Glycobiology vol. 24 no. 8 pp. 728–739, 2014 doi:10.1093/glycob/cwu036 Advance Access publication on April 29, 2014

Rapid milk group classification by 1H NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples

2 Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, NL-9747 AG, Groningen, The Netherlands; 3FrieslandCampina Research, Stationsplein 4, Amersfoort NL-3818 LE, The Netherlands; and 4NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands

Received on March 12, 2014; revised on April 23, 2014; accepted on April 24, 2014

Human milk oligosaccharides (HMOs) are a major constituent of human breast milk and play an important role in reducing the risk of infections in infants. The structures of these HMOs show similarities with blood group antigens in protein glycosylation, in particular in relation to fucosylation in Lewis blood group-type epitopes, matching the maternal pattern. Previously, based on the Secretor and Lewis blood group system, four milk groups have been defined, i.e. Lewis-positive Secretors, Lewis-positive nonSecretors, Lewis-negative Secretors and Lewis-negative non-Secretors. Here, a rapid one-dimensional 1H nuclear magnetic resonance (NMR) analysis method is presented that identifies the presence/absence of (α1-2)-, (α1-3)- and (α1-4)-linked fucose residues in HMO samples, affording the essential information to attribute different HMO samples to a specific milk group. The developed method is based on the NMR structural-reporter-group concept earlier established for glycoprotein glycans. Further evaluation of the data obtained from the analysis of 36 HMO samples shows that within each of the four milk groups the relative levels of the different fucosylation epitopes can greatly vary. The data also allow a separation of the Lewispositive Secretor milk group into two sub-groups. Keywords: human milk oligosaccharides / immunodeterminants / milk group classification

1 To whom correspondence should be addressed: Tel: +31-503632153; Fax: +31-503632154; e-mail: [email protected]

Introduction Human mature milk contains about 70 g/L lactose (Hale and Hartmann 2007) and 5–15 g/L oligosaccharides, of which 10% are acidic oligosaccharides (Bode 2012). Shortly after parturition the human milk oligosaccharides (HMOs) concentration is higher, i.e. 20–25 g/L (Bode 2012). HMOs have diverse roles in relation to health and growth of the infant (Bode 2009), including modifying composition of microbiota ( prebiotics) (Marcobal et al. 2010; Asakuma et al. 2011; Yu et al. 2013), immune-stimulating effects (Bode et al. 2004), influence on brain development (Wang 2009), local effects in the gastro-intestinal tract (Angeloni et al. 2005), anti-adhesive properties (Ruiz-Palacios et al. 2003; Morrow et al. 2004; Newburg et al. 2005) and anti-infective activity. Particularly, preterm infants are vulnerable to pathogens causing diarrhea (Ruiz-Palacios et al. 2003; Morrow et al. 2004; Hu et al. 2012) and necrotizing enterocolitis (Sisk et al. 2007; Neu and Walker 2011; Jantscher-Krenn et al. 2012), and could benefit greatly from human milk. Many mothers of preterm babies, however, are unable to provide sufficient milk (Arslanoglu et al. 2010) and HMO fucosylation is less well regulated (De Leoz et al. 2012), and would benefit from replacement with donor milk. Currently, many human milk donor banks have been established (Leaf and Winterson 2009), and milk-sharing initiatives have started (Akre et al. 2011). Although nowadays over 200 different HMOs have been reported to occur and so far more than 100 have been identified (Urashima et al. 2007, 2009; Kobata 2010; Wu et al. 2010, 2011; Bode 2012), it should be noted that not every woman synthesizes the same ensemble of oligosaccharides. Many HMO structures contain elements also found in protein glycosylation, i.e. fucosylation patterns matching Secretor and Lewis blood group determinants (Kobata 2003; Bode 2012). With respect to the latter, there are notable differences between individuals in the fucosylation of HMOs, mirroring the maternal blood group patterns. Nowadays, four milk groups are distinguished, mostly dependent on the expression and activity of two genes, namely the secretor (Se) gene fut2, coding for α-1,2-fucosyltransferase (FUT2) and the Lewis (Le) gene fut3, coding for α-1,3/1,4-fucosyltransferase (FUT3) (Scheme 1, Table I) (Oriol et al. 1986; Thurl et al. 1997, 2010; Kunz et al. 2000; Newburg et al. 2005; Kobata 2010; Bode 2012). Furthermore, besides FUT3, the Se- and Le-independent α-1,3-fucosyltransferases FUT4, 5, 6, 7 and/or 9 are also of

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Sander S van Leeuwen2, Ruud J W Schoemaker3, Gerrit J Gerwig2,4, Ellen J M van Leusen-van Kan3, Lubbert Dijkhuizen1,2, and Johannis P Kamerling2,4

HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

Table I Expected structural epitopes and relative occurrence are indicated for the four milk groups Milk group

Occurrence (Europe)






1 2 3 4

69% 20% 0% 1%

+ + − −

+ − − −

+ + + +

+ − + −

+ − + −

importance for the final ensemble of Fuc-containing HMOs (Newburg et al. 2005). In the context of fucosyl-HMOs it is worthwhile to mention that in human milk Gal(β1-3)GlcNAc (type 1) chains are dominant over Gal(β1-4)GlcNAc (type 2) chains. Group 1, the largest group, includes Lewis-positive Secretors [Se +Le +; blood type Lewis(a−b+)] and contains HMOs with (α1-2)-, (α1-3)- and (α1-4)-linked Fuc residues, yielding structures with Lea, Leb, (pseudo-)Lex, (pseudo-)Ley and H epitopes. Group 2 includes Lewis-positive non-Secretors [Se −Le +; blood type Lewis(a+b−)] and contains HMOs with (α1-3)- and (α1-4)-linked Fuc residues, yielding Lea and (pseudo-)Lex epitopes. Group 3 includes Lewis-negative Secretors [Se +Le −; blood type Le(a−b−)] and contains HMOs with (α1-2)- and (α1-3)-linked Fuc residues, yielding structures with (pseudo-)Lex, (pseudo-)Ley, and H epitopes. Group 4 includes Lewis-negative non-Secretors [Se −Le−; blood type Lewis(a−b−)] and contains HMOs with only (α1-3)-linked Fuc residues, yielding (pseudo-) Lex epitopes. Despite this simple classification into four milk groups, large variations in HMO composition remain possible between individuals within a specific milk group (Chaturvedi et al. 2001; Newburg et al. 2005; Asakuma et al. 2008; Thurl et al. 2010; Bode 2012), and it is speculated that nutritional and environmental aspects contribute to these variations (Bode 2012).

One of the oldest methods to profile oligosaccharides in human milk samples was based on a combination of gel-filtration and paper chromatography (Kobata et al. 1969; Kobata 2003). During the last years, variations of HMOs in relation to milk groups and lactational periods have been studied with high-pH anion-exchange chromatography-pulsed amperometric detection (Thurl et al. 1996, 1997, 2010), thereby focusing on the presence/absence of 14 generally major oligosaccharides. Also, a matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method for high-throughput analysis of HMOs and classification into milk groups (groups 1, 2 and 3) has been reported (Blank et al. 2011). In this approach, the Fuc linkage types were determined indirectly by measuring MS/MS fragment peak intensity ratios. Considering the close similarity between the Lex/y versus Lea/b structures in the MALDI-TOF-MS analysis, discrimination between these two epitope groups proved to be more difficult, resulting in 2.5% mismatch. More recently, a rapid-throughput LC-CHIP/TOF-MS method for profiling of HMOs, including assigning secretor status and Lewis antigens, has been published (Totten et al. 2012). Here, we present a rapid one-dimensional 1H nuclear magnetic resonance (NMR)-based method for identifying the four HMO milk groups. It involves a further application of the NMR structural-reporter-group concepts for differently linked Fuc residues, as previously developed for the analysis of glycoprotein N- and O-glycans (Vliegenthart et al. 1983; Kamerling and Vliegenthart 1992; Vliegenthart and Kamerling 2007). A total of 36 HMO samples, isolated from small amounts of milk (100 μL), collected from 32 donors, were analyzed. All four different milk groups are represented in the sample collection. Comparison with enzyme-linked immunosorbent assay (ELISA) data of saliva samples against anti-Lea, anti-Leb and anti-H antibodies for 12 donors showed that the 1H NMR structural reporters could distinguish between Lea and Lex epitopes, while ELISA screening could not. 729

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Scheme 1. Biosynthetic pathways for the (α1-2), (α1-3) and (α1-4) fucosylation of type 1 and type 2 elements.

SS van Leeuwen et al.

Results 1 H NMR spectroscopy of reference HMOs A series of 11 commercially available reference HMOs, i.e. 2′-FL, 3-FL, DF-L, F-SL, LNFP I, LNFP III, F-LST a, DF-LNH I, DF-LNH II, LNDFH I and DF-para-LNH, were subjected to 1 H NMR analysis (Figure 1). A survey of the chemical shifts (δ) of the Fuc H-1, H-5 and CH3 signals, the so-called structuralreporter groups of Fuc residues in different microenvironments 730


H NMR analysis of HMO samples The Fuc H-1, Fuc CH3 and NAc regions of the 1H NMR spectra of 36 HMO samples, obtained from 32 volunteers, are presented in Figure 2. Based on the information presented in Table II and Figure 1, the Fuc CH3 region (δ 1.10–1.30) can be separated into three subregions, i.e. Lea/x at δ 1.14–1.19 [Fuc(α1-4) and/or Fuc (α1-3)], H at δ 1.19-1.24 [Fuc(α1-2)] and Leb/y at δ 1.24–1.30 [Fuc(α1-2) with Fuc(α1-4) and/or Fuc(α1-2 with Fuc(α1-3)].

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Fig. 1. Relevant parts (δ 5.00–5.50, δ 1.80–2.20 and δ 1.10–1.30) of the 1D 1H NMR spectra of fucosylated HMO standards. Anomeric signals are annotated: G = Glc, F2 = Fuc(α1-2), F3 = Fuc(α1-3) and F4 = Fuc(α1-4).

(Vliegenthart et al. 1983; Kamerling and Vliegenthart, 1992; Vliegenthart and Kamerling 2007), the GlcNAc NAc signals and the Neu5Ac NAc signals of these HMOs, is presented in Supplementary data, Table SI, together with the available 1H NMR data of 46 other fucosylated HMOs, as reported in the literature (attached to Supplementary data, Table SI). Based on these data, Table II has been compiled, showing the δ regions of the Fuc (H-1, H-5, CH3) and GlcNAc (NAc) structural reporters for the various Fuc-containing immunodeterminants of HMOs. Furthermore, in Figure 1 the relevant Fuc H-1 (δ 5.00–5.50), NAc (δ 1.80–2.20) and Fuc CH3 (δ 1.10–1.30) regions are illustrated by the partial 1H NMR spectra of the 11 reference HMOs. Comparison of the 1H NMR data of Table II and Supplementary data, Table SII shows high similarities for the relevant antigen fragments in both HMOs and glycoprotein glycans. Inspection of these data learns that the occurrence of a terminal H-antigen, i.e. Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3) (LNFP I and DF-LNH I in Figure 1) or Fuc(α1-2)Gal(β1-4)Glc (2′-FL in Figure 1), is reflected by Fuc CH3 doublets at δ 1.22–1.25. Hereby, the Fuc H-1 signal of 2′-FL is found at δ 5.31, whereas all other compounds have the Fuc H-1 signal at δ 5.18–5.19. The Lex/Sialyl-Lex determinant in terminal position, i.e. {Neu5Ac(α2-3)}0/1Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3/6) (LNFP III, DF-LNH I, and DF-LNH II in Figure 1) or the Glc variants (3-FL and F-SL in Figure 1), shows Fuc CH3 doublets at δ 1.16–1.19. When the Lex determinant is present in an internal position, i.e. →GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc (β1-3/6) (DF-para-LNH in Figure 1), a small upfield shift to δ 1.14–1.15 is observed. A further subdifferentiation follows from the Fuc H-1 chemical shifts: roughly, Fuc H-1 of the [Fuc(α1-3)] GlcNAc(β1-3/6) element resonates at δ 5.09–5.14 and Fuc H-1 of the [Fuc(α1→3)]Glc element at δ 5.37/5.44. The Lea/Sialyl-Lea determinant, i.e. {Neu5Ac(α2-3)}0/1Gal (β1-3)[Fuc(α1-4)]GlcNAc(β1-3), reveals Fuc CH3 doublets within the same region as the Lex/Sialyl-Lex determinant, i.e. δ 1.17–1.18 (DF-para-LNH, DF-LNH II, F-LST a shown in Figure 1). However, the Lea/Sialyl-Lea Fuc H-1 signals (δ 5.01–5.03) differ clearly from the Lex/Sialyl-Lex Fuc H-1 signals (δ 5.09–5.14). In case of the Ley determinant, Fuc(α1–2)Gal(β1-4)[Fuc (α1-3)]Glc[NAc(β1-6)], the Fuc(α1-2) CH3 signals resonate at δ 1.26–1.27 and the Fuc(α1-3) CH3 signals at δ 1.23–1.24 (DF-L in Figure 1). For the Leb determinant, Fuc(α1-2)Gal (β1-3)[Fuc(α1-4)]GlcNAc(β1-3), the Fuc(α1-2) CH3 signals resonate at δ 1.27 and the Fuc(α1-4) CH3 signals at δ 1.24–1.26 (LNDFH I in Figure 1). Here, the Ley and Leb Fuc H-1 sets can be used for further discrimination: Ley, Fuc(α1-2) H-1 at δ 5.27–5.28 and Fuc(α1-3) H-1 at δ 5.09 (in case of Glc, δ 5.39/ 5.45); Leb, Fuc(α1-2) H-1 at δ 5.15–5.16 and Fuc(α1-4) H-1 at δ 5.02–5.03.

HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

Table II Regions of structural-reporter-group 1H NMR dataa for Fuc-containing immunodeterminants of human milk oligosaccharides (HMOS), as derived from Supplementary data, Table SI Immunodeterminant

Fuc H-1

Fuc H-5

Fuc CH3

GlcNAc NAc

F2 F2 F2 F2

5.31 5.18–5.19 n.a.c 5.18

4.25/4.23b 4.28–4.30 n.a. 4.31

1.23 1.22–1.24 n.a. 1.25

– 2.04–2.06 n.a. 2.05

Lex (terminal) Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3) Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)

F3 F3

5.12–5.14 5.09–5.11

4.83–4.84 4.82–4.87

1.17–1.18 1.17–1.18

2.02–2.03 2.05–2.07

Lex (internal) →GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3) →GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)

F3 F3

5.10–5.12 5.08–5.09

4.81–4.82 4.80–4.82

1.14–1.15 1.14–1.15

2.01–2.02 2.04–2.05

Sialyl-Lex Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3) Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)

F3 F3

5.12 5.09

4.82 n.a.

1.17 1.16–1.17

2.02 2.03–2.05

Pseudo-(Sialyl-)Lex (terminal and internal) Gal(β1-4)[Fuc(α1-3)]Glc →GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc

F3 F3 F3

5.38/5.44 5.37/5.43 5.38/5.43

4.83/4.84 4.80–4.83 4.81/4.82

1.19/1.18 1.17/1.16 1.18/1.18

– – –

F2 F3 F2 F3

n.a. n.a. 5.27–5.28 5.09

n.a. n.a. 4.26 4.88

n.a. n.a. 1.27 1.23

F2 F3

5.28 5.39/5.45

4.29/4.25 4.87/4.86

1.26 1.24

Lea (terminal) Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3) Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-6)

– –

F4 F4

5.02–5.03 n.a.

4.87–4.88 n.a.

1.17–1.18 n.a.

2.02–2.05 n.a.

Sialyl-Lea Neu5Ac(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3) Neu5Ac(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-6) Neu5Ac(α2-3)Gal(β1-3)[Fuc(α1-4)][Neu5Ac(α2-6)]GlcNAc(β1-3)

F4 F4 F4

5.01 n.a. 5.18

4.86–4.88 n.a. 4.88

1.17 n.a. 1.17

2.04 n.a. 2.03

F2 F4 F2 F4

5.15–5.16 5.02–5.03 n.a. n.a.

4.33–4.34 4.85–4.87 n.a. n.a.

1.27 1.24–1.26 n.a. n.a.

Ley Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3) Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6) Pseudo-Ley Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]Glc

Leb Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3) Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-6)

n.a. 2.05–2.06

2.05–2.06 n.a.

Used abbreviations: F2, Fuc(α1-2); F3, Fuc(α1-3); F4, Fuc(α1-4). Chemical shifts are given in ppm relative to the signal of internal acetone at δ 2.225 in D2O (literature data at 40°C are excluded, see Supplementary data, Table SI). b Two chemical shifts separated by a slash indicate values belonging to α/β anomeric mixture. c n.a., not assigned so far in HMOs. a

Combining the chemical shift information of the Fuc CH3 Lea/x and Leb/y regions with that of the Fuc H-1 regions makes a differentiation of Lewis types possible. All spectra show Fuc H-1 signals in the g-region δ 5.09–5.14, indicative of (α1-3)-linked Fuc residues (Table II). The relatively intense signals in the d-region δ 5.20–5.24 reflect the H-1 atoms of the reducing α-Glc units of the various HMO constituents. The 1H NMR spectra of the HMO samples 1-12 showed Fuc CH3 doublets in the region δ 1.14–1.19, corresponding with the Fuc CH3 region for (Sialyl-)Lex, -4)[Fuc(α1-3)]Glc, and (Sialyl-) Lea epitopes (Figure 2, Table II). No Fuc CH3 signals were observed in the region δ 1.19–1.30, indicating the absence of H, Ley and Leb determinants in these samples (Table II). A further differentiation follows from an inspection of the Fuc H-1 regions of the 12 spectra. The absence of Fuc(α1-2) residues is clear from the absence of Fuc H-1 signals at δ 5.31 (b-region), 5.28

(c-region), 5.18 (e-region) and 5.16 (f-region). In case of samples 1–3, 5, 6 and 8–12, Fuc α-anomeric doublets are found at δ 5.00–5.05 (h-region), corresponding with (α1-4)-linked Fuc; these signals are absent in the spectra of samples 4 and 7. As -4) [Fuc(α1-3)]Glc structural elements are expected to be present in trace amounts [only the minor HMOs 3-FL, DF-L, LNDFH II, LNDFH III, F-SL, F-LST c and FDS-LNT II have been reported (Supplementary data, Table SI)], the absence of Fuc H-1 doublets at δ 5.38/5.44 (a-region) in the spectra should be taken with care. The 1H NMR data of samples 1–12, except 4 and 7, evidently support the presence of (Sialyl-)Lex and (Sialyl-)Lea determinants; for samples 4 and 7 only the presence of (Sialyl-) Lex determinants is supported. The spectra clearly show that the samples may differ in their levels of fucosylation, as evidenced by the intensity of the Fuc CH3 signals in relation to the NAc CH3 signals (Table III). 731

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H Fuc(α1-2)Gal(β1-4)Glc Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3) Fuc(α1-2)Gal(β1-3)GlcNAc(β1-6) Fuc(α1-2)Gal(β1-3)[Neu5Ac(α2-6)]GlcNAc(β1-3)

SS van Leeuwen et al.

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Fig. 2. Relevant parts (δ 5.00–5.50, δ 1.80–2.20 and δ 1.10–1.30) of the 1D 500 MHz 1H NMR spectra of 36 HMO samples isolated from human milk. In the NAc region (δ 1.80–2.20) and the Fuc CH3 region (δ 1.10–1.30) integration boxes are indicated; NAc grey = δ 2.00–2.10, Leb/y orange = δ 1.24–1.30, H green = δ 1.19– 1.24, and Lea/x blue = δ 1.14–1.19. In the Fuc H-1 region (δ 5.00–5.50) relevant peak regions are indicated: (a) ( pseudo-Lex and pseudo-Ley) = δ 5.43–5.46 + δ 5.35–5.38; (b) (H in 2′-FL) = δ 5.30–5.33; (c) (Ley) = δ 5.26–5.29; (d) (α-Glc) = δ 5.20–5.24; (e) (H) = δ 5.16–5.20; (f ) (Leb) = δ 5.14–5.16; (g) (Lex and Ley) = δ 5.09–5.14; and (h) (Lea and Leb) = δ 5.00–5.05. Note: The Fuc anomeric region is not scaled in the same proportion as the NAc and CH3 regions.


HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

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Fig. 2 Continued


SS van Leeuwen et al.

Table III 1D 1H NMR Fuc CH3 region integrals in relation to NAc CH3 (δ 2.00–2.10 = 1.00) for HMOs isolated from 36 milk samples Leb/y δ1.24–1.30

H δ1.19–1.24

Lea/x δ1.14–1.19




Se/Le ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 20 30 31 32 33 34 35 36

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.57 0.56 0.40 0.84 0.51 0.38 0.56 0.55 0.62 0.60 0.72 0.43 1.11 0.72 0.36 0.41 0.26 0.42 0.69 0.40 0.65 1.42 0.62 0.27

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.08 1.60 1.63 1.66 0.79 1.07 1.18 1.22 1.04 1.70 1.64 0.90 1.92 1.60 0.65 0.69 0.43 0.70 1.12 0.64 0.77 1.88 0.90 2.85

0.47 0.49 0.61 0.19 0.86 0.75 0.33 0.69 0.96 0.50 0.60 0.65 0.27 0.36 0.27 0.55 0.36 0.31 0.60 0.58 0.61 0.40 0.26 0.49 0.43 0.57 0.46 0.51 0.53 0.52 0.70 0.51 0.68 0.61 0.61 0.38

0.47 0.49 0.61 0.19 0.86 0.75 0.33 0.69 0.96 0.50 0.60 0.65 1.92 2.52 2.31 3.05 1.66 1.77 2.34 2.35 2.28 2.71 2.62 1.82 3.46 2.88 1.47 1.60 1.22 1.64 2.51 1.55 2.11 3.92 2.13 3.50

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.36 1.88 1.83 2.08 1.04 1.26 1.46 1.49 1.35 2.01 2.00 1.11 2.47 1.96 0.83 0.89 0.56 0.91 1.47 0.84 1.10 2.60 1.21 2.99

0.47 0.49 0.61 0.19 0.86 0.75 0.33 0.69 0.96 0.50 0.60 0.65 0.56 0.64 0.47 0.97 0.61 0.50 0.88 0.85 0.92 0.70 0.62 0.70 0.98 0.92 0.64 0.71 0.66 0.73 1.04 0.71 1.01 1.32 0.92 0.51

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.44 2.95 3.88 2.14 1.70 2.51 1.67 1.75 1.46 2.85 3.21 1.58 2.52 2.11 1.30 1.25 0.84 1.24 1.41 1.18 1.09 1.96 1.32 5.86

Se (Secretory activity) levels are derived from the integral δ 1.19–1.24, adding half of the integral δ 1.24–1.30; the Le (Lewis activity) levels are similarly derived from the integral δ 1.14–1.19, adding half of the integral δ 1.24–1.30.

For further verification of the Fuc CH3 assignments in HMO samples 1–12, the δ value of Fuc H-5 was checked via twodimensional 1H–1H correlation spectroscopy (COSY) measurements (Figure 3A). As is evident from Table II, the position of Fuc H-5 can be used to discriminate (α1-2)-linked (δ 4.23– 4.34) and (α1-3 or α1-4)-linked (δ 4.80–4.88) Fuc residues; see also Supplementary data, Table SII. The COSY spectrum of a pool of samples 1–12 showed only a cross-peak between Fuc CH3 (δ 1.14–1.19) and Fuc H-5 (δ 4.80–4.90), thereby eliminating (α1-2)-linked Fuc residues. The 1H NMR spectra of the HMO samples 13–35 showed, in addition to Fuc CH3 signals in the region δ 1.14–1.19 (Lea and/ or Lex determinants), also signals in the Fuc CH3 regions δ 1.19–1.24 (H-antigen) and δ 1.24–1.30 (Leb and/or Ley epitopes) (Figure 2). Comparison of the peak intensities in the regions δ 1.19–1.24 and δ 1.24–1.30 learns that in samples 13–26 the signals in the H-antigen region occur with a significantly higher relative intensity than in samples 27–35. Also, the signal intensities in the Lea/x region vary considerably between the different samples. The variety in structures in samples 13–35 is also reflected in the anomeric regions of Fuc. Samples 13–26 show relatively 734

low abundances of Lex- and/or Ley-specific signals (g-region, δ 5.09–5.14) and a wide variation in intensity in Lea and/or Leb-related signals (h-region, δ 5.00–5.05). The Ley-related signals (c-region, δ 5.26–5.29) also show a wide variation in intensity in these samples, ranging from nearly none (e.g. sample 15) to medium levels (e.g. sample 23), compared with the Fuc H-1 signal of 2′-FL (b-region, δ 5.30–5.33). Samples 27–35 show a higher level of Lex- and/or Ley-specific signals in the anomeric region of Fuc (δ 5.09–5.14), except for sample 34. This supports observations in the Fuc CH3 regions, where levels of Lea- and/or Lex-related signals are relatively high for samples 27–35, compared with the H-antigen region. For samples 27–35 the Fuc(α1-4)-related signals show a wide range of intensities, as was shown for samples 1–12 and 13–26. Just as was observed for samples 13–26, the intensities of the Ley-related signals (Fuc H-1, δ 5.26–5.29, c-region) vary greatly, from almost zero (e.g. sample 28) up to almost equal to 2′-FL (sample 35). Only few of the samples 13–35 show clear signals for pseudo-Lex/y epitopes (δ 5.37/5.45 ppm), similar to observations for samples 1–12. Sample 35 stands out in this aspect, with levels of pseudo-Lex/y-related signals that are not observed elsewhere. Also Ley- and Leb-related Fuc(α1-2) units

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Milk sample number

HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

show peaks of high intensity (δ 5.26–5.29 and δ 5.14–5.16, respectively), in relation to 2′-FL. COSY measurements were carried out for verification of Fuc CH3 assignments via Fuc H-5 (Figure 3B). The COSY spectrum of a pool of samples 13–35 showed cross-peaks between Fuc CH3 (δ 1.15–1.19)/Fuc CH3 (δ 1.24–1.26) and Fuc H-5 (δ 4.80–4.90), in accordance with (α1-3 or α1-4)-linked Fuc residues, and between Fuc CH3 (δ 1.27–1.30)/Fuc CH3 (δ 1.19–1.24) and Fuc H-5 (δ 4.20–4.30), in accordance with (α1-2)-linked Fuc residues. Finally, the 1H NMR spectrum of HMO sample 36 showed very high Fuc CH3 signals in the region δ 1.19–1.24, specific for (α1-2)-linked Fuc residues (Figure 2). Combined with the very low intensity of the Le-specific Fuc CH3 signals in the regions δ 1.14–1.19 (Lea and/or Lex) and δ 1.24–1.30 (Leb and/ or Ley), it is evident that sample 36 contains high amounts of HMOs with the H-antigen (see also Fuc H-1 signal at δ 5.18, e-region). This statement is supported by the absence of signals in the Fuc H-1 h-region δ 5.00–5.05 (Lea and/or Leb) and f-region δ 5.14–5.16 (Leb), whereas there are minor peaks in the Fuc H-1 region δ 5.09–5.14 (g-region; Lex and/or Ley). Sample 36 contains relatively high amounts of 2′-FL (intense Fuc H-1 signal at δ 5.31, b-region). Discussion Structural-reporter-group signal integration The peak surfaces of the Fuc CH3 peak regions corresponding with the Leb/y (δ 1.24–1.30), H (δ 1.19–1.24), and Lea/x

(δ 1.14–1.19) immuno groups, respectively, represent different types of information (Figure 2). Taken together and expressed in relation to the total peak surface of the NAc peak region (δ 2.00–2.10), it gives an impression of the degree of fucosylation in each sample (Table III). However, in a separate form these three regions yield information about the Se (Secretor activity) and Le (Lewis activity) levels, as presented in Table III. Since the region for Leb/y contains signals for both Fuc(α1-2) and Fuc (α1-3/4) residues, half of the surface area is attributed to Secretor activity (FUT2 activity) and the other half to Lewis activity (combined FUT3 and FUT4, 5, 6, 7 and/or 9 activity). Therefore, the Se levels are derived from the integral of the δ 1.19–1.24 region plus half of the integral of the δ 1.24–1.30 region. In a similar way, the Le levels are derived from the integral of the δ 1.14–1.19 region plus half of the integral of the δ 1.24–1.30 region. Due to limited peak intensity and therefore poorer signalto-noise ratios, the anomeric region (δ 5.00–5.50) is not as readily used for integration, and therefore only used to check presence or absence of signals. Moreover, anomeric peaks closer to the HOD signal may suffer from influence of the WEFT solvent suppression pulse. Because in human milk the type 1 epitope [Gal(β1-3)GlcNAc], which forms the basis for Lea/b determinants (Scheme 1), is present in much higher amounts than the type 2 epitope [Gal(β1-4)GlcNAc], which forms the basis for Lex/y structures (Urashima et al. 2012), it can be expected that Lea/b structures occur in higher abundance than Lex/y epitopes, except for samples from donors that have an inactive fut3 gene (see samples 4, 7 and 36). 735

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Fig. 3. One-dimensional 500 MHz 1H NMR and relevant section of 2D 600 MHz 1H–1H COSY NMR spectra of (A) pool of the HMO samples 1–12 and (B) pool of the HMO samples 13–35.

SS van Leeuwen et al.

In a recent human milk metabolomics study, for quantification purposes use has been made of 1H NMR integration of a set of selected protons for a wide array of metabolites, including 10 HMOs, of which 6 were fucosylated, i.e. 2′-FL, 3-FL, DF-L, LNFP I, LNFP II and LNFP III (Smilowitz et al. 2013). Considering the specific sets of chemical shift regions of Fuc H-1, H-5 and CH3 signals of fucosylated HMOs with H, Lea and/or Lex determinants, as indicated in the present study, focusing on only the compounds mentioned above may lead to overestimation.

HMO samples 13–35 Samples 13–35 show peaks in all three Fuc CH3 regions, i.e. Leb/y, H, and Lea/x, and were classified as belonging to milk group 1 (Lewis-positive Secretor). The observation of an intense Fuc(α1-2) H-1 signal at δ 5.31 supports the presence of major amounts of 2′-FL, one of the major HMO components of Secretors (Thurl et al. 1996; Kunz et al. 2000; Newburg et al. 2004; Bode 2012). 736

HMO sample 36 For sample 36 a unique 1H NMR pattern is shown. The Fuc CH3 region shows a particularly strong H-antigen peak (supported by intense Fuc H-1 peaks in the H-specific regions), flanked by minor Leb/y and Lea/x peaks. Focusing on the anomeric region, no Fuc(α1-4)-related anomeric peaks are observed, excluding Lea and Leb epitopes; only Fuc H-1 peaks of minor intensity, corresponding with Lex and Ley epitopes, are detected. Therefore, this sample is classified as belonging to milk group 3 (Lewis-negative Secretor). Notably, so far only one HMO with a Ley epitope (i.e. DFS-LNnH) has been reported (Grönberg et al. 1989). ELISA screening For 12 donors (HMO samples 4, 10/11/12, 13, 17, 18, 20, 25, 30, 31/32, 34, 35 and 36) a blood group typing by ELISA Table IV Blood group typing by ELISA screening of saliva samples with anti-Lea, anti-Leb and anti-H antibodies Nr




Se status

Predicted type

4 10/11/12 13 17 18 20 25 30 31/32 34 35 36

+ + weak + + + + + + + + +

− − + + + + + + + + + weak

− − + + + + + + + + weak +

Non-Secretor Non-Secretor Secretor Secretor Secretor Secretor Secretor Secretor Secretor Secretor Secretor Secretor

Le(a+b−) Le(a+b−) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a−b+) Le(a+b−)

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HMO samples 1–12 Samples 1–12 do not contain Fuc(α1-2) residues and were classified as non-Secretor samples (Table III). Samples 1-3, 5, 6 and 8– 12 show Fuc H-1 doublets in the region δ 5.00–5.05, which are absent in samples 4 and 7. Therefore, the first group of samples are classified as belonging to milk group 2 (Lewis-positive non-Secretor) and the second group as belonging to milk group 4 (Lewis-negative non-Secretor). It should be noted that in one specific structural element (Table II), when a Fuc(α1-4) residue is flanked by two neighboring Neu5Ac residues, the Fuc(α1-4) H-1 signal is shifted to δ 5.18, thereby overlapping with the H-determinant Fuc(α1-2) H-1 signal. The disialylated structure, however, is only a very minor component and should normally not give a significant signal in the 1H NMR spectrum of an HMO sample mixture. Also the signal of α-Glc in structures containing a pseudo-Lex-epitope, where the Fuc(α1-3) residue is linked to the reducing Glc unit, is shifted from the α-Glc H-1 d-region δ 5.20–5.24 to δ 5.18. Making use of the pseudo-Lex Fuc H-1 signals at δ 5.38/5.44 (a-region), a correction can be carried out for the corresponding reducing α-Glc signal, where necessary. Interestingly, compared with the other samples, sample 9 shows a significant peak at δ 5.18. The relatively large peaks for pseudo-Lex epitopes (δ 5.37/5.45, a-region) in this sample correlate in size with this peak, identifying it as α-Glc H-1, thereby confirming the absence of Fuc(α1-2) residues. Similar peak combinations can be observed in minor intensities in samples 1, 5 and 8. The observation of absence of any Fuc(α1-2) signals in the non-Secretor samples in our study is in accordance with previous findings; the same holds for the Lewis-negative samples, showing the absence of Fuc(α1-4)/Lea structural elements (Kobata 2010; Thurl et al. 2010). Surprisingly, in contrast to all literature data so far and our findings, recent MS studies have shown residual amounts (1–2%) of the Fuc(α1-2) epitope (e.g. 2′-FL, DF-L, LNFP I) in non-Secretor samples when compared with levels in Secretor samples (LC-CHIP/TOF-MS; Totten et al. 2012), but also 2′-FL levels of 20% comparing non-Secretor and Secretor samples (LC-ESI-MS; Hong et al. 2014). Furthermore, Lewis-negative samples were reported to contain up to 30% Lea-containing structures when compared with Lewis-positive samples (Totten et al. 2012).

Inspection of the three Fuc CH3 regions of samples 13–35 shows a wide variety in relative abundances, providing a dynamic range of fucosylated structural elements, as shown previously (Bode 2012). Samples 13–26 show calculated Se/Le ratios between 1.46 and 3.88 (Table III), whereas samples 27–35 generally have a lower ratio between 0.86 and 1.41, except for sample 34 (Se/Le ratio 1.96), which has a unique pattern, with relatively low Lea/x signals and relatively high H-antigen and Leb/y signals. It is speculated that this individual has a particularly high FUT2 activity, combined with lower FUT3 and FUT4, 5, 6, 7 and/or 9 activities, resulting in this unique pattern. Samples 13–26 show a 1D 1H NMR pattern for Fuc CH3 with a higher H-antigen peak, than Leb/y and Lea/x immunodeterminant-related peaks. The 1D 1H NMR pattern for samples 27–35 is noticeably different, with an H-antigen CH3 peak that is of similar intensity as the Le immunodeterminant peaks. It should be noted that sample 34 is unique, in that it has H-antigen and Leb/y immunodeterminant peaks of similar intensity; however, the Lea/x related peak has a much lower intensity. Based on these findings, we propose to make a subdivision within milk group 1, classifying HMO samples in which the H-antigen really dominates over both Leb/y and Lea/x epitopes (samples 13–26), as milk group 1a, and HMO samples wherein this is not the case (samples 27–35) as belonging to milk group 1b.

HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

Differences within HMO ensembles from one donor In this study three donors provided more than one sample. Samples 10–12 were from one donor: sample 10 was taken from the first lactation cycle, and samples 11 and 12 from the second lactation cycle, collected 1 and 3 months post-partum, respectively. Comparing samples 10 and 11, it appears that the fucosylation level, derived from the Fuc/NAc ratios (Table III), is slightly higher in the second lactation cycle. It should be noted that this non-Secretor donor cannot produce HMOs with Fuc(α1-2) residues. Comparing samples 11 and 12, the relative fucosylation levels after 1 and 3 months post-partum are similar. Samples 25–26 and 31–32 are from two other donors, respectively, whereby 25 and 31 are collected from the first lactation cycle, and 26 and 32 from the second lactation cycle. For both donors the fucosylation levels, derived from the Fuc/NAc ratios, are lower during the second lactation cycle. When comparing the Se/Le ratios of these samples, it appears that mostly HMOs with Fuc(α1-2) residues are reduced during the second lactation cycle.

Conclusions The composition of the oligosaccharide fraction of human milk varies greatly between individuals. A major difference is caused by the activity of fut2 and fut3 genes, responsible for the synthesis of Lea, Leb and H-antigen structures. But also the genes encoding the α-1,3-fucosyltransferases FUT4, 5, 6, 7 and/or 9 play a definite role. Here, we have shown that after a single step of table-top solid-phase extraction (SPE) of HMOs, 1D 1H NMR screening provides detailed information on fucosylated oligosaccharides in human milk. Unfortunately, the amount of sialylated HMOs is too low for the detection in intact HMO samples. A total of 36 samples from 32 donors were screened and successfully classified into one of the four milk groups (group 1: Lewis-positive Secretor; group 2: Lewis-positive non-Secretor; group 3: Lewis-negative Secretor; group 4: Lewis-negative non-Secretor). Moreover, the presented approach can differentiate between Lea and Lex structures, whereas ELISA screening of saliva with anti-Lea antibodies cannot make this distinction. Out of the 36 samples, 2 samples were found lacking Fuc(α1-2) and Fuc(α1-4) epitopes, suggesting inactive fut2 and fut3 genes. One sample had a clearly active fut2 gene, as evidenced by the presence of Fuc (α1-2) epitopes, but the lack of Fuc(α1-4) epitopes suggested an inactive fut3 gene. The 1H NMR analysis method was capable of identifying these samples. Beside structural information on the fucosylated elements present, a semi-quantitative estimation can be made of the different structural elements. These quantitative analyses showed a wide range of variation between samples within each milk group, dividing the most abundant group into two subgroups, based on relative abundances of Fuc (α1-2) vs Fuc(α1-3) and (α1-4) epitopes. These data show the power of 1D 1H NMR spectroscopy in analysis of HMO immunodeterminant composition. It is expected that the 1H NMR-based method presented in this paper can also be applied to analyze HMOs in urine and fecal samples, in order to monitor the fate of HMOs in the infant gut.

Materials and methods HMO standards HMO standards were obtained from IsoSep AB (Tullinge, Sweden). Collection of milk samples Human milk was acquired from 32 volunteers, collecting a full feeding of at least 100 mL 1 month post-partum. Three of these individuals collected samples both during the lactation period after birth of their first and their second child. One individual also collected a sample 3 months post-partum. After collection, the milk was stored immediately at −20°C, and after transfer to the laboratory the milk was stored at −80°C until processing. Blood typing test on saliva samples Saliva samples from 12 volunteering mothers were collected and analyzed by Sanquin (Amsterdam, The Netherlands) for Lewis and Secretor status blood typing, using anti-Lea, anti-Leb and anti-H monoclonal antibodies. 737

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screening of saliva samples was carried out by Sanquin (Amsterdam, The Netherlands; Table IV). The two donors of samples 4 and 10/11/12, classified by 1H NMR analysis as a Lewis-negative non-Secretor (milk group 4) and a Lewis-positive non-Secretor sample (milk group 2), respectively, demonstrated both activity against anti-Lea antibodies, but not against anti-H and anti-Leb antibodies, assigning both samples as belonging to Lewis-positive non-Secretors. It has been described that anti-Lea antibodies not only has a strong affinity for Lea epitopes, but also for Lex type structures (Chang et al. 2011). Considering the absence of Fuc(α1-4)-containing HMOs in sample 4 and the presence of Lex epitopes, the activity of anti-Lea antibodies against this sample (Table IV) has to be explained by the presence of Lex structures. The nine donors of samples 13, 17, 18, 20, 25, 30, 31/32, 34 and 35 showed in all cases the Lewis-positive Secretor status, in agreement with the 1H NMR analysis (milk group 1). Sample 35, however, only showed minor interaction with the anti-H antibody, as did sample 13 with the anti-Lea antibody. Sample 36 revealed a weak activity with anti-Leb antibodies, combined with strong interaction with anti-H and anti-Lea antibodies in the ELISA assay, typing it as a Lewis-positive Secretor sample. However, 1H NMR analysis had classified it as a Lewis-negative Secretor sample (milk group 3). Taking into account the NMR data, the anti-Lea interaction can be explained by affinity with Lex epitopes (Chang et al. 2011). As reported, anti-Leb antibodies have medium side-activity against Lea-epitopes and weak interaction against Ley and sialyl-Lea structures (Chang et al. 2011). Taking into account the NMR data, the weak anti-Leb activity in sample 36 has to be explained by residual activity against the Ley epitopes present in the sample. The various observations show that ELISA screening of saliva samples with anti-Lea antibodies particularly, and to a lesser extent with anti-Leb antibodies, has a risk to mismatch in samples derived from FUT3-deficient individuals. It is therefore imaginable that for individuals lacking FUT3 activity, but with a strong FUT4, 5, 6, 7 and/or 9 activity, resulting in relatively high amounts of Lex structures, ELISA would result in a wrong classification.

SS van Leeuwen et al.

Isolation of HMOs Milk was thawed overnight at 4°C; when required further thawing was done at room temperature. Samples of 100 μL human milk were diluted with 100 μL Milli-Q water and applied to Carbograph SPE (300 mg, Grace, Breda, The Netherlands) columns. After washing with 6 × 500 μL water, HMO pools were eluted with 6 × 500 μL 40% acetonitrile, containing 0.05% TFA. Combined eluents were evaporated under a N2 stream, and residues lyophilized from water.

Supplementary Data Supplementary data for this article is available online at http:// Funding This work was financially supported by the European Union/ European Regional Development Fund and by the Dutch Ministry of Economic Affairs, Agriculture and Innovation, the Dutch innovation program ‘Peaks in the Delta’, the Municipality of Groningen, the Provinces of Groningen, Fryslân, and Drenthe, and the Dutch Carbohydrate Competence Center (CCC project WP6a). Acknowledgements We thank the various donors for supplying milk samples and cooperating in ELISA screening of saliva samples. Conflict of interest None declared. Abbreviations COSY, correlation spectroscopy; ELISA, enzyme-linked immunosorbent assay; FUT2, α-1,2-fucosyltransferase; FUT3, α-1,3/ 1,4-fucosyltransferase; FUT4,5,6,7,9, α-1,3-fucosyltransferase 738

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NMR spectroscopy Lyophilized samples were exchanged twice with 99.9%atom D2O (99.9%atom, Cambridge Isotope Laboratories Inc., Andover, MA) with intermediate lyophilization. Samples were dissolved in 650 μL D2O containing acetone (δ 2.225 ppm) as internal standard. One-dimensional 500 MHz 1H NMR spectra were recorded on a Bruker DRX500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University, The Netherlands) at probe temperatures of 300 K, with a spectral width of 5000 Hz and zero filled to 32k. For each sample 64 transients were recorded with a 1.1 s relaxation delay. A presaturation WEFT pulse was applied to suppress the HOD signal. Two-dimensional 600 MHz 1H-1H COSY spectra were recorded on a Varian Inova 600 spectrometer (Groningen Biomolecular Sciences and Biotechnology Institute, NMR Center, University of Groningen, The Netherlands) in 256 increments of 4000 data points with a spectral width of 5000 Hz. Spectra were processed with MestReNova 5.3 (Mestrelabs Research SL, Santiago de Compostella, Spain).

activities; HMO, human milk oligosaccharide; MALDI-TOF-MS, matrix-assisted laser-desorption ionization time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; SPE, solidphase extraction.

HMO immunodeterminants assessed by 1D 1H NMR spectroscopy

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Rapid milk group classification by 1H NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples.

Human milk oligosaccharides (HMOs) are a major constituent of human breast milk and play an important role in reducing the risk of infections in infan...
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