SUPPLEMENTARY INFORMATION
A Comparative Metabolomics Approach Reveals Early Biomarkers for Metabolic Response to Acute Myocardial Infarction Sara E. Ali 1, Mohamed A. Farag 2*, Paul Holvoet 3, Rasha S. Hanafi 4, Mohamed Z. Gad 5 1
Department of Pharmaceutical Biology, Faculty of Pharmacy & Biotechnology, The
German University in Cairo, Egypt 2
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, 11562,
Egypt 3
Department of Cardiovascular Sciences, Atherosclerosis and Metabolism Unit,
Katholieke Universiteit Leuven, Belgium 4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy & Biotechnology, The
German University in Cairo, Egypt 5
Department of Biochemistry, Faculty of Pharmacy & Biotechnology, The German
University in Cairo, Egypt
*Corresponding author:
[email protected],
[email protected], Tel: +20-1004142567
S-1
Table of contents Supplementary Tables Page S-3
S-11
Supplementary Description Table Summary of GC/MS analysis identified metabolites in serum S1 samples from STEMI patients, UA patients and healthy controls. Summary of 1H-NMR analysis identified metabolites in S2 serum samples from STEMI patients, UA patients and healthy controls.
Supplementary Figures Page S-6
S-7
S-8
S-9 S-10 S-14 S-15 S-16
Supplementary Description Figure S1 GC/MS based OPLS-DA of STEMI patients ( ) versus healthy controls ( ) after removing major peaks revealed from the first OPLS analysis. GC/MS based PCA of the fatty acids profile of STEMI S2 patients ( ), before stent samples of unstable angina patients ( ) and healthy controls ( ). GC/MS based OPLS-DA of the fatty acids profile of STEMI S3 patients ( ), before stent samples of unstable angina patients ( ) and healthy controls ( ). SPME-GC/MS of a healthy control ( ) and a STEMI S4 patient ( ). A 600 MHz 1H-NMR spectrum of a healthy human serum S5 with expanded spectral region (B and C). 1 S6 H-NMR based PCA of STEMI patients ( ) versus healthy controls ( ). 1 H-NMR quantification of target compounds in serum of S7 STEMI patients versus healthy controls. S8 NMR based OPLS-DA of STEMI patients ( ) versus healthy controls ( ).
S-2
Table S1 Summary of GC/MS analysis identified metabolites in serum samples from STEMI patients, UA patients and healthy controls. Peak
Metabolite
rt (min)
m/z
RI
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36
unknown Ethanol,2-(methylamino) Hydrogen sulfide (2 TMS) Pentanoic acid (1 TMS) unknown β-Hydroxybutyric acid (2 TMS) Hydrocarbon β-Hydroxybutyric acid (2 TMS) Lactic acid (2 TMS) 2-Aminobutyric acid (2 TMS) Hexanoic acid (1 TMS) Acetic acid (2 TMS) Pyruvic acid (2 TMS) L-Valine (1 TMS) Alanine (2 TMS) Malonic acid (2 TMS) 2-Ketoisocaproic acid (1 TMS) α-Hyroxyisobutyric acid (2 TMS) β-Hydroxybutyric acid (2TMS) α-Hydroxyvaleric acid (2 TMS) L-Valine (2 TMS) Urea (2 TMS) Benzoic acid (1 TMS) Serine (2 TMS) Glycerol (3 TMS) L-Isoleucine (2 TMS) L-Proline (2 TMS) Glycine (3 TMS) Tridecane Glyceric acid (3 TMS) L-Serine (3 TMS) L-Threonine (3 TMS) Aminomalonic acid (3TMS) Aspartic acid (3 TMS) L-Norvaline (2 TMS) Erthyritol (4 TMS)
6.107 7.136 7.612 8.076 8.252 8.67 9.701 10.317 10.358 10.525 10.636 10.757 11.123 11.19 11.591 11.632 11.811 12.306 13.235 13.435 14.909 15.377 15.85 16.116 16.54 17.182 17.334 17.575 17.901 18.19 19.019 19.768 21.955 22.06 22.405 22.973
147 128 171 159 191 117 136 117 191 130 173 205 217 174 190 204 143 131 191 219 218 189 179 132 205 158 142 174 112 189 204 218 218 232 188 217
912 948 964.9 981 984 1011 1037.9 1054 1060.9 1065 1070.7 1075 1087.6 1099 1104 1107 1112 1128.6 1160.5 1165 1220 1240.1 1251.4 1260.5 1275.8 1298.8 1304.4 1313.8 1325.4 1336.1 1366.6 1394.4 1479 1483 1497 1520
RI Golm library
955.7
1036.9 1044.7 1062 1081.7 1087.4
1112.5 1148.4 1207.1 1235.1 1251.3 1254 1262.2 1286.7 1295.7 1302.1 1293.4 1320.3 1351.3 1377.2
1493.3 S-3
M37 M38 M39 M40 M41 M42 M43 M44 M45 M46 M47 M48 M49 M50 M51 M52 M53 M54 M55 M56 M57 M58 M59 M60 M61 M62 M63 M64 M65 M66 M67 M68 M69 M70 M71 M72 M73 M74 M75 M76 M77 M78
Pyroglutamic acid (2 TMS) Erythronic acid (4 TMS) α-Aminoadipic acid (3 TMS) Creatinine (3 TMS) L-Valine (2 TMS) L-Proline (2 TMS) Glutamine (3 TMS) L-Phenylalanine (2 TMS) Dodecanoic acid (1 TMS) N-acetyl glycine (2 TMS) Xylitol (5TMS) Citrulline (4 TMS) Citrulline (4 TMS) D-Mannose (5 TMS) D-Fructose (5 TMS) Citric acid (4 TMS) D-Fructose (5 TMS) Myristic acid (1 TMS) Myo-Inositol (6 TMS) Mannose (5 TMS) Glucopyranose (5 TMS) D-Galactose (5 TMS) D-Mannose (5 TMS) Lysine (4 TMS) Histidine (3 TMS) Pentanoic acid (1 TMS) L-Tyrosine (3 TMS) Glucitol (6 TMS) α-D-Glucopyranose (5 TMS) Palmitelaidic acid (1 TMS) Palmitic acid (1 TMS) N-acetylglutamine (4 TMS) Uric acid (4 TMS) Myo-inositol (6 TMS) Heptadecanoic acid (1 TMS) Linoleic acid (1 TMS) Oleic acid (1 TMS) Stearic acid (1 TMS) L-Tryptophan (3 TMS) Pseudo uridine (5 TMS) Arachidonic acid (1 TMS) Oleamide (1 TMS)
23.29 23.875 24.048 24.276 24.553 24.727 25.318 25.877 26.117 26.79 27.89 29.077 29.077 30.089 30.197 30.21 30.424 30.457 31.4 31.895 31.967 32.141 32.213 32.256 32.322 32.431 32.636 32.768 33.581 33.942 34.38 35.348 35.865 35.947 36.226 37.526 37.6 37.967 38.101 40.052 40.3 40.981
156 292 260 115 186 216 246 218 257 288 217 156 155 204 217 273 204 285 217 147 204 217 204 174 154 299 218 319 204 311 313 203 441 318 327 337 339 341 202 217 129 338
1532.9 1556.9 1564 1572.2 1585 1591.8 1617 1641.1 1651.5 1681 1730 1784 1784 1831 1836.4 1839 1847 1849 1913 1918.5 1922 1931 1934.5 1936.5 1939.8 1946.4 1955.6 1962 2003.1 2022 2045 2096 2124.6 2128.8 2144.4 2215.9 2220 2240.8 2247.5 2355 2368.4 2400
1521.7 1529 1553.7 1583.1 1629.6 1654.1 1660.3 1694.6
1789.8
1880 1879.9 1959.2 1913.6 1914.5 1945.5 1934.1 1959.2
2094.6 2080.3 2144.6 2219.1 2216.6 2243.5 2217.5 2383.7
S-4
M79 M80 M81 M82 M83 M84 M85
Arachidic acid (1 TMS) Monopalmitoyl glycerol (2 TMS) Docosahexanoic acid (1 TMS) Monopalmitoyl glycerol isomer (2 TMS) Cholesterol (1 TMS) 1-Monooleoylglycerol (2 TMS) Monostearin (2 TMS)
41.316 43.191 43.37 43.716 43.868 46.286 46.572
369 218 159 371 368 397 399
2424.3 2527 2537.3 2556 2564.5 2688 2713
2453.8 2575.9 3188.2
S-5
Figure S1 GC/MS based OPLS-DA of STEMI patients ( ) versus healthy controls ( ) after removing major peaks revealed from the first OPLS analysis. (A) OPLS-DA score plot (B) loading S-plot derived from samples of the two groups modeled against each other. The S-plot shows the covariance p[1] against the correlation p(cor)[1] of the variables of the discriminating component of the OPLS-DA model. Selected variables are highlighted in the S-plot with each metabolite denoted by its mass/RT (min) value: M18; αhydroxyisobutyric acid, M41; valine, M48&49; citrulline, M67; palmitic, M69; uric acid. Peak numbers correspond to those listed in (Supplementary Table S1).
A
Control
p(cor) (23.9%)
STEMI
p(27.5%)
B
M49
M48
M67
M41 M18
M69
S-6
Figure S2 GC/MS based PCA of the fatty acids profile of STEMI patients ( ), before stent samples of unstable angina patients ( ) and healthy controls ( ). (A) Score plot of PC1 and PC2 scores (B) Loading plot for PC1 components contributing peaks and their assignments, with each metabolite denoted by its mass/rt (min) value: M67; palmitic acid, M72; linoleic acid, M73; oleic acid, M74; stearic acid. Peak numbers correspond to those listed in (Supplementary Table S1). Control
A
STEMI
PC2 (26.6%)
UA (before stent)
PC1 (48%)
B
M73
M74 M67
M72
S-7
Figure S3 GC/MS based OPLS-DA of the fatty acids profile of STEMI patients ( ), before stent samples of unstable angina patients ( ) and healthy controls ( ). (A) OPLS-DA score plot (B) loading plot derived from samples modeled against each other. The loading plot shows the covariance p[1] against the correlation p(cor)[1] of the variables of the discriminating component of the OPLS-DA model. Selected variables are highlighted in the S-plot with each metabolite denoted by its mass/RT (min) value: M67; palmitic acid. Peak numbers correspond to those listed in (Supplementary Table S1).
A
Control STEMI
p(cor) (4.3%)
UA (before stent)
p (18.7 %)
B
M67
S-8
Figure S4 SPME-GC/MS of a healthy control (
) and a STEMI patient (
).
Intens. x106
STEMI Control
3
2
Acetone STEMI 1
Control
0 2
serum acute-cdf.cdf: TIC +
4
6
8
10
Time [min]
12
SERUM CONTROL-2.cdf: TIC +
S-9
Figure S5 A 600 MHz 1H-NMR spectrum of a healthy human serum with expanded spectral region (B and C).
A
Lactic acid
Sugar region D2O
Glycerol β-hydroxybutyric acid
β-glucose α-glucose
DSS
Chemical shift (ppm)
B
C
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Table S2 Summary of 1H-NMR analysis identified metabolites in serum samples from STEMI patients, UA patients and healthy controls. Number
Metabolite
N1
1-Methylhistidine
N2
1,2-Propanediol
N3 N4
2-Hydroxyisovaleric acid 2-Oxoisocaproic acid
N5 N6
2-Oxoisovaleric acid β-Hydroxybutyric acid
N7 N8 N9 N10 N11 N12 N13
3-Hydroxyisovaleric acid Acetic acid Acetone Acetylcarnitine N-Acetylglycine Alanine Arginine
N14
Arginine/Lysine*
N15
Asparagine
N16 N17 N18 N19
Aspartic acid Betaine Carnitine Choline
N20 N21
Citric acid Creatine
N22
Creatinine
N23 N24 N25
Dimethylglycine Dimethylamine/Sarcosine* Ethanolamine
1
H shift (ppm)
Multiplicity
7.114 7.927 1.144 3.441 3.882 2.017 0.934 2.085 2.612 1.126 1.200 2.305 2.414 4.156 1.269 1.916 2.233 3.194 2.054 1.433 1.664 3.226 1.722 1.734 1.909 2.972 2.929 3.900 3.268 3.235 3.200 3.506 4.052 2.545 3.038 3.933 3.044 4.050 2.942 2.730 3.131
s s d dd m m d m d d d m m m s s s s s d m t m m m d d dd s s s dd m d s s s s s s d
Coupling constant (Hz)
J=6.5 J=11.5, 4.4
J=6.5 J=7.0 J=7.0 J=6.3
J=6.9 J=6.1
J=3.4 J=3.5 J= 14.4, 2.3
J=5.9, 3.5 J=15.1
J=5.8
S-11
N26
α-Glucose
N27
β-Glucose
N28
α/β-Glucose*
N29
Glutamic acid
N30
Glutamine
N31
Glycerol
N32 N33
Glycine Histidine
N34
Isoleucine
N35 N36
Isoleucine/Glycerol* Lactic acid
N37 N38 N39 N40 N41 N42 N43
Lactic acid/Proline/Pyroglutamic acid* Leucine Lysine α-Mannose β-Mannose Methionine N-Methyl-α-aminoisobutyric acid
N44
Proline
N45
Pyruvic acid
N46
Serine
3.842 5.232 3.244 3.900 4.645 3.405 3.458 3.519 3.458 3.526 3.715 3.728 3.829
m d dd dd d m m dd m dd m m m
2.041 2.118 2.349 2.355 3.746 2.131 2.452 3.550 3.569 3.636 3.548 7.205 7.090 8.101 0.966 3.671 3.661 3.680 1.328 4.109 4.151 1.698 1.435 5.181 4.900 3.851 1.597
m m m m dd m m d d d s s d s t d d d d dd dd m m d d dd s
1.949 2.057 2.509 2.479 2.468 2.454 2.442 3.998 3.944
m m s s s s s d d
J=3.8 J=9.3, 8.0 J=12.3, 1.9 J=8.0
J=10.3, 3.8 J=9.5, 3.6
J=7.0, 5.1
J=5.9 J=5.9 J=4.4
J=0.6 J=7.2 J=3.5 J=4.5 J=4.4 J=6.9 J=6.9, 7.0 J=13.4, 6.4
J=1.5 J=0.9 J=7.4, 5.2
J=3.3 J=3.8 S-12
N47 N48 N49
Succinic acid Sucrose Threonine
N50
Tryptophan
N51
Tyrosine
N52 N53 N54 N55
Urea Uridine Valine Xanthine
4.021 2.410 5.421 1.303 1.391 3.592 4.244 7.329 7.546 7.741 7.206 7.179 5.784 5.923 0.994 8.032
d s d d d d m s d d d d s d d s
J=3.1 J=3.7 J=6.3 J=7.2 J=4.4
J=0.7 J=7.9 J=8.6 J=8.5 J=4.8 J=7.0
* Corresponds to overlapping NMR signals
S-13
Figure S6 1
H-NMR based PCA of STEMI patients ( ) versus healthy controls ( ). (A) Score plot of PC1 and PC2 scores (B) Loading plot for PC2 components contributing bin numbers. Signals were assigned in each bin as follows: Bin 44, lactic acid ; bin 94, D-glucose; bin 96, choline and D-glucose; bin 97, glycerol, glycine and D-glucose; bin 105, D-glucose; bin 111, lactic acid; bin 112, β-hydroxybutyric acid ; bin 125, β-glucose.
A
Control
PC2 (23.7%)
STEMI
B
44
0.6
0.8
PC1Loadings ( 40.8%)
94 96 125 105
0.0
97 135 103 9592106 102
-0.2
PC 2
0.2
0.4
High in STEMI
112 111 107 46 104 178 76 56 65 45 58 43 200 179 191 142 141 140 139 184 38 47 80 82 57 39 134 133 149 148 147 146 145 144 143 154 153 162 161 160 159 158 157 156 169 168 167 166 165 176 181 190 193 199 201 204 203 214 213 212 211 210 209 218 217 228 227 226 225 224 223 222 221 220 118 122 121 132 131 152 151 150 155 164 163 172 171 170 175 177 180 195 194 197 202 208 207 206 219 115 114 120 119 205 216 116 138 19 18 17 16 136 174 14 26 25 24 23 22 21 20 52 15 75 78 81 77 79 86 91234567127 867 36 98 130 185 192 198 124 123 129 196 215 183 27 84 137 13 12 54 29 48 83 53 74 11 188 117 128 189 28 55 50 88 437 9893 113 173 186 182 126 187 30 85 66 71 108 90 109 87 99 72 6051 64 8970 110 60 73 59 31 33 40 100 35 101 32 42 91 6334 6169 41 62
-0.4
-0.2
0.0
0.2
0.4
PC 1
S-14
Figure S7 1
H-NMR quantification of target compounds in serum of STEMI patients versus healthy controls. NMR signals (1.33, d, J=6.9) for lactic acid; (5.23, d, J=3.8) for α-glucose and (4.65, d, J=8.0) for β-glucose were used for quantification. Results are expressed as mean ± SEM. A p value ≤ 0.05 was considered statistically significant.
STEMI Control 225,000 5000
***
Mass (μg)
M a s s ( g )
220,000 0000
*** 115,000 5000
110,000 0000
*** 55,000 000
0 0
α-glucose
Lactic acid
L
ac
t
ic
a
d ci
-g
lu
co
β-glucose
se
-
g
lu
co
se
S-15
Figure S8 NMR based OPLS-DA of STEMI patients ( ) versus healthy controls ( ). (A) OPLSDA score plot (B) loading plot derived from samples modeled against each other. The loading plot shows the covariance p[1] against the correlation p(cor)[1] of the variables of the discriminating component of the OPLS-DA model. Signals were assigned in each bin as follows: Bin 92, D-glucose, carnitine and betaine; bin 94 & 95, D-glucose; bin 96, choline and D-glucose; bin 97, D-glucose, glycerol and glycine; bin 103,105 & 106, Dglucose; bin 125, β-glucose; bin 135, α-glucose.
A
Control
p(cor) (20.8 %)
STEMI
p (68.6 %)
B
High in STEMI
S-16
