Clinical Nutrition 30 (2011) 116e123

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Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Original Article

Differences in fat content and fatty acid proportions among colostrum,

transitional, and mature milk from women delivering very preterm,
preterm, and term infants
Carolina Molt-Puigmart a, b, Ana Isabel Castellote a, b, Xavier Carbonell-Estrany c,
M. Carmen Lpez-Sabater a, b, *
a

Department of Nutrition and Food Science, Faculty of Pharmacy, University of Barcelona. Avda. Joan XXIII s/n CE-08028, Barcelona, Spain
CIBER of Epidemiology and Public Health, Spain
Hospital Clnic, Institut de Ginecologia, Obstetrcia i Neonatologia, Agrupaci Sanitria Hospital Clnic e Hospital Sant Joan de Du. IDIBAPS (Institut dInvestigacions Biomdiques
August Pi i Sunyer), University of Barcelona, Barcelona, Spain
b
c

a r t i c l e i n f o

s u m m a r y

Article history:
Received 22 December 2009
Accepted 18 July 2010

Background & aims: Human milk composition changes according to gestational age and stage of lactation,
but infants fed banked human milk often receive pooled milk. We studied the changes in fat content and
fatty acid proportions throughout lactation in very preterm, preterm, and full term milk, and the
differences among gestational age groups.
Methods: Samples from women delivering before 30 (n 10), between 30 and 37 (n 10), and between
38 and 42 (n 23) weeks of gestation were analyzed.
Results: Fat content was higher in very preterm than in preterm and full term samples (p < 0.05).
Medium-chain saturated fatty acids, alpha-linolenic acid, and rumenic acid proportions increased
(p < 0.05) during lactation, while those of most long-chain saturated fatty acids and most long-chain
polyunsaturated fatty acids from the n-3 and n-6 families decreased (p < 0.05). In colostrum and
transitional milk, medium-chain saturated fatty acid proportions were highest in the very preterm group,
and decreased with gestational age (p < 0.05).
Conclusions: The differences in fat and fatty acids of human milk obtained at different gestational ages
and stages of lactation may impact preterm infants health. Therefore they could be taken into account
when feeding newborns banked human milk and when designing infant formulas or human milk
fortiers.
2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords:
Conjugated linoleic acid
DHA
Human milk
Milk banking
Preterm infants
Creamatocrit

1. Introduction
Mothers own milk (MOM), with appropriate fortication for
some premature or very-low birth weight (VLBW) infants, is the
best food for newborns.1e3 However, breastfeeding premature or
Abbreviations: VLBW, very-low birth weight; FT, full term; PT, preterm; VPT,
very preterm; SFAs, saturated fatty acids; MCSFAs, medium-chain saturated fatty
acids; LCSFAs, long-chain saturated fatty acids; MUFAs, monounsaturated fatty
acids; PUFAs, polyunsaturated fatty acids; LCPUFAs, long-chain polyunsaturated
fatty acids; LA, linoleic acid; CLA, conjugated linoleic acid; AA, arachidonic acid;
ALA, alpha-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
* Corresponding author. Department of Nutrition and Food Science, Faculty of
Pharmacy, University of Barcelona. Avda. Joan XXIII s/n CE-08028, Barcelona, Spain.
Tel.: 34 93 402 45 12; fax: 34 93 403 59 31.
E-mail addresses: carolinamolto@ub.edu (C. Molt-Puigmart), aicastellote@ub.
edu (A.I. Castellote), XCARBO@clinic.ub.es (X. Carbonell-Estrany), mclopez@ub.
edu, mclopez@ub.edu (M.C. Lpez-Sabater).

VLBW infants is still a challenge because of their physiological and

neurological immaturity, because they are alert for very short
periods of time, and because they may present inappropriate suck/
swallow/breathe coordination. In spite of this, many neonatal units
help mothers to start and maintain an appropriate milk production,4 not only because some premature newborns may still be able
to breastfeed, but also because those that cannot suck directly at
the breast can still be fed milk extracted from their own mothers.5
When, despite the efforts, a woman is unable to feed her child with
her own milk or cannot supply enough milk, or when breastfeeding
is contraindicated, preterm infants can be fed preterm formula or
pasteurized donor human milk (either as a sole diet or as
a complement of MOM). Two points should be emphasized in
regard to these different feeding strategies for prematures:
a) unfortied MOM and specially donor milk may fail to full the
nutritional requirements of preterm newborns2; b) a higher rate of

0261-5614/$ e see front matter 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
doi:10.1016/j.clnu.2010.07.013

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C. Molt-Puigmart et al. / Clinical Nutrition 30 (2011) 116e123

growth gain has been observed when feeding prematures preterm

formulas instead of unfortied human milk but, in the former case,
they become more prone to suffer from necrotizing enterocolitis
(NEC).6 Therefore, the use of MOM or donor milk with human milk
fortiers could be seen as the currently preferable option. However,
partly because of the high variability in milk composition, there is
no consensus in regard to what the optimal composition and
quantity of these fortiers is. Some neonatal units now opt to apply
individual fortication, in which fortication is done in base of the
specic composition (of macronutrients) of each milk sample or on
the metabolic responses of the infant during feeding. Individual
fortication seems to be a good strategy to follow, but it entails
obvious logistic difculties and it may not be an achievable goal in
milk banks with scarce resources; in addition, some studies warn
about the fact that fortication is not risk-free. In this context, we
consider that a better knowledge on the intrinsic variability of
human milk composition will help neonatologists, human milk
banks, and manufacturers of infant formulas and human milk
fortiers to improve existing feeding strategies, design alternative
ones, and optimize the use of fortiers.
Fat is known to be the most variable macronutrient in milk.
Apart from its role as energy supplier, it is also source of essential
fatty acids and vehicle for fat-soluble vitamins. Many studies have
described the changes in fatty acid composition from colostrum to
mature milk.7e26 However, information on whether these changes
are also found in milk from mothers delivering before the 30th
week of gestation, and on whether very preterm (VPT) milk fatty
acid composition differs from that of preterm (PT) and full term (FT)
milk is scarce. Our aim was, therefore, to study the differences in
human milk fatty acid composition of colostrum, transitional, and
mature milk of Spanish women with VPT, PT, and FT deliveries.
Differences in fat content were also assessed.
2. Participants and methods
2.1. Participants and timing for milk collection
Between June 2006 and May 2008, 50 women living in the
Barcelona metropolitan area who delivered at the Hospital Clnic
from Barcelona participated in the study. Women had to provide
one sample of colostrum (2e4 days post-partum), one of transitional milk (8e12 days post-partum), and one of mature milk
(28e32 days post-partum). Exclusion criteria were: illness of the
mother (including metabolic disorders such as diabetes or gestational diabetes), eclampsia, HIV infection, drug abuse, treatment
with psychoactive drugs or narcotics, and women delivering small
for gestational age babies. Infants weight at birth was recorded.
Milk samples were classied according to the gestational age as
VPT (less than 30 weeks of gestation), PT (30e37 weeks of gestation), and FT (38e42 weeks of gestation). Of these 50 women, 7
abandoned the study for different reasons. A total of 43 women
successfully provided milk samples at the three time points. Of this
total, 23 women delivered FT infants, 10 delivered PT infants, and 10
delivered VPT infants. All procedures were approved by the
hospitals ethical committee, and written informed consent was
obtained from all women.
2.2. Breast milk collection
Colostrum was collected at the hospital by an experienced nurse
and one of the authors (CMP). For the collection of transitional and
mature milk, women were visited at home by the same trained
researcher. Milk was collected between 8:00 and 12:00 a.m., at the
end of the feeding, in sterile polypropylene tubes by mechanical
expression of either or both breasts with a breast pump (Ameda,

117

Zug, Switzerland). Milk was transported to the laboratory in

iceboxes in less than 2 h after collection. Several aliquots were
made from each milk sample, and they were stored at 80  C until
analyzed.
2.3. Creamatocrit analysis and fat estimation
Creamatocrit was determined as described by Lucas et al.27 with
slight modications. In short, milk was drawn by capillarity into
glass capillary tubes, which were then sealed at one end with clay
and centrifuged for 15 min at 12000g (10,921 rpm) and 25  C. The
cream layer and length of total milk column were read under
a magnifying glass with a vernier calliper within 30 min of
centrifugation to prevent the cream column to unpack. The
creamatocrit was calculated as the ratio between the cream layer
and the length of the total milk column and expressed as
percentage. Each sample was analyzed in triplicate. Milk fat content
was calculated from creamatocrit values through the formula
proposed in the same study by Lucas et al.27
2.4. Fatty acid analysis
Fatty acid methyl esters were prepared with sodium methylate
and methanolic boron triuoride and extracted into hexane
following the method developed by Molt-Puigmart et al.28
Subsequently, they were separated and quantied by fast-gas
chromatography with ame ionization detection according to the
same method. Each sample was analyzed in duplicate.
2.5. Statistical analysis
First, a descriptive analysis of clinical and biological data was
performed. According to the KolmogoroveSmirnov test, maternal
and infants clinical characteristics were normally distributed
(except for parity and number of babies in the delivery), as well as
creamatocrit values, fat concentration, and most of the fatty acids.
Normal distribution was assumed for the statistical analyses of fatty
acids, while validity of the obtained results was checked by
repeating the statistical analyses after performing log10 transformation of those following skewed distributions. Creamatocrit
values, fat concentration, fatty acid proportions and maternal
characteristics were expressed as mean  standard deviation.
Differences in maternal and infants clinical data were assessed by
a one-way ANOVA and by the KruskaleWallis non-parametrical
test for normally and not normally distributed variables, respectively. For each of the gestational age groups, a repeated-measures
analysis of variance test (GLM) was used to assess differences in
fatty acid proportions, creamatocrit values, and fat concentrations
with time (stage of lactation). To compare gestational age groups at
each stage of lactation, an ANOVA (one-way) analysis was used.
Differences between gestational age groups were subsequently
identied by using the Bonferroni post-hoc test. Differences associated with p values lower than 0.05 were considered to be statistically signicant. Pearson correlation coefcients were used to
study correlations between fatty acids. The SPSS statistical software
(Version 15, SPSS Inc, Chicago, Illinois, USA) was used for all data
analyses.
3. Results
Mean gestational ages were 26.74 (1.12), 33.49 (1.86) and
40.50 (1.11) weeks for women delivering VPT, PT, and FT infants,
respectively. Maternal age ranged from 22 to 42 years old. Other
clinical data of the participants are shown in Table 1.

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118

C. Molt-Puigmart et al. / Clinical Nutrition 30 (2011) 116e123

Table 1
Characteristics of women and infants included in the study.
Characteristicsc

Very preterm
(n 10)

Maternal age (years)

Maternal weight previous pregnancy (kg)
Maternal height (m)
BMI previous pregnancy (kg/m2)
Weight gain during pregnancy
Parity
Number of babies in the delivery
Number of previous pregnancies
Time from last pregnancy (months)
Weight of the newborns at birth (kg)

33.10
65.90
1.67
23.75
8.4
1.40
1.10
1.33
25.25
0.97

a
b
c
d












3.56
6.85
0.05
1.97
3.75
0.67
0.30
0.96
21.64
0.25

Full term (n 23)

Preterm
(n 10)
32.80
64.15
1.60
25.09
13.55
1.60
1.20
1.25
68.50
2.21












5.33
11.76
0.06
5.25
3.48
0.49
0.40
0.44
53.79
0.58

30.83
55.91
1.60
21.97
11.92
1.61
1.00
1.75
11.05
3.67












4.88
6.48
0.07
2.49
5.04
0.86
0.00
1.73
8.30
0.71

p valued
0.327a
0.004a
0.025a
0.050a
0.050a
0.607b
0.103b
0.795a
0.354a
0.000a

Statistical differences were assessed by one-way ANOVA.

Statistical differences were assessed by the KruskaleWallis non-parametrical test.
Mean  SD.
Signicant differences (p < 0.05) are indicated in bold.

3.1. Creamatocrit and fat content

Differences in creamatocrit values among stages of lactation and
gestational age groups are shown in Fig. 1. Values obtained for VPT,
PT, and FT milk tended to increase from colostrum to transitional
milk, reaching statistical signicance in the VPT and PT groups
(p 0.031 and 0.007, respectively). Values for mature milk were
found to be between those of colostrum and transitional milk. VPT
milk creamatocrit values at each stage of lactation were higher than
those of FT milk at the same stage of lactation (p < 0.05). The same
was true when comparing VPT with PT samples, but it did not reach
statistical signicance in the case of transitional milk (p 0.107).
There was no statistically signicant difference between creamatocrit values of PT and FT milk. Mean fat content (SD) in colostrum, transitional, and mature milk was 4.05  1.62, 4.76  1.62,
and 4.67  1.19 g/100 mL, respectively, in VPT samples; 2.58  1.88,
3.75  1.24, and 2.98  1.75, respectively, in PT samples; and
2.60  1.48, 3.11  1.53, and 3.06  1.29, respectively, in FT samples.
Statistically signicant differences in the fat content among groups
were the same as those observed for the creamatocrit.

Fig. 1. Creamatocrit values (%) in colostrum (black bars), transitional (white bars), and
mature milk (striped bars), from women delivering very preterm (VPT), preterm (PT),
and full term (FT) infants. Lower case letters indicate differences among colostrum,
transitional, and mature milk in each gestational age group (same letters indicate
signicant differences, according to GLM and LSD post-hoc test). Symbols indicate
differences among gestational age groups at each stage of lactation (same symbols
indicate signicant differences, according to ANOVA and Bonferroni post-hoc test).
Signicance: p < 0.05. Error bars represent SD.

3.2. Differences in fatty acid proportions according to stage of

lactation
Several differences were found between colostrum, transitional,
and mature milk saturated fatty acids (SFAs), monounsaturated
fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs)
proportions (Tables 2e4, respectively). The observed differences
were maintained even after log-transforming fatty acids with
skewed distributions.
As shown in Table 2, the sum of SFAs increased in a signicant
way from colostrum to transitional PT and FT milk, and remained
stable in mature milk. Instead, VPT SFAs proportions did not
signicantly vary with the progression of lactation. When looking
at the indivual SFAs, we observed that C10:0 and C12:0 increased
during lactation in the three gestational age groups with
a maximum in transitional milk, SFAs with 20 or more carbon
atoms decreased or tended to decrease during the same period, and
SFAs with 14e18 carbon atoms had a variable behaviour. Differences seen in the sum of medium-chain SFAs (MCSFAs,
C10:0 C12:0) reected the ones just described for the individual
fatty acids (Fig. 2a). When summing up long-chain SFAs proportions (LCSFAs, C14:0), we observed a decrease during lactation in
VPT and PT, but not in FT samples (Fig. 2b).
Despite some oscillations, the sum of MUFAs proportions
remained quite constant during lactation (Table 3). However, more
differences were found when looking at the MUFAs individually. Of
note, percentages of MUFAs with more than 18 carbons (C20:1 n-9,
C22:1 n-9, and C24:1 n-9) considerably decreased, in a signicant
way, from colostrum to mature milk. Instead, oleic acid (C18:1 n-9)
proportions slightly increased in the VPT and PT groups during the
same period.
Regarding PUFAs (Table 4) from the n-6 family, trans isomers of
linoleic acid (t9t12-and c9t12,t9c12-C18:2) and linoleic acid (C18:2
n-6, LA) proportions remained quite stable from colostrum to
mature milk, C18:3 n-6 and c9t11-CLA proportions increased, and
those of PUFAs with more than 18 carbons decreased. In the n-3
family, alpha-linolenic acid (C18:3 n-3, ALA) proportions increased,
those of eicosapentaenoic acid (C20:5 n-3, EPA) did not vary, and
those of C22:5 n-3 and docosahexaenoic acid (C22:6 n-3, DHA)
decreased with progression of lactation. Fig. 3a and b shows the
evolution of arachidonic acid (C20:4 n-6, AA) and DHA throughout
lactation. When n-3 and n-6 fatty acids were summed up, the
percentages of PUFAs from both families (PUFAs n-6 and PUFAs n-3)
decreased during lactation in PT and FT milk, as also did those of the
n-6 and n-3 long-chain PUFAs (LCPUFAs n-6 and LCPUFAs n-3) in all
groups.

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C. Molt-Puigmart et al. / Clinical Nutrition 30 (2011) 116e123

Table 2
Saturated fatty acid proportions according to the stage of lactation and gestational
age groups.
Fatty acid G. age group Colostrum
C8:0

C10:0

C12:0

C14:0

C15:0

C16:0

C17:0

C18:0

C20:0

C22:0

C24:0

SFAs

MCSFAs

LCSFAs

VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT

0.03
0.02
0.02
0.39
0.16
0.13
3.38
1.81
1.33
5.93
4.19
3.69
0.22
0.22
0.23
21.08
23.68
22.71
0.29
0.29
0.29
5.85
6.68
6.21
0.19
0.25
0.24
0.07
0.10
0.10
0.11
0.15
0.18
37.54
37.57
35.13
3.77
1.97
1.46
33.74
35.57
33.65












































0.01a,b
0.01a
0.02a
0.18a,#,
0.07a,b,#
0.08a,b,
1.61a,#,
0.59a,#
0.46a,b,
2.60a,#,
0.45a,#
0.84a,b,
0.05a,b
0.05
0.05a
2.71a,b,#,
2.25a,b,#
2.08a,b,
0.07
0.05a
0.04
1.42
2.20a
1.24
0.04#,
0.09a,b,#
0.06a,
0.02a,#,
0.04a,#
0.04a,
0.05a,#
0.09a
0.10a,#
4.87
4.44a
3.30a,b
1.78a,#,
0.64a,#
0.53a,b,
4.30a
4.25a,b
3.41

Transitional
0.08
0.07
0.08
0.98
0.91
0.80
6.00
5.95
4.28
6.94
7.02
5.28
0.19
0.19
0.26
16.84
19.83
21.70
0.26
0.25
0.30
6.00
5.68
6.45
0.18
0.19
0.20
0.06
0.07
0.07
0.07
0.08
0.07
37.60
40.24
39.48
6.99
6.87
5.09
30.53
33.31
34.32












































0.02a
0.03a
0.02a
0.25a,#
0.19a
0.25a,#
2.04a,#
1.84a,
1.58a,#,
2.79a,b,#
2.16a,
1.96a,#,
0.05a,#
0.06
0.07a,#,
6.06a,#,
2.65a,#
1.66a,
0.04#
0.05a,b,
0.05a,#,
0.96
1.25a,#
0.98#
0.02#
0.05a
0.04a,#
0.01a
0.02a
0.02a
0.04a
0.05a
0.03a
9.64
6.19a
4.56a
2.26a,#
2.01a,
1.80a,#,
7.53#
4.92a
3.24#

Mature
0.08
0.09
0.09
0.86
0.88
0.83
4.41
4.74
4.15
4.95
5.65
4.98
0.19
0.23
0.25
18.64
20.65
21.26
0.25
0.29
0.28
6.15
6.06
6.39
0.18
0.18
0.19
0.04
0.05
0.05
0.05
0.04
0.05
35.79
38.83
38.52
5.28
5.62
4.98
30.44
33.12
33.45












































119

Table 3
Monounsaturated fatty acid proportions according to the stage of lactation and
gestational age groups.
Fatty acid G. age group Colostrum

0.02b
0.03a
0.04a
0.25a
0.22b
0.20b
1.56a
1.19a
1.10b
1.69b
1.58a
1.74b
0.03b,#
0.07
0.11#
1.23b,#,
2.43b,#
2.79b,
0.02
0.06b
0.06a
0.80
0.83
0.93
0.04
0.03b
0.03a
0.02a
0.03a
0.02a
0.02a
0.03a
0.03a
4.71
5.25
5.44b
1.74a
1.40a
1.28b
3.20a,#
4.25b
4.57#

Results expressed as mean  SD. G. age group refers to the three gestational age
groups: very preterm (VPT), preterm (PT), and full term (FT). Lower case letters
indicate differences among colostrum, transitional, and mature milk in each
gestational age group (same letters indicate signicant differences, according to
GLM and LSD post-hoc test). Symbols indicate differences among gestational age
groups, for each stage of lactation (same symbols indicate signicant differences,
according to ANOVA and Bonferroni post-hoc test). Signicance: p < 0.05. MCSFA
refers to the sum of C10:0 and C12:0 and LCSFA to the sum of saturated fatty acids
with 14 carbon atoms or more.

3.3. Differences in fatty acid proportions according to gestational

age groups
Colostrum of VPT, PT, and FT groups had virtually the same
proportions of SFAs. However, when considering MCSFAs and
LCSFAs separately, we found that C10:0 and C12:0 proportions as
well the sum of MCSFAs decreased with gestational age (Table 2
and Fig. 2a), proportions of LCSFAs with 20 or more carbon
atoms increased, those of LCSFAs with 14e18 carbon atoms did not
follow a clear pattern, and the sum of LCSFAs did not signicantly
vary (Table 2 and Fig. 2b). A similar behaviour was observed in
transitional milk. Mature milk SFAs did not differ much among
groups, but lower proportions of C15:0 and C16:0 in mature milk
from women delivering VPT infants led to a lower sum of LCSFAs
in that group in comparison to the PT and FT groups (Table 2 and
Fig. 2b).

C14:1

C16:1 n-9

C16:1 n-7

C17:1

t-C18:1

C18:1 n-9

C18:1 n-7

C20:1 n-9

C22:1 n-9

C24:1

MUFAs

VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT

0.11
0.12
0.12
0.54
0.57
0.56
1.54
1.78
1.48
0.16
0.16
0.14
0.32
0.40
0.45
37.76
36.84
37.11
1.91
2.25
1.95
0.79
0.96
0.96
0.21
0.28
0.27
0.21
0.30
0.32
43.53
43.65
43.37



































Transitional

0.04
0.03a
0.05a,b
0.08a
0.11a,b
0.08a
0.50
0.53a,#
0.38a,b,#
0.04
0.03a
0.02a,b
0.14a,#
0.07a
0.22#
4.52a
3.88
3.51
0.36a,#
0.31a,b,#,
0.48a,b,
0.16a,#,
0.11a,#
0.15a,
0.04a,#,
0.03a,#
0.06a,
0.06a,#,
0.09a,#
0.12a,
4.91
4.37a
3.80a

0.12
0.14
0.18
0.45
0.45
0.43
1.62
1.78
1.82
0.15
0.15
0.17
0.22
0.29
0.51
40.46
35.49
36.12
1.78
1.78
1.72
0.66
0.58
0.52
0.15
0.14
0.12
0.10
0.11
0.08
45.72
40.91
41.68



































0.05#
0.06
0.08a,#
0.07a
0.10a
0.06a
0.46
0.58b
0.49a
0.03#
0.03b,
0.03a,#,
0.09a,#
0.16a,b,
0.37#,
9.78#
5.61a
4.88#
0.35
0.37a
0.27a
0.13a,#,
0.07a,#
0.09a,
0.02a,#
0.02a,
0.02a,#,
0.03a,#
0.04a,
0.02a,#,
9.84
5.97a,b
5.21a

Mature
0.13
0.18
0.21
0.38
0.44
0.39
1.60
2.06
1.99
0.15
0.19
0.18
0.23
0.39
0.43
41.11
39.00
37.45
1.62
1.75
1.66
0.52
0.48
0.47
0.10
0.10
0.10
0.06
0.05
0.05
45.90
44.64
42.92



































0.03#
0.09a
0.13b,#
0.13a
0.06b
0.06a
0.53
0.56a,b
0.64b
0.02#,
0.04a,b,#
0.05b,
0.12#
0.20b
0.30#
5.07a,#
6.47a
5.11#
0.28a
0.17b
0.28b
0.04a
0.10a
0.11a
0.02a
0.01a
0.03a
0.02a
0.01a
0.02a
4.38
6.47b
5.34

Results expressed as mean  SD. G. age group refers to the three gestational age
groups: very preterm (VPT), preterm (PT), and full term (FT). Lower case letters
indicate differences among colostrum, transitional, and mature milk in each
gestational age group (same letters indicate signicant differences, according to
GLM and LSD post-hoc test). Symbols indicate differences among gestational age
groups, for each stage of lactation (same symbols indicate signicant differences,
according to ANOVA and Bonferroni post-hoc test). Signicance: p < 0.05.

The sum of MUFAs was not affected by gestational age. When

considering the different MUFAs individually (Table 3), proportions
in mature milk did not greatly differ among gestational age groups
either, but those in colostrum and transitional milk did. Of note,
MUFAs with more than 18 carbons were higher in PT and FT
colostrum when compared to VPT colostrum, while in transitional
milk it was the other way around (VPT transitional milk had higher
proportions of these fatty acids than PT and FT milk).
In the case of PUFAs (Table 4), proportions in mature milk did
also not vary according to the gestational age group (except for the
lower proportions of c9t12,t9c12-C18:2 in VPT milk). Instead, most
differences were found again among colostrums and transitional
milks at the three gestational age groups. We observed a general
trend of PT samples to have fatty acid proportions in between those
Table 4
Polyunsaturated fatty acid proportions according to the stage of lactation and
gestational age groups.
Fatty acid
t9t12-C18:2

G.age group Colostrum

VPT
PT
FT
c9t12, t9c12- VPT
PT
C18:2
FT
C18:2 n-6
VPT
PT
FT

0.06
0.07
0.07
0.13
0.15
0.18
14.42
12.99
15.95











0.02#
0.01
0.03#
0.02a,#
0.04a
0.07#
6.13
3.41a,#
4.07#

Transitional
0.05
0.06
0.08
0.12
0.13
0.16
13.08
14.79
15.17











Mature

0.02#
0.06  0.02
0.08  0.03a
0.02a,
#,
0.03
0.08  0.05
0.02a,#
0.13  0.03#
0.02a,b, 0.16  0.05b
0.03#,
0.16  0.04#
2.49
15.00  2.35
4.27a,b 13.23  3.51b
4.03
15.24  5.22
(continued on next page)

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120

C. Molt-Puigmart et al. / Clinical Nutrition 30 (2011) 116e123

Table 4 (continued)
Fatty acid

G.age group Colostrum

C18:3 n-6

VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT
VPT
PT
FT

c9t11-CLA

t10c12-CLA

C20:2 n-6

C20:3 n-6

C20:4 n-6

C22:2 n-6

C22:4 n-6

C22:5 n-6

PUFAs n-6

LCPUFA n-6

C18:3 n-3

C20:5 n-3

C22:5 n-3

C22:6 n-3

PUFA n-3

LCPUFA n-3

PUFA

n-6/n-3

LA/ALA

AA/DHA

EPA/DHA

0.09
0.05
0.05
0.12
0.13
0.14
0.010
0.009
0.010
0.76
0.98
1.09
0.82
0.89
0.75
0.78
1.02
0.92
0.15
0.19
0.20
0.31
0.61
0.55
0.12
0.20
0.17
17.61
17.10
19.88
2.78
3.70
3.49
0.33
0.35
0.38
0.06
0.06
0.07
0.24
0.37
0.36
0.51
0.66
0.55
1.13
1.44
1.37
0.80
1.09
0.99
18.91
18.75
21.47
16.18
12.47
15.37
44.61
38.40
45.17
1.61
1.71
1.79
0.11
0.10
0.12




































































0.04a,#,
0.02a,#
0.02a,
0.03
0.03a
0.06a,b
0.001a,b
0.001
0.001
0.22a,#,
0.23a,#
0.23a,
0.25a,b
0.16a,#
0.21a,#
0.14a,#,
0.015a,#
0.19a,
0.04a,#,
0.05a,#
0.04a,
0.10a,#,
0.14a,#
0.24a,
0.09a,#
0.11a,#
0.11a,b
6.12
3.74a
4.10a,b
0.48a,#,
0.60a,#
0.81a,
0.05a
0.09a,b
0.17a,b
0.02
0.01
0.03
0.07a,b,#,
0.10a,#
0.13a,b,
0.14a,#
0.24a,#
0.18a,b
0.21#,
0.33a,#
0.36a,b,
0.21a,#
0.31a,#
0.32a,b
6.13
3.80a
4.22a,b
7.09
4.13a,b
4.59
20.48a,b
9.08a,b
13.12a
0.43
0.57a
0.57
0.02a
0.04a,b,#
0.04a,#

Transitional
0.08
0.08
0.09
0.12
0.14
0.18
0.008
0.007
0.010
0.55
0.59
0.50
0.57
0.60
0.50
0.61
0.72
0.62
0.10
0.11
0.08
0.16
0.22
0.17
0.07
0.10
0.08
15.41
17.44
17.56
1.95
2.23
1.86
0.44
0.50
0.53
0.07
0.06
0.06
0.16
0.19
0.16
0.47
0.48
0.41
1.14
1.24
1.16
0.70
0.74
0.63
16.66
18.82
18.82
14.97
15.04
16.13
32.36
31.66
30.72
1.43
1.65
1.71
0.13
0.15
0.15




































































0.03b
0.03a
0.04a
0.04#
0.06b,
0.06a,#,
0.001a
0.001#
0.000#
0.13a
0.12a,#
0.12a,#
0.15a
0.10a,#
0.13a,#
0.10a,#
0.08a,#,
0.11a,
0.03a
0.03a,#
0.02a,#
0.03a,#
0.06a,#,
0.06a,
0.04a,#
0.03a,#
0.03a
2.58
4.39b
4.16a
0.22a,#
0.28a,#,
0.27a,
0.12a
0.16a
0.18a
0.05
0.05
0.04
0.05a
0.07a
0.05a
0.17b
0.15a
0.15a
0.33
0.36a
0.29a
0.26
0.25a
0.22a
2.50
4.47b
4.22a
5.97
5.87a
5.40
11.68a
13.15a
10.49a
0.50
0.54b
0.59
0.04b
0.07a
0.06a

Mature
0.11
0.12
0.12
0.13
0.17
0.18
0.007
0.009
0.009
0.43
0.40
0.38
0.52
0.53
0.43
0.52
0.54
0.49
0.06
0.06
0.05
0.11
0.13
0.11
0.05
0.06
0.06
17.06
15.42
17.28
1.63
1.65
1.48
0.59
0.52
0.60
0.06
0.05
0.08
0.14
0.13
0.15
0.36
0.29
0.35
1.14
1.00
1.18
0.55
0.47
0.58
18.29
16.50
18.53
15.60
16.39
16.50
28.89
28.55
27.00
1.59
2.02
1.81
0.16
0.19
0.20




































































0.04a,b
0.05a
0.03a
0.03
0.06a,b
0.10b
0.001b
0.001
0.001
0.13a
0.09a
0.10a
0.23b
0.17a
0.08a
0.12a
0.07a
0.08a
0.03a
0.02a
0.02a
0.04a
0.02a
0.03a
0.02a
0.01a
0.07b
2.64
3.69a,b
5.31b
0.52a
0.27a
0.22a
0.27a
0.19b
0.25b
0.02
0.02
0.02
0.03b
0.03a
0.07b
0.15a,b
0.08a
0.27b
0.30
0.25a
0.57b
0.20a
0.12a
0.46b
2.80
3.72a,b
5.49b
3.37
6.21b
6.00
10.04b
14.43b
8.71a
0.48
0.77a,b
0.70
0.03a,b
0.09b
0.09a

Results expressed as mean  SD. G. age group refers to the three gestational age
groups: very preterm (VPT), preterm (PT), and full term (FT). Lower case letters
indicate differences among colostrum, transitional, and mature milk in each gestational age group (same letters indicate signicant differences, according to GLM and
LSD post-hoc test). Symbols indicate differences among gestational age groups, for
each stage of lactation (same symbols indicate signicant differences, according to
ANOVA and Bonferroni post-hoc test). Signicance: p < 0.05. LCPUFA (n-3 or n-6)
refers to the sum of polyunsaturated fatty acids with 20 or more carbon atoms.

of VPT and FT samples, but in the case of several LCPUFAs (including

AA and DHA), PT values were (or tended to be) the highest (Fig. 3a
and b, for differences in AA and DHA among groups).
4. Discussion
In the present study we analyzed colostrum, transitional and
mature milk samples from women delivering between 25.43 and
42 weeks of gestational age, and demonstrated that milk fat
content and fatty acid proportions differ depending on the stage of
lactation and gestational age.
We could only nd one study which did, more than twenty
years ago, a comprehensive investigation on the differences
between VPT, PT, and FT human milk during lactation.9 Among the
other studies on this topic, some of them studied the changes of fat
and/or fatty acid composition during lactation without clearly
differentiating samples according to gestational age.11,13,14 Others
focused solely on the changes in one of these three gestational age
groups10,15,17e22,24e26 or compared two groups.8,23 Some published
studies either did not specify whether the PT group also included
VPT milk samples or put PT and VPT milk samples together in the
same group.12,16,29 Finally, a study compared FT with PT colostrum,
but did not provide information about transitional and mature
milk.7 In addition, some of the above-mentioned studies focused on
specic fatty acids, giving not much detail about the rest or
showing a limited fatty acid prole. The present study provides an
updated and more complete picture of the changes in human milk
fat and fatty acids with the stage of lactation and gestational age.
The descriptive nature of this work makes it not possible to
know whether the observed changes respond to the particular
needs of newborns or are only the result of a different mammary
gland metabolism or fatty acid uptake from maternal tissues at
different gestational ages, and it also does not permit to state
whether the fat and fatty acid composition of VPT milk is the
optimal for VPT newborns. The objective of this work is, instead, to
provide a database for various types of milk and clues as to whether
the found differences may have an impact on infants health.
Our results on the changes of fatty acid proportions throughout
lactation essentially agree with previous works,8e17,19,21,24e26,29
with MCSFAs being lower and LCPUFAs being higher in colostrum
than in transitional and mature milk as a common nding. In some of
these works, lower proportions of MCSFAs early in lactation were
attributed to a still immature metabolism of the mammary gland
(which is responsible for the de novo synthesis of these fatty acids
from glucose). Regarding LCPUFAs, special emphasis must be put on
AA and DHA, which largely accumulate in fetal and infants tissues
during the last trimester of gestation and rst months after
birth.30e32 Both fatty acids are key components of cell membranes,
where they contribute to maintain a good cell uidity and activity.
DHA is especially abundant in brain and retina and is essential for the
proper neurodevelopment of the newborn, while AA is also required
for an adequate growth. Additionally, both fatty acids are precursors
for the synthesis of bioactive lipid mediators. Although AA and DHA
can be endogenously synthesized from the essential fatty acids LA
and ALA, respectively, both in term and preterm infants,33e36 this
synthesis is limited. Therefore, the supply of preformed AA and DHA
is very important. Just after birth, when newborns milk intake is
very low and AA and DHA demands very high, the supply of colostrum, which is percentually richer in preformed AA and DHA than
mature milk, may permit a proper intake of these fatty acids.
In the present work, we also studied the variations of CLA
proportions during lactation and with gestational age. CLA includes
the positional and geometrical isomers of LA with conjugated
double bonds; c9t11- and t10c12-CLA, the major isomers found in
human milk, have been suggested to regulate immune function and

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121

A 1.20
a,#

1.00

AA (%)

0.80

a,

a,#,

a,#,

a,

0.60

a,#

0.40

a
a
a

0.20
0.00
Colostrum

Mature milk

0.70
a,#

0.60
0.50
DHA(%)

Transitional milk

a,b

a
a,#

0.40

0.30

a,b
b
a

0.20
0.10
0.00
Colostrum

Fig. 2. Evolution of medium-chain saturated fatty acids (A) and long-chain saturated
fatty acids (B) throughout lactation according to the gestational age group. Solid,
broken, and dotted lines correspond to very preterm, preterm, and full term samples,
respectively. Lower case letters indicate differences among colostrum, transitional, and
mature milk in each gestational age group (same letters indicate signicant differences, according to GLM and LSD post-hoc test). Symbols indicate differences among
gestational age groups at each stage of lactation (same symbols indicate signicant
differences, according to ANOVA and Bonferroni post-hoc test). Signicance: p < 0.05.
MCSFA refers to the sum of C10:0 and C12:0 and LCSFA to the sum of saturated fatty
acids with 14 carbon atoms or more.

bone formation, and to have anticarcinogenic, antiatherogenic, and

antidiabetic activities [reviewed in Ref. 37]. We only know of one
study on CLA proportions during lactation.22 That study analyzed
transitional and mature milk from women delivering at term and
found no signicant differences between CLA proportions at those
time points, which agrees with our results. In the case of colostrum,
we found c9t11-CLA proportions to be lower than those in transitional and mature milk. However, whether this may have any
physiological implication for the newborn is not known. We additionally determined the proportions of the t10c12-CLA isomer,
which accounted for only a 5% of total CLA.
For MUFAs, we observed a clear trend of those with more than 18
carbons to decrease in percentage with the progression of lactation.
Shorter-chain MUFAs can also be endogenously converted into the
longer-chain ones. However, during the rst days of life, metabolic
pathways may be still not completely functional. Therefore, we
hypothesized (as with LCPUFAs) that during the rst days of life the
supply of colostrum could be important to provide the newborn
with higher proportions of the already preformed longer-chain
MUFAs; among them, nervonic acid (C24:1 n-9), for instance, has
been identied as a typical component of myelin sphingolipids and
therefore a key fatty acid during myelinogenesis.38

Transitional milk

Mature milk

Fig. 3. Evolution of arachidonic (A) and docosahexaenoic (B) acids throughout lactation according to the gestational age group. Solid, broken, and dotted lines correspond
to very preterm, preterm, and full term samples, respectively. Lower case letters
indicate differences among colostrum, transitional, and mature milk in each gestational age group (same letters indicate signicant differences, according to GLM and
LSD post-hoc test). Symbols indicate differences among gestational age groups at each
stage of lactation (same symbols indicate signicant differences, according to ANOVA
and Bonferroni post-hoc test). Signicance: p < 0.05.

Regarding variations among gestational age groups, most differences in the fatty acid composition were found between VPT and FT
groups, while the PT group had a fatty acid composition in between
that of the other two groups. Of note, most differences were found
among colostrums and transitional milks of the three gestational
age groups, while very few differences were observed among
mature milks. This is, at one month post-partum, milks from
women delivering at different gestational ages basically differed in
their fat content (higher in VPT milk) but their fatty acid composition was almost identical. The higher fat content in VPT than in PT
and FT milk, not only in mature milk but also in the other two
stages of lactation is worth mentioning: if considering that milk
intake by VPT infants is very low, the supply of more concentrated
milk (in terms of lipids) may be physiologically important.
For PUFAs and LCPUFAs, we found minimal changes in LA, ALA,
and EPA proportions according to gestational age, which agrees
with the studies from Kovcs et al., Genzel-Boroviczny et al.,
Luukkainen et al., Bitman et al., and Rueda et al.,9,12,16,23,29 all of
them reviewed in Bokor et al.39 According to this same review,
results for other LCPUFAs were more variable among studies: one
study16 could nd higher proportions of DHA, C20:3 n-6, and AA in
PT than in FT colostrum; later in lactation, two out of the ve
studies found higher proportions of C22:5 n-3, DHA, and AA16,29 in
PT than in FT milk, while one of these two also found higher

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122

C. Molt-Puigmart et al. / Clinical Nutrition 30 (2011) 116e123

proportions of C20:3 n-6.16 Our results basically agreed with the

above-mentioned results (higher LCPUFAs in PT than in FT milk),
but statistical signicance was not observed in all cases. When also
studying a VPT group, Bitman et al9 found a correlation between the
level of prematurity and the level of LCPUFAs from both families,
but we did not conrm this nding. In our study, the highest
proportions of LCPUFAs n-3 and n-6 were found in PT instead of
VPT samples. This could reect the fact that PT milk is secreted
during the period when the maximum accretion of LCPUFAs to the
fetus would take place. Instead, in the case of women delivering
VPT infants, it could be possible that their LCPUFAs stores were still
not large enough or still not ready for mobilization into milk.
Milk secreted at lower gestational ages had higher MCSFAs
proportions during the rst 15 days of lactation. This fact could entail
an advantage for VPT and PT newborns due to the properties of
MCSFAs: they constitute a source of energy easily absorbed by
neonates, irrespective of bile acids, they may enhance the absorption
of calcium, magnesium, and amino acids10 and they may have
antimicrobial activity.40 Interestingly, the observation that VPT
colostrum has higher proportions of these MCSFAs than the other
two groups, when one could expect the mammary gland to be more
immature and therefore less ready for the de novo synthesis of these
fatty acids, also suggests that the maturity of the mammary gland
may not be what determines the amount of MCSFAs to be synthesized. We hypothesized that the availability (or non-availability) of
LCPUFAs to be incorporated into milk may be what determines
MCSFA proportions in milk, nding a higher LCPUFA mobilization
into milk during the rst days post-partum than later in lactation
and a reduced mobilization in women with VPT deliveries in
comparison to those delivering PT or FT infants. This could explain
why colostrum (in comparison to transitional and mature milk) has
the lowest proportions of MCSFAs but the highest in LCPUFAs (we
found a Pearson correlation coefcient between MCSFAs and
LCPUFAs in colostrum 0.486, p-value 0.000) and why,
compared to the other two gestational age groups, VPT colostrum
has the highest proportions of MCSFAs but the lowest of LCPUFAs.
The higher MCSFA proportions in VPT milk could also permit
VPT infants to optimize the use of milk LCPUFAs. Rodrguez et al.41
demonstrated that infants fed an MCSFA-enriched formula had the
same plasmatic proportions of LA than when fed a non-supplemented formula, while their plasmatic LCPUFAs proportions
increased. This was probably due to a preferential oxidation of
MCSFA (instead of LA and LCPUFAs) to obtain energy. Therefore, we
speculated that VPT infants receiving VPT milk, richer in MCSFA but
lower in LCPUFAs, could optimize the use of the latter for
membrane formation thanks to the fact that MCSFA (and not PUFA)
would constitute their preferential source of energy.
Finally, among MUFAs, those with more than 18 carbons were
lower in VPT than in PT and FT colostrum, maybe also due (as we
hypothesized with LCPUFAs) to a lower availability of maternal fat
stores or a lower mobilization of these stores in women delivering
VPT infants. In transitional milk, instead, proportions in VPT and PT
milk were higher than in FT milk, leading to a more sustained
supply of these fatty acids in VPT and PT infants during at least the
rst 2 weeks of life.
Considered together, the present results highlight the interest of
controlling the sources of variability of human milk composition,
especially for nutrition of hospitalized newborns, since the differences between various types of milk may impact infants health.
The main limitation of this study is the relatively small sample size,
which entails that reported fatty acid proportions and fat concentrations may not be representative of a larger population. Also, the
collection of only hindmilk may result in an overestimation of the
fat content in all milk samples, due to the increase of the fat content
that naturally occurs during the feeding period.42,43

Further studies on changes of other human milk components

during lactation and with gestational age are warranted. We also
suggest performing clinical studies exploring whether preterm
newborns receiving preterm donor milk have any advantage with
respect to those receiving term donor milk, or whether giving
colostrum instead of pooled milk during the rst days of life can be
benecial. If that would be the case, human milk banks could take
more advantage of the particular characteristics of each different
kind of milk by not pooling together all the milk samples that they
receive but by separating them according to gestational age and
stage of lactation.
Conict of interest
The authors declare no conict of interest.
Acknowledgements
This study received nancial support from the research projects
AGL2005-069401/ALI from the Spanish Ministry of Education and
Science, and AGL2008-04124/ALI and AGL2009-09730/ALI from the
Spanish Ministry of Science and Innovation. CMP was supported by
a predoctoral grant from the Spanish Ministry of Education and
Science.
CMP, AIC, XCE, and MCLS designed the study. CMP collected and
analyzed the data and wrote the manuscript. AIC, XCE, and MCLS
contributed to interpretation of the data and reviewed the manuscript. All authors read and approved the nal manuscript. The
authors thank Toni Monlen Getino for his statistical advice and Dr.
Gloria Moretones, Flor Torres Snchez, Carme Aud, and Chari
Casillas for their help during sample collection. Special thanks to
the women who donated their milk.
References
1. American Academy of PediatricsNutrition Committee of the Canadian Paediatric Societythe Committee on Nutrition of the American Academy of Pediatrics. Breast-feeding. A commentary in celebration of the International Year of
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