SUPPLEMENTARY MATERIAL
Exploring Human Breast Milk composition by NMR-based
Metabolomics
Giulia Praticò1, Giorgio Capuani1, Alberta Tomassini1, Maria Elisabetta
Baldassarre2, Maurizio Delfini1 and Alfredo Miccheli1*
1
Department of Chemistry, Sapienza University of Rome, Italy.
Department of Gynecology, Obstetrics and Neonatology, Section of Neonatology and
NICU, University of Bari, Italy
2
*Corresponding Author: Prof. Alfredo Miccheli, Department of Chemistry, Sapienza
University of Rome, p.le Aldo Moro 5, 00185 Rome, Italy. Tel. ++39 06 49693270
Fax: ++39 06 49913214; e-mail: alfredo.miccheli@uniroma1.it
Exploring Human Breast Milk composition by NMR-based
Metabolomics
Breast milk is a complex fluid evolutionarily adapted to satisfy the nutritional
requirements of growing infants. In addition, milk biochemical and
immunological components protect newborns against infective agents in the
new environment. Human milk oligosaccharides (HMOs), the third most
abundant component of breast milk, are believed to modulate the microbiota
composition, thus influencing a wide range of physiological processes of the
infant. Human milk also contains a number of other bioactive compounds the
functional role of which has not yet been clearly elucidated. In this scenario,
NMR-based metabolic profiling could provide a rapid characterization of
breast milk composition thus allowing a better understanding of its nutritional
properties.
Keywords: NMR, 1H, HMO, milk metabolome, metabolic profiling
Experimental
NMR Spectroscopy
The pulse sequence adopted for 1H NMR spectra acquisition was a presaturation
pulse-single 90° detection pulse-acquire – relaxation delay. The relaxation delay was
7.5 s, while the presaturation pulse was applied for 2 s; the acquisition time needed to
collect the 32 K points was about 5.5 s, therefore the proton nuclei different from the
solvent ones were allowed to relax for 15 s, complying with the full relaxation
condition after a 90 degree pulse. The length of the detection pulse was calibrated
previously to the acquisition of each spectrum, spectral width was set to 5995.02 Hz
(12 ppm), 64 scans were collected for each spectrum preceded by 4 dummy scans.
Double quantum filter 1H-1H Correlation Spectroscopy (COSY) spectrum was
recorded at 298 K with a spectral width of 15 ppm in both dimensions, employing a
matrix of 4k x 400 data points, a repetition time of 2 s and 88 scans.
Total Correlation Spectroscopy (TOCSY) experiment was performed at 298 K
with a spectral width of 12 ppm in both dimensions, employing a matrix of 4k x 256
data points, a repetition time of 2 s, 112 scans. TOCSY spectra were acquired with
several mixing times. In particular, they were optimized to evaluate the scalar
correlation between H-1 and H-6 of the fucosylated moieties, on the basis of the long
range scalar coupling constants.
The 1H-13C Heteronuclear Single Quantum Correlation (HSQC) spectrum was
recorded with the echo–antiecho phase-selective mode, with a spectral width of 12
and 200 ppm in proton and carbon dimensions respectively, employing a matrix of 4k
x 400 data points for f2 and f1 dimensions, a repetition delay of 2 s and 96 scans. The
average heteronuclear coupling constant was 145 Hz.
The 1H-13C Heteronuclear Multiple Bond Correlation (HMBC) spectrum was
obtained with 12 and 220 ppm spectral widths in proton and carbon dimensions
respectively, employing a matrix of 4k x 400 data points for f2 and f1 dimensions, a
repetition delay of 1.5 s and 96 scans. The long range heteronuclear coupling constant
was 8 Hz, while the direct coupling constants comprised between 130 and 170 Hz
were filtered.
The Diffusion Order Spectroscopy DOSY experiment was acquired with a
spectral width of 12 ppm for proton, 64 k data point, 144 scans, 4 dummy scans and a
repetition delay of 5.16 s. The self-diffusion coefficients were evaluated with 32
gradient steps, a diffusion time (∆) of 350 ms and a gradient length (δ) of 2.5 ms.First
paragraph style: use this for the first paragraph in a section.
Table S1. List of metabolites identified in the hydro-alcoholic extract of human milk
by 2D NMR TOCSY, HSQC and HMBC experiments.
N°
Name
1
2-oxoglutarate
Group
Fuc(α1-2)
2
2’FL
Gal(β1-4)
Fuc(α1-3)αGlc
3
3’FL
Fuc(α1-3)βGlc
Neu5Ac(α2-3)
4
3’SL
5
4-hydroxy-phenylacetate
Neu5Ac(α2-6)
Assignment 1H (ppm)
β-CH2
γ-CH2
CH-1
CH-5
CH-4
CH3-6
CH-1
CH-4
CH-2
CH-1
CH-5
CH-3
CH-4
CH3-6
CH-1
CH-5
CH-3
CH-4
CH-2
CH3-6
CH-5
CH-6
CH-3
CH-3’
CH-2,6 ring
CH-3,5 ring
CH-5
CH-6
CH-3
CH-3’
3.00
2.44
5.32
4.23
3.81
1.24
4.53
3.91
3.67
5.39
4.82
3.98
3.86
1.19
5.44
4.81
3.96
3.82
3.68
1.19
3.87
3.70
2.73
1.78
7.15
6.85
3.86
3.72
2.73
1.73
13C
(ppm)
33.58
102.3
69.9
18.34
103.2
101.51
18.34
101.22
69.51
18.34
54.20
42.90
42.90
54.20
6
6’SL
7
Acetic Acid
β-CH3
1.95
8
Alanine
α-CH
β-CH3
3.70
1.48
19.05
9
Carnitine
N(CH3)3
3.23
56.91
10
Choline
N(CH3)3
3.22
56.99
11
Citric Acid
α,γ-CH
α',γ'-CH
CH-1
CH-4
CH-3
CH3-6
2.67
2.54
5.01
3.84
3.58
1.18
47.86
47.86
Fuc(α1-3)GlcNAc
12
Fucose moiety (1)
42.90
42.90
18.34
Fuc(α1-3)GlcNAc
13
14
15
16
17
18
19
20
21
22
CH-1
CH-5
Fucose moiety (2)
CH-3
CH-4
CH3-6
Gal(β1-4)GlcNAc
CH-1
CH-4
Galactose moiety (1)
CH-6
CH-5
Gal(β1-4)GlcNAc
CH-1
CH-4
Galactose moiety (2)
CH-6
CH-5
Gal(β1-4)GlcNAc
CH-1
CH-4
Galactose moiety (3)
CH-6
CH-6’
CH-5
βGlc
CH-1
CH-6
Glucose
CH-6’
CH-3
CH-2
α-CH
γ-CH2
Glutamic Acid
β-CH
β'-CH
CH-2 ring
Histidine
CH-4 ring
α-CH
β-CH
γ'-CH2
Isoleucine
γ-CH2
β-CH3
δ-CH3
α-CH
Lactic Acid
β-CH3
Gal(β1-4)
CH-1
CH-4
CH-6
CH-3
CH-2
αGlc
CH-1
Lactosyl moiety
CH-4
CH-6
CH-5
CH-3
CH-2
βGlc
CH-1
CH-4
5.19
4.30
3.96
3.77
1.24
4.44
4.15
3.73
3.57
4.45
4.10
3.73
3.59
4.45
4.12
3.86
3.69
3.54
4.65
3.93
3.77
3.50
3.30
3.77
2.36
2.14
2.08
7.95
7.05
3.66
1.98
1.47
1.26
1.01
0.91
4.09
1.33
4.47
3.96
3.69
3.68
3.58
5.22
3.96
3.87
3.83
3.67
3.59
4.69
3.96
102.41
69.50
18.34
103.2
71.34
103.21
71.24
103.2
71.50
98.82
36.29
29.89
22.99
105.90
71.39
75.48
73.91
94.92
63.02
62.82
74.17
81.21
98.63
63.02
Fuc(α1-2)
Fuc(α1-3)αGlc
23
LDFT
Fuc(α1-3)βGlc
Fuc(α1-2)
Fuc(α1-4)
24
LNDFHI and branched
GlcNAc(β1-6)
Fuc(α1-4)
Fuc(α1-3)αGlc
25
LNDFHII
Fuc(α1-3)βGlc
Gal(β1-4)
26
LNFPI and branched
Fuc(α1-2)
CH-6
CH-3
CH-2
CH-1
CH-5
CH-4
CH3-6
CH-1
CH-5
CH-3
CH-4
CH3-6
CH-1
CH-3
CH-4
CH3-6
CH-1
CH-5
CH-3
CH-4
CH3-6
CH-1
CH-5
CH-3
CH-4
CH-2
CH3-6
CH-1
CH-3
CH-6
CH-4
CH-5
CH3
CH-1
CH-3
CH-4
CH3-6
CH-1
CH-3
CH-4
CH-1
CH-3
CH-4
CH3-6
CH-1
CH-4
CH-2
CH-1
CH-3
CH3-6
3.81
3.68
3.30
5.29
4.26
3.81
1.28
5.40
4.82
4.00
3.83
1.19
5.46
3.98
3.83
1.23
5.16
4.35
4.01
3.77
1.29
5.03
4.88
3.98
3.82
3.65
1.26
4.61
4.14
3.83-3.96
3.75
3.54
2.08
5.03
3.96
3.82
1.18
5.38
3.98
3.79
5.43
3.98
3.79
1.14
4.51
3.91
3.54
5.32
4.01
1.24
63.16
77.27
76.69
102.30
18.34
101.50
18.34
101.21
18.34
102.70
69.51
18.34
106.23
25.30
100.81
18.34
101.71
101.50
18.34
105.81
GlcNAc
CH3
2.06
Fuc(α1-4)
27
28
29
30
31
32
33
CH-1
5.03
CH-5
3.96
LNFPII
CH-3
3.82
CH3-6
1.18
Fuc(α1-3)GlcNAc
CH-1
5.11
CH-5
4.83
CH-3
3.91-4.01
CH-4
3.82
LNFPIII and branched
CH3-6
1.19
Fuc(α1-3)GlcNAc
CH-1
5.13
CH-5
4.85
CH-3
3.91
CH-4
3.82
CH3-6
1.19
Fuc(α1-3)αGlc
CH-1
5.39
CH-5
4.82
CH-3
3.98
CH-4
3.86
CH3-6
1.19
LNPV
Fuc(α1-3)βGlc
CH-1
5.44
CH-5
4.81
CH-3
3.96
CH-4
3.82
CH-2
3.68
CH3-6
1.19
α-CH
3.68
ε-CH2
3.00
Lysine
β-CH2
1.88
γ-CH2
1.69
δ-CH2
1.47
CH-2 ring
7.85
Me-Histidine
CH-4 ring
7.06
N-CH3
3.09
CH-2 ring
7.80
Me-Histidine
CH-4 ring
7.07
N-CH3
3.12
CH-1
4.06
CH-2,6
3.63
Myo-inositol
CH-3,5
3.54
CH-4
3.28
100.81
18.34
102.40
69.50
18.34
101.51
18.34
101.51
18.34
101.22
69.51
18.34
42.29
29.06
75.12
34
N-acetylcarnitine
N(CH3)3
3.21
56.80
35
N-Acetyl moieties (1)
CH3
2.06-2.08
25.30
36
N-Acetyl moieties (2)
CH3
2.03-2.05
25.04
37
Phenylalanine
38
Succinic Acid
CH-3,4,5 ring 7.38-7.40
CH-2,6 ring 7.22
α-CH
3.92
β-CH
3.6
β’-CH
3.21
β-CH2
2.40
β-CH
4.26
α-CH
3.54
γ-CH3
1.34
CH-2,6 ring 7.20
40
Tyrosine
CH-3,5 ring 6.90
CH-2,6 ring 7.19
41
U1
CH-3,5 ring 6.83
α-CH
2.19
40.40
γ-CH
1.57
42
Valeric Acid
β-CH2
1.29
CH3
0.90
26.69
α-CH
3.59
β-CH
2.32
43
Valine
γ-CH3
1.05
20.85
γ'-CH3
0.99
19.47
List of abbreviations: 2'FL, 2' fucosyllactose; Fuc Fucose; Gal Galactose; 3'FL, 3'
fucosyllactose; Glc Glucose; 3’SL 3’sialyl-lactose; Neu5Ac N-acetylneuraminc acid;
6’SL 6’sialyl-lactose; LDFT, lactodifucotetraose; LNDFHI- II, lacto-N-difucohesaose
I-II, LNFPI –III, V, lacto-N-fucopentaose I-III, V; GlcNAc N-acetylglucosamine.
39
Threonine
Table S2. Quantification of shared metabolites in milk group 1 (Se+/Le+) and
milk group 2 (Se-/Le+) .
Milk Group 1 (Se+/Le+)
(µM)
Isoleucine
Valine
Fuc (α1-3)+Fuc(α1-4)
Lactic Acid
Alanine + Lysine
Acetic Acid
GlcNAc moiety 1
GlcNAc moiety 2
GlcNAc moiety 3
GlcNAc moiety 4
Glutamic Acid
Succinic Acid
Citric Acid
N-acetylcarnitine
Choline
Carnitine
Myoinositol
Lactose
Fucose moiety 1
Fuc(α1-4)
LNFPIII
Fucose moiety 2
Phenylalanine
3’FL
LNDFH II
GlcNAc total
0.8
14.8
2027.1
72.1
139.3
52.5
1384.5
1663.8
1261.8
1575.0
1045.6
20.9
1418.8
108.7
414.8
362.8
1525.9
143800.5
339.0
1260.1
534.7
1430.4
1387.7
1051.6
53.2
8974.8
Milk Group 2 (Se-/Le+)
(µM)
(1.2)
(18.8)
(1057.5)
(50.5)
(68.6)
(37.0)
(963.1)
(755.5)
(606.8)
(854.5)
(416.3)
(20.1)
(697.5)
(61.8)
(274.2)
(116.9)
(585.2)
(23088.2)
(82.3)
(451.6)
(266.9)
0.8
18.6
5875.9
96.5
132.1
56.6
2897.6
1948.3
2162.8
1436.0
966.7
19.4
1633.8
75.2
573.6
364.6
2125.1
147288.8
426.2
1756.0
543.4
(80.8)
(6.6)
(2632.4)**
(70.9)
(29.4)
(17.5)
(1139.6)*
(1213.2)
(1093.1)
(1020.1)
(161.4)
(19.7)
(1049.3)
(46.9)
(202.6)
(171.0)
(863.6)
(57686.1)
(139.8)
(907.2)
(328.7)
(309.6)
(1224.9)
(582.4)
(40.1)
(3064.2)
1163.5
783.9
2571.7
365.5
10293.1
(748.1)
(1151.2)
(1031.7)*
(249.3)*
(3707.3)
Data are presented as medians of concentration (µM) and ranges from the 1st to the 3rd
quartile. *p<0.001; ** p<0.01 between values. List of abbreviations: Fuc Fucose;
3'FL, 3' fucosyllactose; LNDFH II, lacto-N-difucohesaose II, LNFPIII, lacto-Nfucopentaose III; GlcNAc N-acetylglucosamine.
Figure S1. 1H-1H TOCSY NMR experiment of the polar extract of mature breast milk;
the figure displays the TOCSY spectral pattern of fucose units of 2’fucosyllactose
(2’FL) and 3’fucosyllactose (3’FL), whose resonances correspond exactly with those
of lacto-N-fucopentaose V (LNFPV), due to the strong structural similarity.
List of abbreviations: 2'FL, 2' fucosyllactose; 3'FL, 3' fucosyllactose; LDFT,
lactodifucotetraose; LNDFHII, lacto-N-difucohesaose II, LNFPI, lacto-Nfucopentaose I.
Figure S2. 1H-13C HSQC NMR experiment of the polar extract of mature breast milk,
detail of H-1 fucose resonances.
List of abbreviations: 2'FL, 2' fucosyllactose; 3'FL, 3' fucosyllactose; LNDFHI, lactoN-difucohesaose I, LNFPIII, lacto-N-fucopentaose III.