Current methods for the analysis of human milk oligosaccharides (HMOs) and their novel applications

7 Current methods for the analysis of human milk oligosaccharides (HMOs) and their novel applications L. R. Ruhaak and C. B. Lebrilla, University of California, Davis, USA DOI: 10.1533/9780857098818.2.124 Abstract: The microflora in the gastrointestinal tract of an infant plays an important role in the development of the immune system. Human milk oligosaccharides (HMOs) have become the subject of considerable interest because they strongly influence the composition of the gut microflora. The structures of the HMOs are usually very complex and novel techniques such as porous graphitic carbon-liquid chromatography-mass spectrometry (PGC-LC-MS) now facilitate separation and identification of most of the isomers. In contrast, matrix assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis may now generate fast profiles, but does not allow isomer separation. Application of these novel techniques allows more accurate studies of Lewis blood group determinants, phylogenetic differences in milk oligosaccharide composition and bacterial HMO consumption. Such studies will greatly enhance knowledge of the biological functions of HMOs. Key words: milk oligosaccharides, analysis, mass spectrometry, secretor status, Lewis blood group, bacterial consumption, high performance liquid chromatography.

7.1

Introduction

Human milk has evolved to nourish newborns.1,2 As it is the initial source of nutrition, it is regarded as the nutritional gold standard for term infants, conferring several benefits.3 Human milk is highly glycosylated and composed of lactose, glycolipids, free oligosaccharides and glycoproteins. Free oligosaccharides are important constituents of human milk at concentrations ranging from 5 to 23 g/l.4–6 The free oligosaccharides in human milk (HMOs) are linear and branched structures of 3 to 14 monosaccharides, as illustrated in Fig. 7.1.7,8 More than 200 free oligosaccharides have been identified in human milk samples,7–10 and nearly

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Fig. 7.1 Structural characteristics of human milk oligosaccharides. (a) Monosaccharide structures and their symbolic representation; full HMO structure with its symbolic representation. (b) Examples of symbolic representations of typical HMO structures. Reprinted with permission from Wu et al.8

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all of them originate from a lactose (Gal( β1–4)Glc) core that is extended with N-acetyllactosamine (LacNAc) repeats which can be linked as either Gal( β1–3) GlcNAc (type I) or Gal( β1–4)GlcNAc (type II). The linear or branched structures thus formed can be decorated with fucose (Fuc) and/or N-acetylneuraminic acid (NeuAc), where the NeuAc residues may be attached either with an α2–3 or an α2–6 linkage. The Lewis blood group system, and more specifically the secretor gene, is one of the important determinants of the oligosaccharide structures present in an individual’s milk:11–13 fucose residues are attached to HMO according to the expression of both the secretor gene (fucosyltransferase 2) and the Lewis gene (fucosyltransferase 3) .14 Fucose residues may be α1–2 linked to galactose residues when fucosyltransferase 2 is active, while fucose residues may be α1–4 linked when fucosyltransferase 3 is active.15,16 Activity of the fucosyltransferases is regulated by genetic variation, and is thus inherited. While HMOs comprise a large proportion of the oligosaccharide content in human milk, milk also contains other glycoconjugates, such as glycoproteins, which can be decorated with O-linked as well as N-linked glycans and glycolipids. While HMOs are typical for mammalian milk, N- as well as O-glycans are found in tissue (e.g. de Leoz et al.17), blood (e.g. Parekh et al.18) and other excretions such as tears and saliva,19 and have also been found in bacteria and plants. Human N- and O-linked glycans consist of nearly the same monosaccharide ‘building blocks’ as HMOs, but their building template follows a different pathway. N-linked glycans, for example, are characterized by a core structure containing three mannose residues and two N-acetylglucosamine residues. This core may be decorated with mannose, N-acetylglucosamine, fucose and N-acetylneuraminic acid residues. N- and O-glycans have important biological functions in cell–cell and cell–matrix interactions,20 protein folding and protein binding. Moreover, these glycans are associated with several health and disease states, such as autoimmune diseases18,21 and cancer.22–24 Recently, the N-glycans from human milk lactoferrin were shown to affect bacterial binding to epithelial cells.25 Analytical strategies for the evaluation of N- and O-glycans are usually similar to the strategies used for HMOs. The human gastrointestinal tract is not capable of digesting human milk oligosaccharides, and these compounds cannot, therefore, serve any nutritional value. However, the HMOs are the third most abundant component in human milk, which is shaped by long-term evolution,1,2 so they must have important functions. During a child’s development, the HMOs do indeed play important functions (e.g. Zivkovic et al.,1 Newburg et al.,26 Morrow et al.27,28). Milk oligosaccharides promote the growth of probiotic bacteria (e.g. Zivkovic et al.,1 Newburg et al.,26), the prebiotic effect. HMOs, as well as their synthetic counterparts fructooligosaccharide (FOS) and galactooligosaccharide (GOS), have been shown to have anti-adhesive properties, preventing the binding of pathogens to the host’s epithelial cells.26,29 It is proposed that the oligosaccharides mimic the natural ligands of the bacteria, thus occupying their natural binding sites and inhibiting their adhesion. Such anti-adhesive

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properties have been described for several bacteria and viruses, including Streptococcus pneumonia, Listeria monocytogenes, Vibrio cholera, Salmonella fyris, HIV, enteropathogenic Escherichia coli and Campylobacter jejuni.29–32 C. jejuni is one of the major causes of diarrhea and was shown to adhere to 2′-fucosyllactosamine.32 The incidence of diarrhea in breastfed infants was later reported to be directly related to the levels of 2′-fucosyllactosamine in their mothers’ breast milk.33 Recent studies have reported that gut bacteria can grow well on HMO, and this is currently an important focus in HMO research. In particular, several strains of bifidobacteria were reported to grow well on HMO, and HMO structure specificity was observed, as some strains prefer fucosylated oligosaccharides, while others prefer non-fucosylated structures.34 Similarly, different GOS polymers were consumed differently by bifidobacteria strains.35 While a large number of bifidobacteria were shown to consume HMO, recent studies indicate that milk oligosaccharide consumption is not specific for bifidobacteria, but can also be observed for bacteroides species.36 The composition of an infant’s gut microflora is thus largely influenced by the presence or absence of HMOs. It is proposed that a well-balanced intestinal microflora is important for the development of the infant’s immune system,37 indicating that HMOs play an important role in the infant’s well-being. The long-time methods of choice for the analysis of human milk oligosaccharides are technologies such as NMR, anion exchange separation with pulsed amperometric detection (HPAEC-PAD) or lectin affinity. However, with the introduction of mass spectrometry for the analysis of oligosaccharides, additional analytical techniques could be applied (e.g. Ruhaak et al.,38 Pabst and Altmann39), such as hydrophilic interaction liquid chromatography (HILIC)10 and PGC7,8,40 separations with or without coupling to mass spectrometry, or stand-alone MALDI-MS.12 These strategies, which enable the in-depth and large-scale analysis of HMO, allow further evaluation of the role of HMO in an infant’s development. This chapter aims to give an overview of the current state of the art analytical techniques used in milk oligosaccharide analysis, and its biological and clinical implications.

7.2 Analysis of human milk oligosaccharides (HMOs) The structural diversity of the HMOs, originating from the different locations and types of linkages that are formed to link the monosaccharide building blocks, requires comprehensive analytical strategies for their detailed analysis. The introduction of mass spectrometry allowed rapid determination of accurate mass and thus HMO composition. Moreover, structural information may be obtained using tandem mass spectrometry. The coupling of separation techniques such as LC and CE to mass spectrometry provided an ideal analytical tool for profiling of complex mixtures of HMOs. These analytical strategies have been applied for both profiling and in-depth characterization.

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7.2.1 Compositional fingerprinting of HMOs Compositional profiles of HMOs allow the rapid determination of the HMO compositions present in a sample. Offline mass spectrometric analysis of human milk oligosaccharides provides a rapid method for compositional profiling, which may be automated and holds the potential for high-throughput analysis. MALDI-TOF-MS analysis of HMOs was first described by Stahl et al.,41 who were able to examine neutral oligosaccharides in positive mode as monosodium adducts as well as acidic oligosaccharides in both the positive and negative modes. It was noticed that desialylated fragments are observed in the acidic fraction due to the energetics of the ionization method. More recently, a strategy using MALDI-FTICR-MS to monitor bacterial consumption of HMOs was developed.42 Using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, neutral oligosaccharides were observed as sodiated adducts. This analysis is illustrated in Fig. 7.2. The high resolution of the FTICR-MS allowed application of deuterium-labeled internal standards, which was shown to be beneficial for relative quantitation.34,42,43 A similar approach has been applied recently for the determination of Lewis blood group by HMO fingerprinting. Following an automated oligosaccharide purification, HMOs were analyzed using MALDI-TOF with 6-aza-2-thiothymine (ATT) as the matrix.12 Neutral oligosaccharides and sialyllactose were observed as sodium and potassium adducts in the positive mode, while other sialylated

Fig. 7.2 MALDI-FTICR-MS spectrum of reduced human milk oligosaccharides using 2,5-DHB matrix in the positive ionization mode. Signals originating from HMO are marked with a dot. Reprinted with permission from Ninonuevo et al.42

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HMOs were detected as deprotonated molecular ions in the negative mode. Using this method, the correct blood group could be assigned to 93.8% of the samples. In general, direct mass spectrometric strategies are ideal for fast, highthroughput HMO analysis. However, the inability to distinguish isomeric structures limits the applications of these methods, particularly for comprehensive HMO analysis. 7.2.2 HMO profiling at the compound level To allow compounds-specific profiling, which takes the different linkages into account, some mode of separation is required. A non-exhaustive overview of methods used for HMO profiling can be found in Table 7.1. Anion exchange chromatography, particularly high-pH anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD), has traditionally been used for the analysis of HMO.11,44–52 Using anion exchange columns, several HMO isomers may be separated. However, prior separation of the neutral and acidic oligosaccharides may be required, resulting in doubled analysis times. Moreover, the use of PAD detection requires the use of HMO standards for compound identification. This could be overcome by coupling of HPAEC to mass spectrometry, but this requires desalting. Another mode of separation often used for HMO analysis is reverse phase (RP)-HPLC. Native oligosaccharides are not retained on RP material, due to their hydrophilic properties, and therefore derivatization is required. Retention and separation of the HMOs on RP-LC thus mainly depend on the method of derivatization. Labeling of HMOs at their reducing end with chromophoric active tags such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 2-aminopyridine (PA) and 2-aminobenzoic acid (2-AA), as well as perbenzoylation, has been applied for the analysis of HMOs. Besides being hydrophobic tags allowing chromatographic separation on RP stationary phases, they also provide a chromophore used for detection. Additional compounds used for labeling at the reducing end have been used in the analysis of other oligosaccharides (e.g. Ruhaak et al.,38 Anumula53), and may also be applicable for the analysis of HMOs. Permethylation is an alternative derivatization method, which is extensively used in oligosaccharide analysis. It is often used to stabilize oligosaccharides during ionization and to increase sensitivity. However, partially methylated compounds may complicate the analysis. Methods for separation of permethylated oligosaccharides have been developed.54 Standard reverse phase columns such as C18 can provide some isomeric separation, but this, too, lacks comprehensive separation of isomeric species. Native HMOs may be separated using HILIC. This method, which has already been applied extensively for the analysis of N- and O-glycans,55–57 was recently applied to HMO.10 The oligosaccharides are labeled with 2-aminobenzamide (2-AB) using reductive amination to allow fluorescence detection, but retention is mostly based on the oligosaccharide moiety. The elution order is mainly influenced by the number of monosaccharide residues. Several sialylated isomers can be

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HPLC

HILIC

HPEAC

HPEAC

SCX

Derivatization

BMOs, neutral and acidic separated

HMO and FOS/ GOS mixtures49

Reductive amination using 2-AB

–

Oligo- and – monosaccharides from human milk HMO, neutral – and acidic separated

Separation* Sample† Refractive index

Detection Separation of glucose and galactose

Isomer separation

Notes

Oligosaccharides not separated, except fucosyllactose CarboPac Pulsed Separation of Use of salts for elution does not PA-100 amperometric several allow immediate detection isomers (2′- and 3′-FL, coupling to mass 3′- and 6′-SL, spectrometry LNFPs) CarboPac Pulsed Separation of PA-1 amperometric several detection isomers (2′- and 3′-FL, 3′- and 6′-SL, LNFPs) TSK-gel Fluorescence Separation of Retention times amide-80 several can be compared isomers using GU index. Structural assignment confirmed using exoglycosidases and ESI-Q-TOF

Aminex HPX 87 C

Column

Overview of separation methods used in milk oligosaccharide analysis

Ion chromatography

Table 7.1

Bode et al.,44 Coppa et al.,45,46 Erney et al.,47 Moro et al.,49 Nakhla et al.50 Marino et al.10

Thurl et al.,11 Finke et al.,48 Thurl et al.51,52

Coppa et al.5

Ref.

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Neutral HMO

Neutral HMO

Neutral HMO

RP-LC

RP-LC

RP-LC

Carbon-LC HMO

PMP derivatization and reductive amination using PA Reductive amination using PA

Reduction and perbenzoylation

Reduction

Inertsil ODS-3V and ODS100Z Inertsil ODS-3V

Rainin C-8

Porous graphitic carbonchip Separation of most isomers

Library based on mass and retention time, containing 74 structures. Structural assignment confirmed using exoglycosidases and MALDIFTICR UV at 229 nm Limited Elution mostly isomer based on separation increasing molecular weight, linkage has smaller effect UV at 245 nm Partial isomer Separations were and 310 nm, separation different for PMP for PMP compared with and PA, PA derivatization respectively UV at 310 nm Partial isomer separation

nESI-TOF

(Continued)

Sumiyoshi et al.102

Asakuma et al.76

Morrow et al.,33 Chaturvedi et al.,82,101

Wu et al.,7,8 Locascio et al.,43 Ninonuevo et al.60

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Reductive amination using 2-AA

RP-LC

HMO

Reductive amination using APTS

–

–

TSKgel ODS100Z

Column

Isomer separation

Notes

Separation of several isomers (2′- and 3- FL, 3′- and 6′ SL, LNDFHs) UV at 205 nm Separation of Only sialylated several HMO can be isomers separated using this method, since the separation is charge-based Fluorescence Separation of Very fast most isomers separation (10 min). Structural assignment confirmed using ESI-MS-MS

Fluorescence

Detection

Albrecht et al.61–63

Shen et al.,58 Bao et al.59

Leo et al.103,104

Ref.

†

Strong cation exchange (SCX), micellar electrokinetic chromatography (MEKC). Fructooligosaccharides (FOS). BMO, bovine milk oligosaccharide; FL, fucosyllactose; GU, glucose units; LNFP, lacto-N-fucopentaose; SL, sialyllactose; LNDFHs, lacto-N-difucohexaose.

*

CE

Sialylated HMO –

HMO, neutral and acidic separated

Derivatization

Separation* Sample†

Continued

Electromigrative MEKC separations

Table 7.1

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separated, but no extensive isomer separation, which would be necessary for concise profiling of complex HMO mixtures, was observed. Electromigrative separation techniques have also been applied in the analysis of HMO.58,59 Using micellar electrokinetic chromatography, native sialylated milk oligosaccharides were separated, showing good isomer separation. For electromigrative separation techniques charged analytes are necessary. Native neutral human milk oligosaccharides, therefore, cannot be analyzed using such techniques. While the use of separation techniques combined with structural neutral detection such as fluorescence spectrophotometry provides good quantitative repeatability, structural confirmation is only obtained based on the use of standards. HMO standards are expensive and not available for many HMOs. Co-elution/ migration has been observed consistently in most separation techniques and provides a problem with identification of compounds in complex mixtures. Since elution or migration is not perfectly identical in all runs, and HMO samples from different donors may have very different patterns, identification of the signals in each of the samples may be ambiguous. For better identification, coupling of the separation with mass spectrometry has proven to be effective. PGC has only recently been employed in oligosaccharide analysis, but has already been shown to provide a highly versatile stationary phase for oligosaccharide separation. nLC-PGC-chip-TOF-MS in the positive mode was recently introduced for the analysis of HMOs.7,8,60 PGC allows good isomer separation, which is combined with unambiguous identification using mass spectrometry, as shown in Fig. 7.3. Reduction of the reducing end of the oligosaccharides is necessary, since the α- and β-anomers of the aldehyde are separated on the PGC stationary phase. Both neutral and sialylated compounds may be separated in one run, and hundreds of structures may be observed, many of which can be identified using a library containing retention time, accurate masses and fragmentation information.7,8 Electrophoretic methods have also been coupled to mass spectrometry. Capillary electrophoresis with laser-induced fluorescence coupled to mass spectrometry (CE-LIF-MS) of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)labeled milk oligosaccharides has been reported to be efficient for the analysis of HMOs.61–63 Labeling of HMO with APTS introduces a fluorophore, which allows LIF detection, while simultaneously adding the negative charge needed for the separation. Offline CE-LIF provided good separation using very fast runs (around 9 minutes), but both resolution and separation time are usually compromised when coupling CE to mass spectrometry.64 7.2.3 Structural characterization of HMOs While the actual structural information is desirable in HMO fingerprinting studies, structural identification of each of the signals in a given sample is not necessary. Often the structural assignments mostly rely on previous literature or databases, in which HMO structures have been thoroughly characterized. Structural

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Fig. 7.3 Separation of isomers of reduced fucosyl-sialyl-lacto-N-hexaose (FS-LNH) using nLC-PGC-chip-TOF-MS. Extracted ion chromatograms of different isomers of FS-LNH. Isomers were first fractionated on a PGC stationary phase and fractions were analyzed individually using nLC-PGC-chip-TOF-MS. Clearly, different fractions contained signals originating from different isomers, which is illustrated by the different retention times (RT). Reprinted with permission from Wu et al.7

identification is, however, necessary to determine the function of specific HMOs. Pure oligosaccharides are needed for structural characterization and therefore substantial purification is needed, which often results in small (picomolar) amounts of material. Nuclear magnetic resonance yields the most extensive structural information65,66 and is often employed for HMO characterization.

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However, larger amounts of pure oligosaccharides are needed for NMR (typically micromoles of pure compounds), which are often not available. In cases with low amounts of HMO, MS-based fragmentation techniques can be performed on picomolar quantities, and with the proper LC coupling such studies may even be performed in mixtures. Traditionally, fragmentation of HMOs has been performed using collision induced dissociation (CID) on a quadrupole ion trap (Q-IT) MS instrument67,68 and on Fourier transform ion cyclotron resonance (FTICR) MS instruments;69–71 an example of a fragmentation spectrum for three fucosylated HMOs is depicted in Fig. 7.4. Fragmentation behavior of milk oligosaccharides in both positive and negative modes has been reviewed extensively,72,73 and it was observed that cleavages of the glycosidic bond are most common. Cross-ring cleavages are necessary to obtain linkage information and monosaccharide identification (e.g. glucose vs. galactose), but these are not commonly observed using CID. It has to be noted that glycan rearrangements may occur in fragmentation studies performed by CID.74 More recently, structural characterization has also been obtained using Q-TOF-MS instrumentation.7,8 The use of electron capture methods such as electron transfer dissociation (ETD) for characterization of reduced and permethylated milk oligosaccharides was recently reported.75 ETD resulted mainly in cross-ring cleavages, allowing unambiguous linkage identification. So far, however, only simple, linear or minimally branched structures have been analyzed. Although the first results indicate that ETD is a promising complementary fragmentation technique for milk oligosaccharides, further studies using more complicated HMO structures will have to be performed. These methods remain far from routine with oligosaccharides as they require multiply charged species, preferably triply positively charged, which are difficult to produce with non-basic and even acidic milk oligosaccharides. 7.2.4 HMO quantitation For comparison of milk and feces of mother–baby dyads, as well as in bacterial consumption studies, accurate (relative) quantitation of the individual HMOs is necessary. Fluorescence and UV detection are traditionally regarded as being more robust for quantitation,76 but these methods provide structurally neutral detection. Since they provide no structural information, they are less ideal for HMO quantitation in mixtures. HMO quantitation by mass spectrometric detection requires more effort due to matrix effects and ionization suppression. Oligosaccharides in mixtures tend to suppress each other, resulting in different ionization efficiencies for specific components. For example, neutral oligosaccharides (those not containing sialic acids) will tend to suppress sialylated species in the positive mode, while the reverse happens in the negative mode. Ion suppression can be avoided by separating the HMO mixture into individual compounds using, e.g. PGC, as individual components produce responses to ionization and detection that are generally similar. For most cases, therefore, detector response is sufficient in LC/MS.

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Fig. 7.4 Differentiation of LNT and LNnT using CID fragmentation of deprotonated species in the negative ionization mode. MS2 (a, b) and MS3 (c, d) fragmentation spectra of m/z 706 and its fragment m/z 382 are depicted together with fragmentation patterns explaining the most important ions. Clearly, the fragmentation of the fragment at m/z 382 distinguishes LNT from LNnT. Reprinted with permission from Amano et al.67

More accurate quantitation may be obtained using isotopic labeling.34,42,43 The aldehyde may be reduced with sodium borodeuteride, which adds a deuterium to the resulting alditol. A standard mixture of HMOs with deuterium can then be used as an internal standard, when spiked into a complex HMO sample which has been

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reduced with sodium borohydride. Quantitation is obtained using accurate mass instruments such as FTICR and TOF by comparing the monoisotopic peaks of the hydrogenated and deuterated compounds, subtracting for 13C abundances. This method is typically used for quantitating oligosaccharide consumption profiles of bacteria34 or for characterizing enzymatic properties of glycosidases from bacteria.

7.3 Applications of HMO analysis 7.3.1 Determination of secretor status from HMOs Structural characteristics of HMOs are believed to be influenced by an individual’s genetic status. A close relationship exists between the Lewis blood group system and the structures of HMOs found in an individual’s milk.11,12 The genetic background of the Lewis blood group system has been thoroughly reviewed,16 and it is believed that fucose residues may be attached to HMOs depending on the expression of both the Lewis gene (fucosyltransferase 3) and the secretor gene (fucosyltransferase 2). Fucose residues may be α1–2 linked to galactose residues when fucosyltransferase 2 is active, or α1–4 linked when fucosyltransferase 3 is active.15,16 The activities, however, are not only based on the presence of the genes, but also on the expression in the mammary epithelial cells. Over the years, much research has been conducted on the determination of secretor status (FUC2 activity) and Lewis blood group (FUC3 activity). In the early 1990s, a first study was conducted using paper chromatography to determine differences in HMO composition that are associated with secretor status.77 Clear differences were observed and the question was raised whether these different HMO compositions have an influence on the neonate. Indeed, several papers now report on the beneficial effects of α1–2-linked fucose on E. coli infection and the associated incidence of diarrhea.31,78,79 More recently, several studies directed towards the associations of HMO profiles with Lewis blood type have been conducted,9,12,16,28,45,47,52,62,76,77,80–83 and it has been reported more than once that not only secretor status, but also Lewis blood type, can be determined using HMO profiles. In several studies, however, samples were discarded if their HMO profiles did not fit the Lewis blood type as determined by hemagglutination or saliva tests. Therefore, it may be speculated that the Lewis gene is not expressed in the same way in all cell types or tissues. Using the newly developed analytical techniques which make use of both separation and MS detection, it will now be possible to more accurately determine correlations between secretor and Lewis blood type and HMO profile. 7.3.2 Phylogeny based on milk oligosaccharide structures Since human milk is the result of the long trajectory of evolution,1,2 HMO composition is an interesting feature to study the phylogeny of mammals. Indeed, free milk oligosaccharide structures have been characterized in more than 25 mammals.84–92 When comparing placental mammals, monotremes and

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marsupials, it was observed that milk of placental mammals contains large amounts of lactose, which is nearly absent in the other two mammal groups, in which tri- and tetrasaccharides are predominant. Moreover, monotreme milk was observed to be rich in fucose, while this monosaccharide is absent in all marsupials.92 It has to be noted that characterization of the oligosaccharides in most milks was performed using isolation and subsequent structural characterization using NMR spectroscopy. Since this method requires larger amounts of material, usually only around 10 oligosaccharide structures could be identified per species. Milk oligosaccharide profiles for seven primates were recently generated using nLC-PGC-chip-TOF-MS to assess the extent to which HMO compositions reflect ancestral (or primate) patterns relative to more recent evolutionary events.84 This study found 52–116 oligosaccharides for each of the individual primates, but the milk oligosaccharide patterns did not closely reflect the current understanding of primate phylogeny, as illustrated in Fig. 7.5. This indicates that more recent evolutionary processes, such as the prevalence of certain microbes in the gut flora, largely influence the structural characteristics of HMOs.84 7.3.3

Selective consumption of milk oligosaccharides by the gut microflora The interaction of a neonate with the microorganisms growing in the gastrointestinal tract is important for direct survival, but also health in the longer term. Human milk oligosaccharides largely influence the human microflora in the first weeks of a child’s life,1 both by influencing binding of pathogenic organisms to the gut epithelial cells and also by acting as a prebiotic.93,94 Recent studies have focused on the consumption of oligosaccharides by gut bacteria. Several strains of bifidobacteria can grow well on HMOs; however, it was observed that some strains prefer fucosylated oligosaccharides, while others prefer nonfucosylated structures.34 Similarly, different GOS polymers were consumed differently by bifidobacteria strains.35 Marcobal et al. reported recently that milk oligosaccharide consumption is not specific for bifidobacteria, but can also be observed for bacteroides species.36 HMO consumption studies are somewhat complicated. Bacteria can consume HMOs, but in this process they produce smaller saccharide structures, which then in turn may also be consumed. Therefore, the method needs to allow accurate quantitation. So far, spiking of the samples with a deuterated standard has been performed in all cases to improve the quantitation of the HMO consumption, but this procedure not only complicates the sample preparation procedure, but also requires specific data analysis protocols.43 New instrumentation (such as TOF-MS or triple quadrupole mass spectrometers (QQQ-MS)) and sample preparation methods should allow better quantitation, without the need for internal standards. While initial studies were performed using MALDI-FTICR-MS, more recent studies have been performed using nLC-chip-TOF-MS, which allows the identification of linkage-specific oligosaccharide consumption. Using these techniques, it will be possible to screen bacterial HMO consumption much faster, and in a compound-specific manner

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Fig. 7.5 Phylogenetic evaluation of free milk oligosaccharides in seven primate species. (a) Hierarchical cluster analysis of milk oligosaccharides. Oligosaccharides were analyzed using nLC-PGC-TOF-MS and ordered according to size; annotation is given on top according to number of hexose residues, number of HexNAc residues, number of fucose residues and number of sialic acids. Heatmap intensity represents integral values from the MS analysis. Similarities between primates were determined using Pearson’s product and the linkage was determined using the average linkage method. (b) Phylogenetic tree of primates. Reprinted with permission from Tao et al.84

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(with linkage information), allowing better understanding of the processes in which HMOs are involved.

7.4

Conclusion

Recent advances in analytical approaches, especially coupling of PGC-separation to mass spectrometry, allow rapid simultaneous profiling and quantitation of over 100 human milk oligosaccharides in an individual’s milk. These techniques will further advance our understanding of the roles of the free oligosaccharides in human milk. It may now, for instance, be studied whether HMO-consuming bacteria have specificities for specific linkage isomers. Moreover, in-depth studies of the relation between (linkage-specific) HMO profile and Lewis blood group are now feasible. Recent studies also focus on the uptake and clearance of human milk oligosaccharides by the neonate.49,62 Using the recent advances in the analysis of HMOs, it is now feasible to combine data from milk, feces and urine. These results will enhance our knowledge of the clearance of HMOs and their roles at specific locations in the infant. These insights should facilitate further development of infant formulas. While current separations using PGC or CE provide good separation and allow identification of large numbers of milk oligosaccharides, these techniques require relatively long analysis times (10–60 min). Ions can also be separated on a gas interphase under the influence of a weak electric field by ion mobility (e.g. Clemmer and Jarrold95). N-glycans from patients with liver cancer and liver cirrhosis have been separated using ion mobility coupled to mass spectrometry.96,97 Clear differences could be observed in the drift patterns of several N-glycans. While ion mobility mass spectrometry has not yet been applied to HMOs, its use may well result in linkage-specific determinations with very short analysis times (less than 1 min). Selective Reaction Monitoring (SRM) on QQQ-MS is a selective method for quantitative proteomics. The selectivity and sensitivity of SRM methods are superior compared with traditional quantitative proteomics,98 and the application of SRM to HMO is expected to improve their quantitation over current LC-MSbased methods. Currently, applications of SRM for glycomics and glycoproteomics are being developed, and recent studies towards quantitation of fucosylated glycopeptides99 as well as bovine milk oligosaccharides100 have revealed good repeatability data in terms of quantitation. The recent developments in mass spectrometric tools have paralleled their application to the study of HMOs. These advances in analytical tools will greatly enhance our knowledge of the structural properties and biological functions of HMOs. The specificities of bacterial consumption of milk oligosaccharides can now easily be revealed, while better understanding of the interaction between gut microflora (and thus the development of an infant’s immune system) and HMOs may be developed. Such knowledge should facilitate the development and clinical application of better infant formulas, as well as personalized formulas.

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7.5 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19.

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7.6 Appendix: abbreviations 2-AA 2-AB APTS ATT CE CID DHB ETD FOS FTICR GOS

2-aminobenzoic acid 2-aminobenzamide 8-aminopyrene-1,3,6-trisulfonic acid 6-aza-2-thiothymine capillary electrophoresis collision induced dissociation 2,5-dihydroxybenzoic acid electron transfer dissociation fructooligosaccharides Fourier transform ion cyclotron resonance galactooligosaccharides

© Woodhead Publishing Limited, 2013

Current methods for the analysis of HMOs and their novel applications HILIC HMO HPAEC IT LC LIF MALDI MEKC MS PA PAD PGC PMP Q QQQ RP SCX SRM TOF

hydrophilic interaction chromatography human milk oligosaccharides high-pH anion exchange chromatography ion trap liquid chromatography laser induced fluorescence matrix assisted laser desorption ionization micellar electrokinetic chromatography mass spectrometry 2-aminopyridine pulsed amperometric detection porous graphitic carbon 1-phenyl-3-methyl-5-pyrazolone quadrupole triple quadrupole reverse phase strong cation exchange selective reaction monitoring time-of-flight

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