药学学报

PLS-DA model	Data set	Group	Week
DM vs NC	Training set	DM	100.0%	100.0%	100.0%	100.0%
NC	100.0%	100.0%	100.0%	100.0%
Validation set	DM	87.5%	100.0%	100.0%	85.7%
NC	100.0%	100.0%	100.0%	100.0%
D-GBE vs DM	Training set	DM	100.0%	100.0%	100.0%	100.0%
D-GBE	100.0%	100.0%	100.0%	100.0%
Validation set	DM	100.0%	87.5%	100.0%	87.5%
D-GBE	87.5%	87.5%	87.5%	75.0%

PLS-DA model	Data set	Group	Week
DM vs NC	Training set	DM	100.0%	100.0%	100.0%	100.0%
NC	100.0%	100.0%	100.0%	100.0%
Validation set	DM	87.5%	100.0%	100.0%	85.7%
NC	100.0%	100.0%	100.0%	100.0%
D-GBE vs DM	Training set	DM	100.0%	100.0%	100.0%	100.0%
D-GBE	100.0%	100.0%	100.0%	100.0%
Validation set	DM	100.0%	87.5%	100.0%	87.5%
D-GBE	87.5%	87.5%	87.5%	75.0%

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
*PA (20:5/20:0)	3.8↓	6.1×10^-23		3.8↓	6.28×10^-23		3.8↓	3.09×10^-27	3.8↓	4.24×10^-24
*PA (22:6/22:0)	3.8↓	3.53×10^-26	3.8↓	3.57×10^-26	3.8↓	3.68×10^-26	3.8↓	6.59×10^-25
PA (16:0/22:6)		NS	2.3↑	9.27×10^-3		NS		NS
PE (17:2/20:1)		NS		NS		NS	3.8↓	4.04×10^-17
PS (20:5/21:0)	3.3↓	6.35×10^-6		NS		NS		NS
PS (20:4/21:0)	3.3↓	6.2×10^-6		NS		NS		NS
PS (22:4/17:0)		NS		NS	2.3↓	1.01×10^-2		NS
PS (17:0/22:4)		NS		NS	3.3↓	6.74×10^-6	3.8↓	3.33×10^-19
PI (15:0/20:3)		NS	3.8↓	2.90×10^-39		NS		NS
PI (18:0/15:0)	3.8↓	1.18×10^-34		NS		NS		NS
PI (17:2/20:1)	3.8↓	1.55×10^-32		NS		NS		NS
PI (17:2/20:5)		NS	3.8↓	3.60×10^-37		NS		NS
PG (20:4/21:0)		NS		NS		NS	2.6↓	3.00×10^-3
PG (22:2/17:0)		NS	2.2↓	1.58×10^-2		NS		NS
LysoPE (0:0/18:2)		NS	2.5↑	4.12×10^-3		NS		NS
*LysoPE (0:0/20:0)	3.1↑	3.78×10^-5	2.9↑	2.33×10^-4	3.1↑	3.78×10^-5	3.3↑	2.35×10^-6
LysoPE (0:0/22:0)	3.8↑	4.02×10^-23		NS		NS		NS
LysoPE (0:0/20:3)		NS		NS		NS	3.5↑	1.04×10^-7
LysoPE (0:0/24:6)	2.5↑	3.87×10^-3		NS		NS		NS
*TG (16:1/18:3/21:0)		NS	2.5↑	4.33×10^-3	2.5↑	3.76×10^-3	2.5↑	4.54×10^-3
*LacCer (d18:1/12:0)	3.8↑	5.32×10^-18	3.8↑	3.45×10^-17	3.8↑	4.17×10^-17	3.8↑	3.17×10^-17
*N-Oleoacyl histidine	3.6↑	2.19×10^-8	3.5↑	8.35×10^-8	3.3↑	3.80×10^-6	3.0↑	1.22×10^-4
*N-Acrylglycine		NS	2.7↓	1.20×10^-3	2.8↓	5.06×10^-4	2.8↓	4.09×10^-4
Cinnamoyl glycine	3.8↑	2.75×10^-18		NS		NS		NS
L-Asparagine	2.9↑	2.99×10^-4		NS		NS		NS
*Stearic acid	2.3↑	1.20×10^-2	2.3↑	1.21×10^-2	3.8↑	1.17×10^-22	3.8↑	5.88×10^-22
Cholesterol		NS		NS	2.6↑	2.25×10^-3		NS
Oleic acid		NS		NS	3.8↑	1.01×10^-23		NS
Pentadecanoic acid		NS		NS	3.7↑	3.37×10^-11		NS
(6Z, 9Z)-Octadecadienoic acid		NS		NS	2.6↑	1.55×10^-3		NS
*Uric acid	2.8↑	1.30×10^-4	2.8↑	6.36×10^-4	2.7↑	1.36×10^-3	2.8↑	6.36×10^-4
*7-Keto deoxycholic acid	3.2↑	1.20×10^-5	2.6↑	2.66×10^-3	3.2↑	1.20×10^-5	2.5↑	4.05×10^-3
N-Acetyl-D-galactosamine	3.8↑	2.45×10^-18		NS		NS		NS
*Leukotriene C4-d5		NS	2.5↑	4.63×10^-3	3.8↑	7.26×10^-20	3.8↑	4.49×10^-23
1-Deoxy-D-glucosol	3.3↑	6.43×10^-6		NS		NS		NS
*Fumaric acid		NS	2.9↑	3.07×10^-4	3.2↑	2.21×10^-3	3.7↑	5.27×10^-4
27-Nor-5β-cholestane pentol		NS		NS	3.3↑	6.26×10^-6		NS
Pregnenolone sulfate		NS		NS	3.8↓	3.89×10^-24		NS
2E-Tetradecanoacyl-CoA		NS		NS		NS	3.8↑	5.31×10^-29
Palmitic amide		NS		NS		NS	3.8↑	1.26×10^-27

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
*PA (20:5/20:0)	3.8↓	6.1×10^-23		3.8↓	6.28×10^-23		3.8↓	3.09×10^-27	3.8↓	4.24×10^-24
*PA (22:6/22:0)	3.8↓	3.53×10^-26	3.8↓	3.57×10^-26	3.8↓	3.68×10^-26	3.8↓	6.59×10^-25
PA (16:0/22:6)		NS	2.3↑	9.27×10^-3		NS		NS
PE (17:2/20:1)		NS		NS		NS	3.8↓	4.04×10^-17
PS (20:5/21:0)	3.3↓	6.35×10^-6		NS		NS		NS
PS (20:4/21:0)	3.3↓	6.2×10^-6		NS		NS		NS
PS (22:4/17:0)		NS		NS	2.3↓	1.01×10^-2		NS
PS (17:0/22:4)		NS		NS	3.3↓	6.74×10^-6	3.8↓	3.33×10^-19
PI (15:0/20:3)		NS	3.8↓	2.90×10^-39		NS		NS
PI (18:0/15:0)	3.8↓	1.18×10^-34		NS		NS		NS
PI (17:2/20:1)	3.8↓	1.55×10^-32		NS		NS		NS
PI (17:2/20:5)		NS	3.8↓	3.60×10^-37		NS		NS
PG (20:4/21:0)		NS		NS		NS	2.6↓	3.00×10^-3
PG (22:2/17:0)		NS	2.2↓	1.58×10^-2		NS		NS
LysoPE (0:0/18:2)		NS	2.5↑	4.12×10^-3		NS		NS
*LysoPE (0:0/20:0)	3.1↑	3.78×10^-5	2.9↑	2.33×10^-4	3.1↑	3.78×10^-5	3.3↑	2.35×10^-6
LysoPE (0:0/22:0)	3.8↑	4.02×10^-23		NS		NS		NS
LysoPE (0:0/20:3)		NS		NS		NS	3.5↑	1.04×10^-7
LysoPE (0:0/24:6)	2.5↑	3.87×10^-3		NS		NS		NS
*TG (16:1/18:3/21:0)		NS	2.5↑	4.33×10^-3	2.5↑	3.76×10^-3	2.5↑	4.54×10^-3
*LacCer (d18:1/12:0)	3.8↑	5.32×10^-18	3.8↑	3.45×10^-17	3.8↑	4.17×10^-17	3.8↑	3.17×10^-17
*N-Oleoacyl histidine	3.6↑	2.19×10^-8	3.5↑	8.35×10^-8	3.3↑	3.80×10^-6	3.0↑	1.22×10^-4
*N-Acrylglycine		NS	2.7↓	1.20×10^-3	2.8↓	5.06×10^-4	2.8↓	4.09×10^-4
Cinnamoyl glycine	3.8↑	2.75×10^-18		NS		NS		NS
L-Asparagine	2.9↑	2.99×10^-4		NS		NS		NS
*Stearic acid	2.3↑	1.20×10^-2	2.3↑	1.21×10^-2	3.8↑	1.17×10^-22	3.8↑	5.88×10^-22
Cholesterol		NS		NS	2.6↑	2.25×10^-3		NS
Oleic acid		NS		NS	3.8↑	1.01×10^-23		NS
Pentadecanoic acid		NS		NS	3.7↑	3.37×10^-11		NS
(6Z, 9Z)-Octadecadienoic acid		NS		NS	2.6↑	1.55×10^-3		NS
*Uric acid	2.8↑	1.30×10^-4	2.8↑	6.36×10^-4	2.7↑	1.36×10^-3	2.8↑	6.36×10^-4
*7-Keto deoxycholic acid	3.2↑	1.20×10^-5	2.6↑	2.66×10^-3	3.2↑	1.20×10^-5	2.5↑	4.05×10^-3
N-Acetyl-D-galactosamine	3.8↑	2.45×10^-18		NS		NS		NS
*Leukotriene C4-d5		NS	2.5↑	4.63×10^-3	3.8↑	7.26×10^-20	3.8↑	4.49×10^-23
1-Deoxy-D-glucosol	3.3↑	6.43×10^-6		NS		NS		NS
*Fumaric acid		NS	2.9↑	3.07×10^-4	3.2↑	2.21×10^-3	3.7↑	5.27×10^-4
27-Nor-5β-cholestane pentol		NS		NS	3.3↑	6.26×10^-6		NS
Pregnenolone sulfate		NS		NS	3.8↓	3.89×10^-24		NS
2E-Tetradecanoacyl-CoA		NS		NS		NS	3.8↑	5.31×10^-29
Palmitic amide		NS		NS		NS	3.8↑	1.26×10^-27

Name	t_R/min	Formula	MW	[M±H] (m/z)	Error/×10^-6	CE/eV	Ion mode	Main fragment ion (MS/MS)
*PA (20:5/20:0)	12.90	C₄₃H₇₅O₈P	750.519 9	751.539 8	2.65	20	Positive	733.515 9, 449.302 9, 439.223 8, 295.298 8. 285.281 1, 96.968 7
*PA (22:6/22:0)	13.10	C₄₇H₈₁O₈P	804.566 9	805.533 1	-4.20	20	Positive	765.510 8, 465.238 7, 323.329 7, 311.237 1, 98.984 6
PA (16:0/22:6)	8.75	C₄₁H₆₉O₈P	720.473 0	721.502 4	4.08	20	Positive	703.468 9, 465.240 3, 393.239 5, 311.237 1, 239.236 7, 98.984 1
PE (17:2/20:1)	16.16	C₄₂H₇₈NO₈P	755.546 5	756.574 3	3.68	10	Positive	738.543 6, 615.534 9, 490.337 3, 488.357 9
PS (20:5/21:0)	17.72	C₄₇H₈₂NO₁₀P	851.567 6	852.598 6	3.64	10	Positive	831.526 7, 667.566 6, 551.359 1, 527.264 6
PS (20:4/21:0)	19.48	C₄₇H₈₄NO₁₀P	853.583 3	854.561 1	-2.60	10	Positive	833.524 3, 669.588 7, 551.360 2, 529.281 3
PS (22:4/17:0)	19.48	C₄₅H₈₀NO₁₀P	825.552 0	826.578 8	3.25	10	Positive	808.468 9, 669.590 1, 557.311 1, 495.294 7
PS (17:0/22:4)	15.56	C₄₅H₈₀NO₁₀P	825.552 0	826.590 1	4.62	10	Positive	808.470 1, 669.589 5, 557.310 5, 495.298 1
PI (15:0/20:3)	14.73	C₄₄H₇₉O₁₃P	846.525 8	845.511 3	-1.72	10	Negative	827.523 3, 603.293 7, 557.289 1, 259.023 1, 241.232 7, 223.222 9
PI (18:0/15:0)	14.73	C₄₂H₈₁O₁₃P	824.541 5	823.509 7	-3.86	10	Negative	599.668 9, 557.272 5, 419.529 7, 283.467 5, 241.217 3
PI (17:2/20:1)	12.28	C₄₆H₈₃O₁₃P	874.557 1	873.518 2	-4.45	10	Negative	855.547 1, 595.287 1, 563.221 3, 307.261 2, 265.270 3
PI (17:2/20:5)	12.07	C₄₈H₇₉O₁₃P	894.525 8	893.495 1	-3.43	10	Negative	645.303 7, 565.270 3, 329.245 6, 267.212 4
PG (20:4/21:0)	10.38	C₄₇H₈₅O₁₀P	840.588 0	841.548 9	-4.66	10	Positive	823.523 7, 669.562 3, 515.276 1, 309.321 4, 287.235 7, 75.044 7
PG (22:2/17:0)	8.61	C₄₅H₈₅O₁₀P	816.588 0	817.565 1	-2.81	10	Positive	799.582 2, 547.352 1, 481.246 7, 319.287 1, 253.247 8
LysoPE (0:0/18:2)	8.79	C₂₃H₄₄NO₇P	477.285 5	478.301 2	3.28	10	Positive	461.265 1, 263.267 5, 198.053 3, 44.048 8
*LysoPE (0:0/20:0)	16.66	C₂₅H₅₂NO₇P	509.348 1	510.356 6	1.66	10	Positive	492.344 1, 369.337 1, 295.298 8, 198.048 7, 44.049 2
LysoPE (0:0/22:0)	19.34	C₂₇H₅₆NO₇P	537.379 4	538.386 2	1.26	10	Positive	520.375 8, 397.369 1, 323.327 1, 198.041 3, 44.051 1
LysoPE (0:0/20:3)	17.29	C₂₅H₄₆NO₇P	503.301 2	504.309 7	1.69	10	Positive	486.291 2, 363.288 4, 289.251 1, 198.021 3, 44.036 7
LysoPE (0:0/24:6)	9.65	C₂₉H₄₈NO₇P	553.316 8	554.329 7	2.32	10	Positive	536.311 2, 413.301 2, 339.259 7, 198.073 8, 44.053 7
*TG (16:1/18:3/21:0)	10.49	C₅₇H₁₀₀O₆	880.752 0	881.717 3	-3.94	40	Positive	627.501 2, 603.552 4, 571.423 7
*LacCer (d18:1/12:0)	19.11	C₄₂H₇₉NO₁₃	805.555 1	806.5334	-2.70	10	Positive	788.556 7, 644.511 2, 626.491 4, 550.324 1, 464.450 1
*N-Oleoacyl histidine	8.12	C₂₄H₄₁N₃O₃	419.314 8	420.334 3	4.65	10	Positive	402.401 1, 374.421 5, 237.240 1, 154.063 7
*N-Acrylglycine	1.08	C₅H₇NO₃	129.042 6	130.048 8	4.81	10	Positive	112.038 7, 84.041 2, 76.037 8, 59.011 5, 55.018 3
Cinnamoyl glycine	4.22	C₁₁H₁₁NO₃	205.073 9	206.081 3	3.61	10	Positive	188.072 4, 160.077 3, 131.047 8, 76.038 2
L-Asparagine	10.63	C₄H₈N₂O₃	132.053 5	131.047 2	-4.76	20	Negative	114.023 5, 95.301 4, 70.056 7
*Stearic acid	8.54	C₁₈H₃₆O₂	284.271 5	283.264 5	-2.47	10	Negative	265.247 8, 239.274 1
Cholesterol	8.67	C₂₇H₄₆O	386.354 9	387.363 3	2.18	20	Positive	369.350 1, 327.304 7, 297.288 3, 85.102 2, 71.087 1
Oleic acid	8.40	C₁₈H₃₄O₂	282.255 9	281.245 8	-3.57	20	Negative	263.236 9, 237.249 3, 59.014 7, 41.005 7
Pentadecanoic acid	6.75	C₁₅H₃₀O₂	242.224 6	241.217 7	-2.84	20	Negative	223.207 1, 197.228 1, 59.012 4
(6Z, 9Z)-Octadecadienoic acid	8.82	C₁₈H₃₂O₂	280.240 2	279.226 3	-4.97	20	Negative	261.222 3, 235.240 1, 59.124 4
*Uric acid	1.10	C₅H₄N₄O₃	168.028 3	167.022 1	-3.71	20	Negative	124.015 2, 81.011 3, 41.999 2
*7-Keto deoxycholic acid	0.98	C₂₄H₃₈O₅	406.271 9	407.278 3	1.57	20	Positive	389.268 1, 371.257 7, 361.274 5, 343.262 1, 329.248 3
N-Acetyl-D-galactosamine	0.88	C₈H₁₅NO₆	221.089 9	220.081 3	-3.91	10	Negative	174.077 1, 130.052 4, 100.041 2, 89.024 1
*Leukotriene C4-d5	18.49	C₃₀H₄₇N₃O₉S	625.303 3	624.294 3	-1.44	10	Negative	606.285 1, 580.307 1, 351.202 1, 333.190 2, 306.077 1
1-Deoxy-D-glucosol	7.30	C₆H₁₄O₅	166.084 1	165.076 8	-4.41	10	Negative	103.041 1, 91.040 3, 73.028 8
*Fumaric acid	9.10	C₄H₄O₄	116.011 1	115.006 7	-3.67	20	Negative	96.993 1, 71.012 7, 68.998 1, 53.003 1
27-Nor-5β-cholestane pentol	19.87	C₂₆H₄₆O₅	438.334 5	439.353 8	4.40	10	Positive	421.3345, 403.3217, 363.2911, 345.2801, 331.2647
Pregnenolone sulfate	10.39	C₂₁H₃₂O₅S	396.197 0	397.203 5	1.63	10	Positive	381.1752, 379.2013, 299.2381
2E-Tetradecanoacyl-CoA	0.80	C₃₅H₆₀N₇O₁₇P₃S	975.297 9	976.327 3	3.01	10	Positive	958.3013, 469.3132, 286.2212, 136.0647
Palmitic amide	8.07	C₁₆H₃₃NO	255.256 2	256.267 8	4.54	10	Positive	239.2342, 221.2275, 211.2441

Name	t_R/min	Formula	MW	[M±H] (m/z)	Error/×10^-6	CE/eV	Ion mode	Main fragment ion (MS/MS)
*PA (20:5/20:0)	12.90	C₄₃H₇₅O₈P	750.519 9	751.539 8	2.65	20	Positive	733.515 9, 449.302 9, 439.223 8, 295.298 8. 285.281 1, 96.968 7
*PA (22:6/22:0)	13.10	C₄₇H₈₁O₈P	804.566 9	805.533 1	-4.20	20	Positive	765.510 8, 465.238 7, 323.329 7, 311.237 1, 98.984 6
PA (16:0/22:6)	8.75	C₄₁H₆₉O₈P	720.473 0	721.502 4	4.08	20	Positive	703.468 9, 465.240 3, 393.239 5, 311.237 1, 239.236 7, 98.984 1
PE (17:2/20:1)	16.16	C₄₂H₇₈NO₈P	755.546 5	756.574 3	3.68	10	Positive	738.543 6, 615.534 9, 490.337 3, 488.357 9
PS (20:5/21:0)	17.72	C₄₇H₈₂NO₁₀P	851.567 6	852.598 6	3.64	10	Positive	831.526 7, 667.566 6, 551.359 1, 527.264 6
PS (20:4/21:0)	19.48	C₄₇H₈₄NO₁₀P	853.583 3	854.561 1	-2.60	10	Positive	833.524 3, 669.588 7, 551.360 2, 529.281 3
PS (22:4/17:0)	19.48	C₄₅H₈₀NO₁₀P	825.552 0	826.578 8	3.25	10	Positive	808.468 9, 669.590 1, 557.311 1, 495.294 7
PS (17:0/22:4)	15.56	C₄₅H₈₀NO₁₀P	825.552 0	826.590 1	4.62	10	Positive	808.470 1, 669.589 5, 557.310 5, 495.298 1
PI (15:0/20:3)	14.73	C₄₄H₇₉O₁₃P	846.525 8	845.511 3	-1.72	10	Negative	827.523 3, 603.293 7, 557.289 1, 259.023 1, 241.232 7, 223.222 9
PI (18:0/15:0)	14.73	C₄₂H₈₁O₁₃P	824.541 5	823.509 7	-3.86	10	Negative	599.668 9, 557.272 5, 419.529 7, 283.467 5, 241.217 3
PI (17:2/20:1)	12.28	C₄₆H₈₃O₁₃P	874.557 1	873.518 2	-4.45	10	Negative	855.547 1, 595.287 1, 563.221 3, 307.261 2, 265.270 3
PI (17:2/20:5)	12.07	C₄₈H₇₉O₁₃P	894.525 8	893.495 1	-3.43	10	Negative	645.303 7, 565.270 3, 329.245 6, 267.212 4
PG (20:4/21:0)	10.38	C₄₇H₈₅O₁₀P	840.588 0	841.548 9	-4.66	10	Positive	823.523 7, 669.562 3, 515.276 1, 309.321 4, 287.235 7, 75.044 7
PG (22:2/17:0)	8.61	C₄₅H₈₅O₁₀P	816.588 0	817.565 1	-2.81	10	Positive	799.582 2, 547.352 1, 481.246 7, 319.287 1, 253.247 8
LysoPE (0:0/18:2)	8.79	C₂₃H₄₄NO₇P	477.285 5	478.301 2	3.28	10	Positive	461.265 1, 263.267 5, 198.053 3, 44.048 8
*LysoPE (0:0/20:0)	16.66	C₂₅H₅₂NO₇P	509.348 1	510.356 6	1.66	10	Positive	492.344 1, 369.337 1, 295.298 8, 198.048 7, 44.049 2
LysoPE (0:0/22:0)	19.34	C₂₇H₅₆NO₇P	537.379 4	538.386 2	1.26	10	Positive	520.375 8, 397.369 1, 323.327 1, 198.041 3, 44.051 1
LysoPE (0:0/20:3)	17.29	C₂₅H₄₆NO₇P	503.301 2	504.309 7	1.69	10	Positive	486.291 2, 363.288 4, 289.251 1, 198.021 3, 44.036 7
LysoPE (0:0/24:6)	9.65	C₂₉H₄₈NO₇P	553.316 8	554.329 7	2.32	10	Positive	536.311 2, 413.301 2, 339.259 7, 198.073 8, 44.053 7
*TG (16:1/18:3/21:0)	10.49	C₅₇H₁₀₀O₆	880.752 0	881.717 3	-3.94	40	Positive	627.501 2, 603.552 4, 571.423 7
*LacCer (d18:1/12:0)	19.11	C₄₂H₇₉NO₁₃	805.555 1	806.5334	-2.70	10	Positive	788.556 7, 644.511 2, 626.491 4, 550.324 1, 464.450 1
*N-Oleoacyl histidine	8.12	C₂₄H₄₁N₃O₃	419.314 8	420.334 3	4.65	10	Positive	402.401 1, 374.421 5, 237.240 1, 154.063 7
*N-Acrylglycine	1.08	C₅H₇NO₃	129.042 6	130.048 8	4.81	10	Positive	112.038 7, 84.041 2, 76.037 8, 59.011 5, 55.018 3
Cinnamoyl glycine	4.22	C₁₁H₁₁NO₃	205.073 9	206.081 3	3.61	10	Positive	188.072 4, 160.077 3, 131.047 8, 76.038 2
L-Asparagine	10.63	C₄H₈N₂O₃	132.053 5	131.047 2	-4.76	20	Negative	114.023 5, 95.301 4, 70.056 7
*Stearic acid	8.54	C₁₈H₃₆O₂	284.271 5	283.264 5	-2.47	10	Negative	265.247 8, 239.274 1
Cholesterol	8.67	C₂₇H₄₆O	386.354 9	387.363 3	2.18	20	Positive	369.350 1, 327.304 7, 297.288 3, 85.102 2, 71.087 1
Oleic acid	8.40	C₁₈H₃₄O₂	282.255 9	281.245 8	-3.57	20	Negative	263.236 9, 237.249 3, 59.014 7, 41.005 7
Pentadecanoic acid	6.75	C₁₅H₃₀O₂	242.224 6	241.217 7	-2.84	20	Negative	223.207 1, 197.228 1, 59.012 4
(6Z, 9Z)-Octadecadienoic acid	8.82	C₁₈H₃₂O₂	280.240 2	279.226 3	-4.97	20	Negative	261.222 3, 235.240 1, 59.124 4
*Uric acid	1.10	C₅H₄N₄O₃	168.028 3	167.022 1	-3.71	20	Negative	124.015 2, 81.011 3, 41.999 2
*7-Keto deoxycholic acid	0.98	C₂₄H₃₈O₅	406.271 9	407.278 3	1.57	20	Positive	389.268 1, 371.257 7, 361.274 5, 343.262 1, 329.248 3
N-Acetyl-D-galactosamine	0.88	C₈H₁₅NO₆	221.089 9	220.081 3	-3.91	10	Negative	174.077 1, 130.052 4, 100.041 2, 89.024 1
*Leukotriene C4-d5	18.49	C₃₀H₄₇N₃O₉S	625.303 3	624.294 3	-1.44	10	Negative	606.285 1, 580.307 1, 351.202 1, 333.190 2, 306.077 1
1-Deoxy-D-glucosol	7.30	C₆H₁₄O₅	166.084 1	165.076 8	-4.41	10	Negative	103.041 1, 91.040 3, 73.028 8
*Fumaric acid	9.10	C₄H₄O₄	116.011 1	115.006 7	-3.67	20	Negative	96.993 1, 71.012 7, 68.998 1, 53.003 1
27-Nor-5β-cholestane pentol	19.87	C₂₆H₄₆O₅	438.334 5	439.353 8	4.40	10	Positive	421.3345, 403.3217, 363.2911, 345.2801, 331.2647
Pregnenolone sulfate	10.39	C₂₁H₃₂O₅S	396.197 0	397.203 5	1.63	10	Positive	381.1752, 379.2013, 299.2381
2E-Tetradecanoacyl-CoA	0.80	C₃₅H₆₀N₇O₁₇P₃S	975.297 9	976.327 3	3.01	10	Positive	958.3013, 469.3132, 286.2212, 136.0647
Palmitic amide	8.07	C₁₆H₃₃NO	255.256 2	256.267 8	4.54	10	Positive	239.2342, 221.2275, 211.2441

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
N-Oleoacyl histidine	2.9↓	2.36×10^-4		2.4↓	7.08×10^-3		3.0↓	8.27×10^-5	2.3↓	1.26×10^-3
LacCer (d18:1/12:0)	2.5↓	3.10×10^-3	2.8↓	4.44×10^-4	3.2↓	1.69×10^-5	2.6↓	2.43×10^-3
LysoPE (0:0/20:0)	2.6↓	2.43×10^-3	2.3↓	8.38×10^-3	2.7↓	1.40×10^-3	2.8↓	4.44×10^-4
Fumaric acid		NS	2.2↓	3.07×10^-2		NS		NS
Leukotriene C4-d5		NS		NS		NS	2.4↓	7.43×10^-3

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
N-Oleoacyl histidine	2.9↓	2.36×10^-4		2.4↓	7.08×10^-3		3.0↓	8.27×10^-5	2.3↓	1.26×10^-3
LacCer (d18:1/12:0)	2.5↓	3.10×10^-3	2.8↓	4.44×10^-4	3.2↓	1.69×10^-5	2.6↓	2.43×10^-3
LysoPE (0:0/20:0)	2.6↓	2.43×10^-3	2.3↓	8.38×10^-3	2.7↓	1.40×10^-3	2.8↓	4.44×10^-4
Fumaric acid		NS	2.2↓	3.07×10^-2		NS		NS
Leukotriene C4-d5		NS		NS		NS	2.4↓	7.43×10^-3

非靶向血液代谢组学研究糖尿病大鼠发生发展及银杏叶提取物干预的作用机制

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张帆 ¹ , 胡勋秀 ¹ , 李丁香 ¹ , 张染 ¹ , 赵琳琳 ¹ , 杜岩 ¹ , 汤道权 ¹^,²^,^*

药学学报 | 研究论文 2021,56(4): 1127-1136

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药学学报 | 研究论文 2021, 56(4): 1127-1136

非靶向血液代谢组学研究糖尿病大鼠发生发展及银杏叶提取物干预的作用机制

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张帆¹, 胡勋秀¹, 李丁香¹, 张染¹, 赵琳琳¹, 杜岩¹, 汤道权¹^,²^,^*

作者信息

1.徐州医科大学, 江苏省新药研究与临床药学重点实验室, 江苏徐州 221004

2.徐州医科大学药物分析教研室, 江苏徐州 221004

通讯作者:

*汤道权, E-mail: tangdq@xzhmu.edu.cn

Ginkgo biloba extract ameliorates streptozotocin-induced diabetes in rats as measured by non-targeted metabolomics

Fan ZHANG¹, Xun-xiu HU¹, Ding-xiang LI¹, Ran ZHANG¹, Lin-lin ZHAO¹, Yan DU¹, Dao-quan TANG¹^,²^,^*

Affiliations

1. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, China

2. Department of Pharmaceutical Analysis, Xuzhou Medical University, Xuzhou 221004, China

出版时间: 2021-04-12 doi: 10.16438/j.0513-4870.2021-0064

文章导航

摘要

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基于LC-MS代谢组学的方法，研究糖尿病大鼠发生发展及银杏叶提取物干预的作用机制。采用腹腔注射链脲霉素诱导糖尿病大鼠模型，分正常对照组(NC)、正常银杏叶提取物处理组(N-GBE)、糖尿病模型组(DM)、糖尿病模型银杏叶提取物处理组(D-GBE)。各组大鼠持续干预9周，收集第6~9周各组大鼠血浆进行LC-MS检测，动物实验获得徐州医科大学伦理委员会的批准。结果显示，在第6~9周DM组大鼠血浆中共12个代谢产物发生持续性变化，提示脂肪酸、氨基酸、磷脂、胆汁酸、三羧酸循环、嘌呤等6条代谢通路的异常与DM的发生发展密切相关；GBE可显著调节其中的5个代谢产物，提示GBE可能是通过改善脂肪酸、氨基酸、磷脂、三羧酸循环等代谢通路异常而发挥其延缓DM发生发展的作用。

关键词

糖尿病 / 非靶向代谢组学 / 血浆 / 银杏叶提取物 / LC-MS

Abstract

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Metabolomics based on liquid chromatography coupled with mass spectrometry (LC-MS) was used to study the initiation and development of diabetes in rats, and the ability of Ginkgo biloba extract (GBE) to ameliorate this pathology. Diabetes mellitus (DM) was induced by intra-peritoneal injection of streptozotocin. The rats were randomly divided into a normal control group treated with drug-free solution (NC), a normal control group treated with GBE (N-GBE), a DM group treated with drug-free solution (DM), and a DM group treated with GBE (D-GBE); rats were maintained on this protocol for 9 weeks. Rat plasma was collected from the sixth week to the ninth week and then analyzed with LC-MS. Animal experimentation was approved by the Committee on the Ethics of Animal Experiments of Xuzhou Medical University. Twelve plasma metabolites with continuous differentiation were monitored to indicate dysfunction of metabolic pathways including fatty acid metabolism, phospholipid metabolism, amino acid metabolism, tricarboxylic acid cycle activity, bile acid metabolism, and purine metabolism to confirm the occurrence and development of DM. Treatment with GBE partially reversed the changes seen in five metabolites in DM rats, indicating that GBE could prevent the occurrence and development of DM by acting on fatty acid metabolism, phospholipid metabolism, amino acid metabolism, and the tricarboxylic acid cycle.

Key words

diabetes mellitus / metabolomics / plasma / Ginkgo biloba leaves extract / LC-MS

引用本文

张帆, 胡勋秀, 李丁香, 张染, 赵琳琳, 杜岩, 汤道权. 非靶向血液代谢组学研究糖尿病大鼠发生发展及银杏叶提取物干预的作用机制. 药学学报, 2021 , 56 (4) : 1127 -1136 . DOI: 10.16438/j.0513-4870.2021-0064

Fan ZHANG, Xun-xiu HU, Ding-xiang LI, Ran ZHANG, Lin-lin ZHAO, Yan DU, Dao-quan TANG. Ginkgo biloba extract ameliorates streptozotocin-induced diabetes in rats as measured by non-targeted metabolomics[J]. Acta Pharmaceutica Sinica, 2021 , 56 (4) : 1127 -1136 . DOI: 10.16438/j.0513-4870.2021-0064

正文

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糖尿病(diabetes mellitus, DM) 是由环境及遗传因素共同引起的代谢紊乱性疾病, 已渐渐成为全球性的社会健康问题。据世界糖尿病协会报道, 2019年中国糖尿病患者为1.16亿, 发病率约为全国总人口的10%, 是全球糖尿病患者人数最多的国家。胰岛素抵抗、胰岛功能受损等因素导致胰岛素分泌的相对或绝对不足, 引发机体血糖代谢异常进而引发糖尿病^[1]。持续高的血糖水平是驱动DM发生发展的使动因素, 空腹血糖也成为检测DM的重要标志物。但在DM的发生发展过程中, 易发生肾病、眼病、神经病变、冠状动脉病变和外周血管病变等并发症, 其隐藏的代谢通路异常仍未完全阐明。因此, 探究DM发生发展过程中的生物标志物, 对揭示其发生发展的潜在机制具有重要意义。

银杏叶提取物(Ginkgo biloba leaves extract, GBE) 是经现代工艺提取的活性物质群, 主要包括银杏黄酮类及萜内酯类化学成分, 具有抗氧化、扩血管、降血脂、改善血流动力学状态等广泛的药理作用。据文献报道, GBE可明显降低DM患者及动物的糖化血红蛋白、空腹血糖及胰岛素水平^{[2, 3]}, 保护DM大鼠胰岛细胞受损及凋亡^[4], 高胰岛素诱导肝细胞对葡萄糖的耐受能力^[5], 但GBE对DM发生发展过程中代谢通路异常是否影响及潜在的可能机制, 仍未完全阐明。

作为系统生物学的重要平台之一, 代谢组学关注的是生物体系受到外源性刺激或基因变异后, 体液或组织中的小分子代谢产物的整体变化。该整体反应性的特点与中药治疗疾病的多环节、多靶点、协同起效的整体观念十分吻合, 因而成为考察DM发病机制及中药治疗机制的重要手段^[6-9]。但迄今为止, 尚未见针对DM发生发展过程及GBE干预过程中的代谢通路异常, 及其引起的生物标志物持续性变化的相关报道。本研究以链脲霉素(STZ) 诱导的DM大鼠为对象, 选取第6~9周大鼠血浆, 采用LC-MS技术进行非靶向代谢组学分析, 结合PCA及PLS-DA分析, 探寻DM发生发展及GBE干预过程中发生持续性变化的生物标志物及其涉及的代谢通路异常, 以揭示DM发生发展及GBE干预的潜在机制。

材料与方法

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仪器与试剂 Agilent 1290/6550超高效液相色谱-四级杆飞行时间质谱联用仪(美国Agilent公司); XW-80A型微型涡旋仪(上海沪西分析仪器厂有限公司); AB265-S型电子分析天平(瑞士METTLER TOLEDO公司); 5417R型台式高速冷冻离心机(德国Eppendorf公司); Forma 900 Series超低温冰箱和GenPure UV/UF超纯水机(美国Thermo公司); 色谱纯甲醇、乙腈、甲酸(美国MREDA公司); 分析纯丙酮(国药集团化学有限公司); N-芴甲氧羰基-甘氨酸(美国Sigma公司); GBE (批号: 201409) 购自江苏邳州富通生化科技有限公司, 采用本课题组建立的方法进行定量及化学成分分析^{[10, 11]}, 共鉴定出104个化合物(74个黄酮苷、14个黄酮苷元、9个萜内酯、5个酚酸、2个其他类), 芦丁、槲皮素、山柰酚及异鼠李素的含量分别为9.6、20.3、2.7和13.7 mg·g^-1。

动物成年雄性SD大鼠购自上海斯莱克实验动物有限责任公司, 体重190~235 g, 动物生产许可证号: SCXK (沪) 2008-0016。动物实验环境: SPF级动物实验室, 室内温度18~22 ℃, 相对湿度40%~50%, 标准饲料和饮用水, 室内保持12 h光照/12 h避光循环, 适应一周后开始实验。动物实验获得徐州医科大学伦理委员会批准(批准号: XZMULL201612024)。

动物分组、造模、给药及样本收集 雄性SD大鼠40只, 随机取16只设为正常组, 其余大鼠禁食禁水12 h后, 一次性腹腔注射60 mg·kg^-1 STZ柠檬酸缓冲溶液; 正常组给予同等体积的柠檬酸缓冲液, 72 h后空腹血糖值大于13.88 mmol·L^-1者为造模成功。造模成功当日为实验第1天, 将造模成功的大鼠随机分为DM模型对照组(DM)、DM模型GBE给药组(D-GBE, 200 mg·kg^-1), 每组8只。正常组分为正常对照组(NC) 及正常对照GBE给药组(N-GBE, 200 mg·kg^-1), 每组8只。各组大鼠在SPF环境下, GBE组按200 mg·kg^-1·d^-1给予GBE的1% CMC-Na溶液; 对照组给予同体积的溶媒。饲养9周。第2~5周采血, 用试剂盒测定空腹血糖; 第6~9周采血, 分别用于空腹血糖测定及代谢组学研究。血样分析前置-80 ℃冻存, 备用。

样品处理 取大鼠血浆50 μL, 加0.4 mg·mL^-1 N-芴甲氧羰基-甘氨酸甲醇溶液(内标) 5 μL、甲醇200 μL, 涡旋30 s, -20 ℃冻存10 min, 12 000 r·min^-1、4 ℃离心10 min, 取上清液200 μL, 再次12 000 r·min^-1、4 ℃冷冻离心10 min, 取上清进LC-MS分析。

色谱质谱条件及样品分析

色谱条件 Fortis C18柱(100 mm×2.1 mm, 1.7 µm)。流动相A: 0.1%甲酸的水溶液; 流动相B: 0.1%甲酸的甲醇溶液; 流速: 0.3 mL·min^-1。正离子模式梯度洗脱程序: 0~9 min, 25%~85% B; 9~13 min, 85% B; 13~22 min, 85%~95% B; 22~25 min, 95% B; 进样前平衡11 min。负离子模式梯度洗脱程序: 0~8 min, 20% B~95% B; 8~12 min, 95% B; 12~13 min, 95%~98% B; 13~18 min, 98% B; 进样前平衡12 min。柱温40 ℃; 自动进样器4 ℃; 进样量5 μL。

质谱条件 ESI源; 干燥器气流速12 L·min^-1; 干燥气温度280 ℃; 喷雾压力28 psi (1 psi ≈ 6.9 kPa); 鞘气流速12 L·min^-1; 鞘气温度400 ℃ (正离子) 和350 ℃ (负离子); 喷嘴电压500 V; 毛细管电压4 000 V; 扫描范围m/z 50~1 000。

样品分析取各周所有大鼠个体的血浆样品少量, 等体积混合均匀后制得质控(QC) 样本, 预处理方法同样品。样品测定之前先检测6个QC样本, 调整仪器待重现性良好以后开始采集目标样品数据。每检测6个样品检测插入1个QC样本, 通过QC样品的重现性和稳定性以评估仪器状态及方法可靠性。

数据处理与统计分析 将LC-MS原始数据经Profinder 6.0软件进行分子特征提取、背景扣除及数据过滤后, 创建为*.CEF格式, 导入到美国安捷伦公司Mass Profiler Professional (MPP) 软件进行PCA和PLS-DA模式判别分析, 验证分析系统性能的稳定性和分析数据的质量, 并筛选出有统计学意义的代谢物。通过METLIN个人代谢数据库和MPP软件进行代谢物的鉴定。两组间比较采用t检验, 假设检验水准按α = 0.05判定, P < 0.05表示差异有统计学意义。最终相关代谢通路图通过KEGG进行搜索。

结果

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1 大鼠血糖变化

DM大鼠成模后的第2周开始, 每周一次测定大鼠空腹血糖。NC组与N-GBE组之间的大鼠空腹血糖无显著性差异, 提示GBE对正常大鼠空腹血糖无影响; DM组大鼠空腹血糖均显著高于NC组, 提示模型成功; 而给予GBE 7周后, 大鼠空腹血糖明显下降(第8周和第9周), 提示GBE可改善DM大鼠的血糖升高(图 1)。

2 血液代谢轮廓分析

本研究通过研究QC样品中主要色谱峰(正离子: 胞嘧啶、牛磺酸和吡哆醇; 负离子: 脯氨酸、棕榈酸和山梨醇) 的保留时间和峰面积变异, 考察了代谢组学数据的可靠性及重现性。结果显示(图 2), 上述代谢物的保留时间RSD均小于1.0%, 峰面积RSD均小于2.9%, 表明该分析系统稳定。

采用LC-MS进行样本的分离、检测和数据采集。由图 2中可以看出, 无论正离子还是负离子检测模式下, NC组和N-GBE组大鼠血浆的代谢物差异较小; NC组和DM组、DM组和D-GBE组之间的大鼠血浆代谢物均存在差异。

3 差异代谢物筛选

利用MPP软件分别对四组数据建立PCA模型, 由PCA得分图(图 3) 可以看出, 第6~9周的N-GBE组和NC组样品均无法分离, 提示GBE对正常大鼠血浆的代谢轮廓无显著性影响; DM组样品和NC组样品均分别聚集在不同象限, 并随着时间的延长, 分离趋势明显加大, 提示DM改变了大鼠的代谢轮廓, 并随着病程的延长呈现加大趋势; DM组和D-GBE组样品分别集中, 在第6周和第7周的分离趋势不显著, 但随着时间延长, 两组样品分离趋势逐渐增大, 提示随着GBE干预时间的延长, 可明显改变大鼠代谢轮廓。

为了找出DM组与NC组、D-GBE组与DM组之间对分类贡献最大的潜在生物标志物, 利用MPP软件对四组样品数据进行PLS-DA分析, 并对模型得分进行评价。由表 1中数据可以看出, DM和NC组、D-GBE组和DM组之间的PLS-DA模型的预测能力得分均超过85%, 提示所建立的PLS-DA模型稳定可靠。

由PLS-DA得分图可见, NC组与DM组(图 4上)、DM组与D-GBE组(图 4下) 均可明显区分。根据模型中代谢离子响应值的变化倍数(fold change absolute, FC) > 2初步筛选差异性变量。对所得差异性变量进行t检验, 筛选出两组间相对含量存在显著性差异的变量(P < 0.05)。利用METLIN质谱数据库对同时满足FC > 2和P < 0.05的变量进行鉴定, 共检出40种差异代谢物(表 2、3)。其中, DM组大鼠血浆代谢物在第6~9周呈持续性出现变化异常有11种, 分布于磷脂、脂肪酸、氨基酸、嘌呤-嘧啶、胆汁酸等代谢通路中, 提示DM可能对以上通路造成影响, 使整体状态失调; 经GBE干预后, 可发生逆转的代谢物有5种(表 4), 分布于磷脂、氨基酸、三羧酸循环、脂肪酸等代谢通路, 提示GBE可能通过影响上述通路发挥其改善DM的作用(图 5)。

讨论

收起

代谢组学自其诞生以来, 已在T2DM及肥胖的发病机制及药物防治机制的研究中发挥了重要的作用。本课题组前期的细胞动态非靶向代谢组学研究结果显示, 棕榈酸和亚油酸可作为揭示DM向糖尿病肾病(diabetic nephropathy, DN) 发生发展进程的潜在生物标志物; 针对18个氨基酸、25个脂肪酸、19个核酸核碱的靶向代谢组学研究表明, 16个差异代谢物可作为DM向DN发生发展进程的潜在生物标志物, 14个差异代谢物与GBE改善此进程密切相关^{[12, 13]}, 但针对DM发生发展的潜在机制仍需要进一步研究。因此, 本研究采用非靶向代谢组学方法, 针对DM发生发展及GBE干预过程中发生持续性变化的代谢通路紊乱及相应的潜在生物标志物进行研究, 以进一步揭示DM发生发展及GBE干预的潜在机制。

DM发生发展所导致的可能潜在并发症是DM对人类造成伤害的重要因素, 针对此过程进行研究对DM的防治可能具有重要的临床意义。本研究累计发现了40种差异代谢物, 但一些差异代谢物仅在某个检测周出现, 可能与DM的发生发展过程密切相关。如PS (20:5/21:0) 仅在第6周有变化而后期无变化, 可能与DM的相关并发症或实验结果的假阳性有关。因此, 本研究仅关注发生持续性变化的12个差异代谢物, 而不关注偶尔出现差异的代谢物。

磷脂(phospholipids, PLs) 代谢紊乱与DM密切相关, 显著改变的PLs分子被认为是2型DM的生物标志物^[14]。PLs在磷脂酶A2 (phospholipase A2, PLA2) 的作用下, 可生成溶血磷脂1和游离脂肪酸, 如磷脂酰乙醇胺(phosphatidylethanolamine, PE) 在PLA2作用下降解成溶血磷脂酰乙醇胺(LysoPE)。DM状态下, 山梨醇、氧化应激和蛋白激酶C (PKC) 等通路激活, PLA2活性升高, 导致PLs水平降低、溶血磷脂水平增加^[15]。本研究发现, 第6~9周DM大鼠血浆中PA水平降低, LysoPE和甘油三酯的水平升高, 与前文报道一致^[16-18]; GBE干预后, DM大鼠血浆中LysoPE水平降低, 可能与其作用于氧化应激通路进而影响PLA2表达有关^[19]。

乳糖神经酰胺有助于稳定质膜和激活特异性微结构域或筏中的受体分子。DM状态下, 去饱和酶神经酰胺合成酶和丝氨酸棕榈酰转移酶I的表达增加, 导致神经酰胺形成速率加快、水平显著提高; 升高的乳糖神经酰胺可以激活影响细胞增殖、黏附、迁移和血管生成的“氧敏感”信号通路, 且其具有神经毒性可引起胰岛素抵抗, 这些功能障碍可推动DM的发生发展^[20]。本研究也发现了第6~9周DM大鼠血浆中乳糖神经酰胺水平显著提升; 经GBE干预后可显著降低其水平, 但GBE是否改善了神经酰胺合成酶或丝氨酸棕榈酰转移酶I的表达尚未见报道, 该潜在的机制有待进一步研究。

胆汁酸代谢通路异常与DM发生发展密切相关。如CYP7A1是胆汁酸合成的限速酶, DM状态下该酶活性被抑制导致胆汁酸水平升高, 升高的胆汁酸可以激活炎症信号通路从而促进DM的发生发展^[21]。本研究发现第6~9周DM大鼠血浆中7-酮脱氧胆酸均显著高于正常组, 而7-酮脱氧胆酸由初级胆汁酸脱去7α-羟基生成, 因而, 该结果提示DM发生发展过程与胆汁酸代谢通路异常密切相关。

尿酸是嘌呤代谢的最终氧化产物, 由黄嘌呤氧化酶氧化黄嘌呤产生。DM状态下黄嘌呤氧化酶活性升高, 导致尿酸水平升高。因而, 升高的尿酸水平与DM密切相关, 并可能导致DM并发症如DN的发生^[22]。本研究结果也证实了第6~9周DM大鼠血浆中尿酸水平显著高于正常组, 提示嘌呤代谢异常推动了DM大鼠病情的发生发展, 且可能发生肾脏损伤; 而GBE干预对尿酸代谢无显著性影响。

游离脂肪酸水平升高可引起胰岛素抵抗, 并影响糖原分解和葡萄糖的摄取。DM状态下, 参与脂肪酸β氧化过程的左旋肉碱和脂酰CoA水平降低, 可导致硬脂酸的β-氧化障碍、降解减少, 水平升高^[23]。花生四烯酸在5-脂氧化酶作用下代谢生成白三烯, DM状态下5-脂氧化酶表达增加, 导致白三烯类化合物水平上升, 从而促进炎症因子释放和胰岛素抵抗^[24]。本研究结果显示, 第6~9周大鼠DM血浆中硬脂酸及白三烯水平显著升高, GBE干预9周后可逆转白三烯水平的上升但对硬脂酸无影响, 提示GBE可能影响了花生四烯酸的代谢通路异常, 进而降低了DM大鼠血浆中白三烯水平。

尽管大量代谢组学的研究结果显示, DM患者血液中的组氨酸水平显著高或低于正常个体^[25-28], 但作为组氨酸衍生物的N-油酰基组氨酸在DM患者血浆中的变化情况尚未见报道。本研究发现N-油酰基组氨酸在第6~9周DM大鼠血浆的水平持续性高于正常大鼠, 且GBE干预后可逆转此升高, 但其潜在的机制尚有待进一步研究。

三羧酸循环的代谢异常与DM发生发展密切相关, 而富马酸是三羧酸循环的中间产物。研究证实, 血浆中持续升高的富马酸水平, 有可能推动DM的发生发展并导致肾纤维化及视网膜病变^{[29, 30]}。本研究结果也显示, DM大鼠血浆中富马酸水平持续升高可能推动了DM的发生发展; GBE干预后可逆转DM大鼠血浆中富马酸水平的上升, 提示GBE可能通过影响三羧酸循环代谢异常发挥其防治DM发生发展的作用, 但其潜在机制尚有待进一步研究。

综上所述, 磷脂、脂肪酸、氨基酸、胆汁酸、三羧酸循环、嘌呤等代谢通路的异常推动了DM的发生发展; GBE可通过影响脂肪酸、氨基酸、磷脂、三羧酸循环等代谢通路发挥其改善DM发生发展进程的作用。

作者贡献: 张帆负责动物实验和论文初稿的撰写; 胡勋秀负责实验样品的分析与数据处理; 李丁香、张染、赵琳琳参与实验和数据处理; 杜岩参与动物实验、代谢组学实验及实验数据处理; 汤道权负责研究思路的提出、实验总体设计、提供撰写思路及修改论文。所有作者阅读并认可终稿。

利益冲突: 本文的研究内容无任何利益冲突。

基金

收起

江苏省高校自然科学基金重大项目资助(16KJA350001)

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2021年第56卷第4期

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doi: 10.16438/j.0513-4870.2021-0064

接收时间：2021-01-14
首发时间：2025-12-18
出版时间：2021-04-12

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收稿日期：2021-01-14
修回日期：2021-02-24

基金

江苏省高校自然科学基金重大项目资助(16KJA350001)

作者信息

1.徐州医科大学, 江苏省新药研究与临床药学重点实验室, 江苏徐州 221004

2.徐州医科大学药物分析教研室, 江苏徐州 221004

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*汤道权, E-mail: tangdq@xzhmu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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PLS-DA model	Data set	Group	Week
DM vs NC	Training set	DM	100.0%	100.0%	100.0%	100.0%
NC	100.0%	100.0%	100.0%	100.0%
Validation set	DM	87.5%	100.0%	100.0%	85.7%
NC	100.0%	100.0%	100.0%	100.0%
D-GBE vs DM	Training set	DM	100.0%	100.0%	100.0%	100.0%
D-GBE	100.0%	100.0%	100.0%	100.0%
Validation set	DM	100.0%	87.5%	100.0%	87.5%
D-GBE	87.5%	87.5%	87.5%	75.0%

PLS-DA model

Data set

Group

Week

DM vs NC

Training set

100.0%

Validation set

87.5%

100.0%

85.7%

100.0%

D-GBE vs DM

Training set

100.0%

D-GBE

100.0%

Validation set

100.0%

87.5%

100.0%

87.5%

D-GBE

87.5%

75.0%

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
*PA (20:5/20:0)	3.8↓	6.1×10^-23		3.8↓	6.28×10^-23		3.8↓	3.09×10^-27	3.8↓	4.24×10^-24
*PA (22:6/22:0)	3.8↓	3.53×10^-26	3.8↓	3.57×10^-26	3.8↓	3.68×10^-26	3.8↓	6.59×10^-25
PA (16:0/22:6)		NS	2.3↑	9.27×10^-3		NS		NS
PE (17:2/20:1)		NS		NS		NS	3.8↓	4.04×10^-17
PS (20:5/21:0)	3.3↓	6.35×10^-6		NS		NS		NS
PS (20:4/21:0)	3.3↓	6.2×10^-6		NS		NS		NS
PS (22:4/17:0)		NS		NS	2.3↓	1.01×10^-2		NS
PS (17:0/22:4)		NS		NS	3.3↓	6.74×10^-6	3.8↓	3.33×10^-19
PI (15:0/20:3)		NS	3.8↓	2.90×10^-39		NS		NS
PI (18:0/15:0)	3.8↓	1.18×10^-34		NS		NS		NS
PI (17:2/20:1)	3.8↓	1.55×10^-32		NS		NS		NS
PI (17:2/20:5)		NS	3.8↓	3.60×10^-37		NS		NS
PG (20:4/21:0)		NS		NS		NS	2.6↓	3.00×10^-3
PG (22:2/17:0)		NS	2.2↓	1.58×10^-2		NS		NS
LysoPE (0:0/18:2)		NS	2.5↑	4.12×10^-3		NS		NS
*LysoPE (0:0/20:0)	3.1↑	3.78×10^-5	2.9↑	2.33×10^-4	3.1↑	3.78×10^-5	3.3↑	2.35×10^-6
LysoPE (0:0/22:0)	3.8↑	4.02×10^-23		NS		NS		NS
LysoPE (0:0/20:3)		NS		NS		NS	3.5↑	1.04×10^-7
LysoPE (0:0/24:6)	2.5↑	3.87×10^-3		NS		NS		NS
*TG (16:1/18:3/21:0)		NS	2.5↑	4.33×10^-3	2.5↑	3.76×10^-3	2.5↑	4.54×10^-3
*LacCer (d18:1/12:0)	3.8↑	5.32×10^-18	3.8↑	3.45×10^-17	3.8↑	4.17×10^-17	3.8↑	3.17×10^-17
*N-Oleoacyl histidine	3.6↑	2.19×10^-8	3.5↑	8.35×10^-8	3.3↑	3.80×10^-6	3.0↑	1.22×10^-4
*N-Acrylglycine		NS	2.7↓	1.20×10^-3	2.8↓	5.06×10^-4	2.8↓	4.09×10^-4
Cinnamoyl glycine	3.8↑	2.75×10^-18		NS		NS		NS
L-Asparagine	2.9↑	2.99×10^-4		NS		NS		NS
*Stearic acid	2.3↑	1.20×10^-2	2.3↑	1.21×10^-2	3.8↑	1.17×10^-22	3.8↑	5.88×10^-22
Cholesterol		NS		NS	2.6↑	2.25×10^-3		NS
Oleic acid		NS		NS	3.8↑	1.01×10^-23		NS
Pentadecanoic acid		NS		NS	3.7↑	3.37×10^-11		NS
(6Z, 9Z)-Octadecadienoic acid		NS		NS	2.6↑	1.55×10^-3		NS
*Uric acid	2.8↑	1.30×10^-4	2.8↑	6.36×10^-4	2.7↑	1.36×10^-3	2.8↑	6.36×10^-4
*7-Keto deoxycholic acid	3.2↑	1.20×10^-5	2.6↑	2.66×10^-3	3.2↑	1.20×10^-5	2.5↑	4.05×10^-3
N-Acetyl-D-galactosamine	3.8↑	2.45×10^-18		NS		NS		NS
*Leukotriene C4-d5		NS	2.5↑	4.63×10^-3	3.8↑	7.26×10^-20	3.8↑	4.49×10^-23
1-Deoxy-D-glucosol	3.3↑	6.43×10^-6		NS		NS		NS
*Fumaric acid		NS	2.9↑	3.07×10^-4	3.2↑	2.21×10^-3	3.7↑	5.27×10^-4
27-Nor-5β-cholestane pentol		NS		NS	3.3↑	6.26×10^-6		NS
Pregnenolone sulfate		NS		NS	3.8↓	3.89×10^-24		NS
2E-Tetradecanoacyl-CoA		NS		NS		NS	3.8↑	5.31×10^-29
Palmitic amide		NS		NS		NS	3.8↑	1.26×10^-27

Metabolite

The sixth week

The seventh week

The eighth week

The ninth week

P value

*PA (20:5/20:0)

3.8↓

6.1×10^-23

3.8↓

6.28×10^-23

3.8↓

3.09×10^-27

3.8↓

4.24×10^-24

*PA (22:6/22:0)

3.8↓

3.53×10^-26

3.8↓

3.57×10^-26

3.8↓

3.68×10^-26

3.8↓

6.59×10^-25

PA (16:0/22:6)

2.3↑

9.27×10^-3

PE (17:2/20:1)

3.8↓

4.04×10^-17

PS (20:5/21:0)

3.3↓

6.35×10^-6

PS (20:4/21:0)

3.3↓

6.2×10^-6

PS (22:4/17:0)

2.3↓

1.01×10^-2

PS (17:0/22:4)

3.3↓

6.74×10^-6

3.8↓

3.33×10^-19

PI (15:0/20:3)

3.8↓

2.90×10^-39

PI (18:0/15:0)

3.8↓

1.18×10^-34

PI (17:2/20:1)

3.8↓

1.55×10^-32

PI (17:2/20:5)

3.8↓

3.60×10^-37

PG (20:4/21:0)

2.6↓

3.00×10^-3

PG (22:2/17:0)

2.2↓

1.58×10^-2

LysoPE (0:0/18:2)

2.5↑

4.12×10^-3

*LysoPE (0:0/20:0)

3.1↑

3.78×10^-5

2.9↑

2.33×10^-4

3.1↑

3.78×10^-5

3.3↑

2.35×10^-6

LysoPE (0:0/22:0)

3.8↑

4.02×10^-23

LysoPE (0:0/20:3)

3.5↑

1.04×10^-7

LysoPE (0:0/24:6)

2.5↑

3.87×10^-3

*TG (16:1/18:3/21:0)

2.5↑

4.33×10^-3

2.5↑

3.76×10^-3

2.5↑

4.54×10^-3

*LacCer (d18:1/12:0)

3.8↑

5.32×10^-18

3.8↑

3.45×10^-17

3.8↑

4.17×10^-17

3.8↑

3.17×10^-17

*N-Oleoacyl histidine

3.6↑

2.19×10^-8

3.5↑

8.35×10^-8

3.3↑

3.80×10^-6

3.0↑

1.22×10^-4

*N-Acrylglycine

2.7↓

1.20×10^-3

2.8↓

5.06×10^-4

2.8↓

4.09×10^-4

Cinnamoyl glycine

3.8↑

2.75×10^-18

L-Asparagine

2.9↑

2.99×10^-4

*Stearic acid

2.3↑

1.20×10^-2

2.3↑

1.21×10^-2

3.8↑

1.17×10^-22

3.8↑

5.88×10^-22

Cholesterol

2.6↑

2.25×10^-3

Oleic acid

3.8↑

1.01×10^-23

Pentadecanoic acid

3.7↑

3.37×10^-11

(6Z, 9Z)-Octadecadienoic acid

2.6↑

1.55×10^-3

*Uric acid

2.8↑

1.30×10^-4

2.8↑

6.36×10^-4

2.7↑

1.36×10^-3

2.8↑

6.36×10^-4

*7-Keto deoxycholic acid

3.2↑

1.20×10^-5

2.6↑

2.66×10^-3

3.2↑

1.20×10^-5

2.5↑

4.05×10^-3

N-Acetyl-D-galactosamine

3.8↑

2.45×10^-18

*Leukotriene C4-d5

2.5↑

4.63×10^-3

3.8↑

7.26×10^-20

3.8↑

4.49×10^-23

1-Deoxy-D-glucosol

3.3↑

6.43×10^-6

*Fumaric acid

2.9↑

3.07×10^-4

3.2↑

2.21×10^-3

3.7↑

5.27×10^-4

27-Nor-5β-cholestane pentol

3.3↑

6.26×10^-6

Pregnenolone sulfate

3.8↓

3.89×10^-24

2E-Tetradecanoacyl-CoA

3.8↑

5.31×10^-29

Palmitic amide

3.8↑

1.26×10^-27

Name	t_R/min	Formula	MW	[M±H] (m/z)	Error/×10^-6	CE/eV	Ion mode	Main fragment ion (MS/MS)
*PA (20:5/20:0)	12.90	C₄₃H₇₅O₈P	750.519 9	751.539 8	2.65	20	Positive	733.515 9, 449.302 9, 439.223 8, 295.298 8. 285.281 1, 96.968 7
*PA (22:6/22:0)	13.10	C₄₇H₈₁O₈P	804.566 9	805.533 1	-4.20	20	Positive	765.510 8, 465.238 7, 323.329 7, 311.237 1, 98.984 6
PA (16:0/22:6)	8.75	C₄₁H₆₉O₈P	720.473 0	721.502 4	4.08	20	Positive	703.468 9, 465.240 3, 393.239 5, 311.237 1, 239.236 7, 98.984 1
PE (17:2/20:1)	16.16	C₄₂H₇₈NO₈P	755.546 5	756.574 3	3.68	10	Positive	738.543 6, 615.534 9, 490.337 3, 488.357 9
PS (20:5/21:0)	17.72	C₄₇H₈₂NO₁₀P	851.567 6	852.598 6	3.64	10	Positive	831.526 7, 667.566 6, 551.359 1, 527.264 6
PS (20:4/21:0)	19.48	C₄₇H₈₄NO₁₀P	853.583 3	854.561 1	-2.60	10	Positive	833.524 3, 669.588 7, 551.360 2, 529.281 3
PS (22:4/17:0)	19.48	C₄₅H₈₀NO₁₀P	825.552 0	826.578 8	3.25	10	Positive	808.468 9, 669.590 1, 557.311 1, 495.294 7
PS (17:0/22:4)	15.56	C₄₅H₈₀NO₁₀P	825.552 0	826.590 1	4.62	10	Positive	808.470 1, 669.589 5, 557.310 5, 495.298 1
PI (15:0/20:3)	14.73	C₄₄H₇₉O₁₃P	846.525 8	845.511 3	-1.72	10	Negative	827.523 3, 603.293 7, 557.289 1, 259.023 1, 241.232 7, 223.222 9
PI (18:0/15:0)	14.73	C₄₂H₈₁O₁₃P	824.541 5	823.509 7	-3.86	10	Negative	599.668 9, 557.272 5, 419.529 7, 283.467 5, 241.217 3
PI (17:2/20:1)	12.28	C₄₆H₈₃O₁₃P	874.557 1	873.518 2	-4.45	10	Negative	855.547 1, 595.287 1, 563.221 3, 307.261 2, 265.270 3
PI (17:2/20:5)	12.07	C₄₈H₇₉O₁₃P	894.525 8	893.495 1	-3.43	10	Negative	645.303 7, 565.270 3, 329.245 6, 267.212 4
PG (20:4/21:0)	10.38	C₄₇H₈₅O₁₀P	840.588 0	841.548 9	-4.66	10	Positive	823.523 7, 669.562 3, 515.276 1, 309.321 4, 287.235 7, 75.044 7
PG (22:2/17:0)	8.61	C₄₅H₈₅O₁₀P	816.588 0	817.565 1	-2.81	10	Positive	799.582 2, 547.352 1, 481.246 7, 319.287 1, 253.247 8
LysoPE (0:0/18:2)	8.79	C₂₃H₄₄NO₇P	477.285 5	478.301 2	3.28	10	Positive	461.265 1, 263.267 5, 198.053 3, 44.048 8
*LysoPE (0:0/20:0)	16.66	C₂₅H₅₂NO₇P	509.348 1	510.356 6	1.66	10	Positive	492.344 1, 369.337 1, 295.298 8, 198.048 7, 44.049 2
LysoPE (0:0/22:0)	19.34	C₂₇H₅₆NO₇P	537.379 4	538.386 2	1.26	10	Positive	520.375 8, 397.369 1, 323.327 1, 198.041 3, 44.051 1
LysoPE (0:0/20:3)	17.29	C₂₅H₄₆NO₇P	503.301 2	504.309 7	1.69	10	Positive	486.291 2, 363.288 4, 289.251 1, 198.021 3, 44.036 7
LysoPE (0:0/24:6)	9.65	C₂₉H₄₈NO₇P	553.316 8	554.329 7	2.32	10	Positive	536.311 2, 413.301 2, 339.259 7, 198.073 8, 44.053 7
*TG (16:1/18:3/21:0)	10.49	C₅₇H₁₀₀O₆	880.752 0	881.717 3	-3.94	40	Positive	627.501 2, 603.552 4, 571.423 7
*LacCer (d18:1/12:0)	19.11	C₄₂H₇₉NO₁₃	805.555 1	806.5334	-2.70	10	Positive	788.556 7, 644.511 2, 626.491 4, 550.324 1, 464.450 1
*N-Oleoacyl histidine	8.12	C₂₄H₄₁N₃O₃	419.314 8	420.334 3	4.65	10	Positive	402.401 1, 374.421 5, 237.240 1, 154.063 7
*N-Acrylglycine	1.08	C₅H₇NO₃	129.042 6	130.048 8	4.81	10	Positive	112.038 7, 84.041 2, 76.037 8, 59.011 5, 55.018 3
Cinnamoyl glycine	4.22	C₁₁H₁₁NO₃	205.073 9	206.081 3	3.61	10	Positive	188.072 4, 160.077 3, 131.047 8, 76.038 2
L-Asparagine	10.63	C₄H₈N₂O₃	132.053 5	131.047 2	-4.76	20	Negative	114.023 5, 95.301 4, 70.056 7
*Stearic acid	8.54	C₁₈H₃₆O₂	284.271 5	283.264 5	-2.47	10	Negative	265.247 8, 239.274 1
Cholesterol	8.67	C₂₇H₄₆O	386.354 9	387.363 3	2.18	20	Positive	369.350 1, 327.304 7, 297.288 3, 85.102 2, 71.087 1
Oleic acid	8.40	C₁₈H₃₄O₂	282.255 9	281.245 8	-3.57	20	Negative	263.236 9, 237.249 3, 59.014 7, 41.005 7
Pentadecanoic acid	6.75	C₁₅H₃₀O₂	242.224 6	241.217 7	-2.84	20	Negative	223.207 1, 197.228 1, 59.012 4
(6Z, 9Z)-Octadecadienoic acid	8.82	C₁₈H₃₂O₂	280.240 2	279.226 3	-4.97	20	Negative	261.222 3, 235.240 1, 59.124 4
*Uric acid	1.10	C₅H₄N₄O₃	168.028 3	167.022 1	-3.71	20	Negative	124.015 2, 81.011 3, 41.999 2
*7-Keto deoxycholic acid	0.98	C₂₄H₃₈O₅	406.271 9	407.278 3	1.57	20	Positive	389.268 1, 371.257 7, 361.274 5, 343.262 1, 329.248 3
N-Acetyl-D-galactosamine	0.88	C₈H₁₅NO₆	221.089 9	220.081 3	-3.91	10	Negative	174.077 1, 130.052 4, 100.041 2, 89.024 1
*Leukotriene C4-d5	18.49	C₃₀H₄₇N₃O₉S	625.303 3	624.294 3	-1.44	10	Negative	606.285 1, 580.307 1, 351.202 1, 333.190 2, 306.077 1
1-Deoxy-D-glucosol	7.30	C₆H₁₄O₅	166.084 1	165.076 8	-4.41	10	Negative	103.041 1, 91.040 3, 73.028 8
*Fumaric acid	9.10	C₄H₄O₄	116.011 1	115.006 7	-3.67	20	Negative	96.993 1, 71.012 7, 68.998 1, 53.003 1
27-Nor-5β-cholestane pentol	19.87	C₂₆H₄₆O₅	438.334 5	439.353 8	4.40	10	Positive	421.3345, 403.3217, 363.2911, 345.2801, 331.2647
Pregnenolone sulfate	10.39	C₂₁H₃₂O₅S	396.197 0	397.203 5	1.63	10	Positive	381.1752, 379.2013, 299.2381
2E-Tetradecanoacyl-CoA	0.80	C₃₅H₆₀N₇O₁₇P₃S	975.297 9	976.327 3	3.01	10	Positive	958.3013, 469.3132, 286.2212, 136.0647
Palmitic amide	8.07	C₁₆H₃₃NO	255.256 2	256.267 8	4.54	10	Positive	239.2342, 221.2275, 211.2441

Name

t_R/min

Formula

[M±H] (m/z)

Error/×10^-6

CE/eV

Ion mode

Main fragment ion (MS/MS)

*PA (20:5/20:0)

12.90

C₄₃H₇₅O₈P

750.519 9

751.539 8

2.65

Positive

733.515 9, 449.302 9, 439.223 8, 295.298 8. 285.281 1, 96.968 7

*PA (22:6/22:0)

13.10

C₄₇H₈₁O₈P

804.566 9

805.533 1

-4.20

Positive

765.510 8, 465.238 7, 323.329 7, 311.237 1, 98.984 6

PA (16:0/22:6)

8.75

C₄₁H₆₉O₈P

720.473 0

721.502 4

4.08

Positive

703.468 9, 465.240 3, 393.239 5, 311.237 1, 239.236 7, 98.984 1

PE (17:2/20:1)

16.16

C₄₂H₇₈NO₈P

755.546 5

756.574 3

3.68

Positive

738.543 6, 615.534 9, 490.337 3, 488.357 9

PS (20:5/21:0)

17.72

C₄₇H₈₂NO₁₀P

851.567 6

852.598 6

3.64

Positive

831.526 7, 667.566 6, 551.359 1, 527.264 6

PS (20:4/21:0)

19.48

C₄₇H₈₄NO₁₀P

853.583 3

854.561 1

-2.60

Positive

833.524 3, 669.588 7, 551.360 2, 529.281 3

PS (22:4/17:0)

19.48

C₄₅H₈₀NO₁₀P

825.552 0

826.578 8

3.25

Positive

808.468 9, 669.590 1, 557.311 1, 495.294 7

PS (17:0/22:4)

15.56

C₄₅H₈₀NO₁₀P

825.552 0

826.590 1

4.62

Positive

808.470 1, 669.589 5, 557.310 5, 495.298 1

PI (15:0/20:3)

14.73

C₄₄H₇₉O₁₃P

846.525 8

845.511 3

-1.72

Negative

827.523 3, 603.293 7, 557.289 1, 259.023 1, 241.232 7, 223.222 9

PI (18:0/15:0)

14.73

C₄₂H₈₁O₁₃P

824.541 5

823.509 7

-3.86

Negative

599.668 9, 557.272 5, 419.529 7, 283.467 5, 241.217 3

PI (17:2/20:1)

12.28

C₄₆H₈₃O₁₃P

874.557 1

873.518 2

-4.45

Negative

855.547 1, 595.287 1, 563.221 3, 307.261 2, 265.270 3

PI (17:2/20:5)

12.07

C₄₈H₇₉O₁₃P

894.525 8

893.495 1

-3.43

Negative

645.303 7, 565.270 3, 329.245 6, 267.212 4

PG (20:4/21:0)

10.38

C₄₇H₈₅O₁₀P

840.588 0

841.548 9

-4.66

Positive

823.523 7, 669.562 3, 515.276 1, 309.321 4, 287.235 7, 75.044 7

PG (22:2/17:0)

8.61

C₄₅H₈₅O₁₀P

816.588 0

817.565 1

-2.81

Positive

799.582 2, 547.352 1, 481.246 7, 319.287 1, 253.247 8

LysoPE (0:0/18:2)

8.79

C₂₃H₄₄NO₇P

477.285 5

478.301 2

3.28

Positive

461.265 1, 263.267 5, 198.053 3, 44.048 8

*LysoPE (0:0/20:0)

16.66

C₂₅H₅₂NO₇P

509.348 1

510.356 6

1.66

Positive

492.344 1, 369.337 1, 295.298 8, 198.048 7, 44.049 2

LysoPE (0:0/22:0)

19.34

C₂₇H₅₆NO₇P

537.379 4

538.386 2

1.26

Positive

520.375 8, 397.369 1, 323.327 1, 198.041 3, 44.051 1

LysoPE (0:0/20:3)

17.29

C₂₅H₄₆NO₇P

503.301 2

504.309 7

1.69

Positive

486.291 2, 363.288 4, 289.251 1, 198.021 3, 44.036 7

LysoPE (0:0/24:6)

9.65

C₂₉H₄₈NO₇P

553.316 8

554.329 7

2.32

Positive

536.311 2, 413.301 2, 339.259 7, 198.073 8, 44.053 7

*TG (16:1/18:3/21:0)

10.49

C₅₇H₁₀₀O₆

880.752 0

881.717 3

-3.94

Positive

627.501 2, 603.552 4, 571.423 7

*LacCer (d18:1/12:0)

19.11

C₄₂H₇₉NO₁₃

805.555 1

806.5334

-2.70

Positive

788.556 7, 644.511 2, 626.491 4, 550.324 1, 464.450 1

*N-Oleoacyl histidine

8.12

C₂₄H₄₁N₃O₃

419.314 8

420.334 3

4.65

Positive

402.401 1, 374.421 5, 237.240 1, 154.063 7

*N-Acrylglycine

1.08

C₅H₇NO₃

129.042 6

130.048 8

4.81

Positive

112.038 7, 84.041 2, 76.037 8, 59.011 5, 55.018 3

Cinnamoyl glycine

4.22

C₁₁H₁₁NO₃

205.073 9

206.081 3

3.61

Positive

188.072 4, 160.077 3, 131.047 8, 76.038 2

L-Asparagine

10.63

C₄H₈N₂O₃

132.053 5

131.047 2

-4.76

Negative

114.023 5, 95.301 4, 70.056 7

*Stearic acid

8.54

C₁₈H₃₆O₂

284.271 5

283.264 5

-2.47

Negative

265.247 8, 239.274 1

Cholesterol

8.67

C₂₇H₄₆O

386.354 9

387.363 3

2.18

Positive

369.350 1, 327.304 7, 297.288 3, 85.102 2, 71.087 1

Oleic acid

8.40

C₁₈H₃₄O₂

282.255 9

281.245 8

-3.57

Negative

263.236 9, 237.249 3, 59.014 7, 41.005 7

Pentadecanoic acid

6.75

C₁₅H₃₀O₂

242.224 6

241.217 7

-2.84

Negative

223.207 1, 197.228 1, 59.012 4

(6Z, 9Z)-Octadecadienoic acid

8.82

C₁₈H₃₂O₂

280.240 2

279.226 3

-4.97

Negative

261.222 3, 235.240 1, 59.124 4

*Uric acid

1.10

C₅H₄N₄O₃

168.028 3

167.022 1

-3.71

Negative

124.015 2, 81.011 3, 41.999 2

*7-Keto deoxycholic acid

0.98

C₂₄H₃₈O₅

406.271 9

407.278 3

1.57

Positive

389.268 1, 371.257 7, 361.274 5, 343.262 1, 329.248 3

N-Acetyl-D-galactosamine

0.88

C₈H₁₅NO₆

221.089 9

220.081 3

-3.91

Negative

174.077 1, 130.052 4, 100.041 2, 89.024 1

*Leukotriene C4-d5

18.49

C₃₀H₄₇N₃O₉S

625.303 3

624.294 3

-1.44

Negative

606.285 1, 580.307 1, 351.202 1, 333.190 2, 306.077 1

1-Deoxy-D-glucosol

7.30

C₆H₁₄O₅

166.084 1

165.076 8

-4.41

Negative

103.041 1, 91.040 3, 73.028 8

*Fumaric acid

9.10

C₄H₄O₄

116.011 1

115.006 7

-3.67

Negative

96.993 1, 71.012 7, 68.998 1, 53.003 1

27-Nor-5β-cholestane pentol

19.87

C₂₆H₄₆O₅

438.334 5

439.353 8

4.40

Positive

421.3345, 403.3217, 363.2911, 345.2801, 331.2647

Pregnenolone sulfate

10.39

C₂₁H₃₂O₅S

396.197 0

397.203 5

1.63

Positive

381.1752, 379.2013, 299.2381

2E-Tetradecanoacyl-CoA

0.80

C₃₅H₆₀N₇O₁₇P₃S

975.297 9

976.327 3

3.01

Positive

958.3013, 469.3132, 286.2212, 136.0647

Palmitic amide

8.07

C₁₆H₃₃NO

255.256 2

256.267 8

4.54

Positive

239.2342, 221.2275, 211.2441

Metabolite	The sixth week		The seventh week		The eighth week		The ninth week
N-Oleoacyl histidine	2.9↓	2.36×10^-4		2.4↓	7.08×10^-3		3.0↓	8.27×10^-5	2.3↓	1.26×10^-3
LacCer (d18:1/12:0)	2.5↓	3.10×10^-3	2.8↓	4.44×10^-4	3.2↓	1.69×10^-5	2.6↓	2.43×10^-3
LysoPE (0:0/20:0)	2.6↓	2.43×10^-3	2.3↓	8.38×10^-3	2.7↓	1.40×10^-3	2.8↓	4.44×10^-4
Fumaric acid		NS	2.2↓	3.07×10^-2		NS		NS
Leukotriene C4-d5		NS		NS		NS	2.4↓	7.43×10^-3

Metabolite

The sixth week

The seventh week

The eighth week

The ninth week

P value

Pvalue

P value

N-Oleoacyl histidine

2.9↓

2.36×10^-4

2.4↓

7.08×10^-3

3.0↓

8.27×10^-5

2.3↓

1.26×10^-3

LacCer (d18:1/12:0)

2.5↓

3.10×10^-3

2.8↓

4.44×10^-4

3.2↓

1.69×10^-5

2.6↓

2.43×10^-3

LysoPE (0:0/20:0)

2.6↓

2.43×10^-3

2.3↓

8.38×10^-3

2.7↓

1.40×10^-3

2.8↓

4.44×10^-4

Fumaric acid

2.2↓

3.07×10^-2

Leukotriene C4-d5

2.4↓

7.43×10^-3