1H-NMR Metabolomics Analysis of the Effects of Sulfated Polysaccharides from Masson Pine Pollen in RAW264.7 Macrophage Cells
Abstract
1. Introduction
2. Material and Methods
2.1. Materials and Reagents
2.2. Instrument
2.3. Effects of SPPM60-D on RAW264.7 Cell Proliferation
2.4. 1H-NMR Metabolomics Analysis of the Effects of SPPM60-D on RAW264.7 Cells
2.5. Data Analysis
3. Results
3.1. SPPM60-D Characterization
3.2. Effects of PPM60-D and SPPM60-D on RAW264.7 Cell Proliferation
3.3. Intracellular Metabolites Identified by Nuclear Magnetic Resonance Spectroscopy (NMR)
3.4. Data Analysis
3.4.1. Supervised Partial Least Squares Discriminate Analysis (PLS-DA)
3.4.2. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)
3.4.3. Analysis of Differential Metabolites
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples of the compounds PPM60-D are available from the authors. |
No. | Metabolites | Group | δH (Multiplicity) | Loading (SPPM60-D vs. Control) | Loading (LPS Group vs. Control) |
---|---|---|---|---|---|
1 | Isoleucine | CH3 | 0.94 (t) | 64.9 | 9.64 |
CH3 | 0.99 (d) | ||||
CH2 | 1.28 (m) | ||||
CH2 | 1.46 (m) | ||||
CH | 1.99 (m) | ||||
CH | 3.66 (d) | ||||
2 | Leucine | CH3 | 0.96 (d) | 10.73 | 22.66 |
CH3 | 0.97 (d) | ||||
CH | 1.7 (m) | ||||
CH2 | 3.77 (m) | ||||
CH | 3.722 (m) | ||||
3 | Valine | CH3 | 0.99 (d) | 11.7 | 20.34 |
CH3 | 1.05 (d) | ||||
CH | 2.25 (m) | ||||
CH | 3.61 (d) | ||||
4 | Ethanol | CH3 | 1.19 (t) | −150.81 | 146.23 |
CH2 | 3.65 (q) | ||||
5 | (R)-Lactate | CH3 | 1.33 (d) | −1175 | −1020.86 |
CH | 4.11 (q) | ||||
6 | Threonine | CH3 | 1.33 (d) | 41.35 | 40.5984 |
CH | 3.58 (d), | ||||
CH | 4.25 (m) | ||||
7 | Alanine | CH3 | 1.48 (d) | 51.01 | 137.05 |
CH | 3.78 (q) | ||||
8 | Acetate | CH3 | 1.92 (s) | 76.87 | 98.77 |
9 | Proline | CH2 | 2.01 (m) | 476.19 | 22.39 |
CH2 | 2.05 (m) | ||||
CH2 | 2.34 (m) | ||||
CH2 | 3.42 (td) | ||||
CH2 | 3.45 (m) | ||||
CH | 4.12 (m) | ||||
10 | L-Glutamic acid | CH2 | 2.04 (m) | −15.76 | −17.08 |
CH2 | 2.11 (m) | ||||
CH2 | 2.34 (m) | ||||
CH | 3.77 (m) | ||||
11 | L-Glutamine | CH2 | 2.13 (m) | −18.93 | −35.01 |
CH2 | 2.44 (m) | ||||
CH | 3.77 (m) | ||||
12 | Tyrosine | CH2 | 3.06 (dd) | 2.306 | 6.27 |
CH2 | 3.15 (dd) | ||||
CH | 3.94 (dd) | ||||
3,5-CH | 6.91 (d) | ||||
2,6-CH | 7.2 (d) | ||||
13 | Choline | N(CH3)3 | 3.23 (s) | 306.01 | 263.07 |
N CH2 | 3.56 (s) | ||||
O CH2 | 4.07 (t) | ||||
14 | Succinate | CH2 | 2.393 (s) | 10.04 | 17.12 |
15 | Pyruvate | CH3 | 2.46 (s) | 10.48 | 28.31 |
16 | Sarcosine | CH3 | 2.73 (s) | 32.18 | 11.84 |
CH2 | 3.6 (s) | ||||
17 | Aspartate | CH2 | 2.68 (m) | 67.57 | 118.274 |
CH2 | 2.82 (m) | ||||
CH | 3.91 (m) | ||||
18 | Phosphocreatine | CH3 | 3.05 (s) | 201.21 | 142.58 |
CH2 | 3.95 (s) | ||||
19 | Creatine | CH3 | 3.04 (s) | 259.98 | 226.48 |
CH2 | 3.94 (s) | ||||
20 | Taurine | CH2SO3 | 3.25 (t) | −304.83 | −244.785 |
NCH2 | 3.43 (t) | ||||
21 | Trimethylamine N-oxide (TMAO) | CH3 | 3.26 (s) | 54.23 | −189.45 |
22 | Betaine | CH3 | 3.27 (s) | 294.88 | 529.127 |
CH2 | 3.89 (s) | ||||
23 | Methanol | CH3 | 3.352 (s) | −14.48 | 29.20 |
24 | Glycine | CH2 | 3.56 (s) | 306.01 | 263.07 |
25 | Uridine | CH2 | 3.81 (d) | 23.93 | 118.274 |
CH2 | 3.92 (d) | ||||
4-CH | 4.18 (q) | ||||
3-CH | 4.25 (t) | ||||
2-CH | 4.38 (t) | ||||
5-CH | 5.95 (d) | ||||
6-CH | 5.97 (d) | ||||
1-CH | 7.87 (d) | ||||
26 | Uridine diphosphate (UDP) | CH2 | 4.22 (m) | 12.87 | 23.33 |
4-CH | 4.28 (m) | ||||
3-CH | 4.37 (m) | ||||
2-CH | 4.37 (m) | ||||
5-CH | 5.98 (d) | ||||
6-CH | 5.99 (d) | ||||
1-CH | 7.96 (d) | ||||
27 | Fumaric acid | 4,5-CH | 5.52 (s) | 2.66 | 1.91 |
28 | α-glucose | 1-CH | 5.24 (s) | −1.05 | −11.43 |
29 | Cytidine | 3-CH | 6.09 (d) | 1.81 | 2.09 |
2-CH | 7.87 (d) | ||||
30 | Fumarate | CH | 6.522 (s) | 2.20 | 2.07 |
31 | Niacinamide | 5-CH | 7.6 (dd) | −2.28 | 0.75 |
6-CH | 8.72 (d) | ||||
32 | Adenine | 2-CH | 8.11 (s) | 4.47 | 4.78 |
6-CH | 8.12 (s) | ||||
33 | Hypoxanthine | 2-CH | 8.22 (s) | 2.31 | 37.64 |
7-CH | 8.272 (s) | ||||
34 | Formate | CH | 8.46 (s) | 34 | −105.611 |
35 | NADP | 6-CH | 8.84 (d) | 4.54 | 1.71 |
4-CH | 9.15 (d) |
LPS vs. Control | SPPM60-D vs. Control | |||||
---|---|---|---|---|---|---|
NO. | Metabolites | VIP | Trend | Metabolites | VIP | Trend |
1 | Lactate | 25.36 | ↓ | Lactate | 24.06 | ↓ |
2 | Alanine | 14.19 | ↑ | Betaine | 12.84 | ↑ |
3 | Choline | 6.61 | ↑ | Proline | 7.58 | ↑ |
4 | Proline | 5.46 | ↑ | Creatine phosphate | 6.36 | ↑ |
5 | Betaine | 4.27 | ↑ | Creatine | 6.14 | ↑ |
6 | Glycine | 4.23 | ↑ | choline | 5.90 | ↑ |
7 | Creatine | 4.07 | ↓ | Glycine | 5.89 | ↑ |
8 | Taurine | 3.84 | ↓ | Alanine | 4.36 | ↑ |
9 | Acetate | 2.45 | ↑ | Taurine | 3.21 | ↓ |
10 | Aspartate | 1.82 | ↑ | Acetate | 3.10 | ↑ |
11 | Uridine | 1.62 | ↑ | Aspartate | 2.81 | ↑ |
12 | Threonine | 1.67 | ↑ | Trimethylamino oxide (TMAO) | 2.48 | ↑ |
13 | Formate | 1.51 | ↓ | Isoleucine | 2.34 | ↑ |
14 | Trimethylamine N-oxide (TMAO) | 1.43 | ↓ | Threonine | 1.23 | ↑ |
15 | Methanol | 1.40 | ↑ | Ethanol | 1.05 | ↓ |
16 | leucine | 1.31 | ↑ | |||
17 | Ethanol | 1.22 | ↑ | |||
18 | Glutamine | 1.13 | ↓ |
Name | Total | Expected | Hits | Raw p | −LOG (p) |
---|---|---|---|---|---|
Glycine, serine and threonine metabolism | 31 | 0.39379 | 5 | 2.52E-05 | 10.589 |
Methane metabolism | 9 | 0.11433 | 3 | 0.00013807 | 8.8877 |
Aminoacyl-tRNA biosynthesis | 69 | 0.8765 | 5 | 0.001237 | 6.6951 |
Glycolysis or Gluconeogenesis | 26 | 0.33028 | 3 | 0.003731 | 5.5911 |
Nitrogen metabolism | 9 | 0.11433 | 2 | 0.0052073 | 5.2577 |
Arginine and proline metabolism | 44 | 0.55893 | 3 | 0.01646 | 4.1068 |
Pyruvate metabolism | 23 | 0.29217 | 2 | 0.032939 | 3.4131 |
Alanine, aspartate and glutamate metabolism | 24 | 0.30487 | 2 | 0.035665 | 3.3336 |
D-Glutamine and D-glutamate metabolism | 5 | 0.063514 | 1 | 0.062007 | 2.7805 |
Cyanoamino acid metabolism | 6 | 0.076217 | 1 | 0.073964 | 2.6042 |
Pyrimidine metabolism | 41 | 0.52082 | 2 | 0.09334 | 2.3715 |
Taurine and hypotaurine metabolism | 8 | 0.10162 | 1 | 0.097448 | 2.3284 |
Primary bile acid biosynthesis | 46 | 0.58433 | 2 | 0.11353 | 2.1757 |
Valine, leucine and isoleucine biosynthesis | 11 | 0.13973 | 1 | 0.13162 | 2.0278 |
Glyoxylate and dicarboxylate metabolism | 18 | 0.22865 | 1 | 0.20667 | 1.5766 |
Glutathione metabolism | 26 | 0.33028 | 1 | 0.28493 | 1.2555 |
Porphyrin and chlorophyll metabolism | 27 | 0.34298 | 1 | 0.29419 | 1.2235 |
Glycerophospholipid metabolism | 30 | 0.38109 | 1 | 0.32127 | 1.1355 |
Valine, leucine and isoleucine degradation | 38 | 0.48271 | 1 | 0.38878 | 0.94474 |
Purine metabolism | 68 | 0.8638 | 1 | 0.58963 | 0.52826 |
Name | Total | Expected | Hits | Raw p | −LOG (p) |
---|---|---|---|---|---|
Glycine, serine and threonine metabolism | 31 | 0.35004 | 5 | 1.32E-05 | 11.232 |
Glycolysis or Gluconeogenesis | 26 | 0.29358 | 3 | 0.0026238 | 5.9431 |
Methane metabolism | 9 | 0.10162 | 2 | 0.0041113 | 5.494 |
Arginine and proline metabolism | 44 | 0.49682 | 3 | 0.011797 | 4.4399 |
Pyruvate metabolism | 23 | 0.2597 | 2 | 0.026347 | 3.6364 |
Aminoacyl-tRNA biosynthesis | 69 | 0.77911 | 3 | 0.039232 | 3.2383 |
Cyanoamino acid metabolism | 6 | 0.067749 | 1 | 0.065978 | 2.7184 |
Taurine and hypotaurine metabolism | 8 | 0.090332 | 1 | 0.087048 | 2.4413 |
Primary bile acid biosynthesis | 46 | 0.51941 | 2 | 0.092722 | 2.3782 |
Nitrogen metabolism | 9 | 0.10162 | 1 | 0.097415 | 2.3288 |
Glyoxylate and dicarboxylate metabolism | 18 | 0.20325 | 1 | 0.18588 | 1.6827 |
Alanine, aspartate and glutamate metabolism | 24 | 0.271 | 1 | 0.24027 | 1.426 |
Glutathione metabolism | 26 | 0.29358 | 1 | 0.25762 | 1.3563 |
Porphyrin and chlorophyll metabolism | 27 | 0.30487 | 1 | 0.26616 | 1.3236 |
Glycerophospholipid metabolism | 30 | 0.33874 | 1 | 0.29123 | 1.2336 |
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Su, F.; Sun, M.; Geng, Y. 1H-NMR Metabolomics Analysis of the Effects of Sulfated Polysaccharides from Masson Pine Pollen in RAW264.7 Macrophage Cells. Molecules 2019, 24, 1841. https://doi.org/10.3390/molecules24091841
Su F, Sun M, Geng Y. 1H-NMR Metabolomics Analysis of the Effects of Sulfated Polysaccharides from Masson Pine Pollen in RAW264.7 Macrophage Cells. Molecules. 2019; 24(9):1841. https://doi.org/10.3390/molecules24091841
Chicago/Turabian StyleSu, Fangchen, Mengmeng Sun, and Yue Geng. 2019. "1H-NMR Metabolomics Analysis of the Effects of Sulfated Polysaccharides from Masson Pine Pollen in RAW264.7 Macrophage Cells" Molecules 24, no. 9: 1841. https://doi.org/10.3390/molecules24091841
APA StyleSu, F., Sun, M., & Geng, Y. (2019). 1H-NMR Metabolomics Analysis of the Effects of Sulfated Polysaccharides from Masson Pine Pollen in RAW264.7 Macrophage Cells. Molecules, 24(9), 1841. https://doi.org/10.3390/molecules24091841