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ADDITIONAL FILE 2: TABLE S1
List of metabolite integrals and assignments obtained from NMR urine spectra
Metabolite ID
1
2
3
4
5
6
7
8
9
10
11
Pregnanolone-3G
Pregnanediol-3G
UA 0.75(d)
Estrogen metabolite
UA 0.93(s)
Leucine
Isoleucine
Valine
3-hydroxyisobutyrate
4-deoxyerythronic acid
UA 1.15 (d) [3.45, 3.54]
12 3-hydroxybutyrate/3aminoisobutyrate
13 4-deoxythreonic acid
14 UA 1.25
15 3-hydroxyisovalerate
16 UA 1.29(s)
17 Lactate
18 2-hydroxyisobutyrate
19 Alanine
20 UA 1.67(s)
21 Lysine
Abbreviation
Chemical
shift
Integral
range 1
(ppm)
Integral
range 2
(ppm)
Assignment
Metabolic class
Pn3-G
P3-G
Leu
Iso
Val
3-HB
4-DEA
-
0.58 (s)
0.63 (s)
0.75 (d)
0.78 (s)
0.93 (s)
0.96 (t)
1.01 (d)
1.05 (d)
1.08 (d)
1.11 (d)
1.15 (d)
0.55
0.6176
0.741
0.7774
0.9311
0.9503
1.005
1.034
1.063
1.097
1.132
0.57
0.64
0.76
0.79
0.94
0.97
1.03
1.06
1.09
1.12
1.16
Steroid and steroid derivates
Steroid and steroid derivates
Steroid and steroid derivates
Steroid and steroid derivates
Unknown
BCAAs catabolism
BCAAs catabolism
BCAAs catabolism
BCAAs catabolism
Glucose metabolism
Glucose metabolism
3-HB/3-AB
1.20 (ov.)
1.17
1.21
Fraction, spike-in*
Fraction, spike-in, LC-MS/MS
Fraction, Spike-in
2D NMR
2D NMR
2D NMR
2D NMR, fractions, spike-in
2D NMR
2D NMR (organic acid with similar
structure to 3-hydroxyisobutyrate)
2D NMR
4-DTA
3-HV
Lac
2-HB
Ala
Lys
1.24 (d)
1.25 (d)
1.27 (s)
1.222
1.244
1.264
1.282
1.32
1.355
1.472
1.655
1.701
1.24
1.26
1.28
1.29
1.35
1.37
1.51
1.68
1.76
2D NMR
2D NMR
2D NMR
2D NMR, spike-in
2D NMR
2D NMR, Chenomx
Glucose metabolism
Unknown
Glucose metabolism
Unknown
Glucose metabolism
BCAAs catabolism
Glucose metabolism
Unknown
Glucose metabolism
1.33 (d)
1.36 (s)
1.48 (d)
1.67 (s)
1.73 (m)
1
Glucose metabolism
22 Acetate
23 UA 1.99(t)
24 N-acetyl glycoprotein fragments
NAG
1.92 (s)
1.99(t)
2.04 (s)
1.914
1.97
2.022
1.93
2.00
2.05
Literature
Literature
25 N-acetyl neuraminic acid
NAC
2.06 (s)
2.054
2.09
Literature
26
27
28
29
PCS
5-OP
-
2.24 (s)
2.35 (s)
2.40 (m)
2.41 (s)
2.229
2.339
2.387
2.4
2.25
2.355
2.40
2.42
Literature
2D NMR
2D NMR, fractions, spike-in
Literature
Glu
-
2.45 (m)
2.55 (d)
2.43
2.5
2.47
2.58
2D NMR
2D NMR
2.587
2.716
2.774
2.818
2.861
2.9
2.923
3.027
2.64
2.74
2.79
2.85
2.88
2.91
2.94
3.07
2D NMR
Literature
Literature
2D NMR, spike-in
Spike-in
Literature
3.201
3.223
3.261
3.297
3.362
3.21
3.24
3.29
3.31
3.37
Literature
Literature
Literature
Literature
Literature
3.561
3.58
Literature
Acetone
P-cresol sulfate
Pyroglutamate (5-oxoproline)
Succinate
30 Glutamine
31 Citrate
32
33
34
35
36
37
38
39
3-aminoisobutyrate
Dimethylamine
UA 2.78(s)
UA 2.83(s)
Trimethylamine
UA 2.9 (N-acetyl peak)
Dimethylglycine
Creatinine+creatine
3-AB
DMA
TMA
DMG
Creat2
40
41
42
43
44
Choline
Carnitine
Trimethylamine oxide
Proline betaine
Scyllo-inositol
TMAO
PB
SIN
2.62 (m)
2.72 (s)
2.78 (s)
2.83 (s)
2.87 (s)
2.90
2.93 (s)
3.05 (s)
ov.
3.20 (s)
3.23 (s)
3.27 (s)
3.30 (s)
3.36 (s)
Gly
3.57 (s)
45 Glycine
2
TCA cycle
Unknown
Amino sugar and nucleotide
sugar metabolism
Amino sugar and nucleotide
sugar metabolism
Oxidative stress
Bacterial origin
Oxidative stress
TCA cycle and mitochondrial
oxidative stress
Glucose metabolism
TCA cycle and mitochondrial
oxidative stress
BCAAs catabolism
Alkylamines
Unknown
Unknown
Alkylamines/bacterial origin
Oxidative stress
Unknown
Amino Acids and Derivatives
Gluconeogenesis
Oxidative stress
Alkylamines/bacterial origin
Pyrrolidines
Nicotinate and nicotinamide
metabolism
Gluconeogenesis
46
47
48
49
50
51
52
UA 3.8(s)
UA 3.93(s)
UA 3.95(s)
UA 6.48(d)
Fumarate
UA 6.59(s)
Furoylglycine
FG
3.8(s)
3.93(s)
3.95(s)
6.48(d)
6.52 (s)
6.59 (s)
6.64 (dd)
3.792
3.927
3.946
6.477
6.521
6.585
6.636
3.81
3.94
3.96
6.49
6.53
6.61
6.66
Literature
2D NMR, fractions
2PY
6.66 (s)
6.657
6.67
2D NMR
HPA
Tyr
PAG
3-IS
-
6.87 (d)
6.90 (d)
7.32
7.37 (t)
7.51 (d)
7.55 (t)
7.68(s)
8.46 (s)
8.79(d)
6.851
6.89
7.306
7.343
7.494
7.527
7.672
8.453
8.772
6.88
6.92
7.34
7.38
7.52
7.58
7.69
8.47
8.81
2D NMR, STORM**, spike-in
2D NMR
2D NMR
2D NMR, spike-in
2D NMR
Literature
-
63 N-Methylnicotinic acid
NMNA
9.13 (s)
9.113
9.14
2D NMR, spike-in
64 N¹-Methyl-nicotinamide
NMN
9.28 (s)
9.269
9.30
2D NMR, spike-in
53 N-methyl-2-pyridone-5carboxamide
54 p-hydroxyphenylacetate
55 Tyrosine
56 UA 7.32
57 Phenylacetylglutamine
58 3-Indoxylsulfate
59 Hippurate
60 UA 7.68(s)
61 Formate
62 UA 8.79(d)
unknown
Unknown
Unknown
Unknown
Oxidative stress
Unknown
Nicotinate and nicotinamide
metabolism
Nicotinate and nicotinamide
metabolism
Bacterial origin
Amino Acids and Derivatives
Unknown
Bacterial origin
Bacterial origin
Bacterial origin
Unknown
Organic acids
Nicotinate and nicotinamide
metabolism
Nicotinate and nicotinamide
metabolism
Nicotinate and nicotinamide
metabolism
Abbreviations: UA, unassigned
* The method of multiple spike-in of authentic standards in the original sample allowed to validate the identity of NMR signals. The identity of P3G was confirmed by
chromatographic isolation of the target feature (SI Methods) and comparison of MS/MS spectra to an authentic reference compound in accordance with reported
guidelines for metabolite identification (14).
3
**Citation for STORM: Subset Optimization by Reference Matching (STORM): An Optimized Statistical Approach for Recovery of Metabolic Biomarker Structural
Information from 1H NMR Spectra of Biofluids. Joram M. Posma, Isabel Garcia-Perez, Maria De Iorio, John C. Lindon, Paul Elliott, Elaine Holmes, Timothy M. D. Ebbels, and
Jeremy K. Nicholson. Analytical Chemistry 2012 84 (24), 10694-10701 DOI: 10.1021/ac302360v
4