University of Groningen

Brain-selective nutrients in pregnancy and lactation

Stoutjesdijk, Eline

DOI:
10.33612/diss.146373942

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Stoutjesdijk, E. (2020). Brain-selective nutrients in pregnancy and lactation. University of Groningen.
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 4
DHA+EPA supplements during
pregnancy and lactation
 4.1
Fish oil supplemental dose needed to
reach 1 g% DHA+EPA in mature milk

Eline Stoutjesdijk1, Anne Schaafsma2,

D.A.Janneke Dijck-Brouwer1, Frits A.J. Muskiet1

1
University of Groningen and University Medical Center Groningen, Groningen, The Netherlands,
Department of Laboratory Medicine;
2
FrieslandCampina, Amersfoort, the Netherlands

Prostaglandines, Leukotrienes and Essential Fatty Acids 128 (2018) 53-61

Chapter 4
Abstract
Introduction
Erythrocyte (RBC) DHA+EPA is considered optimal at 8 g%. Mothers with lifetime high fish
intakes exhibiting this status produce milk with about 1 g% DHA+EPA. We established
DHA+EPA supplemental dosages needed to augment RBC DHA+EPA to 8 g% and milk
DHA+EPA to 1 g%.
Materials and methods
Pregnant women were randomly allocated to DHA+EPA dosages of: 225+90 (n=9), 450+180
(n=9), 675+270 (n=11) and 900+360 (n=7) mg/day. Samples were collected at 20 and 36
gestational weeks and 4 weeks postpartum.
Results
Linear regression revealed needed dosages rounded at 750 mg/day to reach 8 g% RBC
DHA+EPA and 1,000 mg/day for 1 g% milk DHA+EPA. RBC DHA+EPA increment depended
on baseline values. There was no effect on milk AA, but milk EPA/AA ratio increased.
Conclusion
Women with an RBC DHA+EPA status of 5.5 g% need 750 and 1,000 mg DHA+EPA/day
to reach 8 g% RBC DHA+EPA at the pregnancy end and 1 g% mature milk DHA+EPA,
respectively.
184
DHA+EPA supplements during pregnancy and lactation
Introduction
The health benefits of breastfeeding are widely acknowledged. Long chain
polyunsaturated fatty acids (LCPs), notably eicosapentaenoic- (EPA), docosahexaenoic-
(DHA) and arachidonic- (AA) acids, in milk are important for infant (neuro)development.
Meta analyses of randomized controlled trials (RCT) with prenatal and/or postnatal
LCP supplementation are inconclusive1-5. Failure to demonstrate the importance of LCP
in cognitive and behavioral outcomes may derive from many causes, including: dose,
duration and infant heterogeneity (including different baseline status and variety of post
weaning foods)6,7, while nutrient interactions are usually ignored. Small differences may,
however, result in subtle effects that are difficult to detect but could nevertheless be
relevant7.
4
Breast milk DHA content varies from 0.13-0.37 g per 100 g fatty acids (g%) in the
Netherlands8 to medians of 0.73 g% (Chole, Tanzania)9, 0.96 g% (Ukerewe, Tanzania)10, up
to 1.4 g% (Inuit, Canada)11. Breast milk AA exhibits less variation, ranging from 0.26-0.60 g%
in the Netherlands8 to medians of 0.50 g% (Chole, Tanzania)9, 0.55 g% (Ukerewe, Tanzania)10,
and 0.60 g% (Inuit, Canada)11. The estimated worldwide biological variation of breast milk
DHA is among the highest of all fatty acids (68%), while that of AA is among the lowest
(28%)12. This observation is in line with the dependence of milk DHA on maternal DHA
intake and status, and the independence of milk AA on maternal linoleic acid or AA intakes
and status10,13. Hsieh et al.14,15, showed that, in neonatal baboons, DHA in most tissues,
including the brain, is more sensitive to dietary intake than AA. Taken together, these
observations plead for a sufficient DHA intake to reach an optimal status in both mother
and child.
The Institute of Medicine (IOM) did not define adequate intakes (AI) for EPA and DHA for
0-6 months infants16. The current view is that during the first months of life, term infants
should receive 100 mg DHA/day and 140 mg AA/day, and hence infant formulas should
provide at least 0.3 g% DHA17. Although it has been argued that DHA should be provided
along with similar or higher levels of AA17-19, the European Food Safety Authority (EFSA)
stated that ‘there is no necessity to add AA to infant formula even in the presence of DHA’20.
A recent study with delta-6 desaturase (FADS2) knockout mice showed that, postnatally,
both AA and DHA intakes are important for (brain) growth and motor development21.
Breastfeeding mothers in the Netherlands are advised to achieve a minimum average
daily intake of 200 mg DHA, which translates to one portion of (oily) fish per week22. The
advice is expected to reach 0.3-0.4 g% DHA in milk13. Recently, the Global Organization
for EPA and DHA Omega-3S (GOED) recommended an intake of 700 mg DHA+EPA/day
for pregnant and lactating women23.
185
Chapter 4
Hibbeln et al.24 showed that a daily intake of 1 g DHA+EPA from seafood during pregnancy
associates with lowest offspring risk of low verbal IQ at 8 years. Their studies25 also
indicated that a milk DHA reaching 1 g% is associated to the lowest risk of postpartum
(PP) depression. Other investigators showed that an RBC DHA+EPA of about 8 g% at adult
age associates with lowest risk of cardiovascular disease26, lowest risk of depression27 and
optimal balance between DHA and AA status28. Taken together there is evidence that an
RBC DHA+EPA content of 8 g% in adults should be considered optimal. We argue that
mothers with adequate DHA+EPA status are likely to produce milk with adequate DHA+EPA
contents. Data from our group showed that mothers with lifetime high fish intakes and
an RBC DHA status of 8 g% at delivery, give birth to infants with an RBC DHA of 7-8 g%.
After 3 months exclusive breastfeeding, maternal RBC DHA was 7-8 g%, while infant RBC
DHA had increased to 8 g%. The corresponding breast milk DHA content at 3 months PP
was 1 g%10,29.
In the present study we investigated what DHA+EPA supplemental dose, provided from
20 gestational weeks (GW), augments RBC DHA+EPA to 8 g% and breast milk DHA+EPA to
1 g%. In the FRISO MUM intervention study, conducted earlier by our group, a daily dose
of 220 mg DHA increased RBC DHA from a median of 4.2 g% at 16 GW to a median of
5.5 g% at 36 GW [Van Goor, unpublished]. The median milk DHA contents were 0.60 g%
and 0.39 g% at 2 and 12 weeks PP, respectively13. From their dose-response study, Flock et
al.30 concluded that healthy adults with low RBC DHA+EPA contents, also named omega
3 index, of about 4.3 g% need intakes of 1 g DHA+EPA/day for 5 months to reach an RBC
DHA+EPA of 8 g%. Based on these studies we choose 4 daily supplemental dosages,
ranging from 225+90 to 900+360 mg DHA+EPA. In view of nutrient interaction we also
supplemented the women with a multivitamin and vitamin D. It has e.g. been shown that
the DHA status in rats is dependent on methylation capacity (folate, and vitamins B6 and B12
status)31,32, while EPA, DHA and vitamin D may interact in serotonin biology33. In addition,
we investigated whether the DHA+EPA supplements had adverse effects on the AA status.
186
DHA+EPA supplements during pregnancy and lactation
Methods and materials
This is a randomized controlled trial (ZOOG MUM) that was conducted in Groningen, The
Netherlands. It is part of the ZOOG (‘Zonder Ontsteking Oud en Gezond’) project. The
study was approved by the Ethics Committee of the University Medical Center Groningen
(UMCG) (METc number 2014.263) and was registered in The Netherlands National Trial
Register (Trial ID NTR4959). All women provided us with written informed consent. The
study was in agreement with the Helsinki declaration of 1975, as revised in 2013.
Subjects, supplements, sample collection, storages and analyses.
Forty-three apparently healthy and well nourished Dutch women in their first trimester
of a singleton pregnancy were invited to participate in the study. All expressed their
intension to exclusively breastfeed after birth. They were randomly allocated, by use of
4
block randomization, to four groups (Figure 1). Women with hyperemesis gravidarum,
or a vegetarian or vegan diet were excluded. Pregnancy- or neonatal complications
and premature delivery were criteria for termination of participation. Table 1 depicts the
various supplements, their daily dosages and percentages of the RDA/AI for pregnant and
lactating women. Each participant received a multivitamin (Omega Pharma; Rotterdam,
The Netherlands) that provided 12-125% of the Dutch RDA/AI for vitamins and minerals for
pregnant and lactating women. They also received increasing dosages of DHA-rich fish
oil and vitamin D supplements (both from Bonusan; Numansdorp, The Netherlands). The
total dosages were: 225+90 mg DHA+EPA and 10 µg vitamin D in group A; 450+180 mg
DHA+EPA and 35 µg vitamin D in group B; 675+270 mg DHA+EPA and 60 µg vitamin D in
group C; and 900+360 mg DHA+EPA and 85 µg vitamin D in group D.
187
Chapter 4 DHA+EPA supplements during pregnancy and lactation

Randomly assigned (n = 43)

Table 1. Daily supplemental dosages provided from about 20 gestational weeks to 4 weeks postpartum
First visit:
20 GW
Nutrient Daily dosage Dutch RDA/AI for Percentages of Dutch
Group A Group B Group C Group D
Multivitamin (10 µg vitamin D) Multivitamin (10 µg vitamin D) Multivitamin (10 µg vitamin D) Multivitamin (10 µg vitamin D) pregnancy/lactation1 RDA/AI for pregnancy/
225 mg DHA, 90 mg EPA 25 µg vitamin D 50 µg vitamin D 75 µg vitamin D
(n = 10) 450 mg DHA, 180 mg EPA 667 mg DHA, 270 mg EPA 900 mg DHA, 360 mg EPA lactation1
(n = 11) (n = 11) (n = 11)
Groups A, B, C or D:
Drop out :
Drop out: Disliked supplements DHA+EPA group A (mg) 315 (225+90) 200 157.5
Disliked supplements (n=1)
Drop out:
Disliked supplements
(n=1) DHA+EPA group B (mg) 630 (450+180) 200 315
Excluded:
(n=1)
Second visit: Excluded:
Pregnancy bleeding (n=1)
Use of anticoagulants
(n=1)
DHA+EPA group C (mg) 945 (675+275) 200 450
36 GW
DHA+EPA group D (mg) 1,260 (900+360) 200 630
Attended second visit Attended second visit Attended second visit Attended second visit
(n = 9) (n = 9) (n = 11) (n = 9)
Vitamin D group A (μg) 10 (in multivitamin) 10 100
Excluded: Vitamin D group B (μg) 35 (10+25) 10 350
Pregnancy hypertension
Third visit: (n=1)
Gestational diabetes
Vitamin D group C (μg) 60 (10+50) 10 600
4 weeks PP (n=1)

Completed study Completed study Completed study Completed study

Vitamin D group D (μg) 85 (10+75) 10 850
4
(n=9) (n=9) (n=11) (n=7)

Groups A, B, C and D:
Blood only (n =2) Blood only (n=1)
Milk only (n=1)
Beta carotene (µg) 12002 800/1100 vitamin A 38/27
Vitamin B1 (mg) 1.1 1.4/1.7 79/65
Figure 1. Flow chart of the initially 43 participating women. Pregnant women were supplemented from Vitamin B2 (mg) 1.4 1.4/1.7 100/82
20 gestational weeks (GW) to 4 weeks postpartum (PP). Blood samples were taken at 20 and 36 GW and Vitamin B3 (mg) 16 17/20 94/80
at 4 weeks PP. A milk sample was taken at 4 weeks PP. Vitamin B5 (mg) 6 5/7 120/86
Vitamin B6 (mg) 1.4 1.9 74
Vitamin B8 (µg) 50 40/45 125/111
Data on anthropometrics, socio-economic status and average fish intake were collected
Vitamin B11 (µg) 400 400/ 100
via questionnaires at the study beginning and/or end. Maternal EDTA-anticoagulated Vitamin B12 (µg) 2.5 3.2/3.8 78/66
venous blood samples were taken in the non-fasting state at the study start and at 36 Vitamin C (mg) 40 85/100 47/40
GW and 4 weeks PP. A milk sample was collected at 4 weeks PP. Blood samples were Vitamin D (µg) 10 10 100
Vitamin E (mg) 6 10/11 60/55
processed to plasma by centrifugation. RBC were isolated as described by Luxwolda et
al.10. A 200 µL aliquot of the washed 50% hematocrit RBC suspension was transferred to Calcium (mg) 120 1000 12
a telfon sealable Sovirel tube containing methanol/hydrochloric acid (5:1 v/v), butylated Chromium (µg) 20 30/45 67/44
hydroxytoluene (antioxidant) and 50 µg 17:0 (internal standard). These samples were stored Iodine (µg) 150 175/200 86/78
Copper (µg) 1000 1.0/1.3 100/77
at 4°C until analysis. Breast milk samples from a single completely emptied breast were Magnesium (mg) 56.25 280 20
collected around noon (10.00-14.00 hours) at the day prior to blood sampling. Milk was Manganese (mg) 1 3.0 50
collected manually or by breast milk pump. Following careful swirling it was divided into Molybdenum (µg) 25 65 38
Selenium (µg) 55 60 92
two portions. The participants stored the milk samples in their private freezers. They took
Iron (mg) 16.1 223/15 733/107
the samples with them in a cool transport container for frozen specimens (Sarstedt; mailing Zinc (mg) 10 9/11 111/82
containers) on the following day of blood sampling in the UMCG. The milk samples were
1
Dutch recommended dietary allowance (RDA) or adequate intake (AI) (34)
subsequently stored at -20 °C until analysis. Fatty acids in RBC and milk were measured by 2
Converted to vitamin A (in retinol units) (=300 µg beta-carotene),
capillary gas chromatography with flame ionization detection36,37. RBC DHA+EPA and milk 3
Adequate intake for pregnancy/lactation according to the Institute of Medicine (35)
DHA+EPA were expressed in g/100 g fatty acids (g%).

188 189
Chapter 4 DHA+EPA supplements during pregnancy and lactation

Data analysis and Statistics

p-value

0.896

1.000
0.667

0.892

0.322
0.851

0.355
0.501
0.532

0.476
0.130
0.149
The IBM PASW Statistics 22 software was employed. Since not all data were Gaussian

-
-
distributed we report medians and ranges. Between-group differences were analyzed with
the Kruskal-Wallis test for continuous data. Chi-square test was used for nominal data. A p
value < 0.050 was considered significant. Between-time points differences were analyzed

40 (38 - 42)
30 (21 - 38)
24 (21 - 26)
900+450
D (n = 7)

0.5 (0 - 1)
0.5 (0 -1)

5 (71%)
7 (100)
3 (2-4)
1 (0-2)
2 (1-5)

6 (86)

4 (57)
2 (28)
1 (14)

1 (14)
by Wilcoxon-Signed Rank Test. A p value <0.0167 was considered significant (Bonferroni
correction for 3 time points). The best fits for the analysis of the dose-response curves were
found in this order: cubic (R2=0.496), quadratic (R2=0.478) and linear regression (R2=0.472).
For practical purposes and small differences in fit, we choose the linear regression curves

22 ( 19 - 27)
32 (25 - 36)

40 (37 - 41)
C (n = 11)
675+270

11 (100)

1 (0 - 2)
0 (0 - 2)
8 (75%)
to estimate the dosages needed to reach an RBC DHA+EPA of 8 g% and a milk DHA+EPA

3 (2-4)
2 (0-3)
1 (0-2)

7 (64)
4 (36)

5 (45)
5 (45)
Supplemental group

1 (9)
of 1 g%.

Results 4

40 (38 - 42)
31 (26 - 36)
24 (18 - 28)
450+180

1 (0 - 1.5)
1.5 (0 - 2)
B (n =9)

6 (67%)
9 (100)
3 (2-4)
1 (0-2)
2 (1-4)

4 (44)
2 (22)

2 (22)
7 (78)

3 (33)
Median GW (expressed in weeks+days) at the study entry was 20+3 (range 16+1-21+4). Due
to their stays abroad at 20 GW, two women planned their first visit at GW 16 and 18,
respectively. They were instructed to start taking the supplements at 20 GW. Of the 43

32 (27 - 37)

41 (40 - 42)
24 (19 - 29)

0.5 (0 - 1.5)
women randomly assigned to groups A-D, 36 completed the study (Figure 1). Three

0.5 (0 - 1)
A (n = 9)
225+90

8 (89%)
9 (100)

9 (100)
3 (2-4)
1 (0-2)
2 (1-5)

4 (44)
4 (44)
1 (11)
discontinued voluntarily, two were excluded before 36 GW (pregnancy bleeding and

-
use of anticoagulants) and two were excluded because of late pregnancy complications
(pregnancy induced hypertension, gestational diabetes). All 36 included women reported

0.4 (0.0 - 2.0)

0.8 (0 - 2.0)
24 (18 - 29)

41 (37 - 42)
All (n=36)
compliance with the study protocol. Breast milk samples were available from only 33

31 (21-38)

27 (75%)
36 (100)
2 (0 - 5)

28 (80)
1 (0 -2)

14 (39)
15 (42)
3 (2-4)

7 (20)

7 (20)
mothers, since three discontinued breastfeeding prior to 1 month PP. One mother provided

Table 2. Characteristics of the 36 included mothers and their infants

a milk sample at 4 weeks PP, but did not report for blood sampling.

Dimensions
Characteristics of mothers and their infants

portion
portion
(weeks)
(kg/m )
(years)
2

n (%)

n (%)
n (%)

n (%)
n (%)
n (%)

n (%)
n

n
n
Table 2 shows the characteristics of the 36 included mothers and their infants. The median
maternal age at the study start was 31 (range 21-38) years. Their pre-pregnancy BMIs were
24 (range 18-29 kg/m2). They delivered at term at a median of 41 (range 37-42) GW. Eighty
percent completed college or university and 42% had an annual household income of

Intermediate vocational, High school or less

€50,000 or more. Median fish consumption at the study start was 0.8 (range 0-2) portions

Vitamin supplements before start study

(a portion fish equals 100-150 g fish38) per week, of which 0.4 (range 0-2) consisted of oily
fish. Seventy-five percent of the women took a vitamin supplement before the study start,

College, university or higher

of which 25% contained low dose fish oil (200 mg DHA). Of the 36 infants, 17 were boys

Annual household income

Maternal characteristics

Socio-economic status
Married/living together
and 19 were girls. Their median birth weights were 3,790 (range 2,440-5,020) g. The milk

Dose DHA+EPA (mg)

Weekly oily fish intake

€10,000 – €30.000
€30,000 - €50.000
Household number

€50,000 or more
Pre-pregnancy BMI

Gestation duration
samples were collected at 4.4 (range 3.5-5.3) weeks PP. There were no significant differences

Weekly fish intake

between the characteristics of the various supplemental groups, other then the collection

Education
Gestation
of milk samples, which we consider not to be clinically relevant. Variable

Other
Para
Age
190 191
Chapter 4 DHA+EPA supplements during pregnancy and lactation

Data represent medians (ranges). Groups A-D received supplements from 20 gestational weeks (GW) to 4 weeks postpartum (PP). The nutrients and their dosages are given
Dose needed to reach RBC DHA+EPA of 8 g% at 36 weeks GA
p-value

0.860
0.540

0.530

0.579
0.033
0.671
Figure 2 shows the relations between the DHA+EPA dosages and RBC DHA+EPA at the
study start, at 36 GW and at 4 weeks PP. Groups A-D did not exhibit differences between
RBC DHA+EPA contents at the start (p=0.267). The median RBC DHA+EPA content for the

54.5 (49.5 - 58)

whole group at the study start was 5.5 (range 3.3-8.5) g%. At 36 GW the RBC DHA+EPA

(3440 - 4695)
5.2 (3.6 - 5.6)

4.6 (4 - 5.1)
900+450
D (n = 7)

2 (29%)

3 (43%)
3870
medians had increased to 6.5 (range 5.5-8.6) g% for group A, 7.4 (range 6.2-9.3) g% for
group B, 8.7 (range 8.1-10.4) g% for group C and 9.5 (range 6.0-11.3) g% for group D (p<0.010
for between group differences). The linear relation between the DHA+EPA dosages and
(3070 - 4610) RBC DHA+EPA at 36 GW was y = 5.8 + 2.9 * 10-3 x, where x is the DHA+EPA dosage (mg)
4.4 (4.0 - 5.2)

4 ( 3.6 - 4.9)
51 (48 - 55)
C (n = 11)
675+270

7 (64%)
4 (36%)

and y is the RBC DHA+EPA content (g %). Employing this relation we found that the
Supplemental group

3750

DHA+EPA dose needed to reach the 8 g% RBC DHA+EPA target at 36 GW was 759 mg/
day. At 4 weeks PP the medians of the RBC DHA+EPA contents were 6.5 (range 6.0-7.6) g%
for group A, 7.4 (range 6.5-8.9) g% for group B, 8.6 (range 6.8-9.9) g% for group C and 9.4
4
(2440 - 4640)
4.7 (3.9 - 5.7)

4.4 (3.7 - 5.3)

55 (45 - 59)
450+180

(range 6.7-11.3) g% for group D. Supplemental Table 1 presents the data of RBC DHA+EPA,
B (n =9)

4 (44%)
1 (11%)

3965

DHA, EPA and AA, and the EPA/DHA and EPA/AA ratios for groups A-D at 20 GW, 36 GW
and 4 weeks PP.
51.5 (48 - 55.5)

(2870 - 5020)

4.4 (3.5 - 4.7)

4.4 (3.9 - 5.6)
A (n = 9)
225+90
2 (22%)

3 (33%)
3790
(2440 - 5020)
4.5 (3.6 - 5.7)

4.4 (3.5 - 5.3)

52 (45 - 59)
All (n=36)

17 (47%)
9 (25%)

3790
Characteristics of the 36 included mothers and their infants

Dimensions

(% male)
(weeks)
n (%)

(cm)
(kg)
(g)

in Table 1. A p-value <0.050 is considered significant.

of which contained fish oil
Infant characteristics
Dose DHA+EPA (mg)
Table 2. (Continued)

Lactation duration
Birth weight
Variable

Length2

Gender
Weight

192 193
Chapter 4 DHA+EPA supplements during pregnancy and lactation

Dependence of RBC DHA+EPA increments on baseline status and dose

Daily dose DHA+EPA Figure 4 shows, for each of the supplemented groups A-D, the relation between the
A: 225+90 mg (n=9)#
12
B: 450+180 mg (n=9)
C: 675+270 mg (n=11)
baseline RBC DHA+EPA content (at 20 GW) and the increment of the RBC DHA+EPA content
D: 900+360 mg (n=7)
(∆ DHA+EPA in g%) from 20 to 36 GW. The RBC DHA+EPA increments were found to be
10
dependent on the baseline DHA+EPA status: there were higher RBC DHA+EPA increments
at low baseline status. The increments also gradually diminished with dose, suggested
RBC DHA+EPA (g%)

8
the reach of RBC DHA+EPA saturation at high dose. Although all participants reported
compliance with the protocol, one woman in group D showed no increment of RBC
6 DHA+EPA (triangle in Figure 4). RBC DHA+EPA saturation was estimated to occur at about
10 g%, as suggested by extrapolation of the curves to y=0.
4

Effects of the supplements on milk AA and the milk EPA/DHA and EPA/AA ratios
2 Figure 3 panels D-F show the relations between the DHA+EPA dosages and milk AA
4
content (panel D), EPA/DHA ratio (panel E), and EPA/AA ratio (panel F) for groups A-D at
0
4 weeks PP. Supplemental Table 2 presents the corresponding hard data. There were no
20 GW 36 GW 4 weeks PP
relations between the DHA+EPA dosage and milk AA (panel D; p=0.159) and milk EPA/DHA
Time
ratio (panel E; p=0.195). The median milk AA content was 0.36 (range 0.23-0.63) g% and the
Figure 2. Relations between the DHA+EPA dosages and RBC DHA+EPA at the study start, at 36 gestational
median milk EPA/DHA ratio was 0.25 (range 0.05-0.37) g%. There was, however, a significant
weeks (GW) and at 4 weeks postpartum (PP). The subgroups did not differ in RBC DHA+EPA at the study
start. #Missing data from one women at 4 weeks PP; The linear dose-response relation at 36 GW was dose-dependent increase of the milk EPA/AA ratio (panel F; p<0.010). The medians of the
y = 5.8 + 2.9 * 10 -3 x (x in mg; y in g %). Horizontal line indicates the target of 8 g% RBC DHA+EPA. The milk EPA/AA ratios at 4 weeks PP were 0.23 (range 0.09-0.34) for group A, 0.48 (range 0.33-
calculated DHA+EPA dose needed to reach the RBC DHA+EPA target of 8 g% at 36 weeks GW was 759 0.69) for group B, 0.42 (range 0.16-0.69) for group C and 0.74 (range 0.17-0.88) for group D.
mg/day.

Dose needed to reach milk DHA+EPA of 1 g% at 4 weeks PP

Figure 3 panels A-C show, for each of the groups A-D at 4 weeks PP, the dose-response
curves for milk DHA+EPA (panel A), DHA (panel B) and EPA (panel C), respectively.
Supplemental Table 2 presents the corresponding hard data. The medians of milk
DHA+EPA at 4 weeks PP were 0.36 (range 0.27-0.75) g% for group A, 0.81 range (0.56-
1.06) g% for group B, 1.01 (range 0.71-1.31) g% for group C, and 1.08 (range 0.68-1.68) g%
for group D. The linear relation between the DHA+EPA dosage and milk DHA+EPA was
y = 0.21 + 8.3 * 10-4 x, where x is the DHA+EPA dosage (mg) and y is the milk DHA+EPA
content (g%). The calculated DHA+EPA dose needed to reach the 1 g% milk DHA+EPA
target at 4 weeks PP was 952 mg/day. The milk DHA medians at 4 weeks PP (panel B) were
0.29 (range 0.22-0.60) g% for group A, 0.63 (range 0.42-0.83) g% for group B, 0.83 (range
0.55-1.07) g% for group C, and 0.85 (range 0.57-1.40) g% for group D. The milk EPA medians
at 4 weeks PP (panel C) were 0.08 (range 0.04-0.16) g% for group A, 0.19 (range 0.11-0.23) g%
for group B, 0.18 (range 0.04-0.26) g% for group C, and 0.25 (range 0.11-0.30) g% for group D.

194 195
Chapter 4 DHA+EPA supplements during pregnancy and lactation

1.5

2.00 A B Dose DHA+EPA

225+90 mg DHA+EPA
450+180 mg DHA+EPA
675+270 mg DHA+EPA
1.50 900+360 mg DHA+EPA
Milk DHA+EPA (g%)

1.0
225+90 mg DHA+EPA
6.00 y=7.69-0.73*x

Milk DHA (g%)

450+180 mg DHA+EPA

DHA+EPA (g%) 20 GW - 36 GW
675+270 mg DHA+EPA
y=7.25-0.73*x 900+360 mg DHA+EPA
1.00

.5

.50 4.00

y=4.68-0.44*x

.00 .0
A B C D A B C D
225+90 450+180 675+270 900+360 225+90 450+180 675+270 900+360
(n=7) (n=8) (n=11) (n=7) (n=7) (n=8) (n=11) (n=7)

Daily dose DHA+EPA (mg) Daily dose DHA+EPA (mg) 2.00 y=3-0.35*x

.40 C D
.60 .00

.30
4
Milk EPA (g%)

Milk AA (g%)

3.00 4.00 5.00 6.00 7.00 8.00 9.00

.40

.20 RBC DHA+EPA (g%) at 20 GW (baseline)

Figure 4. Relation between baseline RBC DHA+EPA content (at 20 GW) and the RBC DHA+EPA increment
.20
.10

(∆ DHA+EPA in g%) from 20 to 36 GW. Linear relations are given for the four supplemental groups A-D,
.00 .00
who received supplements from 20 gestational weeks (GW) to 4 weeks postpartum (PP). The nutrients
A B C D A B C D
225+90
(n=7)
450+180
(n=8)
675+270
(n=11)
900+360
(n=7)
225+90
(n=7)
450+180
(n=8)
675+270
(n=11)
900+360
(n=7) and their dosages are given in Table 1.
Daily dose DHA+EPA (mg) Daily dose DHA+EPA (mg)

.40 E 1.00 F Discussion

.80
Ratio milk EPA/AA (g%)

.30
We investigated the DHA+EPA supplemental dose needed to reach an RBC DHA+EPA of
Ratio milk EPA/DHA

.60
8 g% at the pregnancy end and to reach a mature breast milk DHA+EPA content of 1 g%.
.20

.40
An approach by constructing linear dose-response relations estimated these dosages at
.10
759 mg (Figure 2) and 952 mg (Figure 3. Panel A) DHA+EPA, respectively. In view of the
.20
limited number of women in this trial, and for all practical reasons, we consider rounded
dosages of 750 mg and 1,000 mg appropriate to reach these goals. We also found that at
Page 1
.00 .00
A B C D A B C D
225+90 450+180 675+270 900+360 225+90 450+180 675+270 900+360
(n=7) (n=8) (n=11)

Daily dose DHA+EPA (mg)

(n=7) (n=7) (n=8) (n=11)

Daily dose DHA+EPA (mg)

(n=7)
Figure 3. Relations at 4 weeks postpartum (PP) between the DHA+EPA dosages and milk DHA+EPA
(panel A), DHA (panel B), EPA (panel C), AA (panel D), EPA/DHA ratio (panel E), and EPA/AA ratio (panel
F). The linear relation between the DHA+EPA dosages and milk DHA+EPA was y = 0.21 + 8.3 * 10 - 4 x
(x in mg and y in g%; panel A). Horizontal line in panel A indicates the target of 1 g% milk DHA+EPA. The
calculated DHA+EPA dose needed to reach this target was 952 mg/day. There were no relations between
the DHA+EPA dosage and milk AA (p=0.134; panel D) and milk DHA/EPA (p=0.156, panel E).
a given dose, the maternal RBC DHA+EPA increment was inversely related to the baseline
DHA+EPA status (Figure 4). In other words, there was an RBC DHA+EPA ceiling effect:
an RBC DHA+EPA maximum seems to be reached at about 10 g% (Figure 4). Regarding
potential adverse effects, we observed no influence of the employed dosages on the milk
AA content (Figure 3, panel D), no increase of the milk EPA/DHA ratio (Figure 3, panel E),
but increases of the milk EPA content (Figure 3, panel C) and milk EPA/AA ratio (Figure 3,
panel F).

196 197
Chapter 4
Dose needed to reach the 8 g% RBC DHA+EPA target at 36 gestational weeks
We found that 750 mg DHA+EPA/day is sufficient to increase the RBC DHA+EPA to 8
g% at the pregnancy end for women with a median baseline RBC DHA+EPA of 5.5 g%
(range 3.3-8.5). It was previously shown that RBC DHA contents of 7.339 and 7.2 g%40 at the
pregnancy end were reached by supplementing 600 mg DHA for 25 weeks and 1,200
mg for 17 weeks, respectively. Consistent with dependence on baseline, these pregnant
women had somewhat lower baseline RBC DHA of 4.3 and 4.6 g%.
The maternal RBC DHA+EPA contents did not change from 36 GW to 4 weeks PP. However,
within this period, RBC EPA increased, whereas RBC DHA tended to decrease (Supplemental
Table 1). Decreasing postpartum maternal RBC DHA and increasing RBC EPA were previously
demonstrated in supplemented40 and unsupplemented10 lactating women. The observed
trend of decreasing RBC DHA from 36 GW to 4 weeks PP might indicate deteriorating
maternal DHA status due to transplacental transfer and losses via the milk.
Dose needed to reach milk DHA+EPA of 1 g% at 4 weeks PP
A daily dose of 1,000 mg DHA+EPA was needed to reach a milk DHA+EPA of 1 g% at 4
weeks PP. In a previous study, daily DHA dosages of 0, 200, 400, 900 and 1,200 mg, provided
to lactating women 5 days PP resulted in milk DHA contents of 0.21, 0.35, 0.46, 0.86 and
1.13 g% at 12 weeks PP41. This regimen, solely aimed at lactation, produced similar or even
higher milk DHA as compared with the present (median milk DHA of 0.29, 0.63, 0.83 and
0.85 g%, following 225+90, 450+180, 675+270 and 900+360 mg DHA+EPA/day, respectively,
from 20 GW to 4 weeks PP). The INFAT study42 showed a higher mean milk DHA of 1.34
g% at 6 weeks PP in women receiving 1,020+180 mg DHA+EPA/day from 15 GW, as
compared to the median 0.85 g% (range 0.57-1.40) milk DHA in our group D (900+360
mg DHA+EPA/day) at 4 weeks PP. Taken together, the currently available information from
DHA or DHA+EPA supplementation studies indicate differences in the milk DHA contents
that are reached. These may at least in part be explained by differences in baseline status
(see also 4.5), maternal body size and composition (notably fat percentage), supplement
composition (DHA+EPA vs. DHA only), dose, intervention duration and intervention period
(i.e. pregnancy+lactation vs. lactation only). In additionLCP in milk may derive notably from
adipose tissue. It has e.g. been estimated that 70% of linoleic acid (LA) in milk derives from
stores43, while selective mobilization of polyunsaturated fatty acids from adipose tissue has
also been suggested44. The influence of maternal weight gain and losses during pregnancy
and lactation may therefore also be important.
198
DHA+EPA supplements during pregnancy and lactation
Discrepancy between dose needed to reach RBC DHA+EPA of 8 g% and milk
DHA+EPA of 1 g%
Based on our previously published data from women with lifetime high fish intakes we
did not expect to find differences in the needed dosages to reach an RBC DHA+EPA of 8
g% (750 mg/day) and a milk DHA+EPA of 1 g% (1,000 mg/day). Data from these women
showed concomitant 8 g% DHA+EPA in their RBC and 1 g% DHA+EPA in their mature
milk10,29. A possible explanation is that the current women did not reach equilibrium. A new
steady state in adipose tissue may take more than 1-2 years45, while the turnover rate of
DHA in (e.g.) brain is estimated at 2.5 years46. Our current data may therefore be confined
to pregnant women with lifetime suboptimal fish intakes.
Comparison of needed dose with existing recommendations
The need of 750 and 1,000 mg DHA+EPA/day to reach current RBC and milk DHA+EPA
4
targets is in agreement with the recommendation of 1 g DHA+EPA by Flock et al. and
the 700 mg DHA+EPA recommendation for pregnant and lactating women by GOED23.
Not surprisingly, this dosage is also in agreement with optimal secondary prevention
from cardiovascular disease by the American Heart Association47 and for the treatment of
affective disorders by the American Psychiatric Association27,48.
Dependence of RBC DHA+EPA increments on baseline status and ceiling effect
The RBC DHA+EPA increment did not only depend on dose, but also on baseline
RBC DHA+EPA (Figure 4). This finding is in agreement with Flock et al.30, who showed
that participants with lower baseline status had more profound responses upon
supplementation than participants with higher baseline status. Our data also suggested
that a maximum RBC DHA+EPA content may be reached at 10 g%, which is consisted with
our earlier studies reporting on RBC DHA contents in populations with life-long high fish
intakes. In these studies, we observed that the RBC DHA content plateaus at about 9 g%,
with an upper maximum of about 12 g%10.
Effects on milk AA content and milk EPA/DHA and EPA/AA ratios
The synthesis of AA from LA, and of EPA and DHA from ALA, makes use of the same
desaturases and chain elongases. Each of these LCP, endogenously synthesized or from the
diet, may subsequently compete for incorporation into (phospho)lipids49. The outcome of
the latter has functional consequences, since the lipid mediators from the LCPw3 and -w6
series, like eicosanoids, resolvins and (neuro)protectins, often have opposing action. For
instance, in contrast to those from AA, those from EPA lower blood clotting, inflammation
and cell growth, while they lower vascular resistance by vasodilatation50. A low EPA/AA ratio
in adults is strongly related to a pro-inflammatory state51, while a high EPA/AA ratio in (very
low birthweight) infants has been firmly implicated in restricted growth52. Striving at w3/
199
Chapter 4 DHA+EPA supplements during pregnancy and lactation

w6 balance, the 2008 guidelines for LCP in infant formulas and baby foods recommend
that EPA should not exceed DHA and that DHA should not exceed AA53. Like others41,54,55,
we found that the increases of milk DHA and EPA (Figure 3, panels B and C) did not
negatively affect milk AA (Figure 3, panel D), but we did notice a trend. Although EPA never
exceeded DHA (Figure 3, panel E), there was an about 3-fold increase of milk EPA/AA ratio
when the lowest dose was compared with the highest (panel F, 0.23 vs. 0.74 g/g). The
supplement-induced higher EPA/AA ratio is unlikely to be potentially deleterious. These
ratios are well within the physiological range of 0.17-1.08 g/g (unpublished data calculated
from10) of mothers with life-long high fish intakes. The current milk EPA/AA ratio is also more
favorable compared with the seminal study of Carlson et al.56 in which infants exhibited
retarded growth upon feeding with marine oil-supplemented infant formula, as compared
with control formulas. The former contained 0.3 g% EPA, 0.2 g% DHA and no AA, whereas
the control held no LCP. It should also be noted that their study was with very low birth
weight infants weighing 748-1,390 g at delivery56. It seems, on the contrary, possible that,
like in adults51, a higher milk EPA/AA in infants also contributes to an advantageous, less
pro-inflammatory state.
Implications for future studies
The outcome of this study may have implications for future intervention trials. Contemporary
societies are dealing with a conflict between a man-made environment and our only
slowly evolving genome57. The current Western diet is low in fish intake, as compared to our
ancestors who have evolved for a great part in the land-water ecosystem58-61. Various RCTs
with DHA/DHA+EPA or fish oil have been performed during pregnancy and/or lactation,
with subtle outcomes at most1,2,4,7,62. Apart from the above mentioned shortcomings of
these studies (see 4.2), fish also contains relevant amounts of vitamins B12 , A and D and
iodine and selenium, among others63, jointly named brain selective nutrients64. They play
important roles in brain development65, but also interact31,33,66-69. By providing DHA and
EPA, together with vitamin D and a multivitamin with 12-125% of the RDA/AI, we tried to
avoid suboptimal vitamin, mineral and trace element status. Suboptimal micronutrient
status may e.g. affect fatty acid metabolism70 and incorporation into lipids31,32.
Conclusions
Women with a RBC DHA+EPA of about 5.5 g% need about 750 mg DHA+EPA/day to reach
an RBC DHA+EPA of 8 g% at the pregnancy end, and about 1,000 mg DHA+EPA/day to
reach a milk DHA+EPA of 1 g% at 4 weeks PP. The RBC DHA+EPA increment depends on
baseline values. The supplement had no potentially adverse effects on milk AA. The milk
EPA/AA ratio increased but remained within the physiological range. A daily 1,000 mg
DHA+EPA supplement in pregnancy may optimize both mother and child. This dosage is in
excellent agreement with those recommended for secondary prevention of cardiovascular
disease and affective disorders.
Acknowledgements
4
We thank the participants, the participating midwifery practices, ‘Moeders voor Moeders’,
and the UMCG Department of Obstetrics and Gynecology. The UMCG ‘Laboratory for
Special Chemistry’ is gratefully acknowledged for performing the analyses. We also thank
Ms. Ingrid A. Martini, Mr. Herman J.A. Velvis and Master students Eline Hemelt and Wietske
Hemminga for their participation in this study.
This work was supported by Ministry of Economic Affairs, Provinces of Drenthe, Province
of Groningen, the Netherlands. The supplements were kindly provided by Omega Pharma
(Rotterdam, The Netherlands) and Bonusan (Numansdorp, The Netherlands).

Limitations

A limitation of this study is the small number of participants. As previously mentioned in

4.2 the current dose might not be fully reproduced by others, because of the many factors
involved, one of these being the interaction with other nutrients.

200 201
Chapter 4 DHA+EPA supplements during pregnancy and lactation

(16) Institute of Medicine. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty acids,
References Cholesterol, Protein, and Amino Acids (Macronutrients). Washington, D. C: National Academies
Press; 2005.
(1) Gunaratne AW, Makrides M, Collins CT. Maternal prenatal and/or postnatal n-3 long chain poly- (17) Koletzko B, Carlson SE, van Goudoever JB. Should Infant Formula Provide Both Omega-3 DHA and
unsaturated fatty acids (LCPUFA) supplementation for preventing allergies in early childhood. Omega-6 Arachidonic Acid? Ann Nutr Metab 2015;66(2-3):137-138.
Cochrane Database Syst Rev 2015 Jul 22;(7):CD010085. doi(7):CD010085.
(18) Crawford MA, Wang Y, Forsyth S, Brenna JT. The European Food Safety Authority recommendation
(2) Delgado-Noguera MF, Calvache JA, Bonfill Cosp X, Kotanidou EP, Galli-Tsinopoulou A. Supple- for polyunsaturated fatty acid composition of infant formula overrules breast milk, puts infants
mentation with long chain polyunsaturated fatty acids (LCPUFA) to breastfeeding mothers for at risk, and should be revised. Prostaglandins Leukot Essent Fatty Acids 2015 Dec;102-103:1-3.
improving child growth and development. Cochrane Database Syst Rev 2015 Jul 14;(7):CD007901.
(19) Hadley KB, Ryan AS, Forsyth S, Gautier S, Salem N,Jr. The Essentiality of Arachidonic Acid in Infant
doi(7):CD007901.
Development. Nutrients 2016 Apr 12;8(4):216.
(3) Simmer K, Patole SK, Rao SC. Long-chain polyunsaturated fatty acid supplementation in infants
(20) EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA). Scientific Opinion on the essential
born at term. Cochrane Database Syst Rev 2008 Jan 23;(1):CD000376. doi(1):CD000376.
composition of infant and follow-on formulae. EFSA Journal 2014;12(7)(3760).
(4) Simmer K, Patole SK, Rao SC. Long-chain polyunsaturated fatty acid supplementation in infants
(21) Harauma A, Yasuda H, Hatanaka E, Nakamura MT, Salem N,Jr, Moriguchi T. The essentiality of ara-
born at term. Cochrane Database Syst Rev 2011 Dec 7;(12):CD000376. doi(12):CD000376.
chidonic acid in addition to docosahexaenoic acid for brain growth and function. Prostaglandins
(5) Jasani B, Simmer K, Patole SK, Rao SC. Long chain polyunsaturated fatty acid supplementation in Leukot Essent Fatty Acids 2017 Jan;116:9-18.
infants born at term. Cochrane Database Syst Rev 2017 Mar 10;3:CD000376.
(22) Health Council of the Netherlands. Dutch dietary guidelines 2015.. The Hague: Health Council of 4
(6) Innis SM. Omega-3 Fatty acids and neural development to 2 years of age: do we know enough the Netherlands 2015;publication no. 2015/24E(ISBN 978-94-6281-104-1).
for dietary recommendations? J Pediatr Gastroenterol Nutr 2009 Mar;48 Suppl 1:S16-24.
(23) the Global Organi zation of EPA and DHA Omega 3 s. GOED Publishes EPA and DHA Intake Rec-
(7) McCann JC, Ames BN. Is docosahexaenoic acid, an n-3 long-chain polyunsaturated fatty acid, ommendations. April 25 2016; Available at: http://www.goedomega3.com//news/viewnews/59.
required for development of normal brain function? An overview of evidence from cognitive Accessed 02/28, 2017.
and behavioral tests in humans and animals. Am J Clin Nutr 2005 Aug;82(2):281-295.
(24) Hibbeln JR, Davis JM, Steer C, Emmett P, Rogers I, Williams C, et al. Maternal seafood consumption
(8) van Goor SA, Smit EN, Schaafsma A, Dijck-Brouwer DA, Muskiet FA. Milk of women with lifetime in pregnancy and neurodevelopmental outcomes in childhood (ALSPAC study): an observational
consumption of the recommended daily intake of fish fatty acids should constitute the basis for cohort study. Lancet 2007 Feb 17;369(9561):578-585.
the DHA contents of infant formula. J Perinat Med 2008;36(6):548-549.
(25) Hibbeln JR. Seafood consumption, the DHA content of mothers’ milk and prevalence rates of
(9) Kuipers RS, Smit EN, van der Meulen J, Janneke Dijck-Brouwer DA, Rudy Boersma E, Muskiet FA. postpartum depression: a cross-national, ecological analysis. J Affect Disord 2002 May;69(1-3):15-29.
Milk in the island of Chole [Tanzania] is high in lauric, myristic, arachidonic and docosahexaenoic
(26) von Schacky C. Cardiovascular disease prevention and treatment. Prostaglandins Leukot Essent
acids, and low in linoleic acid reconstructed diet of infants born to our ancestors living in tropical
Fatty Acids 2009 Aug-Sep;81(2-3):193-198.
coastal regions. Prostaglandins Leukot Essent Fatty Acids 2007 Apr;76(4):221-233.
(27) McNamara RK. Evaluation of docosahexaenoic acid deficiency as a preventable risk factor for
(10) Luxwolda MF, Kuipers RS, Koops JH, Muller S, de Graaf D, Dijck-Brouwer DA, et al. Interrelationships
recurrent affective disorders: current status, future directions, and dietary recommendations.
between maternal DHA in erythrocytes, milk and adipose tissue. Is 1 wt% DHA the optimal human
Prostaglandins Leukot Essent Fatty Acids 2009 Aug-Sep;81(2-3):223-231.
milk content? Data from four Tanzanian tribes differing in lifetime stable intakes of fish. Br J Nutr
2014 Mar;111(5):854-866. (28) Luxwolda MF, Kuipers RS, Smit EN, Velzing-Aarts FV, Dijck-Brouwer DA, Muskiet FA. The relation
between the omega-3 index and arachidonic acid is bell shaped: synergistic at low EPA+DHA
(11) Innis SM, Kuhnlein HV. Long-chain n-3 fatty acids in breast milk of Inuit women consuming tra-
status and antagonistic at high EPA+DHA status. Prostaglandins Leukot Essent Fatty Acids 2011
ditional foods. Early Hum Dev 1988 Dec;18(2-3):185-189.
Sep-Oct;85(3-4):171-178.
(12) Smit EN, Martini IA, Mulder H, Boersma ER, Muskiet FA. Estimated biological variation of the
(29) Kuipers RS, Luxwolda MF, Sango WS, Kwesigabo G, Dijck-Brouwer DA, Muskiet FA. Maternal DHA
mature human milk fatty acid composition. Prostaglandins Leukot Essent Fatty Acids 2002 May-
equilibrium during pregnancy and lactation is reached at an erythrocyte DHA content of 8 g/100
Jun;66(5-6):549-555.
g fatty acids. J Nutr 2011 Mar;141(3):418-427.
(13) van Goor SA, Dijck-Brouwer DA, Hadders-Algra M, Doornbos B, Erwich JJ, Schaafsma A, et al.
(30) Flock MR, Skulas-Ray AC, Harris WS, Etherton TD, Fleming JA, Kris-Etherton PM. Determinants of
Human milk arachidonic acid and docosahexaenoic acid contents increase following supplemen-
erythrocyte omega-3 fatty acid content in response to fish oil supplementation: a dose-response
tation during pregnancy and lactation. Prostaglandins Leukot Essent Fatty Acids 2009 Jan;80(1):65-
randomized controlled trial. J Am Heart Assoc 2013 Nov 19;2(6):e000513.
69.
(31) Umhau JC, Dauphinais KM, Patel SH, Nahrwold DA, Hibbeln JR, Rawlings RR, et al. The relationship
(14) Hsieh AT, Anthony JC, Diersen-Schade DA, Rumsey SC, Lawrence P, Li C, et al. The influence of
between folate and docosahexaenoic acid in men. Eur J Clin Nutr 2006 Mar;60(3):352-357.
moderate and high dietary long chain polyunsaturated fatty acids (LCPUFA) on baboon neonate
tissue fatty acids. Pediatr Res 2007 May;61(5 Pt 1):537-545. (32) van Wijk N, Watkins CJ, Hageman RJ, Sijben JC, Kamphuis PG, Wurtman RJ, et al. Combined dietary
folate, vitamin B-12, and vitamin B-6 intake influences plasma docosahexaenoic acid concentration
(15) Hsieh AT, Brenna JT. Dietary docosahexaenoic acid but not arachidonic acid influences central
in rats. Nutr Metab (Lond) 2012 May 30;9(1):49-7075-9-49.
nervous system fatty acid status in baboon neonates. Prostaglandins Leukot Essent Fatty Acids
2009 Aug-Sep;81(2-3):105-110.

202 203
Chapter 4
(33) Patrick RP, Ames BN. Vitamin D and the omega-3 fatty acids control serotonin synthesis and action,
part 2: relevance for ADHD, bipolar disorder, schizophrenia, and impulsive behavior. FASEB J 2015
Jun;29(6):2207-2222.
(34) Vitamine Informatie Bureau. Hoeveel heb ik nodig? Available at: http://www.vitamine-info.nl/
hoeveel-heb-ik-nodig/. Accessed 03/01, 2017.
(35) Institute of Medicine. Dietary Reference Intakes (DRIs): Recommended Dietary Allowances and
Adequate Intakes, Elements Available at: https://www.nal.usda.gov/sites/default/files/fnic_up-
loads//RDA_AI_vitamins_elements.pdf. Accessed 03/01, 2017.
(36) Muskiet FA, van Doormaal JJ, Martini IA, Wolthers BG, van der Slik W. Capillary gas chromatographic
profiling of total long-chain fatty acids and cholesterol in biological materials. J Chromatogr 1983
Dec 9;278(2):231-244.
(37) Volmer M, Meiborg G, Muskiet FA. Simultaneous capillary gas chromatographic profiling of me-
dium-and long-chain fatty acid methyl esters with split injection. Correction for injection-related
discrimination by the ‘bracketing’ method. J Chromatogr 1988 Dec 30;434(2):385-394.
(38) Wilson-van den Hooven EC, van Raaij JMA, Ocké MC, van Rossum CTM, Verhagen H. Visuele
weergave van de voedingssituatie in Nederland: een instrument. RIVM Rapport 350060002/2008
2008(http://www.rivm.nl/dsresource?objectid=f9364276-3eaf-4a66-b110-7ec3225e5d93&t-
ype=org&disposition=inline).
(39) Carlson SE, Colombo J, Gajewski BJ, Gustafson KM, Mundy D, Yeast J, et al. DHA supplementation
and pregnancy outcomes. Am J Clin Nutr 2013 Apr;97(4):808-815.
(40) Much D, Brunner S, Vollhardt C, Schmid D, Sedlmeier EM, Bruderl M, et al. Effect of dietary interven-
tion to reduce the n-6/n-3 fatty acid ratio on maternal and fetal fatty acid profile and its relation
to offspring growth and body composition at 1 year of age. Eur J Clin Nutr 2013 Mar;67(3):282-288.
(41) Makrides M, Neumann MA, Gibson RA. Effect of maternal docosahexaenoic acid (DHA) supple-
mentation on breast milk composition. Eur J Clin Nutr 1996 Jun;50(6):352-357.
(42) Much D, Brunner S, Vollhardt C, Schmid D, Sedlmeier EM, Bruderl M, et al. Breast milk fatty acid
profile in relation to infant growth and body composition: results from the INFAT study. Pediatr
Res 2013 Aug;74(2):230-237.
(43) Koletzko B, Rodriguez-Palmero M, Demmelmair H, Fidler N, Jensen R, Sauerwald T. Physiological
aspects of human milk lipids. Early Hum Dev 2001 Nov;65 Suppl:S3-S18.
(44) Raclot T. Selective mobilization of fatty acids from adipose tissue triacylglycerols. Prog Lipid Res
2003 Jul;42(4):257-288.
(45) Katan MB, Deslypere JP, van Birgelen AP, Penders M, Zegwaard M. Kinetics of the incorporation
of dietary fatty acids into serum cholesteryl esters, erythrocyte membranes, and adipose tissue:
an 18-month controlled study. J Lipid Res 1997 Oct;38(10):2012-2022.
(46) Umhau JC, Zhou W, Carson RE, Rapoport SI, Polozova A, Demar J, et al. Imaging incorporation of
circulating docosahexaenoic acid into the human brain using positron emission tomography. J
Lipid Res 2009 Jul;50(7):1259-1268.
(47) Kris-Etherton PM, Harris WS, Appel LJ, American Heart Association. Nutrition Committee. Fish
consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 2002 Nov
19;106(21):2747-2757.
(48) Freeman MP, Hibbeln JR, Wisner KL, Davis JM, Mischoulon D, Peet M, et al. Omega-3 fatty acids: ev-
idence basis for treatment and future research in psychiatry. J Clin Psychiatry 2006 Dec;67(12):1954-
1967.
204
DHA+EPA supplements during pregnancy and lactation
(49) Gibson RA, Muhlhausler B, Makrides M. Conversion of linoleic acid and alpha-linolenic acid to
long-chain polyunsaturated fatty acids (LCPUFAs), with a focus on pregnancy, lactation and the
first 2 years of life. Matern Child Nutr 2011 Apr;7 Suppl 2:17-26.
(50) De Caterina R, Basta G. n-3 Fatty acids and the inflammatory response — biological background.
2001;3(European Heart Journal Supplements, Supplement D, D42–D49).
(51) Rupp H, Wagner D, Rupp T, Schulte LM, Maisch B. Risk stratification by the “EPA+DHA level” and
the “EPA/AA ratio” focus on anti-inflammatory and antiarrhythmogenic effects of long-chain
omega-3 fatty acids. Herz 2004 Nov;29(7):673-685.
(52) Carlson SE. Arachidonic acid status of human infants: influence of gestational age at birth and
diets with very long chain n-3 and n-6 fatty acids. J Nutr 1996 Apr;126(4 Suppl):1092S-8S.
(53) Koletzko B, Lien E, Agostoni C, Bohles H, Campoy C, Cetin I, et al. The roles of long-chain poly-
unsaturated fatty acids in pregnancy, lactation and infancy: review of current knowledge and
consensus recommendations. J Perinat Med 2008;36(1):5-14.
(54) Henderson RA, Jensen RG, Lammi-Keefe CJ, Ferris AM, Dardick KR. Effect of fish oil on the fatty acid
composition of human milk and maternal and infant erythrocytes. Lipids 1992 Nov;27(11):863-869.
(55) Lauritzen L, Jorgensen MH, Hansen HS, Michaelsen KF. Fluctuations in human milk long-chain 4
PUFA levels in relation to dietary fish intake. Lipids 2002 Mar;37(3):237-244.
(56) Carlson SE, Cooke RJ, Werkman SH, Tolley EA. First year growth of preterm infants fed standard
compared to marine oil n-3 supplemented formula. Lipids 1992 Nov;27(11):901-907.
(57) Cordain L, Eaton SB, Sebastian A, Mann N, Lindeberg S, Watkins BA, et al. Origins and evolution of
the Western diet: health implications for the 21st century. Am J Clin Nutr 2005 Feb;81(2):341-354.
(58) Broadhurst CL, Wang Y, Crawford MA, Cunnane SC, Parkington JE, Schmidt WF. Brain-specific lipids
from marine, lacustrine, or terrestrial food resources: potential impact on early African Homo
sapiens. Comp Biochem Physiol B Biochem Mol Biol 2002 Apr;131(4):653-673.
(59) Henn BM, Gignoux CR, Jobin M, Granka JM, Macpherson JM, Kidd JM, et al. Hunter-gatherer
genomic diversity suggests a southern African origin for modern humans. Proc Natl Acad Sci U
S A 2011 Mar 29;108(13):5154-5162.
(60) Marean CW. Pinnacle Point Cave 13B (Western Cape Province, South Africa) in context: The Cape
Floral kingdom, shellfish, and modern human origins. J Hum Evol 2010 Sep-Oct;59(3-4):425-443.
(61) Muskiet FAJ, Kuipers RS. Lessons from shore-based hunter-gatherer diets in East Africa. In: Cunnane
SC, Stewart KM, editors. Human Brain Evolution. 2010; ISBN: 987-0-470-45268-4 ed.: Wiley-Blackwell
Hoboken, New Jersey.
(62) Muhlhausler BS, Yelland LN, McDermott R, Tapsell L, McPhee A, Gibson RA, et al. DHA supple-
mentation during pregnancy does not reduce BMI or body fat mass in children: follow-up of
the DHA to Optimize Mother Infant Outcome randomized controlled trial. Am J Clin Nutr 2016
Jun;103(6):1489-1496.
(63) Bourre JM, Paquotte PM. Contributions (in 2005) of marine and fresh water products (finfish and
shellfish, seafood, wild and farmed) to the French dietary intakes of vitamins D and B12, selenium,
iodine and docosahexaenoic acid: impact on public health. Int J Food Sci Nutr 2008 Sep;59(6):491-
501.
(64) Cunnane SC, Crawford MA. Energetic and nutritional constraints on infant brain development:
implications for brain expansion during human evolution. J Hum Evol 2014 Dec;77:88-98.
(65) Morse NL. Benefits of docosahexaenoic acid, folic acid, vitamin D and iodine on foetal and infant
brain development and function following maternal supplementation during pregnancy and
lactation. Nutrients 2012 Jul;4(7):799-840.
205
206
(69)
(66)

(67)

(68)

(70)
Chapter 4

Fatty Acids 2008 Jan;78(1):11-19.

study. Lancet 2007 Jul 21;370(9583):216.

Endocrinology 1979 Mar;104(3):774-779.

randomized controlled trial. Am J Clin Nutr 2015 Jul;102(1):215-221.
exploration of heterogeneity. J Epidemiol Community Health 2007 Jul;61(7):631-637.

impaired elderly: the importance of long-chain omega-3 fatty acids and B vitamin status in a
Jerneren F, Elshorbagy AK, Oulhaj A, Smith SM, Refsum H, Smith AD. Brain atrophy in cognitively
pression, dementia, and Alzheimer’s disease--but how and why? Prostaglandins Leukot Essent

Dullemeijer C, Durga J, Brouwer IA, Verhoef P. Erythrocyte folate and plasma DHA in the FACIT
Das UN. Folic acid and polyunsaturated fatty acids improve cognitive function and prevent de-

Manku MS, Horrobin DF, Karmazyn M, Cunnane SC. Prolactin and zinc effects on rat vascular
Gilbody S, Lightfoot T, Sheldon T. Is low folate a risk factor for depression? A meta-analysis and

reactivity: possible relationship to dihomo-gamma-linolenic acid and to prostaglandin synthesis.

Supplemental Table 1. DHA+EPA. DHA, EPA and AA, and EPA/DHA and EPA/AA ratios in erythrocytes for groups A-D at 20 gestational weeks (GW), 36 GW and 4
weeks postpartum (PP).

Supplementation group
Variable Group 20 GW p-value 36 GW p-value 4 weeks PP p-value p-value p-value p-value
GW20- GW36 GW20-4WPP GW36-4W PP
RBC – (g%)
DHA+EPA A 5.5 (3.9-8.5) 6.5 (5.5-8.6) 6.5 (6.0-7.6) 0.008 0.069 0.575
B 4.6 (3.6-7.5) 7.4 (6.2-9.3) 7.4 (6.5-8.9) 0.008 0.008 0.859
0.267 0.001 0.000
C 6.0 (4.4-6.9) 8.7 (8.1-10.4) 8.6 (6.8-9.9) 0.005 0.007 0.016
D 6.2 (3.3-7.2) 9.5 (6.0-11.3) 9.4 (6.7-11.3) 0.028 0.018 0.612
DHA A 5.1 (3.6-7.7) 6.1 (5.1-7.8) 5.8 (5.4-6.7) 0.008 0.093 0.263
B 4.2 (3.3-6.9) 6.5 (5.8-8.3) 6.5 (5.7-7.6) 0.008 0.008 0.214
0.324 0.001 0.001
C 5.5 (4.1-6.3) 7.7 (7.1-9.1) 7.4 (5.7-8.2) 0.005 0.007 0.003
D 5.9 (3.0-6.6) 8.2 (5.7-10.0) 7.8(6.0-9.6) 0.028 0.018 0.043
EPA A 0.41 (0.30-0.79) 0.43 (0.37-0.82) 0.61 (0.53-1.01) 0.110 0.012 0.012
B 0.36 (0.28-0.63) 0.76 (0.43-0.99) 1.13 (0.76-1.51) 0.008 0.008 0.008
0.493 0.000 0.000
C 0.44 (0.26-0.69) 1.04 (0.81-1.37) 1.18 (0.78-1.64) 0.005 0.005 0.004
D 0.36(0.28-0.65) 1.21 (0.31-1.37) 1.59 (0.70-1.70) 0.028 0.018 0.018
AA A 13.0 (11.1-14.5) 12.0 (9.9-13.4) 12.1 (10.5-13.7) 0.008 0.012 0.123
B 13.1 (11.7-14.5) 11.3 (10.2-12.4) 11.6 (10.7-12.8) 0.008 0.008 0.139
0.824 0.706 0.169
C 13.3 (12.0-14.7) 11.4 (9.7-12.6) 11.3 (8.4-12.9) 0.005 0.005 0.477
D 12.9 (11.1-14.7) 11.3 (9.6-13.9) 10.7 (9.3-13.7) 0.018 0.008 0.028
EPA/DHA A 0.08 (0.06-0.10) 0.08 (0.05-0.11) 0.11 (0.08-0.15) 0.314 0.012 0.012
B 0.09 (0.06-0.12) 0.12 (0.07-0.14) 0.17 (0.12-0.23) 0.011 0.008 0.011
0.826 0.000 0.002
C 0.09 (0.06-0.12) 0.13 (0.10-0.15) 0.18 (0.10-0.20) 0.005 0.005 0.004
D 0.09 (0.06-0.10) 0.14 (0.05-0.17) 0.20 (0.12-0.22) 0.028 0.018 0.018
207
DHA+EPA supplements during pregnancy and lactation

4
Chapter 4 DHA+EPA supplements during pregnancy and lactation

Supplemental Table 2. Milk DHA+EPA, milk DHA, milk EPA, ratio milk EPA/DHA and ratio milk EPA/AA

GW36-4W PP
p-value in the different groups at 4 weeks PP.

0.004
0.008
0.012

0.018

Data are medians (ranges). A p-value < 0.050 was considered significant between the groups, a p-value < 0.0167 was considered significant between the time points.
Supplementation group
Variable Group 4 weeks PP p-value
GW20- GW36 GW20-4WPP

Milk – (g%)
p-value

0.008
0.005
0.012

0.018 DHA+EPA

Groups A-D received supplements from 20 gestational weeks (GW) to 4 weeks postpartum (PP). The nutrients and their dosages are given in Table 1.
A 0.36 (0.27-0.75)
B 0.81 (0.56-1.06)
<0.01
C 1.01 (0.71-1.31)
D 1.08 (0.68-1.68)
p-value

0.008
0.005
0.011

0.028

DHA A 0.29 (0.22-0.60)

B 0.63 (0.42-0.83)
<0.01
C 0.83 (0.55-1.07)
p-value

4
0.000

D 0.85 (0.57-1.40)
EPA A 0.08 (0.04-0.16)
B 0.19 (0.11-0.23)
0.05 (0.04-0.08)
0.10 (0.06-0.12)

<0.01
0.15 (0.05-0.18)
0.11-0.06-0.15)
4 weeks PP

C 0.18 (0.04-0.26)
D 0.25 (0.11-0.30)
AA A 0.33 (0.26-0.46)
B 0.34 (0.3-0.63)
0.159
p-value
Supplementation group

0.000

C 0.42 (0.23-0.58)
D 0.36 (0.27-0.63)
EPA/DHA A 0.27 (0.15-0.35)
0.04 (0.03-0.06)
0.07 (0.04-0.09)
0.09 (0.06-0.13)
0.11 (0.02-0.14)

B 0.26 (0.23-0.37)
36 GW

0.195
C 0.25 (0.05-0.31)
D 0.22 (0.19-0.32)
EPA/AA A 0.23 (0.09-0.34)
p-value

0.656

B 0.48 (0.33-0.69)
<0.01
C 0.42 (0.16-0.69)
D 0.74 (0.17-0.88)
0.03 (0.02-0.06)

0.03 (0.02-0.06)
0.03 (0.02-0.05)
0.03 (0.02-0.05)

Data are median (range). A p-value < 0.050 is considered significant

20 GW
Supplemental Table 1. (Continued)

Group

D
A

C
B
Variable

EPA/AA

208 209