lipids,
and essential long-chain polyunsaturated fatty acids,
Milk Fat Globules and Complex Lipids
Milk can be characterized as an emulsion of milk fat
globules in an aqueous liquid. Milk fat globules with
markedly variable sizes are formed in the mammary alveolar
cells and contain a core of nonpolar lipids comprised
primarily of triacylglycerols, with added small
amounts of monoglycerides, diglycerides, and nonesterified
fatty acids. These nonpolar lipids are formed in the
endoplasmic reticulum from fatty acids obtained from
the maternal circulation as well as primarily intermediate-
chain fatty acids with 12 and 14 carbon atoms synthesized
from acetyl-CoA. Upon the secretion from the endoplasmic
reticulum of mammary epithelial cells into the
cytosol, this triglyceride-rich core is covered by an inner
membrane derived from the endoplasmic reticulum consisting
of a monolayer primarily of phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, and
cholesterol. When these lipid droplets are further excreted
from mammary epithelial cells into the alveolar space,
they are covered by a piece of the apical plasma membrane,
which results in the addition of another phospholipid
bilayer and hence a phospholipid trilayer, and the
other components of the mammary epithelial cell membrane
such as membrane proteins and glycoproteins
(Fig. 3). This outer layer of the milk fat globule membrane
(MFGM) consists of a bilayer of amphipathic lipids, primarily
phosphatidylcholine, sphingomyelin, and cholesterol,
as well cerebrosides, gangliosides, glycosylated proteins
and polypeptides, filaments, mucins, lactadherin,
butyrophilin, and others; hence, MFGM contain a high
density of bioactive components .
Phospholipids, plasmalogens, and sphingolipids including
ceramides and gangliosides provide about 0.2–
1% of total milk lipids or about 100–400 mg/L . The
concentration of different phospholipids per 100 g milk
were reported as 8.5 mg sphingomyelin, 6.8 mg phosphatidylethanolamine,
6.0 mg phosphatidylcholine, 1.4 mg
phosphatidylserine, and 1.1 mg/100 g for phosphatidylinositol
. Phospholipids serve structural roles as
indispensable components of all plasma membranes of
body cells and organelles, and they have an impact on
membrane functions and metabolism. Complex lipids
also have roles in signal transmission and cell recognition
[2, 3]. Gangliosides contribute some 10% of brain lipids,
with high concentrations in the cerebral cortex.
The biological importance of MFGM is getting increased
attention after several controlled trials reported
benefits of adding bovine MFGM of complex lipid fractions
to infant formula with fat derived predominantly
from vegetable oil. A trial on formula enriched with
sphingomyelin in preterm infants reported neurobehavioral
benefits . In a small trial in Indonesia, the addition
of a ganglioside-rich bovine milk lipid fraction was
reported to improve the hand and eye coordination IQ,
performance IQ, and total IQ assessed with the Griffiths
Mental Developmental Scale at the age of 24 weeks .
Another trial providing a milk formula with addition of a
similar preparation for 12 weeks enrolled 450 infants
aged 8–24 months in India and reported no difference for
rotavirus or for all-cause diarrhea. In a large study that
enrolled more than 500 Peruvian infants, MFGM-supplemented
formula did not affect diarrhea incidence but reduced
longitudinal diarrhea prevalence . A larger trial
that enrolled more than 250 toddlers aged 2.5–6 years
in Belgium reported that a milk preparation enriched
with a phospholipid-rich lipid fraction resulted in less
days with fewer and lower parental scoring of internal,
external, and total behavioral problems . A further
trial enrolled 160 formula-fed infants in Sweden as well
as a breastfed reference group and evaluated effects of
added bovine MFGM, along with reduced formula contents
of energy and protein. The MFGM group achieved
higher cognition scores in the Bayley test at the age of 1
year (Fig. 4) and showed a much lower incidence of acute
otitis media as well as less use of antipyretic drugs [15, 16].
These observations lead to the conclusion that MFGM
and/or the complex lipids provided with the MFGM fraction
may have important biological roles for the development
of nervous and immune functions.
Milk fat globule lipids also provide considerable
amounts of free and esterified cholesterol, resulting in a
total cholesterol content of 90–150 mg/L in human milk
in contrast to typically only 0–4 mg/L in infant formula.
Cholesterol is an indispensable building block for all cell
membranes and is incorporated in considerable amounts
into myelin in the nervous system during the period of
rapid brain growth, and it serves as the substrate for the
synthesis of bile acids, lipoproteins, vitamin D, hormones,
and oxysterols that modulate cholesterol, lipid, and glucose
homeostasis [3, 9, 17–19]. The provision of cholesterol
with breastfeeding is associated with higher plasma
concentrations of total and low-density lipoprotein cholesterol
in breastfed than in formula-fed infants . The
provision of preformed cholesterol is most likely the
cause for the about 3-fold lower endogenous cholesterol
synthesis rate in breastfed than formula-fed infants, since
the synthesis rate is inversely correlated to the daily cholesterol
supply in mg/kg bodyweight . In formula-fed
piglets, dietary cholesterol supply downregulated hepatic
hydroxymethylglutaryl coenzyme A reductase, the rate
regulating enzyme for endogenous cholesterol synthesis
. In human infants aged 4 months, the rate of endogenous
cholesterol synthesis also appeared to be regulated
by dietary cholesterol supply. Breastfed infants with a
high cholesterol and low phytoestrogen intake had the
lowest fractional synthesis rate, while infants receiving
cows’ milk-based formula with low cholesterol and low
phytoestrogen had an intermediate rate, and infants fed
soy-based formula with no cholesterol and high phytoestrogen
had the highest rate of synthesis . When cholesterol
was added to the soy-based infant formula, the
rate of synthesis was changed to similar results as in infants
fed cows’ milk-based formula, which leads to the
conclusion that the amount of dietary cholesterol supply
regulates cholesterol synthesis in infants. Lasting effects
of early feeding on later cholesterol levels were reported
in several studies and reviewed in meta-analyses. A rather
modest lowering of total and low-density lipoprotein
cholesterol was found in adults who had been breastfed
in infancy, compared to previously formula-fed people,
with a greater effect size of exclusive than of partial breastfeeding
[24, 25]. It was proposed that if 30% of infants are
exclusively breastfed, resulting in a blood cholesterol reduction
in adulthood by 0.15 mmol/L, the population
prevalence of cardiovascular disease could be reduced by
as much as 5% . However, Ip et al.  noted that the
analysis reporting reduced serum lipid levels in previously
breastfed adults did not segregate the data according to
gender and did not explicitly analyze potential confounders;
they concluded that in view of the limited methodological
quality of the meta-analysis the relationship between
breastfeeding and adult cholesterol levels cannot
be correctly characterized. Meta-analyses of available
data do not allow definitive conclusions regarding the relationship
between breastfeeding and all-cause mortality
from cardiovascular diseases in adult life, although the
confidence limits around the point estimates and the observed
between-study heterogeneity do not exclude potential
beneficial or adverse cardiovascular effects of
breastfeeding [26, 27] . Therefore, it appears particularly
promising to evaluate the short- and long-term effects of
addition of well bioavailable preparations of cholesterol
to infant formula in randomized controlled trials, which
may shed further light on the potential biological importance
of a dietary cholesterol supply in infancy.
Fatty Acids Provided with Milk Lipids
Triacylglycerols contribute some 98–99% of human
milk fat. The properties of milk triglycerides are very
much influenced by their fatty acid composition. Milk
lipids of European women today typically contain some
35–40% saturated fatty acids, 45–50% monounsaturated
fatty acids, and approximately 15% PUFA (Table 2). The
saturated palmitic acid (C16: 0) provides approximately
25% of all milk fatty acids and hence the major part of the
total saturated fatty acid content. About 70% of human
milk palmitic acid is esterified in the middle position (sn-
2 position) of triacylglycerols which facilitates absorption.
During intestinal digestion, fatty acids in the sn-1
and sn-2 positions are liberated as nonesterified fatty acids
by pancreatic lipases. These nonesterified fatty acids
are quite well absorbed if they are unsaturated and hence
better water soluble. In contrast, liberated long-chain saturated
fatty acids, such as palmitic acid, are poorly water
soluble and poorly absorbed, but rather bind to calcium
and form calcium soaps that are excreted with stools,
thereby reducing both fat and calcium absorption. However,
if palmitic acid is esterified in the sn-2 position, as it
is predominantly the case in human milk lipids, pancreatic
lipolysis yields a palmitoyl-monogylcerol which is
well water soluble and well absorbed, thereby reducing fat
and calcium malabsorption .
The human milk contents of the mono-unsaturated
fatty acid oleic acid (C18: 1n-9) and of the essential PUFA
linoleic acid (C18: 2n-6) and α-linolenic acid (C18: 3n-3)
vary with the maternal dietary intake of these fatty acids.
This is illustrated by the approximately 3-fold increase of
linoleic acid content in mature human milk in the USA
since the mid 1940s, along with the increase of dietary vegetable
oil and linoleic acid consumption in the population,
whereas α-linolenic acid contents have remained rather
constant (Fig. 5) . Thereby the average ratio of the
omega-6 linoleic acid to the omega-3 α-linolenic acid in
human milk has also increased approximately 3-fold. We
studied the transfer of linoleic acid provided to lactating
women into their milk using stable isotope-labelled fatty
acids. An oral dose of 1 mg/kg bodyweight of linoleic acid
uniformly labelled with the stable carbon isotope 13 C was
provided repeatedly during the 2nd, 6th, and 12th week of
lactation . Before and at several times during a 5-day
period after tracer intake, samples of breath and milk were
collected, the volume of daily milk production was assessed,
and dietary nutrient intakes were calculated from
prospective dietary protocols. Some 3.5–4.5% of the ingested
linoleic acid was oxidized to CO 2 and exhaled with
breath, with no significant differences between the studied
time points. Dietary linoleic acid was rapidly transferred
into milk, with a peak enrichment reached about 12 h after
intake (Fig. 6). Linoleic transfer into milk in unchanged
form or as its metabolites did not change during the course
of lactation. The data indicate that about 30% of milk linoleic
acid is derived directly from dietary intake, whereas
about 70% originates from maternal body fat stores. It is
tempting to speculate that this largely indirect transfer of
dietary linoleic via intermediate body stores may represent
a biological benefit to the breastfed infant, since this
mechanism buffers short-term variation of maternal dietary
supply of the parent essential fatty acid and provides
the infant with a relatively stable parent essential fatty acid
supply. However, long-term changes in dietary supply will
also modify maternal body fat stores and thereby explain
the observed marked changes over time (Fig. 5). Only
about 11% of the milk content of the linoleic acid metabolite
dihomo-γ-linolenic acid (C20: 3n-6) in milk originates
from direct endogenous conversion of maternal dietary
linoleic acid, while only 1.2% of the milk arachidonic acid
(ARA, C20: 4n-6) is directly derived from maternal linoleic
acid intake .
Long-Chain Polyunsaturated Fatty Acids
The provision of LC-PUFA with milk, in particular of
omega-6 ARA and omega-3 docosahexaenoic acid (DHA),
has received considerable attention, because many of the
biological effects of the essential fatty acids in early life appear
to be mediated by LC-PUFA rather than the precursor
essential fatty acids. Brenna et al.  performed a
systematic review on 106 studies of human breast milk
worldwide and culled to include only studies that used
modern analysis methods capable of making accurate estimates
of fatty acid contents as well as criteria related to
the completeness of reporting. The final analysis included
65 studies with milk of 2,474 women. The authors found
a milk ARA content of 0.47 ± 0.13% (mean ± SD, % wt/
wt), whereas milk DHA content was lower at 0.32 ± 0.22%
. Higher milk DHA contents were found in coastal
populations and those with regular marine food consumption.
The greater stability of milk ARA levels with a
coefficient of variation of only 29%, as compared to DHA
with a coefficient of variation of 69%, appears to reflect a
greater degree of metabolic regulation of milk ARA content.
Stable isotope studies have led us to the conclusion
that 90% of human milk ARA are not derived directly
from absorbed dietary lipids but rather from maternal
ARA body stores . In contrast, dietary DHA supply is
a key determinant of milk DHA content. We showed that
the dietary DHA intake is linearly correlated to milk DHA
 (Fig. 6). Breastfeeding women need to achieve a daily
DHA intake of at least 200 mg to provide milk with a DHA
content of at least 0.3%, which is required for a fully breastfed
infant to obtain the daily supply of about 100 mg
DHA/day considered desirable to meet metabolic needs
. Given that the regulation of human milk ARA and
DHA content differs, milk DHA and ARA are not closely
correlated (r = 0.25, p = 0.02) , and the ARA/DHA ratio
is not constant. It remains controversial whether the
ratio of ARA to DHA in milk – or rather the amounts of
DHA and of ARA supplied – are of greater relevance for
biological effects in the infant. A balanced supply of both
ARA and DHA appears to be relevant for the adequate
incorporation of ARA and DHA into the growing brain
In view of the marked accretion of ARA and DHA in
the growing brain and the ample experimental evidence
of the impact of LC-PUFA on membrane function, eicosanoid
and docosanoid formation and the resulting regulation
of physiological processes as well as the development
and function of neural and immune tissues, the impact
of LC-PUFA provision with human milk and also
with infant formula has received considerable interest.
The provision of DHA was shown to enhance the early
development of visual acuity. The European Food Safety
Authority (EFSA) concluded that a cause and effect
relationship has been established between the intake of
infant and follow-on formula supplemented with DHA at
levels around 0.3% of total fatty acids and visual function
at 12 months in formula-fed infants born at term from
birth up to 12 months and in breastfed infants after weaning
up to 12 months . However, some controversy
remains with regards to the effects of the supply of preformed
LC-PUFA on neurodevelopment of healthy term
infants. For example, the authors of a meta-analysis on
randomized trials evaluating infant formula with LCPUFA
compared to formula without LC-PUFA concluded
that while some studies showed a significant benefit,
overall no significant effect was detectable [37, 38]. The
authors noted the limitation of their conclusions by a
large degree of heterogeneity of the included studies,
which provided markedly different interventions and
also used a variety of very different outcomes and approaches
to outcome assessment. Of importance, the included
studies did not adjust for the major impact of genetic
variation modulating the rate of endogenous synthesis
of LC-PUFA and related clinical endpoints, in
particular variation in the Fatty Acid Desaturase (FADS)
gene cluster [39, 40]. The lack of adjusting for this major
modulating confounding factor in the included studies
may considerably reduce the sensitivity to detect effects
of dietary LC-PUFA effects. The comparison of breastfed
infants provided with preformed LC-PUFA with infants
fed formula without LC-PUFA in observational studies is
also difficult to interpret, because human milk LC-PUFA
and particularly DHA supply are closely associated with
different dietary and lifestyle choices, including maternal
smoking and parental socioeconomic status, which may
also influence neurodevelopmental outcomes.
Further insight into PUFA effects are offered by considering
the interaction of breastfeeding, which always
supplies preformed LC-PUFA, and the genetic variation
in the FADS gene cluster that predicts the enzyme activities
of fatty acid desaturases 1 and 2. Gene variants of the
FADS gene cluster have a major impact on the fatty acid
composition of blood, tissues, and human milk [39–41] .
We assessed the single-nucleotide polymorphisms in the
FADS genes along with human milk fatty acid composition
in 772 breastfeeding mothers who participated in the
prospective Ulm Birth Cohort study both at 1.5 months
after infant birth and at 6 months postpartum in a subset
of 463 mothers who were still breastfeeding at this time
. At both time points, we found significant associations
of FADS genotype with milk ARA contents and the
ratio of ARA to dihomo-γ-linolenic acid, indicating that
maternal FADS genotypes have an impact on the formation
of LC-PUFA provided with breastmilk . The
variation of FADS genotypes was shown to also modulate
the interaction of breastfeeding and cognitive development.
Genotyping for the rs174575 variant in the FADS2
gene was performed in 5,934 children participating in the
ALSPAC study in whom IQ tests had been performed at
the age of about 8 years . In line with other observational
studies, previously breastfed children had higher
IQ scores than previously formula fed children, but the
relative impact of human milk nutrient supply and of
confounding factors associated with breastfeeding cannot
be easily deciphered from these observational data
alone. Causal inferences on the role of human milk LCPUFA
supply can be drawn from the fact that the beneficial
effect of breastfeeding was much more pronounced,
with an added advantage of about 4.5 IQ points, in the
group of children with a genotype predicting a low ability
of LC-PUFA synthesis . Replication of these findings
was published with the analysis of data from 2 Spanish
birth cohort studies . Since the genotype is considered
to be distributed in the population at random (“Mendelian
randomization”) and unrelated to the parental decision
to breastfeed and to other related lifestyle predictors
of IQ at school age, these data provide powerful evidence
for the causality between early LC-PUFA supply and status
during the breastfeeding period and later IQ achievements.
Galectins are important for cell turnover and immune
regulation. The CRD of galectins is specific for β-gala ctosides.
When cells are desialylated, the density of exposed
galactose moieties on the cell surface increases. For
example, naïve T cells express CD45 with an α-2,6-linked
sialic acid. The amount of α-2,6-linked sialic acid is reduced
following T-cell activation. The decrease in α-2,6-
linked sialic acid renders the activated T cells susceptible
to galectin-1-mediated apoptosis . Thus, binding of
sialyated HMO to cells may prevent apoptosis.
The relevance of LC-PUFA supply for child neurodevelopment
was also demonstrated in a randomized clinical
trial that enrolled 119 breastfeeding women in Texas,
USA . The women were assigned to receive identical
capsules containing either a high-DHA algal oil providing
approximately 200 mg DHA daily or a vegetable oil
without DHA from delivery until 4 months after birth.
Provision of DHA to the mother increased DHA in milk
by about 70%, and in infant plasma phospholipids by
about 20% . At the age of 30 months, child psychomotor
development was significantly better if mothers had
received added DHA during the first 4 months of breastfeeding.
At the age of 5 years, there were no differences in
visual function, but children whose mothers had received
added DHA performed significantly better on the Sustained
Attention Subscale of the Leiter International Performance
Scale (46.5 ± 8.9 vs. 41.9 ± 9.3, p < 0.008). These
results support the conclusion that the DHA supply during
early infancy is of importance for specific aspects of
Mendelian randomization also provided strong support
for the conclusion that the LC-PUFA supply with
breastfeeding is causally linked to protection against a later
manifestation of bronchial asthma. Many studies have
reported a protective effect of breastfeeding on asthma
development, even though results are not consistent .
We evaluated the influence of the FADS1 FADS2 gene
cluster polymorphisms on the association between breastfeeding
and asthma in 2,245 children participating in 2
prospective German birth cohort studies, the GINI and
LISA studies . Logistic regression modelling was used
to analyze the association between exclusive breastfeeding
and doctor-diagnosed asthma occurring up to the age
of 10 years, stratified by genotype. In the stratified analyses,
heterozygous and homozygous carriers of the minor
allele that show a low activity of LC-PUFA synthesis had
a much reduced risk for later asthma if they were breastfed
for 3 or 4 months and hence were provided with preformed
LC-PUFA that can compensate for low endogenous
synthesis (adjusted odds ratio between 0.37 [95%
CI: 0.18–0.80] and 0.42 [95% CI: 0.20–0.88]). Interaction
terms of breastfeeding with genotype were significant and
ranged from –1.17 (p = 0.015) to –1.33 (p = 0.0066). Similarly,
heterozygous and homozygous carriers of the minor
allele who were exclusively breastfed for 5 or 6 months
after birth had a reduced risk of asthma (0.32 [0.18–0.57]
to 0.47 [0.27–0.81]) in the stratified analyses. In contrast,
in individuals carrying the homozygous major allele predicting
a greater degree of endogenous LC-PUFA formation,
breastfeeding with provision of LC-PUFA showed
no significant effect on asthma development. These results
of a Mendelian randomization study demonstrate a
lasting causal protection of breastfeeding for at least 3
months against doctor-diagnosed asthma until school
age in children with a low rate of LC-PUFA synthesis and
a modulating effect of postnatal
A systematic review on human
studies on roles of LCPUFA
and an expert workshop
that reviewed the information
and developed recommendations
was recently performed with support
from the Early Nutrition Academy . It was concluded
that breastfeeding women should get ≥ 200 mg DHA/day
to achieve a human milk DHA content of at least ≈ 0.3%
of fatty acids. Infant formula for term infants should contain
DHA and ARA to provide 100 mg DHA/day and 140
mg ARA/day, and a supply of 100 mg DHA/day should
continue during the second half of infancy. No quantitative
advice on ARA levels in follow-on formula fed after
the introduction of complimentary feeding was provided
due to lack of sufficient data and considerable variation
in ARA amounts provided with complimentary foods.
Should Infant Formula LC-PUFA Composition Be
Guided by Human Milk Composition?
With regards to infant and follow-on formula, the recent
revision of the European legislation that came into
force in 2016 stipulates that all infant and follow-on formula
must contain between 20 and 50 mg DHA/100 kcal
(approx. 0.5–1% of fatty acids), whereas formula without
DHA content will not be allowed any more to be placed
on the European Union market once this legislation is
implemented . To the great surprise of many pediatricians
and of experts in the field, no requirement for a
minimum content of ARA in infant formula has been defined.
This legal regulation is based on advice provided by
the European Food Safety Authority that reviewed a variety
of aspects and nutrients, including LC-PUFA DHA
and ARA. In a first report on nutrient requirements and
dietary intakes of infants and young children published
in 2013, adequate LC-PUFA intakes were defined as 100
mg DHA/day and 140 mg ARA/day from birth to the age
of 6 months, while 100 mg DHA/day were considered adequate
from 6 to 24 months . These conclusions are
in line with many other scientific reports, including the
recent recommendations of the Early Nutrition Academy-
supported global expert group that are based on a systematic
review of the available scientific evidence . In
contrast, the subsequently published EFSA report on the
compositional requirements of infant and follow-on formula
advised that all infant and follow-on formula should
contain relatively high amounts of DHA at 20–50 mg/100
kcal, but without the need to
provide any preformed ARA
. This DHA level stipulated
by EFSA and the new European
legislation is much
higher than the about 0.2–
0.3% DHA found in most LCPUFA-
enriched formulae for term infants currently marketed
around the world, which, however, generally also
contain preformed ARA at levels equal to or often 2-fold
higher than the DHA content. The proposed obligatory
inclusion of DHA in all infant and follow-on formulae is
welcomed by many scientists and pediatricians in view of
the indications for beneficial effects , but the advice
to provide infant formula from birth that supplies DHA
but no ARA has been heavily criticized . During pregnancy
and infancy, both DHA and ARA are deposited in
relatively large amounts in human tissues, including the
brain [51, 52]. Fetal accretion of both DHA and ARA during
pregnancy is facilitated by their active and preferential
maternofetal placental transfer . Pregnant women’s
red blood cell levels of both DHA and ARA were
positively associated with their children’s IQ at school age
. At birth, higher cord blood contents of both DHA
and ARA predicted less later behavioral problems, emotional
difficulties, hyperactivity, and attention deficit at
the age of 10 years . After birth, breastfed infants always
get both preformed DHA and ARA, usually with a
higher provision of ARA than of DHA [31, 56]. DHA
along with ARA have been added to infant formulae since
the 1980s in an attempt to approach the nutrient supply
and functional effects achieved with breastfeeding [57–
59]. The global Codex Alimentarius standard on the compositional
requirements for infant formula stipulates the
optional addition of DHA to infant formula, provided
that the ARA content is equal to or higher than the DHA
content, thus following the model of typical human milk
The advice to provide infant formula
from birth that supplies DHA but no
ARA has been heavily criticized
Infant formulae providing both DHA and ARA have
been evaluated in many controlled trials in infants .
In contrast, the proposed composition of term infant formula
with up to 1% DHA and no ARA is a novel approach
that has not been systematically tested for its suitability
and safety in healthy infants born at term. ARA is an essential
component of all cell membranes. The amount of
ARA incorporated into the developing brain during infancy
exceeds the deposition of DHA. Although humans
can synthesize ARA to some extent from linoleic acid,
infants fed formula without preformed ARA tend to develop
lower ARA levels in blood plasma and erythrocytes
than breastfed infants who receive both DHA and ARA
[51, 57, 61]. In preterm infants, provision of high amounts
of omega-3 LC-PUFA without a concomitant supply of
ARA has been associated with adverse effects on growth
[62, 63]. Further concerns regarding the effects of a high
supply of DHA without increasing ARA intakes on infants
are raised by the findings of a randomized controlled
trial assigning term infants to formula providing
either no LC-PUFA or different levels of 0.32, 0.64, and
0.96% DHA at the same ARA level of 0.64% . The investigators
performed developmental testing of the participating
children up to the age of 6 years. Positive effects
in tests on word production, a card sorting task, and an
intelligence test were observed with the lower DHA dose.
However, performance of children assigned to the highest
DHA dose of 0.96% but with a reduced ratio of dietary
ARA to DHA was attenuated in the MBCDI Word Production
Test and the Dimensional Change Card Sort Test
at the highest DHA level, and it was attenuated at the two
highest DHA levels in the Peabody Picture Vocabulary
Test . Thus, in contrast to what might have been expected,
an increase of formula DHA contents above
0.32% did not further improve or at least stabilize developmental
outcomes, but actually had adverse effects
which might well be due to the reduced dietary ARA to
DHA ratios provided with the higher DHA levels.
The effects of equivalent formulae with similar DHA
and ARA contents on brain composition were tested in
infant baboons. Brain composition in various regions was
analyzed. The formula with about 1% DHA induced a
trend to lower ARA levels in the retina and all the 8 regions
of the brain analyzed, with significantly reduced
ARA values in the globus pallidus and the superior colliculus,
even though the formula contained 0.64% ARA.
These observations raise serious concerns that infant formula
with high contents of DHA but lack of ARA may
induce adverse effects on brain composition and related
These findings in human infants and in nonhuman
primates question the suitability and safety of the compositional
requirements stipulated by the new European
legislation, i.e. to provide infant formula from birth with
up to 1% of fatty acids as DHA without a proportional
increase in the intake of ARA. It is generally agreed upon
that any major change in infant formula composition
should be subjected to a full preclinical and clinical evaluation
of nutritional adequacy and safety prior to the wide
use and market introduction of such a modified formula
[65–70]. Therefore, it appears to be inappropriate and
premature to market formula for term infants from birth
with 20–50 mg/100 kcal DHA without added ARA in the
absence of accountable data on the suitability and safety
from a thorough clinical evaluation of this novel approach
It appears to be inappropriate and
premature to market formula for term
infants from birth with 20–50 mg/
100 kcal DHA without added ARA
In addition to meeting the infant needs for energy and
essential vitamins and PUFA, human milk lipids provide
a mixture of MFGM, complex lipids, and bioactive compounds
that may have important biological roles in the
breastfed infant, for example with regard to the development
of nervous and immune functions. Further studies
defining the specific components responsible for such effects
and the underlying mechanisms could help to design
the best options of nutritional interventions. Methodological
progress in the field of metabolomics and lipidomics
using liquid chromatography coupled with triple
mass spectrometry now allows to determine detailed profiles
of molecular species of complex lipids in milk as well
as in extremely small sample volumes of infant serum or
plasma (e.g. 10 μL) with high quantitative precision [71–
74]. Such lipidomic measurements can serve to provide
markers for tissue composition  and were shown to
be associated with important clinical endpoints in children
and adults [76–78] . It is therefore likely that the use
of these sophisticated and detailed analytical methods, if
combined with appropriate bioinformatics strategies,
provide the opportunity to obtain better insights into the
physiological roles of complex lipids in early life, which
may lead to further improvements in nutritional strategies.
Progress in biotechnology and food technology offers
new avenues for preparing lipid components that can
more closely mimic the complex lipid body provided with
breastfeeding. Careful exploration and evaluation of the
short- and long-term impact in infants could potentially
lead to implementation of major improvements for the
feeding of infants who cannot be breastfed. Opportunity
also exists in improving our understanding of the optimal
supply of LC-PUFA in early and later infancy and in the
underlying mechanisms and mediators of their effects,
e.g. on neurodevelopment and behavior, immune-related
health outcomes, such as allergy and asthma, and pulmonary
The work of the author is financially supported in part by the
Commission of the European Community, the 7th Framework
Programme Early Nutrition (FP7-289346), the Horizon 2020 Research
and Innovation Programme DYNAHEALTH (No 633595),
and the European Research Council Advanced Grant METAGROWTH
(ERC-2012-AdG, No. 322605). This manuscript does
not necessarily reflect the views of the Commission and in no way
anticipates the future policy in this area.
The author declares that no financial or other conflict of interest
exists in relation to the contents of this article. The production
of this paper has been supported by a grant provided by the Nestlé
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