Brain-selective nutrients in pregnancy and lactation
Stoutjesdijk, Eline
DOI:
10.33612/diss.146373942
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Publication date: 2020
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Stoutjesdijk, E. (2020). Brain-selective nutrients in pregnancy and lactation. University of Groningen. https://doi.org/10.33612/diss.146373942
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4
DHA+EPA supplements during
pregnancy and lactation
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
Fish oil supplemental dose needed to
reach 1 g% DHA+EPA in mature milk
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.
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.
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.
186 187
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.
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 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.
Randomly assigned (n = 43) Group A Multivitamin (10 µg vitamin D) 225 mg DHA, 90 mg EPA (n = 10) Group B Multivitamin (10 µg vitamin D) 25 µg vitamin D 450 mg DHA, 180 mg EPA (n = 11) Group C Multivitamin (10 µg vitamin D) 50 µg vitamin D 667 mg DHA, 270 mg EPA (n = 11) Group D Multivitamin (10 µg vitamin D) 75 µg vitamin D 900 mg DHA, 360 mg EPA (n = 11)
Attended second visit
(n = 9) Attended second visit(n = 9) Attended second visit(n = 11) Attended second visit(n = 9)
Completed study
(n=9) Completed study (n=9) Completed study (n=11) Completed study (n=7) Blood only (n =2) Milk only (n=1) Excluded: Pregnancy hypertension (n=1) Gestational diabetes (n=1) Blood only (n=1) First visit: 20 GW Second visit: 36 GW Third visit: 4 weeks PP Drop out : Disliked supplements (n=1) Excluded: Use of anticoagulants (n=1) Drop out: Disliked supplements (n=1) Excluded: Pregnancy bleeding (n=1) Drop out: Disliked supplements (n=1)
Figure 1. Flow chart of the initially 43 participating women. Pregnant women were supplemented from
20 gestational weeks (GW) to 4 weeks postpartum (PP). Blood samples were taken at 20 and 36 GW and at 4 weeks PP. A milk sample was taken at 4 weeks PP.
Data on anthropometrics, socio-economic status and average fish intake were collected via questionnaires at the study beginning and/or end. Maternal EDTA-anticoagulated venous blood samples were taken in the non-fasting state at the study start and at 36 GW and 4 weeks PP. A milk sample was collected at 4 weeks PP. Blood samples were 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
a telfon sealable Sovirel tube containing methanol/hydrochloric acid (5:1 v/v), butylated hydroxytoluene (antioxidant) and 50 µg 17:0 (internal standard). These samples were stored at 4°C until analysis. Breast milk samples from a single completely emptied breast were collected around noon (10.00-14.00 hours) at the day prior to blood sampling. Milk was collected manually or by breast milk pump. Following careful swirling it was divided into two portions. The participants stored the milk samples in their private freezers. They took the samples with them in a cool transport container for frozen specimens (Sarstedt; mailing containers) on the following day of blood sampling in the UMCG. The milk samples were subsequently stored at -20 °C until analysis. Fatty acids in RBC and milk were measured by
capillary gas chromatography with flame ionization detection36,37. RBC DHA+EPA and milk
DHA+EPA were expressed in g/100 g fatty acids (g%).
Table 1. Daily supplemental dosages provided from about 20 gestational weeks to 4 weeks postpartum Nutrient Daily dosage Dutch RDA/AI for
pregnancy/lactation1
Percentages of Dutch RDA/AI for pregnancy/
lactation1 Groups A, B, C or D: DHA+EPA group A (mg) 315 (225+90) 200 157.5 DHA+EPA group B (mg) 630 (450+180) 200 315 DHA+EPA group C (mg) 945 (675+275) 200 450 DHA+EPA group D (mg) 1,260 (900+360) 200 630
Vitamin D group A (μg) 10 (in multivitamin) 10 100
Vitamin D group B (μg) 35 (10+25) 10 350 Vitamin D group C (μg) 60 (10+50) 10 600 Vitamin D group D (μg) 85 (10+75) 10 850 Groups A, B, C and D: Beta carotene (µg) Vitamin B1 (mg) Vitamin B2 (mg) Vitamin B3 (mg) Vitamin B5 (mg) Vitamin B6 (mg) Vitamin B8 (µg) Vitamin B11 (µg) Vitamin B12 (µg) Vitamin C (mg) Vitamin D (µg) Vitamin E (mg) Calcium (mg) Chromium (µg) Iodine (µg) Copper (µg) Magnesium (mg) Manganese (mg) Molybdenum (µg) Selenium (µg) Iron (mg) Zinc (mg) 12002 1.1 1.4 16 6 1.4 50 400 2.5 40 10 6 120 20 150 1000 56.25 1 25 55 16.1 10 800/1100 vitamin A 1.4/1.7 1.4/1.7 17/20 5/7 1.9 40/45 400/ 3.2/3.8 85/100 10 10/11 1000 30/45 175/200 1.0/1.3 280 3.0 65 60 223/15 9/11 38/27 79/65 100/82 94/80 120/86 74 125/111 100 78/66 47/40 100 60/55 12 67/44 86/78 100/77 20 50 38 92 733/107 111/82 1 Dutch recommended dietary allowance (RDA) or adequate intake (AI) (34)
2 Converted to vitamin A (in retinol units) (=300 µg beta-carotene),
3 Adequate intake for pregnancy/lactation according to the Institute of Medicine (35)
190 191
Data analysis and Statistics
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 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 to estimate the dosages needed to reach an RBC DHA+EPA of 8 g% and a milk DHA+EPA of 1 g%.
Results
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 women randomly assigned to groups A-D, 36 completed the study (Figure 1). Three 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 compliance with the study protocol. Breast milk samples were available from only 33 mothers, since three discontinued breastfeeding prior to 1 month PP. One mother provided a milk sample at 4 weeks PP, but did not report for blood sampling.
Characteristics of mothers and their infants
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 €50,000 or more. Median fish consumption at the study start was 0.8 (range 0-2) portions
(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, of which 25% contained low dose fish oil (200 mg DHA). Of the 36 infants, 17 were boys and 19 were girls. Their median birth weights were 3,790 (range 2,440-5,020) g. The milk samples were collected at 4.4 (range 3.5-5.3) weeks PP. There were no significant differences between the characteristics of the various supplemental groups, other then the collection of milk samples, which we consider not to be clinically relevant.
Ta bl e 2 . C ha ra ct er is tic s o f t he 3 6 i nc lu de d m ot he rs a nd t heir i nf an ts Sup pl em en ta l gr oup Variab le D im ens io ns A ll (n =3 6) A ( n = 9 ) B ( n = 9) C ( n = 1 1) D ( n = 7 ) p -v alu e Do se DH A +EP A (m g) 22 5+9 0 45 0+1 80 67 5+2 70 90 0+ 45 0 M at er na l c harac te ris tic s Ag e (y ea rs ) 31 (2 1-38 ) 32 ( 27 - 3 7) 31 ( 26 - 3 6) 32 ( 25 - 3 6) 30 ( 21 - 3 8) 0. 667 Pr e-pr egnan cy BM I (k g/m 2) 24 ( 18 - 2 9) 24 ( 19 - 2 9) 24 ( 18 - 2 8) 22 ( 1 9 - 2 7) 24 ( 21 - 2 6) 0. 89 6 Pa ra n 1 ( 0 -2) 1 ( 0-2 ) 1 ( 0-2 ) 1 ( 0-2 ) 1 ( 0-2 ) 0. 89 2 G es ta tio n n 2 ( 0 - 5 ) 2 ( 1-5) 2 (1 -4 ) 2 ( 0-3 ) 2 ( 1-5) 0. 532 G es ta tio n d ur at io n (w ee ks ) 41 ( 37 - 4 2) 41 ( 40 - 4 2) 40 ( 38 - 4 2) 40 ( 37 - 4 1) 40 ( 38 - 4 2) 0.1 49 So cio -e co no mic s ta tu s M ar rie d/ liv ing to ge th er n ( %) 36 (10 0) 9 ( 10 0) 9 ( 10 0) 11 (1 00 ) 7 ( 10 0) 1. 000 Ho us eh ol d n um b er n 3 ( 2-4) 3 ( 2-4) 3 ( 2-4) 3 ( 2-4) 3 ( 2-4) 0. 851 Ed uc at ion 0. 32 2 In te rm ed ia te v oc at io na l, H ig h s ch oo l o r l es s n ( %) 7 (2 0) -2 ( 22) 4 ( 36) 1 ( 14 ) -Co lle ge , u ni ve rs ity o r h ig he r n ( %) 28 (8 0) 9 ( 10 0) 7 (7 8) 7 ( 64) 6 ( 86) -A nn ua l hou se ho ld inc om e 0. 13 0 € 10 ,000 – € 30 .000 n ( %) 7 (2 0) 1 ( 11 ) 3 ( 33) 1 (9 ) 2 ( 28) € 30 ,000 - € 50 .000 n ( %) 14 (3 9) 4 ( 44) 4 ( 44) 5 ( 45 ) 1 ( 14 ) € 50 ,0 00 o r m or e n ( %) 15 (4 2) 4 ( 44) 2 ( 22) 5 ( 45 ) 4 ( 57 ) O th er W ee kl y fis h i nt ak e p or tion 0. 8 ( 0 - 2 .0 ) 0. 5 ( 0 - 1 .5 ) 1. 5 ( 0 - 2 ) 1 ( 0 - 2 ) 0. 5 ( 0 -1) 0. 355 W ee kl y o ily fi sh i nt ak e p or tion 0. 4 ( 0. 0 - 2 .0 ) 0. 5 ( 0 - 1 ) 1 ( 0 - 1 .5 ) 0 ( 0 - 2 ) 0. 5 ( 0 - 1 ) 0. 501 Vi ta m in s up pl em en ts b ef or e s ta rt s tu dy n ( %) 27 (7 5%) 8 ( 89 %) 6 ( 67 % ) 8 (7 5% ) 5 (7 1% ) 0. 476
4
Ta bl e 2 . C ha ra ct er is tic s o f t he 3 6 i nc lu de d m ot he rs a nd t heir i nf an ts Sup pl em en ta l gr oup Variab le D im ens io ns A ll (n =3 6) A ( n = 9 ) B ( n = 9) C ( n = 1 1) D ( n = 7 ) p -v alu e Do se DH A +EP A (m g) 22 5+9 0 45 0+1 80 67 5+2 70 90 0+ 45 0 of w hi ch c on ta in ed fi sh o il n ( %) 9 ( 25 %) 2 ( 22 %) 1 ( 11 % ) 4 ( 36 %) 2 ( 29 %) 0. 54 0 In fan t c harac te ris tic s Le ng th 2 (c m) 52 ( 45 - 5 9) 51 .5 ( 48 - 5 5. 5) 55 ( 45 - 5 9) 51 ( 48 - 5 5) 54 .5 ( 49 .5 - 5 8) 0. 53 0 We ig ht (kg ) 4. 5 ( 3. 6 - 5 .7 ) 4. 4 ( 3. 9 - 5 .6 ) 4. 7 ( 3. 9 - 5 .7 ) 4. 4 ( 4. 0 - 5 .2 ) 5. 2 ( 3. 6 - 5 .6 ) 0. 671 Bir th we igh t (g ) 37 90 (2 44 0 - 5 02 0) 37 90 (2 87 0 - 5 02 0) 39 65 (2 44 0 - 4 64 0) 37 50 (3 07 0 - 4 61 0) 387 0 (3 44 0 - 4 69 5) 0. 86 0 G en der (% mal e) 17 (4 7%) 3 ( 33 %) 4 (4 4%) 7 ( 64 %) 3 (4 3%) 0. 57 9 La ct at io n d ur at io n (w ee ks ) 4. 4 ( 3. 5 - 5 .3 ) 4. 4 ( 3. 5 - 4 .7 ) 4. 4 ( 3. 7 - 5 .3 ) 4 ( 3 .6 - 4 .9 ) 4. 6 ( 4 - 5 .1) 0. 033 D at a r ep re se nt m ed ia ns ( ra ng es ). G ro up s A -D r ec ei ve d s up pl em en ts f ro m 2 0 g es ta tio na l w ee ks ( G W ) t o 4 w ee ks p os tp ar tu m ( PP ). T he n ut rie nt s a nd t he ir d os ag es a re g ive n in Ta bl e 1 . A p -v alu e < 0. 05 0 i s c on si de re d s ig ni fic an t.
Dose needed to reach RBC DHA+EPA of 8 g% at 36 weeks GA
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 whole group at the study start was 5.5 (range 3.3-8.5) g%. At 36 GW the RBC DHA+EPA 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 RBC DHA+EPA at 36 GW was y = 5.8 + 2.9 * 10-3 x, where x is the DHA+EPA dosage (mg) and y is the RBC DHA+EPA content (g %). Employing this relation we found that the 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 (range 6.7-11.3) g% for group D. Supplemental Table 1 presents the data of RBC DHA+EPA, 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. Ta bl e 2 . ( Con tin ue d)
4
194 195 Time 4 weeks PP 36 GW 20 GW RBC DHA+EPA (g%) 12 10 8 6 4 2 0 D: 900+360 mg (n=7) C: 675+270 mg (n=11) B: 450+180 mg (n=9) A: 225+90 mg (n=9)#
Daily dose DHA+EPA
Page 1
Figure 2. Relations between the DHA+EPA dosages and RBC DHA+EPA at the study start, at 36 gestational
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 y = 5.8 + 2.9 * 10-3x (x in mg; y in g %). Horizontal line indicates the target of 8 g% RBC DHA+EPA. The
calculated DHA+EPA dose needed to reach the RBC DHA+EPA target of 8 g% at 36 weeks GW was 759 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.
Dependence of RBC DHA+EPA increments on baseline status and dose
Figure 4 shows, for each of the supplemented groups A-D, the relation between the baseline RBC DHA+EPA content (at 20 GW) and the increment of the RBC DHA+EPA content (∆ DHA+EPA in g%) from 20 to 36 GW. The RBC DHA+EPA increments were found to be 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 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 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.
Effects of the supplements on milk AA and the milk EPA/DHA and EPA/AA ratios
Figure 3 panels D-F show the relations between the DHA+EPA dosages and milk AA content (panel D), EPA/DHA ratio (panel E), and EPA/AA ratio (panel F) for groups A-D at 4 weeks PP. Supplemental Table 2 presents the corresponding hard data. There were no relations between the DHA+EPA dosage and milk AA (panel D; p=0.159) and milk EPA/DHA ratio (panel E; p=0.195). The median milk AA content was 0.36 (range 0.23-0.63) g% and the median milk EPA/DHA ratio was 0.25 (range 0.05-0.37) g%. There was, however, a significant dose-dependent increase of the milk EPA/AA ratio (panel F; p<0.010). The medians of the milk EPA/AA ratios at 4 weeks PP were 0.23 (range 0.09-0.34) for group A, 0.48 (range 0.33-0.69) for group B, 0.42 (range 0.16-0.33-0.69) for group C and 0.74 (range 0.17-0.88) for group D.
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7) Milk DHA+EPA (g%) 2.00 1.50 1.00 .50 .00 A
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7) Milk DHA (g%) 1.5 1.0 .5 .0 B
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7) Milk EPA (g%) .40 .30 .20 .10 .00 C
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7) Milk AA (g%) .60 .40 .20 .00 D
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7)
Ratio milk EPA/DHA
.40 .30 .20 .10 .00 E
Daily dose DHA+EPA (mg) D 900+360 (n=7) C 675+270 (n=11) B 450+180 (n=8) A 225+90 (n=7)
Ratio milk EPA/AA (g%)
1.00 .80 .60 .40 .20 .00 F
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).
RBC DHA+EPA (g%) at 20 GW (baseline) 9.00 8.00 7.00 6.00 5.00 4.00 3.00 DHA+EPA (g%) 20 GW - 36 GW 6.00 4.00 2.00 .00 y=3-0.35*x y=4.68-0.44*x y=7.25-0.73*x y=7.69-0.73*x 900+360 mg DHA+EPA 675+270 mg DHA+EPA 450+180 mg DHA+EPA 225+90 mg DHA+EPA 900+360 mg DHA+EPA 675+270 mg DHA+EPA 450+180 mg DHA+EPA 225+90 mg DHA+EPA Dose DHA+EPA Page 1
Figure 4. Relation between baseline RBC DHA+EPA content (at 20 GW) and the RBC DHA+EPA increment
(∆ DHA+EPA in g%) from 20 to 36 GW. Linear relations are given for the four supplemental groups A-D, who received supplements from 20 gestational weeks (GW) to 4 weeks postpartum (PP). The nutrients and their dosages are given in Table 1.
Discussion
We investigated the DHA+EPA supplemental dose needed to reach an RBC DHA+EPA of 8 g% at the pregnancy end and to reach a mature breast milk DHA+EPA content of 1 g%. An approach by constructing linear dose-response relations estimated these dosages at 759 mg (Figure 2) and 952 mg (Figure 3. Panel A) DHA+EPA, respectively. In view of the 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 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).
198 199
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.
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 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/
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.
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.
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
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).
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(70) Manku MS, Horrobin DF, Karmazyn M, Cunnane SC. Prolactin and zinc effects on rat vascular reactivity: possible relationship to dihomo-gamma-linolenic acid and to prostaglandin synthesis. Endocrinology 1979 Mar;104(3):774-779. Su pp lem en ta l T abl e 1 . D H A+ EP A . D H A , E PA a nd A A , a nd E PA /D H A a nd E PA /A A r at io s i n e ry thr oc yt es f or g ro up s A -D a t 2 0 g es ta tio na l we ek s ( G W ), 3 6 G W a nd 4 we ek s p os tp ar tu m ( PP ). Sup pl em en ta ti on gr oup Variab le G rou p 20 G W p -v alu e 36 G W p -v alu e 4 w ee ks P P p -v alu e p -v alu e G W 20 - G W 36 p -v alu e G W 20 -4W PP p -v alu e G W 36 -4W PP RB C – ( g% ) DH A +EP A A 5.5 (3 .9 -8 .5 ) 0. 267 6.5 (5 .5-8.6 ) 0. 001 6. 5 ( 6. 0-7. 6) 0. 000 0. 00 8 0. 069 0. 575 B 4.6 (3 .6 -7 .5 ) 7. 4 ( 6. 2-9. 3) 7.4 (6 .5-8. 9) 0. 00 8 0. 00 8 0. 859 C 6. 0 ( 4. 4-6. 9) 8. 7 ( 8. 1-10. 4) 8. 6 ( 6. 8-9. 9) 0. 00 5 0. 007 0. 016 D 6.2 (3 .3 -7 .2 ) 9. 5 ( 6. 0-11 .3 ) 9. 4 ( 6. 7-11 .3 ) 0. 02 8 0. 018 0. 612 D HA A 5. 1 ( 3. 6-7.7 ) 0. 324 6.1 (5 .1 -7 .8 ) 0. 001 5. 8 ( 5. 4-6. 7) 0. 001 0. 00 8 0. 093 0. 26 3 B 4. 2 ( 3. 3-6.9 ) 6. 5 ( 5.8 -8 .3 ) 6.5 (5 .7 -7 .6 ) 0. 00 8 0. 00 8 0. 214 C 5. 5 ( 4.1 -6 .3 ) 7.7 (7. 1-9. 1) 7.4 (5 .7 -8 .2) 0. 00 5 0. 007 0. 00 3 D 5. 9 ( 3. 0-6. 6) 8. 2 ( 5. 7-10. 0) 7. 8( 6. 0-9. 6) 0. 02 8 0. 018 0. 04 3 EPA A 0. 41 ( 0. 30 -0. 79 ) 0. 493 0. 43 ( 0. 37 -0. 82 ) 0. 000 0. 61 ( 0. 53 -1 .0 1) 0. 000 0. 11 0 0. 012 0. 012 B 0. 36 ( 0. 28 -0. 63 ) 0. 76 ( 0. 43 -0. 99 ) 1.1 3 ( 0. 76 -1. 51 ) 0. 00 8 0. 00 8 0. 00 8 C 0. 44 ( 0. 26 -0. 69 ) 1. 04 (0 .8 1-1. 37 ) 1.1 8 ( 0. 78 -1. 64 ) 0. 00 5 0. 00 5 0. 00 4 D 0. 36 (0. 28 -0. 65 ) 1. 21 (0 .3 1-1. 37 ) 1. 59 (0 .7 0-1. 70 ) 0. 02 8 0. 018 0. 018 AA A 13 .0 (11 .1 -1 4. 5) 0. 824 12 .0 (9 .9 -1 3. 4) 0. 70 6 12 .1 ( 10. 5-13 .7 ) 0.1 69 0. 00 8 0. 012 0. 12 3 B 13 .1 (11 .7 -1 4. 5) 11 .3 ( 10. 2-12 .4 ) 11 .6 ( 10. 7-12 .8 ) 0. 00 8 0. 00 8 0. 13 9 C 13 .3 (1 2. 0-14. 7) 11 .4 (9 .7 -1 2. 6) 11 .3 (8 .4 -1 2. 9) 0. 00 5 0. 00 5 0. 477 D 12. 9 ( 11 .1 -1 4. 7) 11 .3 (9 .6 -1 3. 9) 10 .7 (9 .3 -1 3.7 ) 0. 018 0. 00 8 0. 02 8 EP A /DH A A 0. 08 ( 0. 06 -0. 10 ) 0. 82 6 0. 08 ( 0. 05 -0. 11 ) 0. 000 0. 11 ( 0. 08 -0. 15 ) 0. 002 0. 31 4 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. 00 8 0. 011 C 0. 09 ( 0. 06 -0. 12 ) 0. 13 ( 0. 10 -0. 15 ) 0. 18 ( 0. 10 -0. 20 ) 0. 00 5 0. 00 5 0. 00 4 D 0. 09 ( 0. 06 -0. 10 ) 0. 14 ( 0. 05 -0. 17 ) 0. 20 ( 0. 12 -0. 22 ) 0. 02 8 0. 018 0. 018
4
Su pp lem en ta l T abl e 1 . ( Con tin ue d) Sup pl em en ta ti on gr oup Variab le G rou p 20 G W p -v alu e 36 G W p -v alu e 4 w ee ks P P p -v alu e p -v alu e G W 20 - G W 36 p -v alu e G W 20 -4W PP p -v alu e G W 36 -4W PP EPA /A A A 0. 03 ( 0. 02 -0. 06 ) 0. 65 6 0. 04 ( 0. 03 -0. 06 ) 0. 000 0. 05 ( 0. 04 -0. 08 ) 0. 000 0. 011 0. 012 0. 012 B 0. 03 ( 0. 02 -0. 05 ) 0. 07 ( 0. 04 -0. 09 ) 0. 10 ( 0. 06 -0. 12 ) 0. 00 8 0. 00 8 0. 00 8 C 0. 03 ( 0. 02 -0. 05 ) 0. 09 ( 0. 06 -0. 13 ) 0. 11 -0. 06 -0. 15 ) 0. 00 5 0. 00 5 0. 00 4 D 0. 03 ( 0. 02 -0. 06 ) 0. 11 ( 0. 02 -0. 14 ) 0. 15 ( 0. 05 -0. 18 ) 0. 02 8 0. 018 0. 018 D at a a re m ed ia ns ( ra ng es ). A p -v alu e < 0. 05 0 w as c on si de re d s ig ni fic an t b et w ee n t he g ro up s, a p -v alu e < 0. 01 67 w as c on si de re d s ig ni fic an t b et w ee n t he t im e p oi nt s. G ro up s A -D r ec ei ve d s up pl em en ts f ro m 2 0 g es ta tio na l w ee ks ( G W ) t o 4 w ee ks p os tp ar tu m ( PP ). T he n ut rie nt s a nd t he ir d os ag es a re g ive n i n T ab le 1 .
Supplemental Table 2. Milk DHA+EPA, milk DHA, milk EPA, ratio milk EPA/DHA and ratio milk EPA/AA
in the different groups at 4 weeks PP.
Supplementation group
Variable Group 4 weeks PP p-value Milk – (g%) DHA+EPA A 0.36 (0.27-0.75) <0.01 B 0.81 (0.56-1.06) C 1.01 (0.71-1.31) D 1.08 (0.68-1.68) DHA A 0.29 (0.22-0.60) <0.01 B 0.63 (0.42-0.83) C 0.83 (0.55-1.07) D 0.85 (0.57-1.40) EPA A 0.08 (0.04-0.16) <0.01 B 0.19 (0.11-0.23) C 0.18 (0.04-0.26) D 0.25 (0.11-0.30) AA A 0.33 (0.26-0.46) 0.159 B 0.34 (0.3-0.63) C 0.42 (0.23-0.58) D 0.36 (0.27-0.63) EPA/DHA A 0.27 (0.15-0.35) 0.195 B 0.26 (0.23-0.37) C 0.25 (0.05-0.31) D 0.22 (0.19-0.32) EPA/AA A 0.23 (0.09-0.34) <0.01 B 0.48 (0.33-0.69) C 0.42 (0.16-0.69) D 0.74 (0.17-0.88)
Data are median (range). A p-value < 0.050 is considered significant
