Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation

Prasoon Chaturvedi2,3, Christopher D. Warren2,3, Mekibib Altaye4, Ardythe L. Morrow4, Guillermo Ruiz-Palacios5, Larry K. Pickering4 and David S. Newburg1,2,3

2Shriver Center for Mental Retardation, Waltham, MA 02452, USA, 3Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 4Eastern Virginia Medical School, Norfolk, VA 23510, USA, and 5Instituto Nacional de Ciecias Medicas y Nutricion, Mexico City 14000, Mexico

Received on September 14, 2000; revised on December 8, 2000; accepted on January 12, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1–49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of {alpha}1,2 linkages (but contained {alpha}1,3- and {alpha}1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing {alpha}1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of {alpha}1,2-linked fucose. The ratio of {alpha}1,2-linked oligosaccharide concentrations to oligosaccharides devoid of {alpha}1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.

Key words: human milk/oligosaccharide/lactation/HPLC


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human milk is unique in its large amount and complex array of oligosaccharides. In the last four decades, more than 120 oligosaccharides have been isolated and identified, which may represent only a small fraction of the total (Kobata, 1972Go, 1978; Egge et al., 1983Go; Ginsburg and Robbins, 1984Go; Stahl et al., 1994Go; Newburg and Neubauer, 1995Go). Early studies of these oligosaccharides were prompted by their structural homology to erythrocyte glycoconjugates that determine ABO blood group types, their use as tools for studying the specificity of glycosyltransferases and glycosidases, and for raising and characterizing specific antibodies.

There is now a growing interest in the capacity of human milk oligosaccharides, especially fucosyloligosaccharides, to protect infants from enteric and other pathogens, especially during early development (Holmgren et al., 1983Go; Parkkinen and Finne, 1985Go; Andersson et al., 1986Go; Ashkenazi and Mirelman, 1987Go; Newburg et al., 1990Go; Cravioto et al., 1991Go; Schroten et al., 1992Go; Korhonen et al., 1995Go; Newburg, 1996Go). Because milk oligosaccharides are synthesized by glycosyltransferases similar to those that synthesize cell surface glycoproteins and glycolipids, they share common epitopes with host receptors for pathogens (Kunz and Rudloff, 1993Go; Zopf and Roth, 1996Go), and, as soluble homologs or analogs of receptors, may act as decoys, protecting infants against disease. The inhibition of adhesion of Escherichia coli to bladder epithelial cells by milk oligosaccharides supports this hypothesis (Coppa et al., 1990Go); likewise, human milk oligosaccharides with terminal {alpha}1,2-fucosyl linkages have protective activity against Campylobacter jejuni (Ruiz-Palacios et al., 1992Go; Cervantes et al., 1995Go). A fucosylated human milk oligosaccharide interacts with the guanylyl cyclase receptor for the stable toxin of E. coli, thereby inhibiting toxin binding (Crane et al., 1994Go).

Human milk oligosaccharides vary among individuals: They are associated with the same genes that determine Lewis blood type and secretor status (Viverge et al., 1990Go). Indeed, three groups of milk oligosaccharide expression were defined based on amounts of total oligosaccharides produced and the expression of specific {alpha}1,2-linked oligosaccharides. Human milk oligosaccharides also vary over the course of lactation: The amounts of sugar residues derived from the hydrolysis of milk oligosaccharides change longitudinally (Miller et al., 1994Go). Furthermore, fucosyltransferase and fucosidase activities vary in milk specimens, both from different donors and from the same donors at different stages of lactation (Wiederschain and Newburg, 1995Go). Other glycosyltransferases and glycosidases also may vary. As a prelude to studying relationships between specific human milk oligosaccharides and disease in breast-fed infants, it is necessary to define how these oligosaccharides vary over the duration of lactation.

In our previous studies, a reversed-phase high-performance liquid chromatography (HPLC) method was developed to identify and quantitate the perbenzoylated derivatives of intact milk oligosaccharides; large variations in the types and concentrations of human milk neutral oligosaccharides were observed in milk from 50 different donors (Chaturvedi et al., 1997Go). The current study was designed to investigate the degree of variation in expression of individual milk oligosaccharides over the first year of lactation, both within individual donors and between multiple donors.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Milk samples
A total of 84 milk samples were analyzed. Samples were collected at postpartum periods designated as weeks 1, 2, 4, 14, 26, 38, and 49. At least 10 of the 12 donors contributed a sample at each time period. On five occasions two samples were collected from the same subject in a single postpartum time period; these samples were averaged to provide only one data point for each time period, resulting in 79 independent data points for evaluating overall trends in the population. The structures of the oligosaccharides measured in this study are shown in Table I, along with the average content of each oligosaccharide in milk samples of the 12 women over 1 year of lactation. The total neutral oligosaccharide concentration was calculated for each milk sample as the sum of these individual oligosaccharides. For each donor, mean concentrations over the course of lactation appear in Table II for the total milk oligosaccharides, the {alpha}1,2-linked fucosyloligosaccharides (with fucose linked by {alpha}1,2 glycosidic bonds), and the {alpha}1,3/4-linked fucosyloligosaccharides (with fucose linked only by {alpha}1,3 or {alpha}1,4 glycosidic bonds).



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Table I. Major human milk oligosaccharides measureda

aData are expressed as mean ± SE of 84 samples from12 subjects over 1 year of lactation.

bLNF-II and LNF-III co-elute and are measured together as a single peak.

cMinor fucosylated components not included in comparison of {alpha}1,2- and {alpha}1,3/4-linked fucosyloligosaccharides due to inconsistent detection across samples.

 

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Table II. Average concentration of {alpha}1,2-linked, {alpha}1,3/4-linked, and total oligosaccharides throughout 1 year of lactationa
 
Longitudinal pattern of milk oligosaccharides
Of the 12 donors, 11 demonstrated a consistent predominance of {alpha}1,2-linked fucosyloligosaccharides in their milk, and one donor (Table II, donor number 7) initially displayed a lack of 2-linked fucosyloligosaccharides and a predominance of {alpha}1,3/4-linked fucosyloligosaccharides. We therefore analyze and discuss the data on the group of 11 donors as typical of our study population and present the pattern of the other donor’s milk separately as an important variant.

The sum of the individual oligosaccharide concentrations in the milk of each donor at each time point defined the total neutral oligosaccharide concentration of each milk sample. The mean total oligosaccharide concentration for the 11 typical donors (a total of 77 samples) was approximately 9 g/L for the first 14 weeks of lactation followed by a gradual decline to approximately 4 g/L at 1 year postpartum, as shown in Figure 1A. Much of this decline in total oligosaccharide concentration over the course of lactation was due to the decreased 2'-fucosyllactose (2'-FucLac), lacto-N-fucopentaose I (LNF-I), and lacto-N-difucohexaose I (LDFH-I), three of the four major oligosaccharides containing fucose with {alpha}1,2 linkages. Lactodifucotetraose (LDFT) remained at a low, relatively constant concentration. In contrast, the concentration of the three major fucosyloligosaccharides containing only fucosyl {alpha}1,3 and {alpha} 1,4 linkages, that is, 3-fucosyllactose (3-FucLac), lacto-N-fucopentaose II and III (LNF-II/III), and lacto-N-difucohexaose-II (LDFH-II), gradually increased from approximately 1 g/L during the first week of lactation to approximately 2 g/L at 1 year postpartum. 3-FucLac increased fourfold from 300 mg/L to 1.1 g/L (Figure 1A). Early in lactation, {alpha}1,2-linked oligosaccharides predominated over {alpha}1,3/4-linked oligosaccharides, whereas by 1 year, they had converged to approximately equal concentrations. Thus, the ratios of the {alpha}1,2-linked fucosyloligosaccharide concentrations to the {alpha}1,3/4-linked fucosyloligosaccharide concentrations in milk undergo exponential decay over the course of lactation, as shown in Figure 1B, defined by the equation Y = 4.84 x 10–0.013x (regression coefficient [r] = 0.99, p < 0.0001, 95% CI [–0.015, –0.010]).



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Fig. 1. (A) The mean values and standard errors (vertical bars) of the concentrations of {alpha}1,2-linked, {alpha}1,3/4-linked, and total oligosaccharides in human milk from of 11 of the 12 subjects. {alpha}1,2-Linked oligosaccharides clearly predominated over exclusively {alpha}1,3/4-linked oligosaccharides. Each value is the mean ± SE of 10 or 11 data points from individual donors. (B) The ratios of the mean concentrations of {alpha}1,2-linked to {alpha}1,3/4-linked oligosaccharides in the milk of these 11 women.

 
The predominant oligosaccharides for the first few months of lactation were 2'-FucLac and LNF-I (Figure 2A). For the first 3 months of lactation, 2'-FucLac, at approximately 3 g/L, was the oligosaccharide present in the largest concentration. Thereafter, 2'-FucLac declined to 1.2 g/L at 1 year postpartum. Likewise, LNF-I, which had an initial concentration of 2 g/L in the first month postpartum, declined eightfold to only 250 mg/L by 1 year postpartum. In contrast, 3-FucLac, which had an initial concentration of only 300 mg/L, rose over the course of lactation to a concentration of 1.1 g/L. These three oligosaccharides accounted for most of the change in {alpha}1,2-linked and {alpha}1,3/4-linked oligosaccharides (Figure 2A).



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Fig. 2. (A) The mean concentrations of specific oligosaccharides in milk of the 11 mothers in which {alpha}1,2-linked fucosyloligosaccharides predominated over exclusively {alpha}1,3/4-linked fucosyloligosaccharides. Each value is the mean of 10 or 11 data points from individual donors. (B) The oligosaccharide profile of milk in which {alpha}1,3/4-linked oligosaccharides predominated over {alpha}1,2-linked oligosaccharides; only one subject produced milk with this profile.

 
Covariation of oligosaccharide concentrations
The correlation between concentrations of structurally related fucosyloligosaccharides was examined. In the 11 donors whose milk initially contained a preponderance of {alpha}1,2-linked fucosyloligosaccharides, the concentrations of specific {alpha}1,2-linked fucosyloligosaccharides generally correlated with each other, with r-values (calculated over time and subjects) ranging from 0.4 to 0.6. Similarly, the concentrations of specific fucosyloligosaccharides lacking {alpha}1,2-linked fucose (but containing {alpha}1,3- or {alpha}1,4-linked fucose) generally correlated with each other, with r-values close to 0.5.

However, correlations within these two groups of oligosaccharides (that is, those containing {alpha}1,2-linked fucose and those whose fucose was not {alpha}1,2-linked) varied greatly during the course of lactation and between subjects. Over the course of lactation the relationships between specific {alpha}1,2-linked fucosyloligosaccharides demonstrated considerable heterogeneity, with correlations ranging from r = –0.13 to r = 0.97. Similarly, the correlations between the same {alpha}1,2-linked fucosyloligosaccharides in milk from different donors displayed considerable heterogeneity (r = –0.18 to r = 0.98). Within the group of fucosyloligosaccharides devoid of {alpha}1,2 linkages, correlations between individual oligosaccharide concentrations were stable over the course of lactation (r = 0.28 to r = 0.97) but varied considerably between subjects, with r-values ranging from –0.58 to 0.96.

The relationships between concentrations of the {alpha}1,2-linked fucosyloligosaccharides taken as a group and the {alpha}1,3/4-linked fucosyloligosaccharides taken as a group varied between subjects (r = – 0.66 to r = 0.93) and over the course of lactation (r = 0.21 to r = 0.93). However, at 26 weeks, a significant positive correlation between {alpha}1,2-linked and {alpha}1,3/4-linked fucosyloligosaccharides became apparent and became stronger over the remainder of the study, coincident with the decreasing {alpha}1,2-linked fucosyloligosaccharide and the increasing {alpha}1,3/4-linked fucosyloligosaccharide concentrations in the milk of these 11 donors.

The exceptional oligosaccharide profile
These general trends for the 11 donors were true in the inverse for the donor with the unique oligosaccharide profile, displayed in Figure 2B. For the first 9 months of lactation, her milk was almost devoid of {alpha}1,2-linked oligosaccharides. During the first month, it contained unusually high levels of {alpha}1,3/4-linked oligosaccharides, so that the oligosaccharides consisted almost entirely of 3-FucLac, LNF-II/III, and LDFH-II. At 9 months the concentrations of 2'-FucLac and LDFT, both of which contain {alpha}1,2-linked fucose, started to rise; combined, they approached the concentrations of the {alpha}1,3/4-linked fucosyloligosaccharides (whose concentrations had diminished) at the end of 1 year of lactation. Thus, the ratio of {alpha}1,2-linked fucose molecules to those devoid of {alpha}1,2-linked fucose, which was close to zero until the 38th week of lactation, rose rapidly toward unity by the 49th week, as shown in Figure 3.



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Fig. 3. The ratios of the mean concentrations of {alpha}1,2-linked to {alpha}1,3/4-linked oligosaccharides in the milk of the subject in whose milk {alpha}1,3/4-linked oligosaccharides predominated.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Total oligosaccharides
The mean total neutral oligosaccharide value of 9 g/L during the first 14 weeks of lactation in this study is lower than the 12 g/L value typically reported for mature milk by earlier studies that used classical analytical techniques. This difference could result from differences in the populations studied or could be a consequence of our use of reversed-phase HPLC of the perbenzoylated oligosaccharides. Unlike classical techniques, this HPLC technique permits the absolute resolution of oligosaccharides from lactose and from each other while providing accurate quantitation. Total oligosaccharides are calculated as the sum of individual, fully resolved and defined chromatographic peaks. This HPLC technique does not measure acidic oligosaccharides, large oligosaccharides with a degree of polymerization of nine or more sugars, or small unidentified peaks; thus our values might be slightly lower than the actual total oligosaccharides present in milk. However, the omission of these minor components could account for only a limited portion of the discrepancy between our results and those of classical techniques. It is more likely that the exclusion of residual lactose from our total oligosaccharide values accounts for most of this difference. In this context, it is interesting to note that the concentrations of the individual oligosaccharides averaged over 1 year of lactation across 12 women reported herein were very close to the values reported by Erney et al. (2000)Go for Latin American women. Their method also excludes lactose and other irrelevant constituents, but through a very different type of chromatographic resolution.

Earlier reports indicated that colostrum (1–3 days postpartum) contains double the concentration of oligosaccharides found in mature milk (Montreuil and Mullet, 1960Go) and that the concentration of total oligosaccharides decreases in the first 120 days of lactation (Coppa et al., 1993Go). As measured by our technique, the total oligosaccharides in milk decreased from the original 9 g/L to 4 g/L by the end of 1 year. These results suggest that the decreases in most oligosaccharide concentrations noted during early lactation continue throughout lactation for at least 1 year; this finding appears to disprove, at least with regard to oligosaccharides, the assumption that the composition of mature milk is stable.

Variation among individuals
Earlier studies, often based on qualitative or semi-quantitative data, concluded that the milk oligosaccharide patterns of individuals could be classified into three or four distinct categories based on the amounts of {alpha}1,2-linked fucosyloligosaccharides, presumably reflecting variation in the {alpha}1,2-fucosyltransferase activity in the mammary gland. Because a major {alpha}1,2-fucosytransferase in human milk is the product of the secretor gene (now designated fucosyltransferase II or FucT II), variation in secretor gene expression was thought to account for these different human milk oligosaccharide patterns (Viverge et al., 1990Go).

We found that the largest contributors to the variation in different donors’ milk oligosaccharides, especially in the first several months of lactation, were 2'-FucLac, LNF-I, and LDFH-I, which is consistent with control by the {alpha}1,2-fucosyltransferases. The extreme difference between the unique oligosaccharide pattern of one mother who initially lacked {alpha}1,2-linked fucose moieties and the patterns of the other 11 donors is reminiscent of described differences between nonsecretors, who lack specific {alpha}1,2-linked moieties in their secretions due to a genetic inability to express 2-fucosyltransferase, and secretors, who express this {alpha}1,2-fucosyltransferase. However, though the predominant types of individual oligosaccharides found in human milk were consistent with control by the secretor gene, the relative amounts of oligosaccharides varied between mothers more than can be accounted for by this gene alone. The more detailed quantitative profiles provided by our method revealed further complexity. Expression of the exclusively {alpha}1,3/4-linked fucosyloligosaccharides (those with fucoses linked only by {alpha}1,3 or {alpha}1,4 glycosidic bonds) also varied between individuals. This variation, which was most clearly seen in later stages of lactation when the levels of the {alpha}1,2-linked fucosyloligosaccharides had diminished, may indicate differences in the abilities of individual mothers to express {alpha}1,3/4-fucosyltransferase (fucosyltransferase III, the product of the Lewis gene) as well.

Variation over time
Contrary to earlier conclusions that were based on measurements of monosaccharides, total oligosaccharides, and lactose over the first 4 months of lactation (Coppa et al., 1993Go) the results of our 1-year study indicate that individual neutral human milk oligosaccharides vary greatly during lactation, qualitatively as well as quantitatively. For the 11 donors, 2'-FucLac, at 3 g/L, was the dominant oligosaccharide early in lactation, consistent with early reports (Kuhn et al., 1956Go). However, by 1 year of lactation, the concentration had markedly decreased; LNF-I had a similar pattern. In contrast, 3-FucLac increased fourfold over the year of lactation.

The {alpha}1,2-linked and exclusively {alpha}1,3/4-linked oligosaccharides tended to vary as two classes, or families. The concentrations of individual oligosaccharides containing {alpha}1,2-linked fucose tended to covary with one another over the course of lactation. This covariance is consistent with one Fuc{alpha}1,2-transferase activity, perhaps the secretor gene product, controlling their biosynthesis. Likewise, oligosaccharides containing exclusively {alpha}1,3/4-linked fucose covaried with one another, implying that a Fuc{alpha}1,3/4-transferase activity, perhaps the Lewis gene product (or distinct but linked Fuc{alpha}1,3- and Fuc{alpha}1,4-transferases) control biosynthesis of these oligosaccharides. These two families of oligosaccharides did not covary during the first 14 weeks of lactation, implying that the {alpha}1,2-transferase activities and {alpha}1,3/4-transferase activities may each be under separate control.

Oligosaccharide patterns during the second 6 months of lactation (after 26 weeks) had not been studied previously; our results indicate that this may be a period of dramatic changes. In the 11 donors whose oligosaccharide patterns were initially dominated by {alpha}1,2-linked fucosyl moieties, the ratio of {alpha}1,2- and {alpha}1,3/4-linked fucosyloligosaccharides approached unity late in lactation (1 year). Likewise, in the donor whose milk initially lacked {alpha}1,2-linked fucose moieties, {alpha}1,2-linked fucosyloligosaccharides appeared late in lactation to the extent that the ratios of {alpha}1,2-linked to exclusively {alpha}1,3/4-linked structures also approached unity by one year. This phenomenon has not been described to date, although the population data at random time points published by Erney et al. (2000)Go are consistent with such a shift in oligosaccharide patterns late in lactation (Newburg, 2000Go).

These patterns suggest that after 26 weeks a shift occurs in the types of fucosyltransferases used in the synthesis of milk oligosaccharides or that the mechanism of anabolic control of fucoslyoligosaccharides in the mammary gland changes, or both. A shift in anabolic control could either be intrinsic to this period of lactation or could be secondary to the beginning of ablactation, stimulated by decreased frequency of feeding by the infant. However, the feeding records of the 12 mothers indicate that 1 of them provided two or more feedings per day through the 40th week of lactation, 2 provided four or more feedings per day through the 40th week, and the other 9 provided an average of four or more feedings for an average of 83 (range 55–110) weeks. Thus, for most of these mothers, ablactation did not coincide with this shift in oligosaccharide profiles. Therefore, we hypothesize that the changes in milk oligosaccharide patterns are an intrinsic characteristic of human lactation after 26 weeks. Furthermore, for the 11 donors with the prevalent oligosaccharide profiles, the ratio of the {alpha}1,2-linked fucoslyoligosaccharides to those whose fucose is linked in only the {alpha}1,3/4 position closely follows the exponential decay function shown in Figure 1B. This reciprocal relationship between the products of {alpha}1,2-fucosyltransferase and {alpha}1,3/4-fucosyltransferase activities over the entire first year of lactation suggests some form of coordinated control in the synthesis of the two families of fucosyloligosaccharides that has not been described in human milk heretofore. Such control mechanisms might include competition for substrates, reciprocal transcriptional control, translational control, or subcellular compartmentation of the transferases and/or substrates.

Possible clinical importance of variation
Because these oligosaccharides are synthesized by transferases similar to those that synthesize glycoproteins, glycolipids, and other glycoconjugates, qualitative and quantitative changes in the milk oligosaccharides probably reflect similar changes in the glycoconjugates of the breast acinar cells, where the milk components are formed. Glycoconjugate structures vary in other tissues also. In rat intestine, variation in the activities of a sialyltransferase and a fucosyltransferase seems to correlate with the expression of glycoconjugate receptors for various pathogens (Bouhours et al., 1983Go; Torres-Pinedo and Mahmood, 1984Go; Pang et al., 1987Go). Other intestinal glycosyltransferase activities, such as galactosyltransferase and N-acetylglucosaminyltransferase, vary with the age of the rat (Biol et al., 1987Go; Ozaki et al., 1989Go). These variations, in both host cell target glycoconjugates and in the milk oligosaccharides that may serve as target decoys, suggest the possibility of a coordinated type of defense against pathogens by glycoconjugate expression.

Regardless of the oligosaccharide pattern of a donor’s milk, the neutral fucosyloligosaccharides were at their highest concentrations in the first 2–4 weeks of lactation. At this time, the nursing infant might be expected to be most susceptible to infections due to the immaturity of its immune system and porosity of its intestinal epithelial barrier. After this initial period, the concentrations of the larger, more complex oligosaccharides decreased, although the levels of the smaller molecules, that is, the fucosylated trisaccharides, were at appreciable levels even after a year of lactation. Similarly, decreases have been reported in total sialylated (that is, the acidic) oligosaccharides of human milk; the mean daily uptake of sialic acid in the form of acidic oligosaccharides declines from about 170 mg/kg body weight during the first 2 weeks of lactation to just 20 mg/kg after 10 weeks (Carlson, 1985Go). Many pathogens utilize sialylated receptors in the human intestinal mucosa. However, biologically active oligosaccharides, found in relatively low concentrations late in lactation may nonetheless contribute significantly to the intestinal contents of older infants, because both the size of the gut and the amounts of milk consumed have increased with time. Furthermore, we have found that oligosaccharides are poorly digested and absorbed from the infant gut. Oligosaccharides at low concentrations in milk may reach concentrations in the gut capable of inhibiting pathogen binding to the intestinal mucosa.

In almost 80% of the Caucasian population, milk oligosaccharides containing the Fuc{alpha}1,2Gal epitope are the preponderant species (Kunz and Rudloff, 1993Go). Also, the milks of most mammals contain 2'-FucLac, the simplest oligosaccharide with the Fuc{alpha}1,2Gal moiety. We speculate that this epitope in human milk might have evolved as a defense against many widespread enteric pathogens. Among the fucosyloligosaccharides containing Fuc{alpha}1,2Gal, one protects suckling mice against the stable toxin of E. coli (Newburg et al., 1990Go), and another, containing the Fuc{alpha}1,2Galß1,4GlcNAc moiety, shows protective activity against C. jejuni (Cervantes et al., 1995Go).


    Conclusions
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 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The idea that mature milk has a typical, stable profile clearly does not apply to the oligosaccharides of human milk and may be legitimately questioned in regard to milk of other species and, perhaps, other glycoconjugate components of human milk. Oligosaccharide patterns in human milk during the second 6 months of lactation had not been studied previously. Our results indicate that this may be a period of dramatic changes in fucosyloligosaccharide concentrations. Clinically, if different fucosyloligosaccharide moieties offer different protective effects, then qualitative and quantitative variation in fucosyloligosaccharides may affect the capacity of milk to protect a nursing infant. This raises questions regarding use of milk from donors other than an infant’s mother or milk obtained at different stages of lactation, even from an infant’s own mother. These data suggest also that further studies are warranted regarding the metabolic control of fucosyloligosaccharide synthesis and regarding the relationship between variation in milk oligosaccharide levels and infant health and development.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sample collection
Human milk samples from the Instituto Nacional de Ciecias Medicas y Nutricion in Mexico City were stored at –70°C until use. The 12 mothers included in this study were a subset of women who were enrolled in a previously described longitudinal study (Newburg et al., 1998Go) and from whom a set of milk samples was available from 1 week to 1 year postpartum. Milk samples used in this study had been collected at postpartum weeks 1, 2, 4, 12–16 (designated for analysis as week 14), 24–28 (designated as 26), 35–41 (designated as 38), and 44–54 (designated as 49).

Reagents
Sodium borohydride and dimethylaminopyridine (DMAP) were purchased from Aldrich Chemical Company (Milwaukee, WI). Benzoic acid was obtained from Sigma Chemical Company (St. Louis, MO), and the AG50WX8 (H+) and AG1-X8 (OAc) ion exchange resins (100–200 mesh) were obtained from Bio-Rad (Hercules, CA). Before use, the AG50 resin was converted to the pyridinium form by treatment with aqueous pyridine. HPLC-grade acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA). Double-distilled deionized water was used for all HPLC analyses. All the other chemicals and solvents were analytical-grade. Human milk oligosaccharide standards were purchased from Oxford GlycoSystems (Rosedale, NY).

Isolation of oligosaccharides
Milk oligosaccharides were isolated as described previously (Chaturvedi et al., 1997Go). Briefly, milk samples (0.1 ml) were thawed immediately before use. After addition of water (0.9 ml), the samples were centrifuged at 4000 x g for 45 min at 4°C. The solidified layer of fats and lipids was removed by pipetting from the lower, aqueous layer. The proteins and a portion of lactose were precipitated overnight at 4°C after the addition of ethanol to a final concentration of 66.7%. The precipitate was removed by centrifugation at 4000 x g for 15 min at 4°C. The clear supernatant was then dried under nitrogen and lyophilized.

The samples were reduced with excess aqueous sodium borohydride and separated into acidic and neutral species using AG1 anion exchange resin. The oligosaccharides were perbenzoylated as described earlier (Chaturvedi et al., 1997Go). Neutral, reduced oligosaccharides were thoroughly dried under vacuum over phosphorus pentoxide for 4–6 h then perbenzoylated using a solution of benzoic anhydride and 4-dimethylaminopyridine. The perbenzoylated oligosaccharides were isolated from the reactants by adsorption on a C-18 Bond-Elut column (Varian, Walnut Creek, CA) followed by elution with methanol; after drying, samples were dissolved in HPLC acetonitrile. The perbenzoylated oligosaccharides were resolved by HPLC on a Rainin Microsorb C-8 column (Varian) (3 µ; 4.6 mm x 10 cm) at a flow rate of 1 ml/min with a 15-min linear gradient of acetonitrile and water, from 80% acetonitrile to 100% acetonitrile, and then holding at final conditions for an additional 10 min. Samples were detected at 229 nm. The milk oligosaccharides were identified by co-elution with perbenzoylated authentic standards run concurrently. Peaks were collected as individual fractions, and their structures confirmed by O-debenzoylation and permethylation followed by electrospray mass spectrometry. Peak areas corresponding to the milk oligosaccharides were integrated by computer. The molar response for each oligosaccharide, determined using known amounts of authentic standard, was used to calculate the concentration of each major oligosaccharide (tri- to octasaccharides) in the samples.

Statistical methods
The individual and mean concentrations of specific and total milk oligosaccharides measured at each lactation time point were calculated and graphed. The central tendencies and variability in quantity of oligosaccharides over all subjects was examined for all time points. The quantity of {alpha}1,2-linked oligosaccharide was calculated by summing the quantities of 2'-FucLac, LNF-I, LDFH-I and LDFT. Similarly, the quantity of {alpha}1,3/4-linked oligosaccharide was calculated by summing the quantities of 3-FucLac, LNF-II/III, and LDFH-II. The covariation and relationships among oligosaccharides were examined using correlation analysis.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank the mothers at San Pedro Martir for their participation in this study and the nurses and field workers of the Instituto Nacional de Ciecias Medicas y Nutricion, under the direction of Dr. Maria de Lordes Guerrero Almeida, for scrupulous collection of samples and data. We gratefully acknowledge the editorial assistance of Louise Kittredge and Kathryn J. Newburg. Supported by the National Institute of Child Health and Human Development grant HD13021.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2'-FucLac, 2'-fucosyllactose; 3-FucLac, 3-fucosyllactose; DMAP, dimethylaminopyridine; HPLC, high-performance liquid chromatography; LNF-I, lacto-N-fucopentaose I; LDFH-I, lacto-N-difucohexaose I; LDFT, lactodifucotetraose; LNF-II/III, lacto-N-fucopentaose II and lacto-N-fucopentaose-III; LDFH-II, lacto-N-difucohexaose-II.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Conclusions
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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