http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–4 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2015.1020785

ORIGINAL ARTICLE

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Is there a difference in breast milk fatty acid composition of mothers of preterm and term infants? Esther Granot1,3, Keren Ishay-Gigi1, Lea Malaach1, and Orna Flidel-Rimon2,3 1

Department of Pediatrics and 2Department of Neonatology, Kaplan Medical Center, Rehovot and Hebrew University-Hadassah Medical School, Jerusalem, Israel, and 3Hadassah Medical School, Hebrew University, Jerusalem, Israel

Abstract

Keywords

Objective: Arachidonic acid (ARA) (c20:4 w6) and docosahexanoic acid (DHA) (c22:6 w3) are of major importance for neural maturation and retinal function in infancy. Requirements in preterm infants are increased due to accelerated growth and limited body stores. Data regarding human milk fatty acid composition after preterm and full-term delivery is inconsistent. This study compared fatty acid composition in breast milk from full-term and preterm infants. Findings were correlated with maternal dietary intake. Methods: Human milk was obtained 4–5 days after full-term delivery (20 infants) and 4–5, 10–11 and 14–15 days after preterm delivery (21 infants, of whom 6 were born before 30 weeks). For fatty acid analysis, lipids were extracted, transesterified and separated by gas liquid chromatography. Results: Total fat content was similar in the two groups. FA composition including LCPUFA and specifically ARA & DHA were similar in full-term and preterm infants and in the sub-set born before 30 weeks. In preterm infants, postnatal age did not influence LCPUFA content. Conclusions: This study did not detect any effect of gestational age or postnatal age on milk LCPUFA content. Accordingly, the increased demand for LCPUFA and specifically DHA in preterm infants need to be met by other supplementation.

Arachidonic acid, creamatocrit, docosahexanoic acid, long-chain polyunsaturated fatty acids, medium chain fatty acids

Introduction Long-chain polyunsaturated fatty acids (LCPUFA), and specifically arachidonic acid (ARA c:20:4 w6) and docosahexanoic acid (DHA c:22:6 w3), are major components of brain and retinal cell membrane phospholipids, and therefore of pivotal importance for neural maturation and retinal function in infancy [1–3]. LCPUFA and especially DHA are preferentially transported across the placenta to the developing fetus and accumulate extensively in the fetal brain during the last trimester of pregnancy [4,5]. Thus, infants born prematurely are at a disadvantage as their body stores of LCPUFA are limited while at the same time requirements for fatty acid deposition in their rapidly growing tissues are high [6]. After delivery, as endogenous LCPUFA synthesis is relatively low, supply of LCPUFA to the breast-fed infant depends on the amount of these fatty acids in breast milk. Human milk fatty acid composition is influenced by factors such as parity, maternal diet and stage of lactation [7,8]. As infants born prematurely have increased requirements for Address for correspondence: Esther Granot, Department of Pediatrics, Kaplan Medical Center, Rehovot and Hebrew University-Hadassah Medical School, Jerusalem, Israel. Tel: 972-505708504. Fax: 972-89411942. E-mail: [email protected]

History Received 14 January 2015 Accepted 16 February 2015 Published online 11 March 2015

LCPUFA, it is of interest to determine whether human milk fatty acid composition is also influenced by duration of pregnancy. To date, studies relating to LCPUFA content in milk of mothers giving birth to preterm infants have yielded conflicting results [9–12]. We therefore compared fatty acid composition in human milk of mothers who gave birth to full-term and pre-term infants. In addition, we assessed the effect of postnatal age in the preterm group by studying breast milk FA composition during the first two weeks after delivery.

Subjects and methods Subjects The study included nursing mothers of term and preterm infants born at Kaplan Medical Center, Rehovot, Israel. The study was approved by the Hospital Ethics Committee. Methods Human milk samples were obtained from mothers of both term and preterm infants at age 4–5 days. In the preterm group, repeat samples were obtained at ages 10–11 and 14–15 days. All milk samples were hindmilk obtained at the end of the first morning feed.

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J Matern Fetal Neonatal Med, Early Online: 1–4

Fat analysis

Table 1. Breast milk fatty acid composition.

Fat content was estimated by creamatocrit, measured in a capillary tube centrifuged at 8000 rpm for 5 min. The fatty acid analysis included extraction of lipids by the Folch method (chloroform/methanol 2:1 v/v) [13], in the presence of butylated hydroxytoluene as antioxidant. The lipid fraction was dried under nitrogen, dissolved in chloroform and frozen at 70  C until analysis. Lipids were transesterified with methanolic trimethylammonium hydroxide (Meth Prep II, Applied Science, Deerfield, IL) [14]. After transesterification, fatty acid methyl esters were dried under nitrogen and redistributed in chloroform for gas chromatography analysis. Fatty acid methyl esters were separated and quantified by high-resolution gas liquid chromatography (Migal Analytical Service Laboratory, Kiryat Shmona, Israel). Results were expressed as a percentage of total fatty acids by weight. All of the study mothers completed dietary questionnaires that focused on the amount and type of oil and fish consumed each week.

Fatty acid

Statistical analysis Sample size was calculated based on the anticipated fatty acid concentrations based on the study of Kovacs et al. [12]. Assuming that 50% of the preterm group would have LC-PUFA levels below 0.4%, with a 1:1 ratio between the groups, alpha ¼ 90% and beta ¼ 80%, a sample size of 20 infants per group was required. Analyses were performed with InStat GraphPad INC, San Diego, CA, using the two-tailed Student t test and ANOVA for continuous variables and Wilcoxon rank sum test and a Friedman two-factor analysis for categorical variables. A p value of less than 0.05 was considered significant. All results expressed as mean ± standard deviation.

Results Description of study sample The term group consisted of 20 full-term infants. The mean age of the mothers was 31.6 ± 5.2 years, mean parity was 4 ± 2.72 and, at birth, mean gestational age was 39 ± 1.2 weeks and mean birth weight was 3157 ± 382 g. The preterm group consisted of 21 preterm infants. The mean age of the mothers was 29.9 ± 6.8 years, mean parity was 2 ± 1.38 and, at birth, mean gestational age was 31 ± 2.9 weeks and mean birth weight was 1615 ± 496 g. Of the preterm infants, 6 were born at 24–30 weeks gestation (mean gestational age 27 ± 2 weeks and mean birth weight was 930 ± 230 g. Total fat content Total fat content, as measured by creamatocrit, was similar in the two groups (term  6.95 ± 3.1% versus 6.75 ± 3.05% preterm, p ¼ ns). In the preterm group, creamatocrit values rose slightly between days 4–5 (8.45 ± 3.59) and 14–15 (8.26 ± 2.72), but this finding was not statistically significant.

c8:0 Capric acid (c10:0) Lauric acid (c12:0) Myristic acid (c14:0) Palmitic acid (c16:0) Palmitoleic acid (c16:1) Stearic acid (c18:0) Oleic acid (c18:1w9) Linoleic acid (c18:2w6) Linolenic acid (c18:3w3) c18:3 w6 c20:2 w6 c20:3 w3 c20:3 w6 Arachidonic acid (ARA) (c20:4 w6) Eicosapentanoic acid (EPA) (c20:5w3) Docosahexanoic acid (DHA) (c22:6 w3)

Full-term

Preterm

0.08 ± 0.08 0.69 ± 0.38 4.06 ± 1.89 6.04 ± 1.25 25.05 ± 2.1* 2.07 ± 0.66 7.36 ± 1.08 29.39 ± 3.76 16.39 ± 2.53 1.37 ± 0.67 0.13 ± 0.057 1.24 ± 0.34 0.17 ± 0.08 0.89 ± 0.23 0.97 ± 0.20 0.11 ± 0.15 0.68 ± 0.36

0.08 ± 0.09 0.785 ± 0.58 3.85 ± 2.28 6.31 ± 2.54 21.67 ± 2.2 1.97 ± 0.67 7.56 ± 1.67 29.89 ± 3.60 17.36 ± 3.05 1.6 ± 0.47 0.15 ± 0.09 1.25 ± 0.42 0.18 ± 0.09 0.91 ± 0.30 0.99 ± 0.26 0.15 ± 0.26 1.1 ± 1.05

Fatty acid composition (expressed as % of total FA by weight, mean ± SD) in breast milk samples of mothers giving birth to full term or premature infants. Samples were obtained on the 4th–5th day post delivery. *p50.001.

Medium chain fatty acids Levels of C8:0, C10:0, C12:0 (lauric) and C14:0 (myristic) fatty acids did not differ between the groups. Palmitic acid (c16:0) Palmitic acid was higher in the term group (term  25.05 ± 2/1% versus preterm  21.67 ± 2.2%, p50.001). In addition, a comparison was performed between the extremely preterm infants (24–30 weeks, n ¼ 6) and larger preterm infants (430 weeks) and no significant difference was detected (extremely preterm  21.03 ± 1.7% versus larger preterm term 21.67 ± 2.2%). Levels of additional fatty acids including stearic, oleic, the essential fatty acids linoleic and linolenic acids and LCPUFA including ARA, EPA and DHA did not differ between the groups. Effect of postnatal age In the preterm group, except for the increase in medium chain fatty acid levels at age 4–5, 10–11 and 14–15 days no statistically significant differences were detected in levels of essential fatty acids and ARA & DHA (Table 2). In addition, no significant differences were found between the milk of mothers of extremely preterm and larger preterm infants in fatty acid composition. Specifically, ARA levels in term, larger preterm and extremely preterm infants were 0.97 ± 0.20, 0.99 ± 0.26, 1.15 ± 0.28, respectively (p ¼ 0.29) Similarly, DHA levels did not differ between the groups (term, larger preterm or extremely premature infants  0.68 ± 0.36, 1.1 ± 1.05, 1.06 ± 0.49, respectively, p ¼ 0.21). Mothers’ dietary intake

Milk fatty acid composition Fatty acid levels (as a % of total FA by weight) were compared in the two groups at age 4–5 days (Table 1).

Similar dietary habits were observed in mothers of both groups. Weekly intake of fish, poultry meat and nuts was similar in the two groups (Table 3). Type of cooking oil and

Milk fatty acids after preterm and term delivery

DOI: 10.3109/14767058.2015.1020785

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Table 2. Fatty acid composition of milk samples of mothers giving birth to preterm infants, according to stage of lactation.

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Fatty acid

Preterm sample I

c8:0 Capric acid (c10:0) Lauric acid (c12:0) Myristic acid (c14:0) Palmitic acid (c16:0) Palmitoleic acid (c16:1) Stearic acid (c18:0) Oleic acid (c18:1w9) Linoleic acid (c18:2w6) Linolenic acid (c18:3w3) c18:3 w6 c20:2 w6 c20:3 w3 c20:3 w6 Arachidonic acid (ARA) (c20:4 w6) Eicosapentanoic acid (EPA) (c20:5w3) Docosahexanoic acid (DHA) (c22:6 w3)

ab

0.08 ± 0.09 0.79 ± 0.58cd 3.85 ± 2.28ef 6.31 ± 2.54 21.7 ± 2.2g 1.97 ± 0.67 7.49 ± 1.68 29.9 ± 3.49 17.36 ± 3.05 1.6 ± 0.67 0.09 ± 0.10 1.25 ± 0.42 0.18 ± 0.09 0.91 ± 0.30 0.99 ± 0.26 0.15 ± 0.27 1.1 ± 1.05

Preterm sample II a

0.24 ± 0.10 1.50 ± 0.60c 6.10 ± 2.18e 7.73 ± 2.69 20.5 ± 2.37 2.43 ± 0.63 7.72 ± 1.73 27.8 ± 3.10 17.45 ± 3.30 1.9 ± 0.71 0.12 ± 0.16 0.94 ± 0.32 0.15 ± 0.06 0.82 ± 0.35 0.95 ± 0.29 0.13 ± 0.06 0.85 ± 0.43

Preterm sample III 0.195 ± 0.10b 1.43 ± 0.64d 5.96 ± 2.77f 7.13 ± 2.89 19.9 ± 1.78h 2.03 ± 0.72 7.48 ± 0.77 28.45 ± 3.60 18.35 ± 3.67 1.99 ± 0.70 0.14 ± 0.17 0.79 ± 0.24 0.13 ± 0.06 0.68 ± 0.25 0.83 ± 0.20 0.11 ± 0.08 0.77 ± 0.41

Fatty acid composition (expressed as % of total FA by weight, mean ± SD) of milk samples of mothers giving birth to preterm infants, according to stage of lactation (sample I; 4–5 days, sample II; 10–11days, and sample III; 14–15 days, post delivery). ab p50.001; cdp50.01; ef, g versus hp50.05.

Table 3. Mothers’ dietary intake.

Poultry and meat servings /week Fish servings/week Servings of nuts/week

Full term infants

Premature infants

3.70 (±1.89) 0.70 (±0.54) 0.72 (±0.76)

3.80 (±1.93) 0.69 (±0.55) 0.76 (±0.58)

Mothers’ dietary intake, based on data obtained from questionnaires. p ¼ n.s.

salad oil consumed were also similar as canola oil and olive oil were used by approximately 90% of mothers in both groups.

Discussion This study found no significant differences in the fat content of breast milk from mothers of term or preterm infants. Total fat content as measured by creamatocrit (6.95 ± 3.1% in the term group) is similar to that reported by Kedem et al. (5.4 ± 4.0%) [15] in breast milk of 17 mothers of a similar age group and similar population. In the Kedem et al. study, creamatocrit levels correlated inversely with the gestational age. This is in contrast to the results of our study in which total fat content did not differ between milk of mothers giving birth to term, preterm or extremely preterm infants. As for changes in fat content during the first two weeks of lactation, when measured in 3 breast milk samples from mothers giving birth to preterm infants, no differences in fat content were observed. This is in agreement with findings of Kovacs et al. [12], who similarly did not note any differences in fat content between milk samples from the 1st and 2nd weeks post delivery. As for the medium chain and intermediate chain fatty acids, specifically lauric acid (c12: 0) and myristic acid (c14:0), both Bitman et al. [9] and Genzel-Boroviczeny et al. [11] did show levels to be higher in preterm milk samples and hypothesized that as MCFA may be more readily absorbed

than longer fatty acids absorbed via chylomicrons, higher MCFA in preterm infants may be of functional significance. We did not observe differences in the levels of lauric acid or myristic acid in milk samples of term or preterm infants at age 4–5 days, although at age 10–11 and 14–15 days preterm infants did have higher c10:0 and c12:0 levels. Myristic acid levels (c14:0) were also higher, but these differences did not reach statistical significance. In our study, sequential breast milk samples from mothers giving birth to full term infants were not available, thus precluding conclusions regarding MCFA levels after term delivery. Essential fatty acids, linoleic (c18:2 w6) and alpha linoleic acid (c18:3 w3) levels were not influenced by either gestational or postnatal age and these results are in accordance with those of Kovacs et al. [12] who also did not observe differences in essential fatty acid levels in milk of 10 mothers giving birth to full term infants and that of 8 mothers giving birth to preterm infants throughout a 28-day post delivery study period. Luukkainen et al. [10] similarly did not observe differences in the proportions of essential fatty acids in preterm and term milk. Genzel-Boroviczeny et al. [11] studying milk samples from 30 mothers of term infants and 19 mothers of preterm infants also observed that term and preterm milk does not differ in the percent content of linoleic and alpha linolenic acids, but in contrast to our results they did note a significant increase in these fatty acid levels in milk samples from day 5 as compared to levels in samples obtained on day 10, post delivery. The comparison of the levels of arachidonic acid and docosahexanoic acid in term and preterm milk is of special interest. ARA and DHA both play a pivotal role in brain development and visual function maturation. The critical period of placental transfer of these fatty acids is believed to be in the 3rd trimester of pregnancy. It has been estimated that approximately 60 mg of DHA per day accrues in the fetal brain during this time period [16,17]. Thus, in infants born prematurely, the depleted DHA stores, coupled with limited ability to synthesize LCPUFA [18] and enhanced needs due to

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accelerated growth places them at high risk for ARA and DHA deficiency. When comparing breast milk of mothers giving birth at term to those delivering preterm infants, we did not observe differences in the proportion of either ARA or DHA. Nor did these levels increase in breast milk of mothers giving birth to preterm infants during the two weeks post delivery period, studied (Table 2). Furthermore, even in the extremely preterm group, breast milk ARA and DHA levels did not differ from mean levels in breast milk of mothers giving birth to term infants; ARA term  0.97 ± 0.2% versus preterm 1.15 ± 0.28% and DHA term 0.68 ± 0.36 versus preterm 1.06 ± 0.49%. Indeed, Genzel Boroviczeny et al. [11] similarly did not observe a difference in the percent content of LCPUFA in term and preterm milk, and Luukkainen et al [10] also showed no differences in the proportion of ARA & DHA in term and preterm milk samples at one week post delivery. In contrast, Kovacs et al. [12] showed almost two-fold higher levels of both ARA & DHA in preterm versus term milk in samples obtained during the first week post delivery. Bitman et al. [9] found ARA levels to be higher in 1st–4th day milk samples from mothers giving birth to term infants as compared to levels in milk samples of mothers giving birth to premature (31–36 weeks of gestation) or ‘‘very small’’ premature (26– 30 weeks gestation) infants. In later post delivery samples no differences in ARA levels were observed. These authors did not analyze milk DHA content. It is noteworthy that Luukkainen et al. did observe a gradual decrease in the proportion of ARA & DHA which was more marked in term milk samples, so by 6 months of lactation, the levels of ARA & DHA were 1.5–2-fold higher in preterm milk. We studied milk samples from mothers giving birth to premature infants only during the first two weeks of lactation and in this short time period no change in percent content of these fatty acids was observed. Based on our findings we conclude that breast milk of mothers of preterm infants does not compensate for the increased requirements of LCPUFA, which result from prematurity. In milk of mothers delivering preterm infants, we did not observe adaptive changes in breast milk composition of either ARA or DHA levels required for the special needs of preterm infants. The efficacy and long-term benefits of LCPUFA-supplemented formulas, in preterm infants, are as yet controversial [19,20]. A recent comprehensive review by Lapillonne et al. [21] addressed the relevant literature and recommendations related to the specific needs of LCPUFAs in preterm infants. The authors reiterated the superiority of human milk while emphasizing the highly variable content of DHA in human milk, which may not be adequate to meet requirements of preterm and especially those of extremely preterm infants. They further urged that future studies should evaluate various DHA dose–response relationships and elucidate safety issues and potential immediate and long-term benefits of DHA supplementation. The optimal daily amounts of ARA & DHA, required in preterm infants, remain be defined so that they can be adequately supplemented in infant formulas and also provided to breast-fed preterm infants via dietary

J Matern Fetal Neonatal Med, Early Online: 1–4

supplementation to the lactating mother or as an enriched fortifier of breast milk.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Is there a difference in breast milk fatty acid composition of mothers of preterm and term infants?

Arachidonic acid (ARA) (c20:4 w6) and docosahexanoic acid (DHA) (c22:6 w3) are of major importance for neural maturation and retinal function in infan...
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