What Is Known
Human milk oligosaccharides belong to the third most abundant group of milk components.
They have a high potential for gastrointestinal and systemic functions.
Small human milk oligosaccharides can be produced on a commercial scale for clinical studies.
Human milk oligosaccharides are unique structures and not to compare with common prebiotics.
What Is New Quantifying 16 single human milk oligosaccharides revealed
No difference in the total amount between term and preterm milk.
An increasing concentration of lacto-N-tetraose (core structure) during the first month.
Approximately 35% to 45% less total human milk oligosaccharides in Lewis blood group (a+b−), “nonsecretor” milk as compared to Lewis (a−b+), “secretor” milk.
Human milk oligosaccharides (HMOs) belong to the third most abundant group of milk components, besides lactose and fat. Among bioactive components HMOs constitute one of the main gap between human milk and infant formula as most of these oligosaccharides are not present in bovine milk, the common basis for infant formula. The great interest in HMOs in recent years is reflected by studies on diverse aspects such as HMOs’ potential in health and disease, microbial activity and metabolism, new analytical approaches, or metabolism in animals and humans (1–8) (9)
Regarding HMOs’ metabolism it is interesting that approximately 1% to 2% of HMOs are excreted via the infants’ urine (10–12) (13–16) (17–23) (24–29) (25,27–28,30)
In the present study we aimed to investigate whether differences exist in the total amount of HMOs and individual oligosaccharides between term and preterm milk, from mothers with secretor or nonsecretor status and milk with a specific Lewis blood group pattern.
METHODS
Subjects and Design
A longitudinal study to characterize and quantitate HMOs in 96 milk samples from 32 voluntary mothers was conducted (Fig. 1 ). Inclusion criteria were healthy women who were exclusively breast-feeding. Participants were recruited after delivery at the Hospital Clínico Universitario of Valencia, Spain. Clinical records from mothers including maternal age, parity, gestational age, mode of delivery, antibiotic administration, and weight gain during pregnancy were recorded.
FIGURE 1: Study design, subjects, and characteristics. ∗ One sample excluded as designation to a Lewis blood group was not possible. HMO = human milk oligosaccharide.
Within the prospective longitudinal collection of breast milk samples, 14 mothers delivering at term (≥37 weeks of gestations) and 18 mothers delivering preterm (<37 weeks of gestations) were recruited. The study protocol was approved by the Hospital's Ethics Committee and fully complied with the Declaration of Helsinki as revised in 2000. The participating women received written, complete, and detailed information about the study, and before providing samples, all donors signed an informed consent form for participation in the study.
Samples
Mothers were given written instructions for standardized collection of samples in the morning. Breast milk samples were collected within the first month of exclusive breast-feeding during 3 stages of lactation, that is, colostrum from day 1 to 7, transitional milk from day 8 to 15 and mature milk from day 16 to 30. Breast milk was collected with a sterile automatic breast milk pump (Medela Symphony, Baar, Switzerland), polystyrene suction funnels, and screw-top bottles adapted to suction funnels for the direct milk collection. Breast milk samples were aliquoted using sterile material, frozen, and stored at −80°C for later analysis.
Oligosaccharide Extraction
HMOs were extracted as described in (31)
Oligosaccharide Analysis and Quantification
After elution, oligosaccharides were dried for at least 3 and up to 12 hours in a vacuum centrifuge, and resuspended in ultrapure water. An HPAEC-PAD (Dionex ICS-5000) equipped with a Carbo Pac PA-1 column was operated using the Chromeleon 6.80 software (Thermo Fisher Scientific, Dreieich, Germany). The gradient parameters at a constant flow rate of 1.0 mL/min were as follows: the mobile phase consisted of 0.1 mol/L NaOH at 1.0 mL/min with a 34-minute gradient from 0 to 0.25 mol/L sodium acetate; the lag time was 7.5 minutes and the total running time including equilibration 52.5 minutes. For each target component, an individual 4-point calibration curve with quadratic equation was obtained injecting defined concentrations of single standard oligosaccharides (Carbosynth Ltd, Bershire, United Kingdom; Dextra Laboratories, West Berkshire, United Kingdom; Elicityl, Crolles, France). All oligosaccharide standards were of highest quality with a proven purity between 90% and 95%. Oligosaccharides, which were quantified by HPAEC-PAD are listed in Table 1 .
TABLE 1: Oligosaccharides in human milk quantified by high pH anion exchange chromatography with pulsed amperometric detection
The presence or absence of secretor/nonsecretor or Lewis blood group–specific components in milk by HPAEC-PAD was used for the identification of groups 1–3 as shown in Table 2 . We were not able to identify any samples from group 4 as their frequency is <1% (5,32–33)
TABLE 2: Assignment of milk samples to a secretor/nonsecretor or a Lewis blood group pattern according to the presence or absence of specific oligosaccharides
Statistical Analysis
Comparisons of several oligosaccharides and oligosaccharide fractions among groups were done by GraphPadPrism software. For each group median and interquartile range (IQR) were reported. Significant differences between delivery groups (term vs preterm) and between secretor status (secretor vs non-secretor) were assessed by Mann-Whitney U test. Friedman test with Dunn multiple comparison was used to compare values between lactational stages (colostrum vs transitional vs mature). The abundance of HMO fractions among the different Lewis blood groups (Lewis (a−b+), Lewis (a+b−), Lewis (a−b−) were compared by Kruskal-Wallis test with Dunn multiple comparison. Differences were considered significant if P value <0.05.
RESULTS
We collected 96 human breast milk samples from 32 mothers at 3 different time points within the first 30 days of lactation and analyzed their HMO content and composition. For quantification, oligosaccharides in term and preterm milk samples were subjected to HPAEC-PAD.
Human Milk Oligosaccharides in Term and Preterm Milk
Based on the quantification of 16 single components in colostrum, transitional and mature milk the median level of the total HMO content in term milk from 14 mothers was 7.49 g/L in colostrum (IQR 6.59–10.68), 9.14 g/L in transitional milk (IQR 7.18–10.21), and 8.17 g/L in mature milk (IQR 7.17–10.02). In preterm milk from 18 mothers, the median level did not differ from term milk , neither for colostrum with 8.71 g/L (IQR 5.14–10.65), nor for transitional milk with 8.59 g/L (IQR 5.45–9.92) or mature milk with 8.57 g/L (IQR 6.01–9.06) (Fig. 2 and Supplemental Digital Content 1, Tables 1.1 and 1.2, https://links.lww.com/MPG/A779 Fig. 2 A−C; see also Supplemental Digital Content 1, Table 1, https://links.lww.com/MPG/A779 https://links.lww.com/MPG/A779
FIGURE 2: Concentration of total, neutral, and acidic HMO in term and preterm milk (A–C), secretor and nonsecretor milk (D–F), and Lewis blood type milk (G–I). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Black bars represent median levels with interquartile ranges. HMO = human milk oligosaccharide.
Human Milk Oligosaccharides in “Secretor” and “Nonsecretor” Milk
Because we did not observe any difference between term and preterm milk, we classified all samples according to their HPAED-PAD milk pattern, that is, depending on the presence or absence of secretor/nonsecretor specific individual HMOs (Table 2 ). Representative HPAEC-PAD separations are shown in Figure 3 . A clear separation of all 16 individual oligosaccharides used for quantification is obvious with the exception of 3FL, which was not separated well from lacto-N-difucohexaose (LNDFH I). Their content is therefore given as the sum of 3FL and LNDFH I. The difference between both groups is the presence or absence of secretor-specific components, for example of 2’FL, lactodifucotetrose, and lacto-N-fucopentaose I (LNFP I) (Fig. 3 ).
FIGURE 3: High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) chromatogram of oligosaccharides in mature milk of 2 different donors. Black: secretor, Lewis (a−b+); red: nonsecretor, Lewis (a+b−). Secretor and Lewis-specific human milk oligosaccharides (HMOs) are highlighted. DSLNT = disialyllacto-N-tetraose; 2’FL = 2’-fucosyllactose; LDFT = difucosyllactose; LNDFH = lacto-N-difucohexaose; LNFP = lacto-N-fucopentaose; LNT = lacto-N-tetraose; LST = sialyl-acto-N-tetraose; 6'SL = 6'sialyllactose.
Here, we found major differences between both groups of donors. In “secretor” milk, the total HMO concentration was significantly higher compared with “nonsecretor” colostrum, transitional, and mature milk (median, 9.67, 9.47, and 8.67 g/L vs 5.17, 5.61, and 5.54 g/L, respectively; Table 3 A and B and Fig. 2 D−F). These differences were mainly caused by high levels of 2’FL, LNFP I, and 3FL/LNDFH I in “secretor” milk. These components were not detectable in “nonsecretor” milk. In addition, the concentration of LNT, the core structure in human milk, was only half of the concentration or less in secretor milk compared with nonsecretor milk (Table 3 A and B).
TABLE 3: Concentrations (g/L) of single oligosaccharides in human milk from “secretors” (n = 21) (A) and “nonsecretors (n = 11) (B) at different stages of lactation
Table 3 also demonstrates that despite the separation into “secretor”/“nonsecretor” milk we still found large variations in the concentrations of individual oligosaccharides (eg, for LNT, 2’FL, LNFP I, and LNFP II).
Core Lacto-N-Tetraose and other Individual Human Milk Oligosaccharides
As can be seen in Figure 4 A, LNT— the core structure in human milk—is present in approximately 5 to 15 times higher concentrations compared to LNnT “in nonsecretor” milk and 3 to 5 times higher in “secretor” milk. In the latter, the major oligosaccharide is 2’FL with median concentrations between 2.76 g/L (mature milk) and 3.99 g/L (colostrum), followed by LNFP I which can be as high as 1.30 g/L (transitional milk, median) (Table 3 A and Fig. 4 B). In “nonsecretor” milk, the major single oligosaccharide is LNT with a median concentration between 1.65 and 2.92 g/L (Table 3 B and Fig. 4 A) followed by LNFP II with median levels ranging from 1.14 to 1.26 g/L. LNFP III, a component with the terminal Gal linked in β1-4 position to GlcNAc, characteristic for type 2 structures and not related to secretor or nonsecretor status, was detected in all samples with large IQRs, for example, between approximately 0.16 and 0.32 g/L in mature milk of secretors.
FIGURE 4: Concentration of specific human milk oligosaccharides (HMOs, g/L) in mature milk of secretor and nonsecretor donors. ∗∗ P < 0.01, ∗∗∗ P < 0.001; Black bars represent median levels. 2’FL = 2’-fucosyllactose; LNDFH = lacto-N-difucohexaose; LNT = lacto-N-tetraose; LNFP = lacto-N-fucopentaose.
Neutral Human Milk Oligosaccharides in “Lewis Blood Group” Milk
Because the pattern of neutral HMOs also depends on the Lewis blood group status, we further classified the samples into the following 3 milk groups depending on specific components as shown in Table 2 : group 1 as Lewis (a−b+), “secretor,” group 2 as Lewis (a+b−), “nonsecretor” and group 3 as Lewis (a−b−), “secretor.”
A comparison of the total HMO content in Lewis-specific mature milk samples revealed a much lower total HMO concentration in group 2 compared with groups 1 and 3 (group 1: median total HMOs: 8.46 g/L, IQR 7.95–9.42; group 2: median total HMOs: 5.54 g/L, IQR 4.10–8.20; group 3: median total HMOs: 9.62 g/L, IQR 7.57–10.77). This overall picture can also be found in the neutral HMO fractions of the 3 Lewis groups (see Supplemental Digital Content 2, Table 2, https://links.lww.com/MPG/A780
The distribution of individual HMOs within the 3 groups was different (Fig. 5 ). LNT together with the secretor-specific 2’FL, LNFP I, and difucosyllactose (LDFT) comprised approximately 50% of the total HMO content in mature milk in group 1, with 2’FL being the most abundant oligosaccharide (>30%). Despite the lack of Lewis-specific components in group 3, almost the same composition as in group 1 was observed. In group 3, 2’FL together with LNT and LNFP I comprised approximately 80% of the total oligosaccharides. In milk from Lewis a+b− individuals (group 2); however, the HMO pattern was different. Here, LNT concentrations were 2 to 3 times higher compared with the other groups and, together with LNFP II, LNT comprised approximately 55% of the total HMO content (see also Supplemental Digital Content 3, Table 3, https://links.lww.com/MPG/A781
FIGURE 5: Distribution of human milk oligosaccharides (HMOs, %) in mature milk of group 1, 2, and 3 donors. Others = 3FL + LNDFH I. DSLNT = disialyllacto-N-tetraose; 2’FL = 2’-fucosyllactose; LDFT = difucosyllactose; LNDFH = lacto-N-difucohexaose; LNFP = lacto-N-fucopentaose; LNT = lacto-N-tetraose; LST = sialyl-acto-N-tetraose; 6'SL = 6'sialyllactose.
Acidic Human Milk Oligosaccharides in “Lewis Blood Group” Milk
The concentrations of acidic components are neither related to the secretor nor to the Lewis blood group status of the mother. Individual acidic HMOs such as sialyllactose varied within their isoforms, that is, the concentration of 6'SL was about 3 times higher compared with 3'SL (Table 3 and Fig. 6 ). Among the 3 isoforms of sialyl-LNT, LSTb, and LSTc were present in similar but higher concentrations than LSTa. No difference, however, was found between the 3 groups of donors.
FIGURE 6: Concentration of acidic human milk oligosaccharides (HMOs, g/L) in mature milk of Lewis (a−b+), Lewis (a+b−), and Lewis (a−b−) milk; ∗∗ P < 0.01. Black bars represent median. DSLNT = disialyllacto-N-tetraose; LST = sialyllacto-N-tetraose; 6'SL = 6'sialyllactose.
Among the acidic component, which do not depend on the secretor status we found 4 milk samples without any disialyllacto-N-tetraose (Fig. 6 ). Because this is an unusual observation we tried to relate these data to the other acidic oligosaccharides in the same milk samples but could not find any association.
DISCUSSION
The recent commercial production of some smaller components on a large scale is already leading to the initiation of clinical studies with natural identical HMOs. Therefore, questions of how much of a single HMO or which combinations of oligosaccharides should be added to an infant formula need to be addressed. Hence, data about the amount of oligosaccharides in human milk are needed.
Comparing term and preterm milk samples, we did not find any difference in the total amount of HMOs neither in colostrum nor in transitional or in mature milk of both groups (Fig. 2 A–C, Supplemental Digital Content 1, Table 1, https://links.lww.com/MPG/A779 (26) (27) (33,34) (8)
Classifying our samples from Spain according to the secretor and nonsecretor status of the mother, we found large differences in the total amount of HMOs and also in the amount of individual components (Fig. 4 and Table 3 ). This was expected from the activity of the secretor-specific glycosyltransferase FUT-2 which catalyzes the linkage of fucose in α1–2 position to a backbone, for example, to the galactose moiety of lactose which in that case leads to the formation of 2’FL. The absence of α1–2-fucosylated components such as 2’FL, LNFP I, LDFT, and LNDFH I in “non-secretor” milk explains the lower total amount of HMOs (Fig. 2 and Table 3 ). Particularly 2’FL currently receives much attention because it has been used in the first human study in infants receiving an energy-reduced infant formula supplemented with 2’FL. Comparing their growth with that of breast-fed infants as control group revealed the same growth pattern (9) Campylobacter pylori , rotavirus, or norovirus infections (17,18,21,22)
Our data on high amounts of 2’FL in “secretor” milk, particularly in colostrum, and a decline within the first few weeks postpartum are in agreement with most of the published data, although we did not detect as much 2’FL as has been reported by others (27,28,33,35) Table 3 ). Although only the first 3 days have been investigated by Asakuma et al (36)
Regarding LNT, the major core structure for oligosaccharides in human milk, one can get the impression that the concentration in general is approximately 1 g/L in secretor milk with 2 outliers with much higher concentrations (Fig. 4 A). In nonsecretor milk, there is a group with roughly the same concentration as in secretor milk (n = 5) and another group (n = 6) with a concentration similar to the 2 outliers in this group. It is intriguing to speculate that the various LNT concentrations may be due to a different production and not dependent on Lewis blood group or secretor status. When we compared our data in term and preterm milk the same picture with 2 groups can be found. Further studies are certainly needed to make an unequivocal statement about the reasons for our observations.
Not focusing on quantitative but qualitative data De Leoz et al (37)
In addition to differences between “secretor” and “nonsecretor” milk, the HMO pattern also depends very much on the Lewis blood group system. The presence or absence of an active FUT 3 gene in the mammary gland expressing fucosyltransferases, which attach fucose either in α1–3 or α1–4 linkages to subterminal GlcNAc within HMOs together with a secretor-specific enzyme such as FUT 2 (responsible for α1–2 linkages) can lead to 4 different milk patterns shown in Table 1 . In our sample cohort we did not have any samples from group 4 (Lewis negative and nonsecretor). This is not surprising as the incidence of this pattern is <1% (5,25,32) Table 1 , Supplemental Digital Content 2, Table 2, https://links.lww.com/MPG/A780 https://links.lww.com/MPG/A781
Among the acidic components, which do not depend on the secretor status we found 4 milk samples without disialyllacto-N-tetraose not only in mature milk (Fig. 6 ) but also in colostrum and transitional milk of the same women. Although this is an unexpected observation, we could not find a clear relation to the other acidic oligosaccharides in the same milk samples. LSTa was detected at the lower concentration range in all 4 samples, but due to the large variation of LSTa in milk, a statistical evaluation cannot be made.
To summarize our data with regard to the effect of the secretor status of the mothers, it can be concluded that
In mature milk of “nonsecretors” compared to “secretors” the ratio of neutral to acidic oligosaccharides is 71/29 and 81/19.
In “nonsecretor” milk, the major HMOs were found to be LNT (53%) and LNFP II (21%), comprising >70% of the total HMO content (mature milk).
The higher amount of neutral HMOs in “secretor” milk was mainly caused by 2’FL (32%), LNFP I (13%), and LNT (11%) in mature milk.
Among the acidic components, 6'SL was the predominant HMO.
To summarize our observations with regard to different Lewis blood group specificities , we came to the following conclusions:
In milk of groups 1 (Lewis (a−b+), “secretors”) and 3 (Lewis (a−b−), “secretors”) there was
a similar HMO pattern in colostrum, transitional, and mature milk,
about the same total content of neutral and acidic HMOs among groups,
2’FL as the major component with a similar content of LNT,
a much lower, but similar content of LDFT, LNnT, LNFP III, and acidic HMOs,
in “Le (a−b+)” milk, Lewis-specific components LNFP II and LNDFH II are of minor importance in comparison to secretor-specific HMOs (2’FL, LDFT, LNFP I) in group 3.
In milk of group 2 (Le (a+b−), “nonsecretor” there was a clearly distinct HMO pattern as
LNT is the major component, its content being 2 to 3 times higher than that in groups 1 and 3.
There was a high content of the Lewis-specific components LNFP II and LNDFH II,
The percentage contribution of acidic HMO is much higher compared with groups 1 and 3 because the total HMO content is much lower compared the other 2 groups (Fig. 5 ).
An important issue in obtaining quantitative data is that no standardized methods are available for HMOs, which would allow an easy comparison of the data of different laboratories. Other factors that complicate a direct comparison of published data are that samples from different time points during lactation were analyzed, potential variations of HMO concentration during the day may occur, or different collection methods (manual expression vs breast pump) may have been used.
We come to the conclusion that the total amount of HMOs in term milk does not differ from that in preterm milk. In general, there were large individual variations of almost all components and changes during lactation. Differences in the total amount of HMOs, between neutral and acidic oligosaccharides and depending on the secretor and the Lewis blood group status should be taken into account when considering studies to prevent or influence various diseases.
Acknowledgments
The authors would like to thank all the mothers participating in the study. The authors gratefully acknowledge Parisa Kodhayar-Pardo, MD and Virtudes Molina Sevilla, MSc for assistance in conducting the clinical study and recruitment and for assistance with the samples processing.
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