Journal of Human Lactation http://jhl.sagepub.com/
Human Milk Oligosaccharide Composition Differs between Donor Milk and Mother's Own Milk in the NICU Carolin Marx, Renee Bridge, Alison K. Wolf, Wade Rich, Jae H. Kim and Lars Bode J Hum Lact 2014 30: 54 originally published online 26 November 2013 DOI: 10.1177/0890334413513923 The online version of this article can be found at: http://jhl.sagepub.com/content/30/1/54
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JHLXXX10.1177/0890334413513923Journal of Human LactationMarx et al
Original Research
Human Milk Oligosaccharide Composition Differs between Donor Milk and Mother’s Own Milk in the NICU
Journal of Human Lactation 2014, Vol 30(1) 54–61 © The Author(s) 2013 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0890334413513923 jhl.sagepub.com
Carolin Marx, MS1, Renee Bridge, RN1, Alison K. Wolf, RN, NNP1, Wade Rich, RRT, CCRC1, Jae H. Kim, MD, PhD1, and Lars Bode, PhD1
Abstract Background: Human milk oligosaccharides (HMO) represent the third most abundant component of human breast milk. More than a hundred structurally distinct HMO have been identified, and the HMO composition varies between mothers as well as over the course of lactation. Some newborn infants receive donor milk (DM) when their mother’s own milk (MOM) volume is inadequate or unavailable. Objective: This study aimed to compare HMO content between DM and MOM. Methods: We used high performance liquid chromatography analysis of fluorescently labeled HMO to analyze the variation in HMO amount and composition of 31 different batches of DM (each pooled from 3 individual donors) provided by the Mothers’ Milk Bank in San Jose, California, and compared it to 26 different MOM samples donated by mothers with infants in our neonatal intensive care unit (NICU). Results: Total HMO amount as well as concentrations of lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose 1, and disialyllacto-N-tetraose were significantly lower in DM than in MOM, whereas the concentrations of 3’-sialyllactose and 3-fucosyllactose were significantly higher in DM. Conclusion: Our data show that infants in our NICU who receive DM are likely to ingest HMO at different total amounts and relative composition from what they would receive with their MOM. Recent in vitro and animal studies have started to link individual HMO to infant health and disease. Future studies are needed to assess the importance of a mother-infant match with regard to HMO composition. Keywords breastfeeding, breast milk, human milk, milk banking, neonatal nutrition, oligosaccharides, preterm infants
Well Established Human milk oligosaccharides (HMO) are the third most abundant component in breast milk, and their composition varies from 1 mother to another. Donor breast milk (DM) is widely used in neonatal intensive care units (NICUs), but the HMO composition of DM has not yet been analyzed.
Newly Expressed The amount and composition of HMO are significantly different between DM and the milk that infants in the NICU receive from their own mothers.The physiological and clinical relevance of this potential mismatch needs further investigation.
Background Breastfeeding is considered the optimum nutrition for the first 6 months of life.1 When mother’s own milk (MOM) is unavailable or in short supply, donor breast milk (DM) is increasingly
used in neonatal intensive care units (NICUs) to provide infants with some of the benefits of breast milk.2 Studies by Perrine and Scanlon3 and Parker and colleagues4 indicate that 22% to 42% of the NICUs in the United States use DM. In addition to traditional nutrients, breast milk contains a variety of bioactive compounds, and their amount and composition often depend on genetic and environmental factors and can change over the course of lactation. Amount and composition of milk bioactive compounds can change when DM is processed (ie, pasteurized) to protect the recipient infant against disease 1
Division of Neonatology, Department of Pediatrics, University of California, San Diego, San Diego, CA, USA Date submitted: August 29, 2013; Date accepted: October 30, 2013. Corresponding Author: Lars Bode, PhD, Division of Neonatology and Division of Gastroenterology and Nutrition, Department of Pediatrics, University of California, San Diego, 200 West Arbor Drive, MC 8450, San Diego, CA 92103-8450, USA. Email: [email protected]
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Marx et al transmission.5-8 In North America and the United Kingdom, DM is pasteurized at 62.5°C for 30 minutes (Holder pasteurization), which partially inactivates some but not all of the milk bioactive compounds.9 As a most recent example, Holder pasteurization was shown to reduce adiponectin and insulin concentrations by 32.8% and 46.1%, respectively.10 Human milk oligosaccharides (HMO) are a group of milk bioactive compounds thought to benefit the human milk-fed infant on multiple levels.11-16 HMO are prebiotics and antiadhesive antimicrobials that shape the infant’s intestinal microbiota composition.11,12,16,17 They may also directly interact with the infant’s intestinal epithelial cells18-20 or modulate immune cell responses21-23 and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition.24 After lactose and lipids, HMO are the third most abundant component in human breast milk.11 HMO concentrations decline over the course of lactation with 20 to 30 g/L in colostrum and 10 to 15 g/L in mature milk.11 The milk of mothers delivering preterm infants has higher HMO concentrations than term milk.25 More than 100 different HMO have been identified so far, but not every woman synthesizes the same set of oligosaccharides.26 HMO composition is in part genetically determined and mirrors the woman’s Secretor and Lewis blood group status.11,16,27 More than 70% of women are Secretors,27 which means that they express the α1-2-fucosyltransferase FUT2 and their milk contains characteristic α1-2-fucosylated HMO like 2’-fucosyllactose (2’FL) or lacto-N-fucopentaose 1 (LNFP1). The milk of non-Secretor women does not contain these specific HMO, and infants fed non-Secretor milk are at higher risk for diarrhea caused by certain viral and bacterial infections.28 Women with Lewis positive blood group status express the α1-3/4-fucosyltransferase FUT3 and their milk contains characteristic α1-4-fucosylated HMO like LNFP2. The beneficial effects reported for HMO are based on total HMO amount as well as distinct structural composition.21,22,28-30 Animal model data from our own group, for example, suggest that 1 specific HMO, disialyllacto-N-tetraose (DSLNT), protects from necrotizing enterocolitis,30 1 of the most serious intestinal disorders in preterm infants. DM is widely and increasingly fed to preterm infants in the NICU, but the health outcomes are not always comparable to infants who receive their mother’s own milk.31,32 We therefore aimed to investigate whether total HMO amount and composition differ between DM and MOM fed to infants in our NICU at the University of California, San Diego (UCSD).
Methods
1 milk sample, which was collected by hand-expression after the feed in the morning between 8 and 11 AM. Fresh milk samples were transported to the lab on ice and stored at –80°C prior to HMO analysis. To assess the effects of pasteurization on HMO amount and composition, 5 of the fresh milk samples were split in 2 aliquots and 1 aliquot was heated at 62.5°C for 30 minutes. Afterward, HMO amount and composition in both aliquots were analyzed in parallel.
Donor Milk The Mothers’ Milk Bank, San Jose, California, provided 1-mL aliquots from 31 different DM batches. Each batch was a pool of milk from 3 individual donors. The pooled milk was further processed by Holder pasteurization following the guidelines of the Human Milk Banking Association of North America.7 Samples were shipped on dry ice and stored at –80°C prior to HMO analysis.
Human Milk Oligosaccharide Analysis Oligosaccharides from MOM or DM were analyzed as previously described.30,33 Briefly, raffinose was added to 40 μL milk to serve as an internal standard through sample processing and analysis. Lipids and proteins were removed from the samples by centrifugation and chloroform/methanol extraction. Lactose was removed by overnight incubation on lactase-immobilized beads (Invitrogen, Carlsbad, California, USA) at 37°C. Residual peptides and salt were removed over Sep-Pak C18 cartridges followed by porous graphitized carbon (PGC) cartridges. The reducing end of the dried oligosaccharides was labeled with the fluorescent tag 2-aminobenzamide (2AB) for 2 hours at 65°C. Free 2AB label was separated from the 2AB-labeled oligosaccharides using silica gel cartridges. 2AB-labeled oligosaccharides were analyzed by high performance liquid chromatography (HPLC) on an amide-80 column (4.6 mm ID × 25 cm, 5 µm; Tosoh Bioscience, Tokyo, Japan) with a 50 mM ammonium formate/acetonitrile buffer system. Separation was performed at 25°C and monitored with a fluorescence detector at 360 nm excitation and 425 nm emission. Peak annotation was based on standard retention times and mass spectrometric (MS) analysis on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano-ESI source. Total oligosaccharides were calculated as the sum of the 20 most abundant individual oligosaccharides.
Statistical Analysis
Mother’s Own Milk We recruited 26 mothers with infants in the NICU at UCSD Medical Center, San Diego, California, after approval by UCSD’s institutional review board.Each mother provided
Differences in oligosaccharide concentrations between MOM and DM were calculated by 2-tailed Mann-Whitney test. Correlations between oligosaccharide concentrations in MOM and their respective lactation time postpartum were
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Figure 1. Characteristics of Mother’s Own Milk (Top) and Donor Milk (Bottom) Based on Infant Maturity at Birth (Left) and Maternal Race/Ethnicity (Right).
(n=26) Preterm (76.9%)
Caucasian/White (Non-Hispanic) (46.2%)
Term (23.1%)
Black/ African American (7.7%)
Other (Hispanic) (42.3%)
Asian/ Pacific Islander (3.8%)
Donor Milk (DM) (n=31)
Preterm (21.5%)
Term (78.5%)
Other (Hispanic) (21.5%)
Caucasian/White (Non-Hispanic) (41.9%)
Asian/ Pacific Islander (36.6%)
calculated by 2-tailed Spearman test with 95% confidence interval. Significance was defined as P values of less than .05.
Results We randomly collected 26 human breast milk samples from mothers with infants in the NICU who were receiving their mother’s own milk. We analyzed the HMO composition from all 26 milk samples and compared them to 31 different DM batches that were available at the time we collected the MOM. Whereas more than 75% of the infants in the NICU were born preterm, almost 80% of the mothers who donated milk for the DM batches had term deliveries (Figure 1). Distributions of race and ethnicity were also different between the mothers with infants in the NICU in San Diego (Southern California) and the 93 mothers who contributed to the 31 pooled milk samples that we received from the Mothers’ Milk Bank in San Jose (Northern California).
HMO analysis confirmed interpersonal variations in HMO composition as exemplified by the different chromatograms in Figure 2. The HMO profile of a Secretor woman is characterized by a high abundance of α1-2-fucosylated HMO (Figure 2a) that are absent from the milk of non-Secretor women (Figure 2b). The HMO profile in the DM sample (Figure 2c) looks somewhat different, contains high amounts of 2’FL but low amounts of LNFP1, and has higher concentrations of 3FL and 3’SL compared to most MOM samples. Figure 3 compares the concentrations of individual HMO between MOM and DM. The concentration of total HMO is significantly lower in DM compared to MOM (Figure 3a). Although the concentration of 2’FL was not significantly different between the 2 groups (Figure 3b), there was a difference in the number of non-Secretor samples, milk samples that lack 2’FL and other α1-2-fucosylated HMO. Whereas 4 out of the 26 MOM samples were from non-Secretor mothers, only 1 out of the 31 DM samples was a non-Secretor, indicating that all 3 individual donors who contributed to this
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Marx et al Figure 2. Representative High Performance Liquid Chromatography Chromatograms of Oligosaccharides from Mother’s Own Milk (a and b) and Donor Milk (c).
(a) Relative Intensity [mV]
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LNnT, LNFP1, and DSLNT were significantly lower in DM than in MOM (Figures 3e-3h). To exclude the possibility that the differences in HMO composition were due to alterations during the pasteurization process, we separated 5 fresh MOM samples in 2 aliquots each, pasteurized 1 of the 2 aliquots, and analyzed HMO composition of both aliquots in parallel. Differential processing, however, did not result in significant differences in total HMO amount and composition (data not shown). Although information on the postpartum lactation time was not available for the DM samples, MOM samples were collected as early as 2 days postpartum and as late as 169 days postpartum [median, 7.5 days; interquartile range, 16.75 (20.75-4.00)]. Because HMO concentrations are known to decline over the course of lactation, we plotted the concentrations of individual HMO against postpartum lactation time (Figure 4). Concentrations of 2’FL, LNnT, and LNFP1 negatively correlated with the postpartum lactation time, and there was a nonsignificant trend in the association of total HMO and the postpartum lactation time (P = .083). Differences in the time of lactation may be 1 explanation for some of the HMO differences we report for DM and MOM.
DM
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Mother A is a Secretor and her milk is abundant in 2’-fucosyllactose (peak 1) and lacto-N-fucopentaose 1 (peak 6), but there is hardly any 3-fucosyllactose (peak 2). Mother B is a non-Secretor and her milk contains no α1-2fucosylated HMO but more 3-fucosyllactose. The donor milk batch is a hybrid of 3 individual milk samples and contains both 2’-fucosyllactose and 3-fucosyllactose as well as fairly high amounts of 3’-sialyllactose (peak 4). 0: raffinose (internal standard); 1: 2’-fucosyllactose (2’FL); 2: 3-fucosyllactose (3FL); 3: lacto-N-neotetraose (LNnT); 4: 3’-sialyllactose (3’sLac); 5: lacto-N-tetraose (LNT); 6: lacto-N-fucopentaose 1 (LNFP1); 7: lacto-N-fucopentaose 2 (LNFP2); 8: sialyllacto-N-tetraose b (LST b); 9: sialyllacto-N-tetraose c (LST c); 10: lacto-N-difucohexaose (LNDFH); 11: disialyllacto-N-tetraose (DSLNT).
DM batch were likely non-Secretors. The concentrations of 3FL and 3’SL were significantly higher in DM than in MOM (Figures 3c and 3d). In contrast, concentrations of LNT,
Discussion We report that HMO amount and composition are significantly different between DM and the milk that infants in the NICU receive from their own mothers. Accumulating data from in vitro, ex vivo, and in vivo studies strongly suggest that infants benefit from HMO on multiple levels.11,12,14,16 They serve as prebiotics that help shape the microbial composition in the infant’s intestine, as anti-infective agents that reduce pathogen adhesion to epithelial surfaces, or as immune modulators. The beneficial effects are often structure-specific and correlate with the concentration of individual HMO.21,22,28-30,33 Campylobacter jejuni, for example, is 1 of the most common causes of bacterial diarrhea and infant mortality.34,35 α12-fucosylated HMO like 2’FL or LNFP1 inhibit C. jejuni attachment to intestinal epithelial cells in vitro and ex vivo,29 and high concentrations of α1-2-fucosylated HMO in the mother’s milk reduced the infant’s risk of developing C. jejuni diarrhea.28 Although there was no significant difference in the concentration of 2’FL between DM and MOM (Figure 3b), LNFP1 was less abundant in DM than in MOM (Figure 3g). Because more than 70% of women are Secretors27 and each DM sample contained milk from 3 different women, the number of non-Secretor samples was lower in the DM group than in the MOM group. It remains unknown whether there are any health consequences for infants who are fed Secretor DM rich in α1-2-fucosylated HMO but would otherwise receive milk from a non-Secretor mother who lacks these specific HMO. Other anti-adhesive antimicrobial effects have been described for HMO and they are often highly structuredependent and not limited to viruses and bacteria.11,12 For example, the protozoan parasite Entamoeba histolytica
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Figure 3. Comparison of Human Milk Oligosaccharide Concentrations between Mother’s Own Milk and Donor Milk.
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a, Total HMO as the sum of individual HMO detected as follows. b, 2’-fucosyllactose (2’FL). c, 3-fucosyllactose (3FL). d, 3’-sialyllactose (3’sLac). e, Lacto-N-tetraose (LNT). f, Lacto-N-neotetraose (LNnT). g, Lacto-N-fucopentaose 1 (LNFP1). h, Disialyl-lacto-N-tetraose (DSLNT). Dots represent concentrations of individual samples. Bars and lines represent median and interquartile range. Symbols above each graph represent the respective HMO structure containing glucose (dark circle), galactose (light circle), N-acetyllactosamine (square), fucose (triangle), and sialic acid (diamond). Abbreviations: DM, donor milk; HMO, human milk oligosaccharide; MOM, mother’s own milk; ns, not significant. *P < .05. **P < .01. ***P < .001 (2-tailed Mann-Whitney test).
causes amebiasis, which is the third leading cause of death by parasitic diseases after malaria and schistosomiasis.36 Specific neutral HMO like LNT and LNnT reduce E. histolytica attachment to and killing of human intestinal epithelial cells.33 Here, we show that both LNT and LNnT are less abundant in DM than in MOM (Figures 3e and 3f). Based on our results, DM may contain less anti-adhesive antimicrobial HMO and therefore be less effective in protecting the neonate from certain viral, bacterial, or protozoan infections.
In an example most relevant to the NICU, our lab has recently shown that an individual HMO, disialyllacto-Ntetraose (DSLNT), protects from necrotizing enterocolitis in an animal model.30 Here, we report that, on average, DSLNT is less abundant in DM than in MOM. However, whether or not DSLNT concentrations in some DM batches are insufficient to protect the preterm neonate against necrotizing enterocolitis remains unknown and needs further investigation.
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Marx et al Figure 4. Correlation between Human Milk Oligosaccharide Concentrations and Lactation Times Postpartum in Mother’s Own Milk.
r: -0.3557 (ns)
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a, Total HMO as the sum of individual HMO detected as follows. b, 2’-fucosyllactose (2’FL). c, 3-fucosyllactose (3FL). d, 3’-sialyllactose (3’sLac). e, Lacto-N-tetraose (LNT). f, Lacto-N-neotetraose (LNnT). g, Lacto-N-fucopentaose 1 (LNFP1). h, Disialyl-lacto-N-tetraose (DSLNT). Dots represent concentrations of individual samples. Spearman r values and significances are given above each graph. Abbreviations: HMO, human milk oligosaccharide; ns, not significant; pp, postpartum. *P < .05. **P < .01. ***P < .001 (2-tailed Mann-Whitney test).
The question remains why HMO amount and composition differ between DM and MOM. HMO are not affected by pasteurization,37 which we confirmed in this study with a limited set of samples (n = 5). Differences in postpartum lactation time, however, may be 1 explanation. MOM samples were randomly collected from mothers with infants in the NICU who would otherwise receive DM, and the lactation age ranges from 2 to 169 days postpartum. Half of the MOM samples were collected within the first week postpartum. Although information on the lactation stage was not available for the DM samples, it is likely that most women donate milk later when lactation is fully established and the amount exceeds their infants’ needs. HMO concentrations decline over the course of lactation, which we confirmed even with the limited number of MOM samples analyzed in this study (Figure 4). It needs to be further investigated whether feeding mature DM to infants in the first few days of life has any health consequences with respect to HMO amount and composition.
Lactation time may explain some but not all of the HMO differences between DM and MOM. Milk donors had a slightly different racial/ethnic background from the mothers with infants in the NICU (Figure 1), but there are currently no data that link HMO composition to race and ethnicity. Inherent to milk sampling in the NICU, the majority (> 75%) of all MOM samples were from mothers with preterm infants. Less than 22% of the individual donors, however, had preterm deliveries. A most recent report has shown that the milk of mothers delivering preterm infants has higher HMO concentrations than term milk.25 Therefore, it is likely that some of the HMO differences between DM and MOM stem from the use of mostly term DM in a NICU population with predominately preterm infants.
Conclusion Our data show that HMO composition differs significantly between DM and the milk of mothers with infants in the
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NICU. These differences may have important implications for the health outcomes of term and preterm infants. Further, it raises the question of whether DM should be more thoroughly selected to match the infant’s gestational age and lactation time and maybe even the mother’s Secretor and Lewis blood status. Acknowledgments We thank Pauline Sakamoto and Dr Sean Xiong from the Mothers’ Milk Bank, San Jose, California, for providing us with donor milk aliquots, and Dr Donna Geddes, from the University of Western Australia, for comments and suggestions on the article.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The project was supported by divisional funds to LB and a German Academic Exchange Service research fellowship to CM.
References 1. Gartner LM, Morton J, Lawrence RA, et al. Breastfeeding and the use of human milk. Pediatrics. 2005;115(2):496-506. 2. Tully MR. A year of remarkable growth for donor milk banking in North America. J Hum Lact. 2000;16(3):235-236. 3. Perrine CG, Scanlon KS. Prevalence of use of human milk in US advanced care neonatal units. Pediatrics. 2013;131(6):10661071. 4. Parker MG, Barrero-Castillero A, Corwin BK, Kavanagh PL, Belfort MB, Wang CJ. Pasteurized human donor milk use among US level 3 neonatal intensive care units. J Hum Lact. 2013;29(3):381-389. 5. Springer S. Human milk banking in Germany. J Hum Lact. 1997;13(1):65-68. 6. Springer S. News about human milk banking in Germany. Adv Exp Med Biol. 2000:478:441-442. 7. Human Milk Banking Association of North America. Guidelines for the Establishment and Operation of a Donor Human Milk Bank. 15th ed. Fort Worth, TX: Human Milk Banking Association of North America; 2009. 8. Grovslien AH, Gronn M. Donor milk banking and breastfeeding in Norway. J Hum Lact. 2009;25(2):206-210. 9. Tully DB, Jones F, Tully MR. Donor milk: what’s in it and what’s not. J Hum Lact. 2001;17(2):152-155. 10. Ley SH, Hanley AJ, Stone D, O’Connor DL. Effects of pasteurization on adiponectin and insulin concentrations in donor human milk. Pediatr Res. 2011;70(3):278-281. 11. Kunz C, Rudloff S, Baier W, Klein N, Strobel S. Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annu Rev Nutr. 2000;20:699-722. 12. Newburg DS, Ruiz-Palacios GM, Morrow AL. Human milk glycans protect infants against enteric pathogens. Annu Rev Nutr. 2005;25:37-58.
13. Bode L. Recent advances on structure, metabolism, and function of human milk oligosaccharides. J Nutr. 2006;136(8):2127-2130. 14. German JB, Freeman SL, Lebrilla CB, Mills DA. Human milk oligosaccharides: evolution, structures and bioselectivity as substrates for intestinal bacteria. Nestle Nutr Workshop Ser Pediatr Program. 2008;62:205-218. 15. Bode L. Human milk oligosaccharides: prebiotics and beyond. Nutr Rev. 2009;67(2):S183-S191. 16. Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012;22(9):1147-1162. 17. Sela DA, Mills DA. Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides. Trends Microbio. 2010;18(7):298-307. 18. Angeloni S, Ridet JL, Kusy N, et al. Glycoprofiling with micro-arrays of glycoconjugates and lectins. Glycobiology. 2005;15(1):31-41. 19. Kuntz S, Kunz C, Rudloff S. Oligosaccharides from human milk induce growth arrest via G2/M by influencing growthrelated cell cycle genes in intestinal epithelial cells. Br J Nutr. 2009;101(9):1306-1315. 20. Kuntz S, Rudloff S, Kunz C. Oligosaccharides from human milk influence growth-related characteristics of intestinally transformed and non-transformed intestinal cells. Br J Nutr. 2008;99(3):462-471. 21. Bode L, Kunz C, Muhly-Reinholz M, Mayer K, Seeger W, Rudloff S. Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides. Thromb Haemost. 2004;92(6):1402-1410. 22. Bode L, Rudloff S, Kunz C, Strobel S, Klein N. Human milk oligosaccharides reduce platelet-neutrophil complex formation leading to a decrease in neutrophil beta 2 integrin expression. J Leukoc Biol. 2004;76(4):820-826. 23. Eiwegger T, Stahl B, Schmitt J, et al. Human milk-derived oligosaccharides and plant-derived oligosaccharides stimulate cytokine production of cord blood T-cells in vitro. Pediatr Res. 2004;56(4):536-540. 24. Wang B. Sialic acid is an essential nutrient for brain development and cognition. Annu Rev Nutr. 2009;29:177-222. 25. Gabrielli O, Zampini L, Galeazzi T, et al. Preterm milk oligosaccharides during the first month of lactation. Pediatrics. 2011;128(6):e1520-e1531. 26. Kobata A. Structures and application of oligosaccha rides in human milk. Proc Jpn Acad Ser B Phys Biol Sci. 2010;86(7):731-747. 27. Stahl B, Thurl S, Henker J, Siegel M, Finke B, Sawatzki G. Detection of four human milk groups with respect to Lewisblood-group-dependent oligosaccharides by serologic and chromatographic analysis. Adv Exp Med Biol. 2001;501:299-306. 28. Morrow AL, Ruiz-Palacios GM, Altaye M, et al. Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants. J Pediatr. 2004;145(3):297-303. 29. Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez-Munguia B, Newburg DS. Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem. 2003;278(16):14112-14120. 30. Jantscher-Krenn E, Zherebtsov M, Nissan C, et al. The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats. Gut. 2012;61(10):1417-1425.
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Marx et al 31. Boyd CA, Quigley MA, Brocklehurst P. Donor breast milk versus infant formula for preterm infants: systematic review and meta-analysis. Arch Dis Child Fetal Neonatal Ed. 2007;92(3):F169-F175. 32. Quigley MA, Henderson G, Anthony MY, McGuire W. Formula milk versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database Syst Rev. 2007;17(4):CD002971. 33. Jantscher-Krenn E, Lauwaet T, Bliss LA, Reed SL, Gillin FD, Bode L. Human milk oligosaccharides reduce Entamoeba histolytica attachment and cytotoxicity in vitro. Br J Nutr. 2012;108(10):1839-1846.
34. Samuel MC, Vugia DJ, Shallow S, et al. Epidemiology of sporadic Campylobacter infection in the United States and declining trend in incidence, FoodNet 1996-1999. Clin Infect Dis. 2004;38(3):S165-S174. 35. Marcus R. New information about pediatric foodborne infections: the view from FoodNet. Curr Opin Pediatr. 2008;20(1):79-84. 36. Pritt BS, Clark CG. Amebiasis. Mayo Clin Proc. 2008;83(10):1154-1160. 37. Bertino E, Coppa GV, Giuliani F, et al. Effects of Holder pasteurization on human milk oligosaccharides. Int J Immunopathol Pharmacol. 2008;21(2):381-385.
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Human Milk Oligosaccharide Composition Differs between Donor Milk and Mother's Own Milk in the NICU Carolin Marx, Renee Bridge, Alison K. Wolf, Wade Rich, Jae H. Kim and Lars Bode J Hum Lact 2014 30: 54 originally published online 26 November 2013 DOI: 10.1177/0890334413513923 The online version of this article can be found at: http://jhl.sagepub.com/content/30/1/54
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research-article2013
JHLXXX10.1177/0890334413513923Journal of Human LactationMarx et al
Original Research
Human Milk Oligosaccharide Composition Differs between Donor Milk and Mother’s Own Milk in the NICU
Journal of Human Lactation 2014, Vol 30(1) 54–61 © The Author(s) 2013 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0890334413513923 jhl.sagepub.com
Carolin Marx, MS1, Renee Bridge, RN1, Alison K. Wolf, RN, NNP1, Wade Rich, RRT, CCRC1, Jae H. Kim, MD, PhD1, and Lars Bode, PhD1
Abstract Background: Human milk oligosaccharides (HMO) represent the third most abundant component of human breast milk. More than a hundred structurally distinct HMO have been identified, and the HMO composition varies between mothers as well as over the course of lactation. Some newborn infants receive donor milk (DM) when their mother’s own milk (MOM) volume is inadequate or unavailable. Objective: This study aimed to compare HMO content between DM and MOM. Methods: We used high performance liquid chromatography analysis of fluorescently labeled HMO to analyze the variation in HMO amount and composition of 31 different batches of DM (each pooled from 3 individual donors) provided by the Mothers’ Milk Bank in San Jose, California, and compared it to 26 different MOM samples donated by mothers with infants in our neonatal intensive care unit (NICU). Results: Total HMO amount as well as concentrations of lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose 1, and disialyllacto-N-tetraose were significantly lower in DM than in MOM, whereas the concentrations of 3’-sialyllactose and 3-fucosyllactose were significantly higher in DM. Conclusion: Our data show that infants in our NICU who receive DM are likely to ingest HMO at different total amounts and relative composition from what they would receive with their MOM. Recent in vitro and animal studies have started to link individual HMO to infant health and disease. Future studies are needed to assess the importance of a mother-infant match with regard to HMO composition. Keywords breastfeeding, breast milk, human milk, milk banking, neonatal nutrition, oligosaccharides, preterm infants
Well Established Human milk oligosaccharides (HMO) are the third most abundant component in breast milk, and their composition varies from 1 mother to another. Donor breast milk (DM) is widely used in neonatal intensive care units (NICUs), but the HMO composition of DM has not yet been analyzed.
Newly Expressed The amount and composition of HMO are significantly different between DM and the milk that infants in the NICU receive from their own mothers.The physiological and clinical relevance of this potential mismatch needs further investigation.
Background Breastfeeding is considered the optimum nutrition for the first 6 months of life.1 When mother’s own milk (MOM) is unavailable or in short supply, donor breast milk (DM) is increasingly
used in neonatal intensive care units (NICUs) to provide infants with some of the benefits of breast milk.2 Studies by Perrine and Scanlon3 and Parker and colleagues4 indicate that 22% to 42% of the NICUs in the United States use DM. In addition to traditional nutrients, breast milk contains a variety of bioactive compounds, and their amount and composition often depend on genetic and environmental factors and can change over the course of lactation. Amount and composition of milk bioactive compounds can change when DM is processed (ie, pasteurized) to protect the recipient infant against disease 1
Division of Neonatology, Department of Pediatrics, University of California, San Diego, San Diego, CA, USA Date submitted: August 29, 2013; Date accepted: October 30, 2013. Corresponding Author: Lars Bode, PhD, Division of Neonatology and Division of Gastroenterology and Nutrition, Department of Pediatrics, University of California, San Diego, 200 West Arbor Drive, MC 8450, San Diego, CA 92103-8450, USA. Email: [email protected]
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Marx et al transmission.5-8 In North America and the United Kingdom, DM is pasteurized at 62.5°C for 30 minutes (Holder pasteurization), which partially inactivates some but not all of the milk bioactive compounds.9 As a most recent example, Holder pasteurization was shown to reduce adiponectin and insulin concentrations by 32.8% and 46.1%, respectively.10 Human milk oligosaccharides (HMO) are a group of milk bioactive compounds thought to benefit the human milk-fed infant on multiple levels.11-16 HMO are prebiotics and antiadhesive antimicrobials that shape the infant’s intestinal microbiota composition.11,12,16,17 They may also directly interact with the infant’s intestinal epithelial cells18-20 or modulate immune cell responses21-23 and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition.24 After lactose and lipids, HMO are the third most abundant component in human breast milk.11 HMO concentrations decline over the course of lactation with 20 to 30 g/L in colostrum and 10 to 15 g/L in mature milk.11 The milk of mothers delivering preterm infants has higher HMO concentrations than term milk.25 More than 100 different HMO have been identified so far, but not every woman synthesizes the same set of oligosaccharides.26 HMO composition is in part genetically determined and mirrors the woman’s Secretor and Lewis blood group status.11,16,27 More than 70% of women are Secretors,27 which means that they express the α1-2-fucosyltransferase FUT2 and their milk contains characteristic α1-2-fucosylated HMO like 2’-fucosyllactose (2’FL) or lacto-N-fucopentaose 1 (LNFP1). The milk of non-Secretor women does not contain these specific HMO, and infants fed non-Secretor milk are at higher risk for diarrhea caused by certain viral and bacterial infections.28 Women with Lewis positive blood group status express the α1-3/4-fucosyltransferase FUT3 and their milk contains characteristic α1-4-fucosylated HMO like LNFP2. The beneficial effects reported for HMO are based on total HMO amount as well as distinct structural composition.21,22,28-30 Animal model data from our own group, for example, suggest that 1 specific HMO, disialyllacto-N-tetraose (DSLNT), protects from necrotizing enterocolitis,30 1 of the most serious intestinal disorders in preterm infants. DM is widely and increasingly fed to preterm infants in the NICU, but the health outcomes are not always comparable to infants who receive their mother’s own milk.31,32 We therefore aimed to investigate whether total HMO amount and composition differ between DM and MOM fed to infants in our NICU at the University of California, San Diego (UCSD).
Methods
1 milk sample, which was collected by hand-expression after the feed in the morning between 8 and 11 AM. Fresh milk samples were transported to the lab on ice and stored at –80°C prior to HMO analysis. To assess the effects of pasteurization on HMO amount and composition, 5 of the fresh milk samples were split in 2 aliquots and 1 aliquot was heated at 62.5°C for 30 minutes. Afterward, HMO amount and composition in both aliquots were analyzed in parallel.
Donor Milk The Mothers’ Milk Bank, San Jose, California, provided 1-mL aliquots from 31 different DM batches. Each batch was a pool of milk from 3 individual donors. The pooled milk was further processed by Holder pasteurization following the guidelines of the Human Milk Banking Association of North America.7 Samples were shipped on dry ice and stored at –80°C prior to HMO analysis.
Human Milk Oligosaccharide Analysis Oligosaccharides from MOM or DM were analyzed as previously described.30,33 Briefly, raffinose was added to 40 μL milk to serve as an internal standard through sample processing and analysis. Lipids and proteins were removed from the samples by centrifugation and chloroform/methanol extraction. Lactose was removed by overnight incubation on lactase-immobilized beads (Invitrogen, Carlsbad, California, USA) at 37°C. Residual peptides and salt were removed over Sep-Pak C18 cartridges followed by porous graphitized carbon (PGC) cartridges. The reducing end of the dried oligosaccharides was labeled with the fluorescent tag 2-aminobenzamide (2AB) for 2 hours at 65°C. Free 2AB label was separated from the 2AB-labeled oligosaccharides using silica gel cartridges. 2AB-labeled oligosaccharides were analyzed by high performance liquid chromatography (HPLC) on an amide-80 column (4.6 mm ID × 25 cm, 5 µm; Tosoh Bioscience, Tokyo, Japan) with a 50 mM ammonium formate/acetonitrile buffer system. Separation was performed at 25°C and monitored with a fluorescence detector at 360 nm excitation and 425 nm emission. Peak annotation was based on standard retention times and mass spectrometric (MS) analysis on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano-ESI source. Total oligosaccharides were calculated as the sum of the 20 most abundant individual oligosaccharides.
Statistical Analysis
Mother’s Own Milk We recruited 26 mothers with infants in the NICU at UCSD Medical Center, San Diego, California, after approval by UCSD’s institutional review board.Each mother provided
Differences in oligosaccharide concentrations between MOM and DM were calculated by 2-tailed Mann-Whitney test. Correlations between oligosaccharide concentrations in MOM and their respective lactation time postpartum were
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Journal of Human Lactation 30(1)
Figure 1. Characteristics of Mother’s Own Milk (Top) and Donor Milk (Bottom) Based on Infant Maturity at Birth (Left) and Maternal Race/Ethnicity (Right).
(n=26) Preterm (76.9%)
Caucasian/White (Non-Hispanic) (46.2%)
Term (23.1%)
Black/ African American (7.7%)
Other (Hispanic) (42.3%)
Asian/ Pacific Islander (3.8%)
Donor Milk (DM) (n=31)
Preterm (21.5%)
Term (78.5%)
Other (Hispanic) (21.5%)
Caucasian/White (Non-Hispanic) (41.9%)
Asian/ Pacific Islander (36.6%)
calculated by 2-tailed Spearman test with 95% confidence interval. Significance was defined as P values of less than .05.
Results We randomly collected 26 human breast milk samples from mothers with infants in the NICU who were receiving their mother’s own milk. We analyzed the HMO composition from all 26 milk samples and compared them to 31 different DM batches that were available at the time we collected the MOM. Whereas more than 75% of the infants in the NICU were born preterm, almost 80% of the mothers who donated milk for the DM batches had term deliveries (Figure 1). Distributions of race and ethnicity were also different between the mothers with infants in the NICU in San Diego (Southern California) and the 93 mothers who contributed to the 31 pooled milk samples that we received from the Mothers’ Milk Bank in San Jose (Northern California).
HMO analysis confirmed interpersonal variations in HMO composition as exemplified by the different chromatograms in Figure 2. The HMO profile of a Secretor woman is characterized by a high abundance of α1-2-fucosylated HMO (Figure 2a) that are absent from the milk of non-Secretor women (Figure 2b). The HMO profile in the DM sample (Figure 2c) looks somewhat different, contains high amounts of 2’FL but low amounts of LNFP1, and has higher concentrations of 3FL and 3’SL compared to most MOM samples. Figure 3 compares the concentrations of individual HMO between MOM and DM. The concentration of total HMO is significantly lower in DM compared to MOM (Figure 3a). Although the concentration of 2’FL was not significantly different between the 2 groups (Figure 3b), there was a difference in the number of non-Secretor samples, milk samples that lack 2’FL and other α1-2-fucosylated HMO. Whereas 4 out of the 26 MOM samples were from non-Secretor mothers, only 1 out of the 31 DM samples was a non-Secretor, indicating that all 3 individual donors who contributed to this
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Marx et al Figure 2. Representative High Performance Liquid Chromatography Chromatograms of Oligosaccharides from Mother’s Own Milk (a and b) and Donor Milk (c).
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LNnT, LNFP1, and DSLNT were significantly lower in DM than in MOM (Figures 3e-3h). To exclude the possibility that the differences in HMO composition were due to alterations during the pasteurization process, we separated 5 fresh MOM samples in 2 aliquots each, pasteurized 1 of the 2 aliquots, and analyzed HMO composition of both aliquots in parallel. Differential processing, however, did not result in significant differences in total HMO amount and composition (data not shown). Although information on the postpartum lactation time was not available for the DM samples, MOM samples were collected as early as 2 days postpartum and as late as 169 days postpartum [median, 7.5 days; interquartile range, 16.75 (20.75-4.00)]. Because HMO concentrations are known to decline over the course of lactation, we plotted the concentrations of individual HMO against postpartum lactation time (Figure 4). Concentrations of 2’FL, LNnT, and LNFP1 negatively correlated with the postpartum lactation time, and there was a nonsignificant trend in the association of total HMO and the postpartum lactation time (P = .083). Differences in the time of lactation may be 1 explanation for some of the HMO differences we report for DM and MOM.
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Mother A is a Secretor and her milk is abundant in 2’-fucosyllactose (peak 1) and lacto-N-fucopentaose 1 (peak 6), but there is hardly any 3-fucosyllactose (peak 2). Mother B is a non-Secretor and her milk contains no α1-2fucosylated HMO but more 3-fucosyllactose. The donor milk batch is a hybrid of 3 individual milk samples and contains both 2’-fucosyllactose and 3-fucosyllactose as well as fairly high amounts of 3’-sialyllactose (peak 4). 0: raffinose (internal standard); 1: 2’-fucosyllactose (2’FL); 2: 3-fucosyllactose (3FL); 3: lacto-N-neotetraose (LNnT); 4: 3’-sialyllactose (3’sLac); 5: lacto-N-tetraose (LNT); 6: lacto-N-fucopentaose 1 (LNFP1); 7: lacto-N-fucopentaose 2 (LNFP2); 8: sialyllacto-N-tetraose b (LST b); 9: sialyllacto-N-tetraose c (LST c); 10: lacto-N-difucohexaose (LNDFH); 11: disialyllacto-N-tetraose (DSLNT).
DM batch were likely non-Secretors. The concentrations of 3FL and 3’SL were significantly higher in DM than in MOM (Figures 3c and 3d). In contrast, concentrations of LNT,
Discussion We report that HMO amount and composition are significantly different between DM and the milk that infants in the NICU receive from their own mothers. Accumulating data from in vitro, ex vivo, and in vivo studies strongly suggest that infants benefit from HMO on multiple levels.11,12,14,16 They serve as prebiotics that help shape the microbial composition in the infant’s intestine, as anti-infective agents that reduce pathogen adhesion to epithelial surfaces, or as immune modulators. The beneficial effects are often structure-specific and correlate with the concentration of individual HMO.21,22,28-30,33 Campylobacter jejuni, for example, is 1 of the most common causes of bacterial diarrhea and infant mortality.34,35 α12-fucosylated HMO like 2’FL or LNFP1 inhibit C. jejuni attachment to intestinal epithelial cells in vitro and ex vivo,29 and high concentrations of α1-2-fucosylated HMO in the mother’s milk reduced the infant’s risk of developing C. jejuni diarrhea.28 Although there was no significant difference in the concentration of 2’FL between DM and MOM (Figure 3b), LNFP1 was less abundant in DM than in MOM (Figure 3g). Because more than 70% of women are Secretors27 and each DM sample contained milk from 3 different women, the number of non-Secretor samples was lower in the DM group than in the MOM group. It remains unknown whether there are any health consequences for infants who are fed Secretor DM rich in α1-2-fucosylated HMO but would otherwise receive milk from a non-Secretor mother who lacks these specific HMO. Other anti-adhesive antimicrobial effects have been described for HMO and they are often highly structuredependent and not limited to viruses and bacteria.11,12 For example, the protozoan parasite Entamoeba histolytica
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Journal of Human Lactation 30(1)
Figure 3. Comparison of Human Milk Oligosaccharide Concentrations between Mother’s Own Milk and Donor Milk.
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a, Total HMO as the sum of individual HMO detected as follows. b, 2’-fucosyllactose (2’FL). c, 3-fucosyllactose (3FL). d, 3’-sialyllactose (3’sLac). e, Lacto-N-tetraose (LNT). f, Lacto-N-neotetraose (LNnT). g, Lacto-N-fucopentaose 1 (LNFP1). h, Disialyl-lacto-N-tetraose (DSLNT). Dots represent concentrations of individual samples. Bars and lines represent median and interquartile range. Symbols above each graph represent the respective HMO structure containing glucose (dark circle), galactose (light circle), N-acetyllactosamine (square), fucose (triangle), and sialic acid (diamond). Abbreviations: DM, donor milk; HMO, human milk oligosaccharide; MOM, mother’s own milk; ns, not significant. *P < .05. **P < .01. ***P < .001 (2-tailed Mann-Whitney test).
causes amebiasis, which is the third leading cause of death by parasitic diseases after malaria and schistosomiasis.36 Specific neutral HMO like LNT and LNnT reduce E. histolytica attachment to and killing of human intestinal epithelial cells.33 Here, we show that both LNT and LNnT are less abundant in DM than in MOM (Figures 3e and 3f). Based on our results, DM may contain less anti-adhesive antimicrobial HMO and therefore be less effective in protecting the neonate from certain viral, bacterial, or protozoan infections.
In an example most relevant to the NICU, our lab has recently shown that an individual HMO, disialyllacto-Ntetraose (DSLNT), protects from necrotizing enterocolitis in an animal model.30 Here, we report that, on average, DSLNT is less abundant in DM than in MOM. However, whether or not DSLNT concentrations in some DM batches are insufficient to protect the preterm neonate against necrotizing enterocolitis remains unknown and needs further investigation.
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Marx et al Figure 4. Correlation between Human Milk Oligosaccharide Concentrations and Lactation Times Postpartum in Mother’s Own Milk.
r: -0.3557 (ns)
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a, Total HMO as the sum of individual HMO detected as follows. b, 2’-fucosyllactose (2’FL). c, 3-fucosyllactose (3FL). d, 3’-sialyllactose (3’sLac). e, Lacto-N-tetraose (LNT). f, Lacto-N-neotetraose (LNnT). g, Lacto-N-fucopentaose 1 (LNFP1). h, Disialyl-lacto-N-tetraose (DSLNT). Dots represent concentrations of individual samples. Spearman r values and significances are given above each graph. Abbreviations: HMO, human milk oligosaccharide; ns, not significant; pp, postpartum. *P < .05. **P < .01. ***P < .001 (2-tailed Mann-Whitney test).
The question remains why HMO amount and composition differ between DM and MOM. HMO are not affected by pasteurization,37 which we confirmed in this study with a limited set of samples (n = 5). Differences in postpartum lactation time, however, may be 1 explanation. MOM samples were randomly collected from mothers with infants in the NICU who would otherwise receive DM, and the lactation age ranges from 2 to 169 days postpartum. Half of the MOM samples were collected within the first week postpartum. Although information on the lactation stage was not available for the DM samples, it is likely that most women donate milk later when lactation is fully established and the amount exceeds their infants’ needs. HMO concentrations decline over the course of lactation, which we confirmed even with the limited number of MOM samples analyzed in this study (Figure 4). It needs to be further investigated whether feeding mature DM to infants in the first few days of life has any health consequences with respect to HMO amount and composition.
Lactation time may explain some but not all of the HMO differences between DM and MOM. Milk donors had a slightly different racial/ethnic background from the mothers with infants in the NICU (Figure 1), but there are currently no data that link HMO composition to race and ethnicity. Inherent to milk sampling in the NICU, the majority (> 75%) of all MOM samples were from mothers with preterm infants. Less than 22% of the individual donors, however, had preterm deliveries. A most recent report has shown that the milk of mothers delivering preterm infants has higher HMO concentrations than term milk.25 Therefore, it is likely that some of the HMO differences between DM and MOM stem from the use of mostly term DM in a NICU population with predominately preterm infants.
Conclusion Our data show that HMO composition differs significantly between DM and the milk of mothers with infants in the
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Journal of Human Lactation 30(1)
NICU. These differences may have important implications for the health outcomes of term and preterm infants. Further, it raises the question of whether DM should be more thoroughly selected to match the infant’s gestational age and lactation time and maybe even the mother’s Secretor and Lewis blood status. Acknowledgments We thank Pauline Sakamoto and Dr Sean Xiong from the Mothers’ Milk Bank, San Jose, California, for providing us with donor milk aliquots, and Dr Donna Geddes, from the University of Western Australia, for comments and suggestions on the article.
Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The project was supported by divisional funds to LB and a German Academic Exchange Service research fellowship to CM.
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