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Human milk arachidonic acid and docosahexaenoic acid contents increase following supplementation during pregnancy and lactation
ARTICLE IN PRESS Prostaglandins, Leukotrienes and Essential Fatty Acids 80 (2009) 65–69
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Human milk arachidonic acid and docosahexaenoic acid contents increase following supplementation during pregnancy and lactation$ Saskia A. van Goor a,, D.A. Janneke Dijck-Brouwer b, Mijna Hadders-Algra c, Bennard Doornbos d, Jan Jaap H.M. Erwich e, Anne Schaafsma f, Frits A.J. Muskiet b a
Pathology and Laboratory Medicine, UMCG, University Medical Center Groningen, The Netherlands Laboratory Center, UMCG, University Medical Center Groningen, The Netherlands c Department of Pediatrics, UMCG, University Medical Center Groningen, The Netherlands d Department of Psychiatry, UMCG, University Medical Center Groningen, The Netherlands e Department of Obstetrics and Gynecology, UMCG, University Medical Center Groningen, The Netherlands f Friesland Foods, Leeuwarden, The Netherlands b
a r t i c l e in fo
abstract
Article history: Received 3 June 2008 Received in revised form 3 November 2008 Accepted 7 November 2008
Introduction: Docosahexaenoic acid (DHA) and arachidonic acid (AA) are important for neurodevelopment. Maternal diet influences milk DHA, whereas milk AA seems rather constant. We investigated milk AA, DHA and DHA/AA after supplementation of AA plus DHA, or DHA alone during pregnancy and lactation. Subjects and methods: Women were supplemented with AA+DHA (220 mg each/day), DHA (220 mg/day) or placebo during pregnancy and lactation. Milk samples were collected at 2 (n ¼ 86) and 12 weeks (n ¼ 69) postpartum. Results: Supplementation of AA+DHA elevated milk AA (week 2, 14%; week 12, 23%) and DHA (43% and 52%) as compared to placebo. DHA tended to decrease milk AA and vice versa. Milk AA, DHA and DHA/ AA decreased from 2 to 12 weeks postpartum. Conclusions: Milk AA and in particular DHA are sensitive to maternal supplementation. It seems that maternal AA and notably DHA status decline with advancing lactation. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Arachidonic acid Docosahexaenoic acid Long-chain polyunsaturated fatty acids Supplementation Human milk Pregnancy
1. Introduction The long-chain polyunsaturated fatty acids (LC-PUFA) docosahexaenoic acid (DHA) and arachidonic acid (AA) are important structural components of phospholipids. DHA is a modulator of gene expression, neurotransmitter metabolism, membrane-bound processes [1] and of immunity and inflammation through its precursorship of resolvins and protectins [2]. AA is important in growth and cell signaling, in part through its precursorship of eicosanoids and lipoxins [1]. Both DHA and AA are considered important for development, including that of the central nervous system [3]. Highest brain DHA contents are encountered in grey matter, notably motor areas and high energy consumption areas, and in the retina [3,4]. From animal and human studies, it can be concluded that neonatal brain DHA is positively related to cognitive and behavioral performance. Small differences in brain
$
This study was financially supported by Friesland Foods, The Netherlands.
Corresponding author at: CMC-V, Room Y3.181, internal postal code EA22,
Groningen University Hospital, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Tel.: +31 50 361 0399/361 9228; fax: +31 50 361 2290. E-mail address: [email protected] (S.A. van Goor). 0952-3278/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2008.11.002
DHA content may result in subtle effects that are currently difficult to detect but may be relevant [5]. Arachidonic acid and DHA derive either from the diet, or from endogenous synthesis from their respective parent essential fatty acids (EFA) linoleic acid (LA) and alpha-linolenic acid. The current Western diet is characterized by a high LA intake from vegetable oils, and by low intakes of eicosapentaenoic acid (EPA) and DHA [6]. Fish, and especially fatty fish, is a rich source of EPA and DHA. Preformed dietary DHA and AA are the most efficient sources of DHA and AA, since synthesis from their parent EFA is limited [7,8]. A recent short-term intervention with baboons during the perinatal period indicates that brain DHA is sensitive to dietary intake, whereas AA seems rather constant [4]. In contrast, a recent long-term intervention study with more extreme diets in pregnant mice shows that fetal brain AA is lower at higher fetal brain DHA contents [9]. The consequences of low brain AA are less certain than those of low DHA. Maintenance of a dietary DHA/AA balance is however widely acknowledged [10], since o3 and o6 fatty acids compete for desaturation and elongation, and for their incorporation into at least some body compartments [11]. The relative contributions of preformed dietary AA and endogenously synthesized AA to the total milk AA pool are as
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yet unknown. Supplementation of lactating women in Jerusalem with 300 mg AA per day for 1 week did not increase their milk AA content [12]. The biological variation of milk AA content is among the lowest of all milk fatty acids (CV 28%; median 0.42 mol%; range 0.19–0.99), while those of EPA (100%; 0.05 mol%; 0.00–1.18) and DHA (86%; 0.21 mol%; 0.08–1.63) constitute the highest [13]. The apparently low biological variation of AA may however derive from a sampling bias, that is caused by studying samples from populations with relatively low AA intakes [14]. To illustrate, milk from women living in Doromoni (Tanzania), exhibit both high milk AA (median 0.70 mol%; range 0.50–0.93) and high DHA (0.75 mol%; 0.25–1.47). These women have high lifetime intakes of local AA and DHA rich fish as their only source of animal fat, which may also explain the positive correlation between milk AA and DHA found in their milk [15]. Tracer studies indicated that the vast majority of milk EFA do not derive directly from the diet, but from maternal stores [16]. For instance, 30% of milk LA has been estimated to derive directly from the diet and 70% from maternal stores [16,17]. Arachidonic acid might be subjected to a large distribution volume, because of its high tendency to become incorporated into membrane phospholipids, and to a lesser extent in plasma and adipose tissue triacylglycerols [18,19]. Supplementation studies in adults show that supplemental AA up to 6 g/day causes higher AA status, that supplements up to 1.5 g/day are likely to be safe, and that serum phospholipids and triacylglycerols reach steady-state AA contents after 2 weeks [19]. As compared with a 210 mg daily intake, the intake of 1.7 g AA/day for 50 days greatly increased AA and its chain elongation product 22:4o6 in plasma phospholipids. Moderate AA increases occurred in plasma cholesterol esters, fasting plasma triacylglycerols, fasting plasma free fatty acids and erythrocyte phospholipids, while no changes were noticed in adipose tissue phospholipids and triacylglycerols [18]. The apparently low biological variation of milk AA but nevertheless high milk AA in populations with high AA intakes, the predominant derivation of milk LCP from maternal stores, the large distribution volume of AA and the inability to augment milk AA by short-term supplementation, suggest that human milk AA is mainly dependent on long term dietary AA intake. In view of these long-term effects, we investigated the influence of supplementation of AA plus DHA, or DHA alone from early pregnancy up to 12 weeks of lactation on milk AA, DHA, and the DHA/AA ratio.
Table 1 Daily intakes of fatty acids (in mg) from the supplements.
LA ALA AA EPA DHA
Placebo
DHA
DHA+AA
535 60 0 0 0
274 32 15 34 220
46 7 220 36 220
Abbreviations: LA, linoleic acid, 18:2o6; ALA, alpha-linolenic acid, 18:3o3; AA, arachidonic acid, 20:4o6; EPA, eicosapentaenoic acid, 20:5o3; DHA, docosahexaenoic acid, 22:6o3.
allowances. The women were instructed to take 2 capsules once daily from enrollment till 12 weeks postpartum. The AA+DHA group received 220 mg AA (Wuhan Alking Bioengeneering Co., Ltd., Wuhan, China) and 220 mg DHA (Marinol D40, Lipid Nutrition B.V., Wormerveer, The Netherlands). The DHA group received 220 mg DHA and one capsule containing soy bean oil (Wuhan Alking Bioengeneering Co., Ltd., Wuhan, China) and the placebo group received two capsules containing soy bean oil. Table 1 shows the daily fatty acid intakes from the capsules. The daily dosages of AA and DHA are within the range of typical intakes by Western adults [20]. The research protocol was approved by the Central Committee on Research Involving Human Subjects (CCMO, Den Haag, The Netherlands; protocol number P03.1071C). All women gave written informed consent. 2.3. Sample collection and analytical methods Women were instructed to collect 5 ml of milk at 2 and 12 weeks postpartum. Clock time and sampling within one feeding were not standardized since, in contrast to the milk fat content, the milk fatty acid composition does not vary to an appreciable extent during the day or during a single feeding [21,22]. One hundred microlitres of milk was transferred to a Sovirel tube containing 2 ml methanol/HCl (5:1, v/v) and 5 mg butylated hydroxytoluene. Following transmethylation, the milk fatty acid compositions were determined by using our previously described capillary gas chromatographic method with flame-ionization detection [23]. Fatty acids are presented in relative amounts, i.e. g/100 g fatty acids (wt%).
2. Materials and methods
2.4. Statistical methods
2.1. Subjects
Statistical analyses were performed using SPSS 14.0. The data were evaluated on the basis of intention to treat. Between-group differences in milk fatty acid compositions at 2 and 12 weeks postpartum were calculated using Mann–Whitney U tests. To correct for multiple testing, po0.017 was considered to be significant. Milk fatty acid contents at 2 and 12 weeks were compared using the non-parametric Wilcoxon Signed Rank test for related samples. po0.05 was considered significant.
This study was part of a double-blind placebo-controlled randomized trial with apparently healthy pregnant women who were recruited by midwives or gynecologists in and around the city of Groningen (The Netherlands) from December 2004 till December 2006. We included women with a low risk, first or second, singleton pregnancy. Women using a vegetarian or vegan diet and women with diabetes mellitus were excluded. Women were enrolled between 14 and 20 weeks of pregnancy, with the majority (80%) being enrolled between 15.6 and 17.4 weeks postmenstrual age (mean: 16.5 weeks). Criteria for termination of participation were any maternal or neonatal complications. 2.2. Study design At enrollment, the women were randomized into three groups using block randomization. All women received a supplement of vitamins and minerals according to Dutch recommended dietary
3. Results From the 182 pregnant women who were included in this trial, 57 women dropped out due to lack of motivation to fill in questionnaires and take capsules every day. Two women dropped out because of pregnancy complications (1 threatened preterm labor and 1 preterm birth). From the remaining 123 women, 88 chose to breastfeed their infants, of whom 69 continued breastfeeding for at least 12 weeks. The number of women who collected samples at 2 weeks postpartum amounted to 24
ARTICLE IN PRESS S.A. van Goor et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 80 (2009) 65–69
67
higher 22:4o6 as compared to the DHA group (+17%; data available in Supplementary table). The DHA/AA ratio was increased at 2 and 12 weeks postpartum after supplementation with DHA alone, as compared to placebo. At 2 weeks, women receiving DHA also had a higher milk DHA/AA ratio compared to those receiving AA+DHA, while at 12 weeks those receiving AA+DHA had higher milk DHA compared to placebo. Although not significant, supplementation with DHA tended to decrease milk AA, and probably vice versa, especially at 2 weeks postpartum. There were no other between-group differences in milk fatty acids (data available in supplementary table). Table 3 shows longitudinal changes in milk fatty acids of the 67 women who provided milk samples both at 2 and 12 weeks. In each supplementation group, milk AA and DHA decreased from 2 to 12 weeks postpartum. In the placebo group, the DHA/AA ratio
(placebo group), 34 (DHA group) and 28 (AA+DHA group), two women did not provide samples. At 12 weeks postpartum, 19 women had discontinued breastfeeding. The remaining participants provided 19 (placebo), 30 (DHA) and 20 (AA+DHA) milk samples. The characteristics of the 88 breastfeeding mothers are described in Table 2. Fig. 1 shows that supplementation of AA+DHA significantly elevated milk AA both at 2 (+22%; difference between medians) and 12 (+23%) weeks postpartum when compared to the DHA group. At 2 weeks postpartum, milk AA was also higher in the AA+DHA group compared to the placebo group (+14%). Compared with placebo, supplementation of DHA or AA+DHA significantly increased milk DHA both at 2 (+59% and +43%, respectively) and 12 (+56% and +52%, respectively) weeks postpartum. In addition, at 2 weeks postpartum, the AA+DHA group showed significantly
Table 2 Perinatal maternal and infant characteristics.
Maternal age (yr) Prepregnancy BMI (kg/m2) Weight gain during pregnancy (kg) Gestational age at birth (w) Birth weight (g) Firstborn
Placebo (n ¼ 25)
DHA (n ¼ 34)
DHA+AA (n ¼ 29)
33.5 (26.0–40.3) 21.5 (17.5–31.7) 14 (0–20) 40.4 (34.9–41.9)a 3520 (2850–4840) 16 (64)
32.3 (22.3–43.3) 23.1 (18.7–41.8) 13 (2–27) 40.3 (37.0–42.1) 3610 (2620–4470) 21 (62)
31.5 (24.8–41.4) 23.1 (17.2–41.4) 18 (3–30) 40.0 (37.0–41.9) 3610 (3050–4310) 18 (62)
Data are presented as median (range) or as n (%). DHA, docosahexaenoic acid; AA, arachidonic acid; BMI, body mass index. Different from placebo (p ¼ 0.003) and DHA (p ¼ 0.004). a One woman in the placebo group gave preterm birth. Her milk fatty acid composition did not differ from that of the rest of the group, both at 2 and 12 weeks.
Fig. 1. Milk DHA, AA and DHA/AA ratio at 2 (left) and 12 (right) weeks postpartum. Women were supplemented with either placebo, DHA (220 mg/day) or DHA (220 mg/ day)+AA (220 mg) from week 17 of pregnancy till 12 weeks postpartum. The boxes show the interquartile range (25–75th percentile). The horizontal line displays the median, and the whiskers 1.5 times the interquartile range; outliers not shown. Interconnected boxes were significantly different at po0.017 (Bonferroni-adjusted p-value). DHA, docosahexaenoic acid; AA, arachidonic acid; DHA/AA, ratio of DHA and AA; FA, fatty acids.
Table 3 Longitudinal changes of milk arachidonic acid (AA), docosahexaenoic acid (DHA) and the DHA/AA ratio. DHA
Placebo (n ¼ 18) DHA (n ¼ 30) DHA+AA (n ¼ 19)
AA
DHA/AA
2 weeks p.p.
12 weeks p.p.
2 weeks p.p.
12 weeks p.p.
2 weeks p.p.
12 weeks p.p.
Median
Range
Median
Median
Range
Median
Median
Range
Median
Range
0.21–0.74 0.33–2.58 0.17–1.07
0.25 0.39 0.38
0.34–0.78 0.36–1.09 0.49–0.83
0.39 0.39 0.49
0.49–1.31 0.43–4.27 0.32–1.43
0.59
0.31–0.94 0.44–6.54 0.18–2.63
0.40 0.60 0.51
Range 0.13–0.37 0.16–1.46 0.08–1.04
0.58 0.57 0.66
Range 0.26–0.60 0.22–0.56 0.37–0.73
0.68 1.12 0.81
0.97 0.75
Data, presented as wt%, are from the 67 women of whom milk samples were available at both 2 and 12 weeks postpartum. p.p., postpartum. Indicates that the content in week 12 is lower compared with week 2, as tested with a Wilcoxon signed rank test for paired samples at po0.05.
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decreased, which is most likely caused by a stronger decrease of DHA as compared to AA.
4. Discussion This study is the first to show that milk AA increases after supplementation of a relatively low dose of AA, i.e. 220 mg/day, during pregnancy and lactation. Our study confirmed that DHA supplementation increases milk DHA. DHA seemed more responsive to supplementation than AA, since its increase was higher in both absolute (wt%) and relative (difference between medians) amounts. Some degree of competition between DHA and AA is conceivable since supplementation with DHA tended to reduce milk AA, and probably vice versa. Milk from women receiving AA+DHA contained insignificantly lower DHA at both 2 and 12 weeks postpartum compared to milk from women receiving DHA alone. Furthermore, 2 weeks postpartum, the women in the DHA group had insignificantly lower milk AA compared to the placebo group. A tendency of supplemental AA to reduce milk LCPo3 levels was previously noted [12], although supplementation of fish oil or DHA alone did not lower milk AA [24–26]. Because of the apparent competition between o3 and o6 LCP for incorporation into milk lipids, it seems important to supplement these at the appropriate ratio, which is as yet subject to discussion. The decline of both milk DHA and AA with advancing lactation has been previously noted [27]. This decline is likely to be caused by the increasing milk triacylglycerol/phospholipid ratio that accompanies the increase of milk fat globular size and triacylglycerol concentration with advancing lactation [27–29]. Alternatively, it may also reflect maternal AA and DHA depletion, and the currently supplemented dosages proved unable to prevent this alleged depletion. Interestingly, the DHA/AA ratio also declined over time, at least in the placebo group, suggesting that maternal DHA stores become more severely depleted than AA. A lactationinduced maternal DHA depletion is conceivable, since Otto et al. [30] showed a more pronounced postpartum decline of the DHA status in lactating as compared to nonlactating women. The outcome of this study is different from our previous study in which we supplemented 300 mg AA daily for 1 week [12]. The present AA dosage was lower (220 mg/day) but was administered for 26 (up to 2 weeks postpartum) and 36 weeks (up to 12 weeks postpartum), respectively. It seems that milk AA is notably dependent on long-term AA intake, which is conceivable from both the origins of milk LCP and the distribution of AA. Milk PUFA are known to derive mainly from maternal stores and only small amounts of milk AA and DHA originate from endogenous synthesis from their parent EFA precursors [16,17]. Moreover, supplemental AA is notably incorporated into phospholipids, such as those in circulating plasma lipoproteins and red blood cell membranes, and to a limited extent into plasma triacylglycerols, resulting in a large distribution volume [18]. The stronger increase of milk DHA as compared to AA suggests a smaller distribution volume of DHA. It is conceivable that the present supplementation periods of 26 and 36 weeks did not cause steady-state milk AA contents. In other words, the apparent milk AA increase of about 0.12 wt% (22%) at 2 weeks postpartum and 0.09 wt% (23%) at 12 weeks postpartum may underestimate the response of a prolonged supplemental intake of 220 mg AA/day. However, short-term DHA supplementation does not seem to cause a steady state either, as may be concluded from the few data on the DHA intake-milk DHA dose-response curves available in literature. These reveal a steeper DHA dose–response curve from a long-term DHA intake [31] as compared to the dose-response curve from a 12-week DHA supplement [25]. Translation of
currently recommended intakes of LCP during pregnancy and lactation into milk LCP contents may therefore await the construction of dose–response relationships that correlate milk LCP contents with long-term LCP intakes as estimated from reliable food frequency questionnaires. We conclude that breast milk AA content is sensitive to dietary AA intake, and that milk DHA seems more responsive to dietary intake than milk AA. The influence of dietary AA intake becomes noticeable after prolonged supplementation, which is in line with the notion that milk PUFA derive notably from maternal body stores. More studies are needed to determine optimal human milk and formula DHA, AA and DHA/AA.
Conflict of interest None of the authors had any conflict of interest.
Acknowledgments We kindly thank R.S. Kuipers, I.B.M. Meijer and I.A. Martini for their valuable aid in the analysis of the milk fatty acids.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.plefa.2008.11.002.
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Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Human milk arachidonic acid and docosahexaenoic acid contents increase following supplementation during pregnancy and lactation$ Saskia A. van Goor a,, D.A. Janneke Dijck-Brouwer b, Mijna Hadders-Algra c, Bennard Doornbos d, Jan Jaap H.M. Erwich e, Anne Schaafsma f, Frits A.J. Muskiet b a
Pathology and Laboratory Medicine, UMCG, University Medical Center Groningen, The Netherlands Laboratory Center, UMCG, University Medical Center Groningen, The Netherlands c Department of Pediatrics, UMCG, University Medical Center Groningen, The Netherlands d Department of Psychiatry, UMCG, University Medical Center Groningen, The Netherlands e Department of Obstetrics and Gynecology, UMCG, University Medical Center Groningen, The Netherlands f Friesland Foods, Leeuwarden, The Netherlands b
a r t i c l e in fo
abstract
Article history: Received 3 June 2008 Received in revised form 3 November 2008 Accepted 7 November 2008
Introduction: Docosahexaenoic acid (DHA) and arachidonic acid (AA) are important for neurodevelopment. Maternal diet influences milk DHA, whereas milk AA seems rather constant. We investigated milk AA, DHA and DHA/AA after supplementation of AA plus DHA, or DHA alone during pregnancy and lactation. Subjects and methods: Women were supplemented with AA+DHA (220 mg each/day), DHA (220 mg/day) or placebo during pregnancy and lactation. Milk samples were collected at 2 (n ¼ 86) and 12 weeks (n ¼ 69) postpartum. Results: Supplementation of AA+DHA elevated milk AA (week 2, 14%; week 12, 23%) and DHA (43% and 52%) as compared to placebo. DHA tended to decrease milk AA and vice versa. Milk AA, DHA and DHA/ AA decreased from 2 to 12 weeks postpartum. Conclusions: Milk AA and in particular DHA are sensitive to maternal supplementation. It seems that maternal AA and notably DHA status decline with advancing lactation. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Arachidonic acid Docosahexaenoic acid Long-chain polyunsaturated fatty acids Supplementation Human milk Pregnancy
1. Introduction The long-chain polyunsaturated fatty acids (LC-PUFA) docosahexaenoic acid (DHA) and arachidonic acid (AA) are important structural components of phospholipids. DHA is a modulator of gene expression, neurotransmitter metabolism, membrane-bound processes [1] and of immunity and inflammation through its precursorship of resolvins and protectins [2]. AA is important in growth and cell signaling, in part through its precursorship of eicosanoids and lipoxins [1]. Both DHA and AA are considered important for development, including that of the central nervous system [3]. Highest brain DHA contents are encountered in grey matter, notably motor areas and high energy consumption areas, and in the retina [3,4]. From animal and human studies, it can be concluded that neonatal brain DHA is positively related to cognitive and behavioral performance. Small differences in brain
$
This study was financially supported by Friesland Foods, The Netherlands.
Corresponding author at: CMC-V, Room Y3.181, internal postal code EA22,
Groningen University Hospital, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Tel.: +31 50 361 0399/361 9228; fax: +31 50 361 2290. E-mail address: [email protected] (S.A. van Goor). 0952-3278/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2008.11.002
DHA content may result in subtle effects that are currently difficult to detect but may be relevant [5]. Arachidonic acid and DHA derive either from the diet, or from endogenous synthesis from their respective parent essential fatty acids (EFA) linoleic acid (LA) and alpha-linolenic acid. The current Western diet is characterized by a high LA intake from vegetable oils, and by low intakes of eicosapentaenoic acid (EPA) and DHA [6]. Fish, and especially fatty fish, is a rich source of EPA and DHA. Preformed dietary DHA and AA are the most efficient sources of DHA and AA, since synthesis from their parent EFA is limited [7,8]. A recent short-term intervention with baboons during the perinatal period indicates that brain DHA is sensitive to dietary intake, whereas AA seems rather constant [4]. In contrast, a recent long-term intervention study with more extreme diets in pregnant mice shows that fetal brain AA is lower at higher fetal brain DHA contents [9]. The consequences of low brain AA are less certain than those of low DHA. Maintenance of a dietary DHA/AA balance is however widely acknowledged [10], since o3 and o6 fatty acids compete for desaturation and elongation, and for their incorporation into at least some body compartments [11]. The relative contributions of preformed dietary AA and endogenously synthesized AA to the total milk AA pool are as
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yet unknown. Supplementation of lactating women in Jerusalem with 300 mg AA per day for 1 week did not increase their milk AA content [12]. The biological variation of milk AA content is among the lowest of all milk fatty acids (CV 28%; median 0.42 mol%; range 0.19–0.99), while those of EPA (100%; 0.05 mol%; 0.00–1.18) and DHA (86%; 0.21 mol%; 0.08–1.63) constitute the highest [13]. The apparently low biological variation of AA may however derive from a sampling bias, that is caused by studying samples from populations with relatively low AA intakes [14]. To illustrate, milk from women living in Doromoni (Tanzania), exhibit both high milk AA (median 0.70 mol%; range 0.50–0.93) and high DHA (0.75 mol%; 0.25–1.47). These women have high lifetime intakes of local AA and DHA rich fish as their only source of animal fat, which may also explain the positive correlation between milk AA and DHA found in their milk [15]. Tracer studies indicated that the vast majority of milk EFA do not derive directly from the diet, but from maternal stores [16]. For instance, 30% of milk LA has been estimated to derive directly from the diet and 70% from maternal stores [16,17]. Arachidonic acid might be subjected to a large distribution volume, because of its high tendency to become incorporated into membrane phospholipids, and to a lesser extent in plasma and adipose tissue triacylglycerols [18,19]. Supplementation studies in adults show that supplemental AA up to 6 g/day causes higher AA status, that supplements up to 1.5 g/day are likely to be safe, and that serum phospholipids and triacylglycerols reach steady-state AA contents after 2 weeks [19]. As compared with a 210 mg daily intake, the intake of 1.7 g AA/day for 50 days greatly increased AA and its chain elongation product 22:4o6 in plasma phospholipids. Moderate AA increases occurred in plasma cholesterol esters, fasting plasma triacylglycerols, fasting plasma free fatty acids and erythrocyte phospholipids, while no changes were noticed in adipose tissue phospholipids and triacylglycerols [18]. The apparently low biological variation of milk AA but nevertheless high milk AA in populations with high AA intakes, the predominant derivation of milk LCP from maternal stores, the large distribution volume of AA and the inability to augment milk AA by short-term supplementation, suggest that human milk AA is mainly dependent on long term dietary AA intake. In view of these long-term effects, we investigated the influence of supplementation of AA plus DHA, or DHA alone from early pregnancy up to 12 weeks of lactation on milk AA, DHA, and the DHA/AA ratio.
Table 1 Daily intakes of fatty acids (in mg) from the supplements.
LA ALA AA EPA DHA
Placebo
DHA
DHA+AA
535 60 0 0 0
274 32 15 34 220
46 7 220 36 220
Abbreviations: LA, linoleic acid, 18:2o6; ALA, alpha-linolenic acid, 18:3o3; AA, arachidonic acid, 20:4o6; EPA, eicosapentaenoic acid, 20:5o3; DHA, docosahexaenoic acid, 22:6o3.
allowances. The women were instructed to take 2 capsules once daily from enrollment till 12 weeks postpartum. The AA+DHA group received 220 mg AA (Wuhan Alking Bioengeneering Co., Ltd., Wuhan, China) and 220 mg DHA (Marinol D40, Lipid Nutrition B.V., Wormerveer, The Netherlands). The DHA group received 220 mg DHA and one capsule containing soy bean oil (Wuhan Alking Bioengeneering Co., Ltd., Wuhan, China) and the placebo group received two capsules containing soy bean oil. Table 1 shows the daily fatty acid intakes from the capsules. The daily dosages of AA and DHA are within the range of typical intakes by Western adults [20]. The research protocol was approved by the Central Committee on Research Involving Human Subjects (CCMO, Den Haag, The Netherlands; protocol number P03.1071C). All women gave written informed consent. 2.3. Sample collection and analytical methods Women were instructed to collect 5 ml of milk at 2 and 12 weeks postpartum. Clock time and sampling within one feeding were not standardized since, in contrast to the milk fat content, the milk fatty acid composition does not vary to an appreciable extent during the day or during a single feeding [21,22]. One hundred microlitres of milk was transferred to a Sovirel tube containing 2 ml methanol/HCl (5:1, v/v) and 5 mg butylated hydroxytoluene. Following transmethylation, the milk fatty acid compositions were determined by using our previously described capillary gas chromatographic method with flame-ionization detection [23]. Fatty acids are presented in relative amounts, i.e. g/100 g fatty acids (wt%).
2. Materials and methods
2.4. Statistical methods
2.1. Subjects
Statistical analyses were performed using SPSS 14.0. The data were evaluated on the basis of intention to treat. Between-group differences in milk fatty acid compositions at 2 and 12 weeks postpartum were calculated using Mann–Whitney U tests. To correct for multiple testing, po0.017 was considered to be significant. Milk fatty acid contents at 2 and 12 weeks were compared using the non-parametric Wilcoxon Signed Rank test for related samples. po0.05 was considered significant.
This study was part of a double-blind placebo-controlled randomized trial with apparently healthy pregnant women who were recruited by midwives or gynecologists in and around the city of Groningen (The Netherlands) from December 2004 till December 2006. We included women with a low risk, first or second, singleton pregnancy. Women using a vegetarian or vegan diet and women with diabetes mellitus were excluded. Women were enrolled between 14 and 20 weeks of pregnancy, with the majority (80%) being enrolled between 15.6 and 17.4 weeks postmenstrual age (mean: 16.5 weeks). Criteria for termination of participation were any maternal or neonatal complications. 2.2. Study design At enrollment, the women were randomized into three groups using block randomization. All women received a supplement of vitamins and minerals according to Dutch recommended dietary
3. Results From the 182 pregnant women who were included in this trial, 57 women dropped out due to lack of motivation to fill in questionnaires and take capsules every day. Two women dropped out because of pregnancy complications (1 threatened preterm labor and 1 preterm birth). From the remaining 123 women, 88 chose to breastfeed their infants, of whom 69 continued breastfeeding for at least 12 weeks. The number of women who collected samples at 2 weeks postpartum amounted to 24
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higher 22:4o6 as compared to the DHA group (+17%; data available in Supplementary table). The DHA/AA ratio was increased at 2 and 12 weeks postpartum after supplementation with DHA alone, as compared to placebo. At 2 weeks, women receiving DHA also had a higher milk DHA/AA ratio compared to those receiving AA+DHA, while at 12 weeks those receiving AA+DHA had higher milk DHA compared to placebo. Although not significant, supplementation with DHA tended to decrease milk AA, and probably vice versa, especially at 2 weeks postpartum. There were no other between-group differences in milk fatty acids (data available in supplementary table). Table 3 shows longitudinal changes in milk fatty acids of the 67 women who provided milk samples both at 2 and 12 weeks. In each supplementation group, milk AA and DHA decreased from 2 to 12 weeks postpartum. In the placebo group, the DHA/AA ratio
(placebo group), 34 (DHA group) and 28 (AA+DHA group), two women did not provide samples. At 12 weeks postpartum, 19 women had discontinued breastfeeding. The remaining participants provided 19 (placebo), 30 (DHA) and 20 (AA+DHA) milk samples. The characteristics of the 88 breastfeeding mothers are described in Table 2. Fig. 1 shows that supplementation of AA+DHA significantly elevated milk AA both at 2 (+22%; difference between medians) and 12 (+23%) weeks postpartum when compared to the DHA group. At 2 weeks postpartum, milk AA was also higher in the AA+DHA group compared to the placebo group (+14%). Compared with placebo, supplementation of DHA or AA+DHA significantly increased milk DHA both at 2 (+59% and +43%, respectively) and 12 (+56% and +52%, respectively) weeks postpartum. In addition, at 2 weeks postpartum, the AA+DHA group showed significantly
Table 2 Perinatal maternal and infant characteristics.
Maternal age (yr) Prepregnancy BMI (kg/m2) Weight gain during pregnancy (kg) Gestational age at birth (w) Birth weight (g) Firstborn
Placebo (n ¼ 25)
DHA (n ¼ 34)
DHA+AA (n ¼ 29)
33.5 (26.0–40.3) 21.5 (17.5–31.7) 14 (0–20) 40.4 (34.9–41.9)a 3520 (2850–4840) 16 (64)
32.3 (22.3–43.3) 23.1 (18.7–41.8) 13 (2–27) 40.3 (37.0–42.1) 3610 (2620–4470) 21 (62)
31.5 (24.8–41.4) 23.1 (17.2–41.4) 18 (3–30) 40.0 (37.0–41.9) 3610 (3050–4310) 18 (62)
Data are presented as median (range) or as n (%). DHA, docosahexaenoic acid; AA, arachidonic acid; BMI, body mass index. Different from placebo (p ¼ 0.003) and DHA (p ¼ 0.004). a One woman in the placebo group gave preterm birth. Her milk fatty acid composition did not differ from that of the rest of the group, both at 2 and 12 weeks.
Fig. 1. Milk DHA, AA and DHA/AA ratio at 2 (left) and 12 (right) weeks postpartum. Women were supplemented with either placebo, DHA (220 mg/day) or DHA (220 mg/ day)+AA (220 mg) from week 17 of pregnancy till 12 weeks postpartum. The boxes show the interquartile range (25–75th percentile). The horizontal line displays the median, and the whiskers 1.5 times the interquartile range; outliers not shown. Interconnected boxes were significantly different at po0.017 (Bonferroni-adjusted p-value). DHA, docosahexaenoic acid; AA, arachidonic acid; DHA/AA, ratio of DHA and AA; FA, fatty acids.
Table 3 Longitudinal changes of milk arachidonic acid (AA), docosahexaenoic acid (DHA) and the DHA/AA ratio. DHA
Placebo (n ¼ 18) DHA (n ¼ 30) DHA+AA (n ¼ 19)
AA
DHA/AA
2 weeks p.p.
12 weeks p.p.
2 weeks p.p.
12 weeks p.p.
2 weeks p.p.
12 weeks p.p.
Median
Range
Median
Median
Range
Median
Median
Range
Median
Range
0.21–0.74 0.33–2.58 0.17–1.07
0.25 0.39 0.38
0.34–0.78 0.36–1.09 0.49–0.83
0.39 0.39 0.49
0.49–1.31 0.43–4.27 0.32–1.43
0.59
0.31–0.94 0.44–6.54 0.18–2.63
0.40 0.60 0.51
Range 0.13–0.37 0.16–1.46 0.08–1.04
0.58 0.57 0.66
Range 0.26–0.60 0.22–0.56 0.37–0.73
0.68 1.12 0.81
0.97 0.75
Data, presented as wt%, are from the 67 women of whom milk samples were available at both 2 and 12 weeks postpartum. p.p., postpartum. Indicates that the content in week 12 is lower compared with week 2, as tested with a Wilcoxon signed rank test for paired samples at po0.05.
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decreased, which is most likely caused by a stronger decrease of DHA as compared to AA.
4. Discussion This study is the first to show that milk AA increases after supplementation of a relatively low dose of AA, i.e. 220 mg/day, during pregnancy and lactation. Our study confirmed that DHA supplementation increases milk DHA. DHA seemed more responsive to supplementation than AA, since its increase was higher in both absolute (wt%) and relative (difference between medians) amounts. Some degree of competition between DHA and AA is conceivable since supplementation with DHA tended to reduce milk AA, and probably vice versa. Milk from women receiving AA+DHA contained insignificantly lower DHA at both 2 and 12 weeks postpartum compared to milk from women receiving DHA alone. Furthermore, 2 weeks postpartum, the women in the DHA group had insignificantly lower milk AA compared to the placebo group. A tendency of supplemental AA to reduce milk LCPo3 levels was previously noted [12], although supplementation of fish oil or DHA alone did not lower milk AA [24–26]. Because of the apparent competition between o3 and o6 LCP for incorporation into milk lipids, it seems important to supplement these at the appropriate ratio, which is as yet subject to discussion. The decline of both milk DHA and AA with advancing lactation has been previously noted [27]. This decline is likely to be caused by the increasing milk triacylglycerol/phospholipid ratio that accompanies the increase of milk fat globular size and triacylglycerol concentration with advancing lactation [27–29]. Alternatively, it may also reflect maternal AA and DHA depletion, and the currently supplemented dosages proved unable to prevent this alleged depletion. Interestingly, the DHA/AA ratio also declined over time, at least in the placebo group, suggesting that maternal DHA stores become more severely depleted than AA. A lactationinduced maternal DHA depletion is conceivable, since Otto et al. [30] showed a more pronounced postpartum decline of the DHA status in lactating as compared to nonlactating women. The outcome of this study is different from our previous study in which we supplemented 300 mg AA daily for 1 week [12]. The present AA dosage was lower (220 mg/day) but was administered for 26 (up to 2 weeks postpartum) and 36 weeks (up to 12 weeks postpartum), respectively. It seems that milk AA is notably dependent on long-term AA intake, which is conceivable from both the origins of milk LCP and the distribution of AA. Milk PUFA are known to derive mainly from maternal stores and only small amounts of milk AA and DHA originate from endogenous synthesis from their parent EFA precursors [16,17]. Moreover, supplemental AA is notably incorporated into phospholipids, such as those in circulating plasma lipoproteins and red blood cell membranes, and to a limited extent into plasma triacylglycerols, resulting in a large distribution volume [18]. The stronger increase of milk DHA as compared to AA suggests a smaller distribution volume of DHA. It is conceivable that the present supplementation periods of 26 and 36 weeks did not cause steady-state milk AA contents. In other words, the apparent milk AA increase of about 0.12 wt% (22%) at 2 weeks postpartum and 0.09 wt% (23%) at 12 weeks postpartum may underestimate the response of a prolonged supplemental intake of 220 mg AA/day. However, short-term DHA supplementation does not seem to cause a steady state either, as may be concluded from the few data on the DHA intake-milk DHA dose-response curves available in literature. These reveal a steeper DHA dose–response curve from a long-term DHA intake [31] as compared to the dose-response curve from a 12-week DHA supplement [25]. Translation of
currently recommended intakes of LCP during pregnancy and lactation into milk LCP contents may therefore await the construction of dose–response relationships that correlate milk LCP contents with long-term LCP intakes as estimated from reliable food frequency questionnaires. We conclude that breast milk AA content is sensitive to dietary AA intake, and that milk DHA seems more responsive to dietary intake than milk AA. The influence of dietary AA intake becomes noticeable after prolonged supplementation, which is in line with the notion that milk PUFA derive notably from maternal body stores. More studies are needed to determine optimal human milk and formula DHA, AA and DHA/AA.
Conflict of interest None of the authors had any conflict of interest.
Acknowledgments We kindly thank R.S. Kuipers, I.B.M. Meijer and I.A. Martini for their valuable aid in the analysis of the milk fatty acids.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.plefa.2008.11.002.
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