Int. J. Devl Neuroscience 33 (2014) 115–121
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Preeclampsia alters milk neurotrophins and long chain polyunsaturated fatty acids Kamini Dangat a , Anitha Kilari a , Savita Mehendale b , Sanjay Lalwani c , Sadhana Joshi a,∗ a b c
Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune 411043, India Department of Obstetrics and Gynecology, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune 411043, India Department of Pediatrics, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune 411043, India
a r t i c l e
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Article history: Received 11 September 2013 Received in revised form 6 December 2013 Accepted 17 December 2013 Keywords: DHA NGF BDNF Anthropometry Lactation
a b s t r a c t Objective: To examine the levels of breast milk neurotrophins 1.5, 3.5, and 6 months of lactation and long chain polyunsaturated fatty acids (LCPUFA) at day 3, 1.5, 2.5, 3.5 and 6 months of lactation in mothers with preeclampsia and compare them with normotensive women. Their associations with growth parameters in children are also examined. Methods: Women with preeclampsia (n = 101) and normotensive women (n = 144) with singleton pregnancies were recruited for this study. Milk samples were collected and anthropometry was recorded at the first 6 months. The LCPUFA composition of milk samples was analyzed by using gas chromatography at all time points and neurotrophins were analyzed at 1.5, 3.5 and 6 months by Emax Immuno Assay System using Promega kits. Results: Milk DHA levels were higher at day 3 (9.5%), and 1.5 (23%) and 3.5 (40%) months in mothers with preeclampsia as compared to controls. Milk nerve growth factor (NGF) levels were lower in preeclampsia group as compared to control group at 1.5 (20%) and 3.5 months (27.7%). Milk brain derived neurotrophic factor (BDNF) levels were lower at 1.5 months (10.5%) in the preeclampsia group as compared to control group. Conclusion: The present study suggests that there is a differential regulation of DHA and neurotrophins in breast milk in preeclampsia and are associated with growth parameters of children. Future studies should explore the associations between milk LCPUFA, neurotrophins with neurodevelopment in children. © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Human milk has been reported to contain wide range of nutrients and several components like hormones (Kulski and Hartmann, 1981), growth factors (Donovan and Odle, 1994), cytokines (Srivastava et al., 1996), immunological components (Jackson and Nazar, 2006), immunomodulatory factors (Bertino et al., 2012) and fatty acids (Agostoni et al., 2005) which play a vital role in the growth and development of the infant (Serpero et al., 2012). Pregnancy complications such as pregnancy induced hypertension (PIH) (Cunningham et al., 1993); preeclampsia (Erba˘gci et al., 2005) and gestational diabetes (Butte et al., 1987) are known to affect lactogenesis (Kaushik et al., 2002). Babies born to mothers
Abbreviations: AA, arachidonic acid; BDNF, brain derived neurotrophic factor; DHA, docosahexaenoic acid; LCPUFA, long chain polyunsaturated fatty acids; NGF, nerve growth factor. ∗ Corresponding author. Tel.: +91 020 24366929/24366931; fax: +91 020 24366929. E-mail address: [email protected] (S. Joshi). 0736-5748/$36.00 © 2014 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.12.007
with preeclampsia are at high risk for metabolic and cognitive disorders (Many et al., 2003) due to neurodevelopmental impairment in later life (Streimish et al., 2012). Evidence suggests that long-term breastfeeding is associated with lower hyperactivity symptoms (Julvez et al., 2007); improved neuropsychological and socio-behavioral outcomes and cognition in children (Keim et al., 2012; Hadders-Algra, 2011; Jensen and Lapillonne, 2009; Daniels and Adair, 2005; Clark et al., 2006; Hüppi, 2008). A review has suggested that milk long chain polyunsaturated fatty acids (LCPUFA), specifically docosahexaenoic acid (DHA) and arachidonic acid (AA), many growth factors and hormones influence infant’s growth, development and cognitive performance (Yum, 2007). Human milk polyunsaturated fatty acids (PUFA) are either derived from the diet or are synthesized from endogenous precursor fatty acids (Sauerwald et al., 2001) and are required in adequate quantities for optimal growth and development of infants (Xiang et al., 2005). However, reports on milk LCPUFA influence on infant growth are controversial (Tinoco et al., 2009; Scholtens et al., 2009). Reports also indicate that LCPUFA especially DHA regulates the levels of neurotrophins (Rao et al., 2007; Wu et al., 2004). Milk
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Fig. 1. Flow chart for number of samples analyzed at various time points. PE: preeclampsia; BDNF: brain derived neurotrophic factor; NGF: nerve growth factor.
growth factors such as brain derived neurotrophic factor (BDNF) (Li et al., 2011) and nerve growth factor (NGF) (Banapurmath et al., 1996) are known to play a critical role in the development and maintenance of the nervous system. Recent reports also indicate that the milk components change over the course of lactation due to time dependent programmed requirement for the child development (Ballard and Morrow, 2013; Erba˘gci et al., 2005; Li et al., 2011). Milk fatty acid profile found to be affected by maternal dietary and life style factors of mother (Antonakou et al., 2012). We have earlier reported that milk fatty acids and neurotrophins levels are altered at d3 of lactation in preeclampsia (Dangat et al., 2010, 2013) which may affect the milk quality and postnatal programming of child. However, it is unclear whether these vital constituents remain altered during the complete duration of lactation. Thus, the present study for the first time examines simultaneously the levels of human milk fatty acids and neurotrophins in mothers with preeclampsia and compares them with controls at various time points during lactation and also examines their association with growth of the infant.
Preeclampsia was diagnosed by the criteria discussed by us in a number of our studies reported earlier (Kilari et al., 2011; Kulkarni et al., 2010). Briefly, preeclampsia was defined by systolic and diastolic blood pressures greater than 140 and 90 mmHg respectively with proteinuria in a dipstick test. The oil consumption per month has been recorded at the time of recruitment. Treatment of preeclamptic women included antihypertensive drugs and arginine supplementation. In severe preeclamptic cases, MgSO4 was given intravenously. It is unlikely that these would affect BDNF status. 2.1. Collection of milk samples Human milk sampling was done at all time points during lactation. The samples were collected when the babies come for vaccination at 1.5, 2.5 and 3.5 months of age. Samples of breast milk (2 ml) were taken at the end of the baby’s being breast-fed and collected into cryo vials and were stored at −80 ◦ C. 2.2. Fatty acid analysis
2. Materials and methods This study was conducted at the Departments of Pediatrics and Obstetrics & Gynecology, Bharati Hospital, Pune during the year 2011–2012. Singleton pregnant women with preeclampsia (n = 101) and without preeclampsia (controls) (n = 144) were recruited at the time of delivery for this prospective study. Mothers and their babies were followed-up at day 3, and 1.5, 2.5, 3.5 and 6 months post delivery. The anthropometry (weight and length, head and chest circumference) of the infant was recorded at all time points during lactation. Fig. 1 shows the number of milk samples analyzed for fatty acids and neurotrophins at various time points during lactation. This study was approved by Institutional Ethical Committee and a written informed consent was taken from each subject. Subjects were excluded from the study if there was evidence of other pregnancy complications like multiple gestation, chronic hypertension, type I or type II diabetes mellitus, seizure disorder, renal or liver disease. Pregnant women with alcohol or drug abuse were also excluded from the study. The control group consisted of pregnant women with no medical or obstetrical complications.
Milk samples were analyzed at all time points by the gas chromatography and have been described by us earlier (Dangat et al., 2010). Fatty acids were expressed as g/100 g fatty acid. A total of 15 fatty acids were estimated. Saturated fatty acids include myristic acid, palmitic acid and stearic acid while monounsaturated fatty acids include myristoleic, palmitoleic, oleic and nervonic acids. The omega 3 fatty acids included alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) and DHA while omega 6 fatty acids included linoleic acid (LA), gamma linolenic acid (GLA), di-homogammalinolenic acid (DGLA), docosapentaenoic acid (DPA) and AA. 2.3. Neurotrophins analysis Milk neurotrophins at day 3 has been published recently (Dangat et al., 2013). Milk neurotrophins at 1.5, 3.5 and 6 months of lactation were analyzed by Emax Immuno Assay System using the Promega kits for BDNF (Hornbeck et al., 1994) and NGF (Zettler et al., 1996) which has been described by us earlier (Dangat et al., 2013). The amount of BDNF and NGF was expressed as pg/ml.
K. Dangat et al. / Int. J. Devl Neuroscience 33 (2014) 115–121 Table 1 Maternal characteristics at the time of recruitment. Control (n = 144) Age (years) Income (INR) Age at marriage (years) Body mass index (BMI) Gestation (weeks) Systolic blood pressure at delivery (mmHg) Diastolic blood pressure at delivery (mmHg) **
24.0 7784 20.1 25.7 39.2 120.1
± ± ± ± ± ±
3.7 4720 2.9 3.88 1.1 9.2
77.6 ± 6.1
Preeclampsia (n = 101) 23.7 7593 20.8 22.64 36.9 151.2
± ± ± ± ± ±
3.6 5086 2.9 4.6 2.8** 18.5**
99.2 ± 10.4**
p < 0.01.
2.4. Statistical methods Values are expressed as mean ± SD. The data were analyzed using SPSS/PC+ package for MS Windows (Version 20, Chicago, IL). Skewed variables (DHA at 1.5 months, NGF at 1.5 months) were transformed to satisfy the underlying assumption of normality using natural logarithm transformation (Loge ). Mean values of the estimates of milk fatty acids and neurotrophins were compared by Student’s “t” test. Correlations between milk fatty acids, baby weight, height, head and chest circumferences were studied using Pearson’s correlation analysis after adjusting for maternal age, body mass index (BMI) and duration of gestation as they are possible confounders. Frequency of consumption of oil was analyzed using chi square test.
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NGF levels were significantly lower at 1.5 (20%) and 3.5 (27.7%) months in preeclampsia group as compared to controls. However, milk NGF levels remain lower but not significant between the groups at 6 months. In contrast, Milk BDNF levels were significantly lower (10.5%) at 1.5 months in preeclampsia as compared to controls (Fig. 4). However, there was no significant difference in milk neurotrophins levels between groups at 6 months of lactation. 3.4. Associations of milk fatty acids with baby characteristics during lactation Milk AA levels were negatively associated with baby weight (r = −0.284, p = 0.017, n = 68) at 1.5 month and 3.5 months (r = −0.396, p = 0.003, n = 52) in the preeclampsia group but were positively associated with baby length (r = 0.231, p = 0.045, n = 74) at 3.5 months in controls. Milk DHA levels were negatively associated with baby length (r = −0.363, p = 0.007, n = 52) at 3.5 months only in the preeclampsia group. Saturated fatty acid levels were negatively associated with baby weight at 3.5 months (r = −0.268, p = 0.050, n = 52) in preeclampsia group. However, these associations were not seen at 6 months of lactation. 3.5. Associations of milk neurotrophins with baby characteristics There was negative association between milk NGF and baby length at 1.5 months only in control group (r = −0.337, p = 0.019, n = 48). There was no association observed between milk BDNF and baby characteristics.
3. Results
3.6. Associations of milk neurotrophins with milk fatty acids
3.1. Maternal and baby characteristics
There was a negative association between milk NGF and milk DHA levels (r = −0.311, p = 0.033, n = 47) in the control group while milk NGF was negatively associated with milk AA (r = −0.463, p = 0.008, n = 32) in preeclampsia group at 1.5 months of lactation. Similarly, milk NGF levels were negatively associated with milk omega 6 fatty acids (r = −0.543, p = 0.001, n = 32) in control group at 3.5 months. However, milk BDNF levels did not show any association with milk fatty acids.
Table 1 shows the maternal characteristics. Systolic and diastolic blood pressures were higher (p < 0.01) and gestation was shorter (p < 0.01) in the preeclampsia group as compared to controls. Baby weight, length, head and chest circumferences at birth were significantly lower in preeclampsia group as compared to controls which has been reported by us earlier (Dangat et al., 2013). The baby weight, length, head and chest circumferences increased in both groups with almost similar trends but these remained lower in preeclampsia group at all-time points, i.e. day 3, 1.5, 2.5, 3.5 and 6 months as compared to control group (Fig. 2). 3.2. Milk fatty acid levels during lactation Fig. 3 shows the levels of milk fatty acids in both the groups. Milk LA (a precursor of AA) levels were lower but not significant except at 2.5 months (7.1%) in mothers with preeclampsia as compared to controls at all other time points; day 3, 1.5, 3.5 and 6 months of lactation. Whereas, milk AA levels were similar in the preeclampsia group as compared to controls at all time points. The levels of ALA (a precursor of DHA) were similar or slight trend toward higher in preeclampsia compared to controls. In contrast, milk DHA levels were higher (p < 0.05) at day 3 (9.5%), 1.5 (23%) months and 3.5 (40%) months in women with preeclampsia as compared to controls. However, the levels of omega-3 fatty acids, monounsaturated fatty acids and saturated fatty acids did not differ much between groups while total omega-6 fatty acid levels were lower at 2.5 months in preeclampsia throughout the study period. Milk fatty acid levels were similar between groups at 6 months of lactation. 3.3. Levels of milk neurotrophins The levels of both NGF and BDNF in controls as wells as preeclampsia also show very different longitudinal patterns. Milk
3.7. Dietary intake of oil consumption Oil consumption has been divided into 1.5/month. Oil intake was significantly higher (p < 0.01) in preeclampsia group as compared to control group. In control group, 63.92% of women had consumed 1.5 kg per month. In preeclampsia group, 47.73% had consumed 1.5 kg per month. The women in the cohort mainly consumed soyabean (41%), sunflower (32%) and ground nut oil (15%). 4. Discussion This study for the first time simultaneously reports the levels of LCPUFA (primarily LA, AA, ALA and DHA) and neurotrophins (NGF and BDNF) in human milk from Indian women at various time points during lactation. All mothers exclusively breastfed their babies. The key findings of our study are (1) milk DHA levels were higher in preeclampsia as compared to controls at day 3, 1.5 and 3.5 months, (2) milk NGF levels were generally lower in preeclampsia group as compared to control group throughout lactation, (3) milk BDNF levels were lower at 1.5 months of lactation in the preeclampsia group as compared to control group, (4) baby weight was negatively associated with milk AA levels at 1.5 and 3.5 months while milk DHA levels were negatively associated
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Fig. 2. Baby growth charts from birth to 6 months. Mon: months
with baby length at 3.5 months in preeclampsia group and (5) there was a negative association between milk NGF and baby length at 1.5 months in control group. However, there was no association observed between milk BDNF and baby characteristics. Our data has shown decrease in AA and DHA levels from day 3 to 6 months of lactation and is consistent with a reported longitudinal study which has also demonstrated that the milk DHA and AA gradually decrease from colostrum to mature milk (Chen et al., 1997). Furthermore, the levels of both the precursors (LA and ALA) were lower and the products (AA and DHA) levels were higher in milk in both the groups at day 3 (closer to colostrum) compared to milk at later time points (mature milk). Increased levels of milk AA at day 3 may improve the growth of the newborns. It is well known that AA is an important nutrient factor for growth in early postnatal life (Much et al., 2013). Reports indicate that milk AA levels may not be mediating inflammation because human milk contains high anti-inflammatory than pro-inflammatory lipid mediators (Takamoto et al., 1995). LCPUFA such as AA and DHA are
synthesized by elongation/desaturation of precursors like LA and ALA, respectively. Desaturases are the enzymes which regulate the unsaturated fatty acid biosynthesis and are mainly present in liver. It has been reported that fatty acid desaturase enzyme activity in newborns is low (Innis, 2007). Current data shows increased milk levels of both LCPUFA precursors (LA and ALA) and reduced products (AA and DHA) from day 3 to 6 months of lactation. At later time points, the levels of AA and DHA decrease possibly because of reduced need for products (AA and DHA) due to increased conversion ability of the infant. It has been mentioned that the infants probably can synthesize additional LCPUFAs from the LA and ALA contents of human milk (Heird, 2001). Similarly, the levels of BDNF are highest at 3 days whereas the NGF levels steadily increase but the levels of BDNF decrease at 1.5 month and there was a trend toward increase until 6 months. We have earlier reported increased levels of milk BDNF at day 3 in the same cohort (Dangat et al., 2013). The significant quantitative differences in BDNF at several time points during lactation between controls and preeclampsia
Fig. 3. Milk fatty acid levels during lactation. Mon: months; *p < 0.05
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Fig. 4. Milk neurotrophins levels during lactation. Mon: months; *p < 0.05; **p < 0.01; BDNF: brain derived neurotrophic factor; NGF: nerve growth factor; pg/ml: picograms/milliliter.
probably indicate that the milk programming by mother’s breast is altered by preeclampsia. It is likely that these changes are most likely adaptive changes of mother and some of these changes are not normalized even up to 6 months. The levels of DHA in human milk levels remain higher in mothers with preeclampsia as compared to controls throughout lactation except at 2.5 month levels were almost similar. We have earlier reported higher milk DHA levels at postnatal day 3 in preeclampsia in spite of its lower plasma DHA levels in mother which is consistent with the data reported by us earlier in another cohort (Dangat et al., 2010). The mean milk DHA concentrations in current study are similar to the levels reported in countries like Nepal (Glew et al., 2001) and Bangladesh (Yakes et al., 2011). Milk DHA levels in our study is similar to Sudanese mothers whose diet is devoid of fish products (Nyuar et al., 2010) and American Indian women with low DHA intake (Glew et al., 2011). In contrast, milk DHA levels were slightly higher in Cuba (Krasevec et al., 2002). A study on milk samples from 9 different countries has revealed that milk saturated and MUFA composition is constant but PUFA like DHA is highly variable (Yuhas et al., 2006). A study from China reveals that mothers in coastal area of china showed higher DHA status of milk (Peng et al., 2009). Milk DHA showed a positive correlation with dietary DHA intake (Glew et al., 2011). This suggests maternal diet and fatty acid metabolism plays a critical role in determining the DHA status of mother. Frequency of consumption of omega 3 fatty acid rich foods was similar which has been reported by us earlier in another cohort (Kulkarni et al., 2011). Increased milk DHA levels in current study levels probably due to increased expression of Sterol regulatory element binding proteins (SREBP-1) in the mammary glands which in turn increases the expression of delta-5 desaturase and delta-6 desaturase ultimately resulting into increased DHA synthesis. Our study subjects are predominantly vegetarian and their lower levels compared to higher levels in countries with higher sea food diet may explain the differences. In our study, the mean milk DHA levels decreased from day 3 to 6 months of lactation in both preeclampsia and control groups. Higher milk DHA levels in colostrum compared to mature milk have been reported in both term and preterm mothers (Berenhauser et al., 2012). This is also consistent with the reports from lactating Italian mothers (Agostoni, 2005). The levels of AA in the present study were in the ideal range which has been recently reported (Antonakou et al., 2012). Our study suggests that the need of LCPUFAs, especially DHA, can be satisfied in healthy normotensive mothers by breastfeeding
during early lactating period. Reports suggest that long term (DelPrado et al., 2001) and short term (Innis, 2007) maternal diet and fatty acid status influences the milk fatty acid status. Total omega 3 fatty acid intakes were similar in both controls as well as in preeclampsia groups which was reported by us earlier in another cohort (Kulkarni et al., 2011). In current study, higher milk DHA levels in mothers with preeclampsia may be due to altered metabolism of fatty acids. In addition to maternal diet, breast milk fatty acid composition is also dependent on mobilization of endogenous stores of fatty acids and de novo synthesis of fatty acids by liver or breast tissue (Sabel et al., 2012). In our study, milk AA levels were negatively associated with baby weight at 1.5 and 3.5 months and milk DHA levels were negatively associated with baby length at 3.5 months during lactation in the preeclampsia group. In contrast, few studies have shown that total milk omega 3 PUFA in normal population was positively associated with infant weight gain (Tinoco et al., 2009), motor, mental and behavioral developments (Sabel et al., 2012). A recent review discusses the controversial reports on fatty acid supplementation effects on neurodevelopment in both animal and human studies (Davis-Bruno and Tassinari, 2011). Further works are required to examine the association of milk fatty acids with the neurodevelopment of babies born to mothers with preeclampsia. This is the first study to report milk BDNF and NGF concentrations across the lactation in women with preeclampsia and normotensive women. In our study, NGF levels increase across lactation. Since NGF is known to play a crucial role in the process of angiogenesis (Karatzas et al., 2013), cell development as well as tissue proliferation (De Nicoló et al., 2013), increments in milk NGF may play a role in infant growth and development during lactation. In this study, milk NGF levels were lower in preeclampsia group as compared to control group throughout lactation. Composition of bioactive components like growth factors of breast milk are suggested to be lower based in mothers associated with oxidative stress (Gutikova, 2007). Our earlier study shows that preeclampsia is associated with increased oxidative stress (Mehendale et al., 2008) which may partly explain the lower levels of milk NGF in preeclampsia group. Our data also shows that the milk BDNF levels show a higher trend throughout lactation except at 1.5 months in preeclampsia group as compared to controls. There is a report which indicates no change in milk BDNF levels at days 3, 10, 30, and 90 after parturition (Li et al., 2011). The levels of BDNF in the present study are lower than that reported by Li et al. (2011) and may be attributed to the
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difference in methodology. In the present study, BDNF levels were analyzed from whole milk. There has been no evidence that BDNF in human milk are derived entirely from the serum or mammary gland cells (Li et al., 2011). In our earlier preliminary study we have reported that milk NGF levels were similar while milk BDNF levels were higher at day 3 in the preeclampsia group as compared to controls (Dangat et al., 2013). This study indicates that there is differential altered regulation of milk NGF and BDNF levels in women with preeclampsia. 5. Conclusions Our data suggests that milk LCPUFAs and neurotrophins are altered in preeclampsia. Further, LCPUFA could plausibly influence the growth especially in children born to mothers with preeclampsia. Future studies should explore the associations between milk LCPUFA, neurotrophins with neurodevelopmental parameters in children. Furthermore, animal studies should investigate the mechanisms involved in the mammary gland synthesis of BDNF, NGF and LCPUFAs and their role in growth and development of offspring. This may eventually provide important clues for the better management and optimization of the levels of these vital constituents in mother’s milk or proper supplementation to reduce the risk for adult disorders. Role of the funding source This study was funded by Indian Council of Medical Research (ICMR), Government of India. However, ICMR has not been involved in the study design; collection of samples, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Conflict of interest None. Acknowledgments We would like to thank ICMR for financial support. We thank all the subjects for their participation in the study. We also thank the hospital staff for their cooperation in collecting the samples. References Agostoni, C., 2005. Small-for-gestational-age infants need dietary quality more than quantity for their development: the role of human milk. Acta Paediatr. 94, 827–829. Agostoni, C., Brunetti, I., Marco, Di A., 2005. Polyunsaturated fatty acids in human milk and neurological development in breastfed infants. Curr. Pediatr. Rev. 1, 25–30. Antonakou, A., Skenderi, K.P., Chiou, A., Anastasiou, C.A., Bakoula, C., Matalas, A.L., 2012. Breast milk fat concentration and fatty acid pattern during the first six months in exclusively breastfeeding Greek women. Eur. J. Nutr. 52, 963–973. Ballard, O., Morrow, A.L., 2013. Human milk composition: nutrients and bioactive factors. Pediatr. Clin. North Am. 60, 49–74. Banapurmath, C.R., Banapurmath, S., Kesaree, N., 1996. Developing brain and breastfeeding. Indian Pediatr. 33, 35–38. Berenhauser, A.C., Pinheiro do Prado, A.C., da Silva, R.C., da Silva, R.C., Gioielli, L.A., Block, J.M., 2012. Fatty acid composition in preterm and term breast milk. Int. J. Food Sci. Nutr. 63, 318–325. Bertino, E., Arslanoglu, S., Martano, C., Di Nicola, P., Giuliani, F., Peila, C., Cester, E., Pirra, A., Coscia, A., Moro, G., 2012. Biological, nutritional and clinical aspects of feeding preterm infants with human milk. J. Biol. Regul. Homeost. Agents, 9–13. Butte, N.F., Garza, C., Burr, R., Goldman, A.S., Kennedy, K., Kitzmiller, J.L., 1987. Milk composition of insulin-dependent diabetic women. J. Pediatr. Gastroenterol. 6, 936–941. Chen, Z.Y., Kwan, K.Y., Tong, K.K., Ratnayake, W.M., Li, H.Q., Leung, S.S., 1997. Breast milk fatty acid composition: a comparative study between Hong Kong and Chongqing Chinese. Lipids 32, 1061–1067.
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Preeclampsia alters milk neurotrophins and long chain polyunsaturated fatty acids Kamini Dangat a , Anitha Kilari a , Savita Mehendale b , Sanjay Lalwani c , Sadhana Joshi a,∗ a b c
Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune 411043, India Department of Obstetrics and Gynecology, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune 411043, India Department of Pediatrics, Bharati Medical College and Hospital, Bharati Vidyapeeth University, Pune 411043, India
a r t i c l e
i n f o
Article history: Received 11 September 2013 Received in revised form 6 December 2013 Accepted 17 December 2013 Keywords: DHA NGF BDNF Anthropometry Lactation
a b s t r a c t Objective: To examine the levels of breast milk neurotrophins 1.5, 3.5, and 6 months of lactation and long chain polyunsaturated fatty acids (LCPUFA) at day 3, 1.5, 2.5, 3.5 and 6 months of lactation in mothers with preeclampsia and compare them with normotensive women. Their associations with growth parameters in children are also examined. Methods: Women with preeclampsia (n = 101) and normotensive women (n = 144) with singleton pregnancies were recruited for this study. Milk samples were collected and anthropometry was recorded at the first 6 months. The LCPUFA composition of milk samples was analyzed by using gas chromatography at all time points and neurotrophins were analyzed at 1.5, 3.5 and 6 months by Emax Immuno Assay System using Promega kits. Results: Milk DHA levels were higher at day 3 (9.5%), and 1.5 (23%) and 3.5 (40%) months in mothers with preeclampsia as compared to controls. Milk nerve growth factor (NGF) levels were lower in preeclampsia group as compared to control group at 1.5 (20%) and 3.5 months (27.7%). Milk brain derived neurotrophic factor (BDNF) levels were lower at 1.5 months (10.5%) in the preeclampsia group as compared to control group. Conclusion: The present study suggests that there is a differential regulation of DHA and neurotrophins in breast milk in preeclampsia and are associated with growth parameters of children. Future studies should explore the associations between milk LCPUFA, neurotrophins with neurodevelopment in children. © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Human milk has been reported to contain wide range of nutrients and several components like hormones (Kulski and Hartmann, 1981), growth factors (Donovan and Odle, 1994), cytokines (Srivastava et al., 1996), immunological components (Jackson and Nazar, 2006), immunomodulatory factors (Bertino et al., 2012) and fatty acids (Agostoni et al., 2005) which play a vital role in the growth and development of the infant (Serpero et al., 2012). Pregnancy complications such as pregnancy induced hypertension (PIH) (Cunningham et al., 1993); preeclampsia (Erba˘gci et al., 2005) and gestational diabetes (Butte et al., 1987) are known to affect lactogenesis (Kaushik et al., 2002). Babies born to mothers
Abbreviations: AA, arachidonic acid; BDNF, brain derived neurotrophic factor; DHA, docosahexaenoic acid; LCPUFA, long chain polyunsaturated fatty acids; NGF, nerve growth factor. ∗ Corresponding author. Tel.: +91 020 24366929/24366931; fax: +91 020 24366929. E-mail address: [email protected] (S. Joshi). 0736-5748/$36.00 © 2014 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.12.007
with preeclampsia are at high risk for metabolic and cognitive disorders (Many et al., 2003) due to neurodevelopmental impairment in later life (Streimish et al., 2012). Evidence suggests that long-term breastfeeding is associated with lower hyperactivity symptoms (Julvez et al., 2007); improved neuropsychological and socio-behavioral outcomes and cognition in children (Keim et al., 2012; Hadders-Algra, 2011; Jensen and Lapillonne, 2009; Daniels and Adair, 2005; Clark et al., 2006; Hüppi, 2008). A review has suggested that milk long chain polyunsaturated fatty acids (LCPUFA), specifically docosahexaenoic acid (DHA) and arachidonic acid (AA), many growth factors and hormones influence infant’s growth, development and cognitive performance (Yum, 2007). Human milk polyunsaturated fatty acids (PUFA) are either derived from the diet or are synthesized from endogenous precursor fatty acids (Sauerwald et al., 2001) and are required in adequate quantities for optimal growth and development of infants (Xiang et al., 2005). However, reports on milk LCPUFA influence on infant growth are controversial (Tinoco et al., 2009; Scholtens et al., 2009). Reports also indicate that LCPUFA especially DHA regulates the levels of neurotrophins (Rao et al., 2007; Wu et al., 2004). Milk
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Fig. 1. Flow chart for number of samples analyzed at various time points. PE: preeclampsia; BDNF: brain derived neurotrophic factor; NGF: nerve growth factor.
growth factors such as brain derived neurotrophic factor (BDNF) (Li et al., 2011) and nerve growth factor (NGF) (Banapurmath et al., 1996) are known to play a critical role in the development and maintenance of the nervous system. Recent reports also indicate that the milk components change over the course of lactation due to time dependent programmed requirement for the child development (Ballard and Morrow, 2013; Erba˘gci et al., 2005; Li et al., 2011). Milk fatty acid profile found to be affected by maternal dietary and life style factors of mother (Antonakou et al., 2012). We have earlier reported that milk fatty acids and neurotrophins levels are altered at d3 of lactation in preeclampsia (Dangat et al., 2010, 2013) which may affect the milk quality and postnatal programming of child. However, it is unclear whether these vital constituents remain altered during the complete duration of lactation. Thus, the present study for the first time examines simultaneously the levels of human milk fatty acids and neurotrophins in mothers with preeclampsia and compares them with controls at various time points during lactation and also examines their association with growth of the infant.
Preeclampsia was diagnosed by the criteria discussed by us in a number of our studies reported earlier (Kilari et al., 2011; Kulkarni et al., 2010). Briefly, preeclampsia was defined by systolic and diastolic blood pressures greater than 140 and 90 mmHg respectively with proteinuria in a dipstick test. The oil consumption per month has been recorded at the time of recruitment. Treatment of preeclamptic women included antihypertensive drugs and arginine supplementation. In severe preeclamptic cases, MgSO4 was given intravenously. It is unlikely that these would affect BDNF status. 2.1. Collection of milk samples Human milk sampling was done at all time points during lactation. The samples were collected when the babies come for vaccination at 1.5, 2.5 and 3.5 months of age. Samples of breast milk (2 ml) were taken at the end of the baby’s being breast-fed and collected into cryo vials and were stored at −80 ◦ C. 2.2. Fatty acid analysis
2. Materials and methods This study was conducted at the Departments of Pediatrics and Obstetrics & Gynecology, Bharati Hospital, Pune during the year 2011–2012. Singleton pregnant women with preeclampsia (n = 101) and without preeclampsia (controls) (n = 144) were recruited at the time of delivery for this prospective study. Mothers and their babies were followed-up at day 3, and 1.5, 2.5, 3.5 and 6 months post delivery. The anthropometry (weight and length, head and chest circumference) of the infant was recorded at all time points during lactation. Fig. 1 shows the number of milk samples analyzed for fatty acids and neurotrophins at various time points during lactation. This study was approved by Institutional Ethical Committee and a written informed consent was taken from each subject. Subjects were excluded from the study if there was evidence of other pregnancy complications like multiple gestation, chronic hypertension, type I or type II diabetes mellitus, seizure disorder, renal or liver disease. Pregnant women with alcohol or drug abuse were also excluded from the study. The control group consisted of pregnant women with no medical or obstetrical complications.
Milk samples were analyzed at all time points by the gas chromatography and have been described by us earlier (Dangat et al., 2010). Fatty acids were expressed as g/100 g fatty acid. A total of 15 fatty acids were estimated. Saturated fatty acids include myristic acid, palmitic acid and stearic acid while monounsaturated fatty acids include myristoleic, palmitoleic, oleic and nervonic acids. The omega 3 fatty acids included alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) and DHA while omega 6 fatty acids included linoleic acid (LA), gamma linolenic acid (GLA), di-homogammalinolenic acid (DGLA), docosapentaenoic acid (DPA) and AA. 2.3. Neurotrophins analysis Milk neurotrophins at day 3 has been published recently (Dangat et al., 2013). Milk neurotrophins at 1.5, 3.5 and 6 months of lactation were analyzed by Emax Immuno Assay System using the Promega kits for BDNF (Hornbeck et al., 1994) and NGF (Zettler et al., 1996) which has been described by us earlier (Dangat et al., 2013). The amount of BDNF and NGF was expressed as pg/ml.
K. Dangat et al. / Int. J. Devl Neuroscience 33 (2014) 115–121 Table 1 Maternal characteristics at the time of recruitment. Control (n = 144) Age (years) Income (INR) Age at marriage (years) Body mass index (BMI) Gestation (weeks) Systolic blood pressure at delivery (mmHg) Diastolic blood pressure at delivery (mmHg) **
24.0 7784 20.1 25.7 39.2 120.1
± ± ± ± ± ±
3.7 4720 2.9 3.88 1.1 9.2
77.6 ± 6.1
Preeclampsia (n = 101) 23.7 7593 20.8 22.64 36.9 151.2
± ± ± ± ± ±
3.6 5086 2.9 4.6 2.8** 18.5**
99.2 ± 10.4**
p < 0.01.
2.4. Statistical methods Values are expressed as mean ± SD. The data were analyzed using SPSS/PC+ package for MS Windows (Version 20, Chicago, IL). Skewed variables (DHA at 1.5 months, NGF at 1.5 months) were transformed to satisfy the underlying assumption of normality using natural logarithm transformation (Loge ). Mean values of the estimates of milk fatty acids and neurotrophins were compared by Student’s “t” test. Correlations between milk fatty acids, baby weight, height, head and chest circumferences were studied using Pearson’s correlation analysis after adjusting for maternal age, body mass index (BMI) and duration of gestation as they are possible confounders. Frequency of consumption of oil was analyzed using chi square test.
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NGF levels were significantly lower at 1.5 (20%) and 3.5 (27.7%) months in preeclampsia group as compared to controls. However, milk NGF levels remain lower but not significant between the groups at 6 months. In contrast, Milk BDNF levels were significantly lower (10.5%) at 1.5 months in preeclampsia as compared to controls (Fig. 4). However, there was no significant difference in milk neurotrophins levels between groups at 6 months of lactation. 3.4. Associations of milk fatty acids with baby characteristics during lactation Milk AA levels were negatively associated with baby weight (r = −0.284, p = 0.017, n = 68) at 1.5 month and 3.5 months (r = −0.396, p = 0.003, n = 52) in the preeclampsia group but were positively associated with baby length (r = 0.231, p = 0.045, n = 74) at 3.5 months in controls. Milk DHA levels were negatively associated with baby length (r = −0.363, p = 0.007, n = 52) at 3.5 months only in the preeclampsia group. Saturated fatty acid levels were negatively associated with baby weight at 3.5 months (r = −0.268, p = 0.050, n = 52) in preeclampsia group. However, these associations were not seen at 6 months of lactation. 3.5. Associations of milk neurotrophins with baby characteristics There was negative association between milk NGF and baby length at 1.5 months only in control group (r = −0.337, p = 0.019, n = 48). There was no association observed between milk BDNF and baby characteristics.
3. Results
3.6. Associations of milk neurotrophins with milk fatty acids
3.1. Maternal and baby characteristics
There was a negative association between milk NGF and milk DHA levels (r = −0.311, p = 0.033, n = 47) in the control group while milk NGF was negatively associated with milk AA (r = −0.463, p = 0.008, n = 32) in preeclampsia group at 1.5 months of lactation. Similarly, milk NGF levels were negatively associated with milk omega 6 fatty acids (r = −0.543, p = 0.001, n = 32) in control group at 3.5 months. However, milk BDNF levels did not show any association with milk fatty acids.
Table 1 shows the maternal characteristics. Systolic and diastolic blood pressures were higher (p < 0.01) and gestation was shorter (p < 0.01) in the preeclampsia group as compared to controls. Baby weight, length, head and chest circumferences at birth were significantly lower in preeclampsia group as compared to controls which has been reported by us earlier (Dangat et al., 2013). The baby weight, length, head and chest circumferences increased in both groups with almost similar trends but these remained lower in preeclampsia group at all-time points, i.e. day 3, 1.5, 2.5, 3.5 and 6 months as compared to control group (Fig. 2). 3.2. Milk fatty acid levels during lactation Fig. 3 shows the levels of milk fatty acids in both the groups. Milk LA (a precursor of AA) levels were lower but not significant except at 2.5 months (7.1%) in mothers with preeclampsia as compared to controls at all other time points; day 3, 1.5, 3.5 and 6 months of lactation. Whereas, milk AA levels were similar in the preeclampsia group as compared to controls at all time points. The levels of ALA (a precursor of DHA) were similar or slight trend toward higher in preeclampsia compared to controls. In contrast, milk DHA levels were higher (p < 0.05) at day 3 (9.5%), 1.5 (23%) months and 3.5 (40%) months in women with preeclampsia as compared to controls. However, the levels of omega-3 fatty acids, monounsaturated fatty acids and saturated fatty acids did not differ much between groups while total omega-6 fatty acid levels were lower at 2.5 months in preeclampsia throughout the study period. Milk fatty acid levels were similar between groups at 6 months of lactation. 3.3. Levels of milk neurotrophins The levels of both NGF and BDNF in controls as wells as preeclampsia also show very different longitudinal patterns. Milk
3.7. Dietary intake of oil consumption Oil consumption has been divided into 1.5/month. Oil intake was significantly higher (p < 0.01) in preeclampsia group as compared to control group. In control group, 63.92% of women had consumed 1.5 kg per month. In preeclampsia group, 47.73% had consumed 1.5 kg per month. The women in the cohort mainly consumed soyabean (41%), sunflower (32%) and ground nut oil (15%). 4. Discussion This study for the first time simultaneously reports the levels of LCPUFA (primarily LA, AA, ALA and DHA) and neurotrophins (NGF and BDNF) in human milk from Indian women at various time points during lactation. All mothers exclusively breastfed their babies. The key findings of our study are (1) milk DHA levels were higher in preeclampsia as compared to controls at day 3, 1.5 and 3.5 months, (2) milk NGF levels were generally lower in preeclampsia group as compared to control group throughout lactation, (3) milk BDNF levels were lower at 1.5 months of lactation in the preeclampsia group as compared to control group, (4) baby weight was negatively associated with milk AA levels at 1.5 and 3.5 months while milk DHA levels were negatively associated
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Fig. 2. Baby growth charts from birth to 6 months. Mon: months
with baby length at 3.5 months in preeclampsia group and (5) there was a negative association between milk NGF and baby length at 1.5 months in control group. However, there was no association observed between milk BDNF and baby characteristics. Our data has shown decrease in AA and DHA levels from day 3 to 6 months of lactation and is consistent with a reported longitudinal study which has also demonstrated that the milk DHA and AA gradually decrease from colostrum to mature milk (Chen et al., 1997). Furthermore, the levels of both the precursors (LA and ALA) were lower and the products (AA and DHA) levels were higher in milk in both the groups at day 3 (closer to colostrum) compared to milk at later time points (mature milk). Increased levels of milk AA at day 3 may improve the growth of the newborns. It is well known that AA is an important nutrient factor for growth in early postnatal life (Much et al., 2013). Reports indicate that milk AA levels may not be mediating inflammation because human milk contains high anti-inflammatory than pro-inflammatory lipid mediators (Takamoto et al., 1995). LCPUFA such as AA and DHA are
synthesized by elongation/desaturation of precursors like LA and ALA, respectively. Desaturases are the enzymes which regulate the unsaturated fatty acid biosynthesis and are mainly present in liver. It has been reported that fatty acid desaturase enzyme activity in newborns is low (Innis, 2007). Current data shows increased milk levels of both LCPUFA precursors (LA and ALA) and reduced products (AA and DHA) from day 3 to 6 months of lactation. At later time points, the levels of AA and DHA decrease possibly because of reduced need for products (AA and DHA) due to increased conversion ability of the infant. It has been mentioned that the infants probably can synthesize additional LCPUFAs from the LA and ALA contents of human milk (Heird, 2001). Similarly, the levels of BDNF are highest at 3 days whereas the NGF levels steadily increase but the levels of BDNF decrease at 1.5 month and there was a trend toward increase until 6 months. We have earlier reported increased levels of milk BDNF at day 3 in the same cohort (Dangat et al., 2013). The significant quantitative differences in BDNF at several time points during lactation between controls and preeclampsia
Fig. 3. Milk fatty acid levels during lactation. Mon: months; *p < 0.05
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Fig. 4. Milk neurotrophins levels during lactation. Mon: months; *p < 0.05; **p < 0.01; BDNF: brain derived neurotrophic factor; NGF: nerve growth factor; pg/ml: picograms/milliliter.
probably indicate that the milk programming by mother’s breast is altered by preeclampsia. It is likely that these changes are most likely adaptive changes of mother and some of these changes are not normalized even up to 6 months. The levels of DHA in human milk levels remain higher in mothers with preeclampsia as compared to controls throughout lactation except at 2.5 month levels were almost similar. We have earlier reported higher milk DHA levels at postnatal day 3 in preeclampsia in spite of its lower plasma DHA levels in mother which is consistent with the data reported by us earlier in another cohort (Dangat et al., 2010). The mean milk DHA concentrations in current study are similar to the levels reported in countries like Nepal (Glew et al., 2001) and Bangladesh (Yakes et al., 2011). Milk DHA levels in our study is similar to Sudanese mothers whose diet is devoid of fish products (Nyuar et al., 2010) and American Indian women with low DHA intake (Glew et al., 2011). In contrast, milk DHA levels were slightly higher in Cuba (Krasevec et al., 2002). A study on milk samples from 9 different countries has revealed that milk saturated and MUFA composition is constant but PUFA like DHA is highly variable (Yuhas et al., 2006). A study from China reveals that mothers in coastal area of china showed higher DHA status of milk (Peng et al., 2009). Milk DHA showed a positive correlation with dietary DHA intake (Glew et al., 2011). This suggests maternal diet and fatty acid metabolism plays a critical role in determining the DHA status of mother. Frequency of consumption of omega 3 fatty acid rich foods was similar which has been reported by us earlier in another cohort (Kulkarni et al., 2011). Increased milk DHA levels in current study levels probably due to increased expression of Sterol regulatory element binding proteins (SREBP-1) in the mammary glands which in turn increases the expression of delta-5 desaturase and delta-6 desaturase ultimately resulting into increased DHA synthesis. Our study subjects are predominantly vegetarian and their lower levels compared to higher levels in countries with higher sea food diet may explain the differences. In our study, the mean milk DHA levels decreased from day 3 to 6 months of lactation in both preeclampsia and control groups. Higher milk DHA levels in colostrum compared to mature milk have been reported in both term and preterm mothers (Berenhauser et al., 2012). This is also consistent with the reports from lactating Italian mothers (Agostoni, 2005). The levels of AA in the present study were in the ideal range which has been recently reported (Antonakou et al., 2012). Our study suggests that the need of LCPUFAs, especially DHA, can be satisfied in healthy normotensive mothers by breastfeeding
during early lactating period. Reports suggest that long term (DelPrado et al., 2001) and short term (Innis, 2007) maternal diet and fatty acid status influences the milk fatty acid status. Total omega 3 fatty acid intakes were similar in both controls as well as in preeclampsia groups which was reported by us earlier in another cohort (Kulkarni et al., 2011). In current study, higher milk DHA levels in mothers with preeclampsia may be due to altered metabolism of fatty acids. In addition to maternal diet, breast milk fatty acid composition is also dependent on mobilization of endogenous stores of fatty acids and de novo synthesis of fatty acids by liver or breast tissue (Sabel et al., 2012). In our study, milk AA levels were negatively associated with baby weight at 1.5 and 3.5 months and milk DHA levels were negatively associated with baby length at 3.5 months during lactation in the preeclampsia group. In contrast, few studies have shown that total milk omega 3 PUFA in normal population was positively associated with infant weight gain (Tinoco et al., 2009), motor, mental and behavioral developments (Sabel et al., 2012). A recent review discusses the controversial reports on fatty acid supplementation effects on neurodevelopment in both animal and human studies (Davis-Bruno and Tassinari, 2011). Further works are required to examine the association of milk fatty acids with the neurodevelopment of babies born to mothers with preeclampsia. This is the first study to report milk BDNF and NGF concentrations across the lactation in women with preeclampsia and normotensive women. In our study, NGF levels increase across lactation. Since NGF is known to play a crucial role in the process of angiogenesis (Karatzas et al., 2013), cell development as well as tissue proliferation (De Nicoló et al., 2013), increments in milk NGF may play a role in infant growth and development during lactation. In this study, milk NGF levels were lower in preeclampsia group as compared to control group throughout lactation. Composition of bioactive components like growth factors of breast milk are suggested to be lower based in mothers associated with oxidative stress (Gutikova, 2007). Our earlier study shows that preeclampsia is associated with increased oxidative stress (Mehendale et al., 2008) which may partly explain the lower levels of milk NGF in preeclampsia group. Our data also shows that the milk BDNF levels show a higher trend throughout lactation except at 1.5 months in preeclampsia group as compared to controls. There is a report which indicates no change in milk BDNF levels at days 3, 10, 30, and 90 after parturition (Li et al., 2011). The levels of BDNF in the present study are lower than that reported by Li et al. (2011) and may be attributed to the
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difference in methodology. In the present study, BDNF levels were analyzed from whole milk. There has been no evidence that BDNF in human milk are derived entirely from the serum or mammary gland cells (Li et al., 2011). In our earlier preliminary study we have reported that milk NGF levels were similar while milk BDNF levels were higher at day 3 in the preeclampsia group as compared to controls (Dangat et al., 2013). This study indicates that there is differential altered regulation of milk NGF and BDNF levels in women with preeclampsia. 5. Conclusions Our data suggests that milk LCPUFAs and neurotrophins are altered in preeclampsia. Further, LCPUFA could plausibly influence the growth especially in children born to mothers with preeclampsia. Future studies should explore the associations between milk LCPUFA, neurotrophins with neurodevelopmental parameters in children. Furthermore, animal studies should investigate the mechanisms involved in the mammary gland synthesis of BDNF, NGF and LCPUFAs and their role in growth and development of offspring. This may eventually provide important clues for the better management and optimization of the levels of these vital constituents in mother’s milk or proper supplementation to reduce the risk for adult disorders. Role of the funding source This study was funded by Indian Council of Medical Research (ICMR), Government of India. However, ICMR has not been involved in the study design; collection of samples, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Conflict of interest None. Acknowledgments We would like to thank ICMR for financial support. We thank all the subjects for their participation in the study. We also thank the hospital staff for their cooperation in collecting the samples. References Agostoni, C., 2005. Small-for-gestational-age infants need dietary quality more than quantity for their development: the role of human milk. Acta Paediatr. 94, 827–829. Agostoni, C., Brunetti, I., Marco, Di A., 2005. Polyunsaturated fatty acids in human milk and neurological development in breastfed infants. Curr. Pediatr. Rev. 1, 25–30. Antonakou, A., Skenderi, K.P., Chiou, A., Anastasiou, C.A., Bakoula, C., Matalas, A.L., 2012. Breast milk fat concentration and fatty acid pattern during the first six months in exclusively breastfeeding Greek women. Eur. J. Nutr. 52, 963–973. Ballard, O., Morrow, A.L., 2013. Human milk composition: nutrients and bioactive factors. Pediatr. Clin. North Am. 60, 49–74. Banapurmath, C.R., Banapurmath, S., Kesaree, N., 1996. Developing brain and breastfeeding. Indian Pediatr. 33, 35–38. Berenhauser, A.C., Pinheiro do Prado, A.C., da Silva, R.C., da Silva, R.C., Gioielli, L.A., Block, J.M., 2012. Fatty acid composition in preterm and term breast milk. Int. J. Food Sci. Nutr. 63, 318–325. Bertino, E., Arslanoglu, S., Martano, C., Di Nicola, P., Giuliani, F., Peila, C., Cester, E., Pirra, A., Coscia, A., Moro, G., 2012. Biological, nutritional and clinical aspects of feeding preterm infants with human milk. J. Biol. Regul. Homeost. Agents, 9–13. Butte, N.F., Garza, C., Burr, R., Goldman, A.S., Kennedy, K., Kitzmiller, J.L., 1987. Milk composition of insulin-dependent diabetic women. J. Pediatr. Gastroenterol. 6, 936–941. Chen, Z.Y., Kwan, K.Y., Tong, K.K., Ratnayake, W.M., Li, H.Q., Leung, S.S., 1997. Breast milk fatty acid composition: a comparative study between Hong Kong and Chongqing Chinese. Lipids 32, 1061–1067.
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