Prediction of gastric cancer metastasis through urinary metabolomic investigation using GC/MS

Table 1 Urine metabolites of mice in the three groups (normal, non-metastasis and metastasis)

Peak No.	Retention time	Metabolites	Match percent (%)
1	7.196	Lactic acid	91
2	7.508	Acetic acid	90
3	8.105	Alanine	90
4	8.518	Glycine	91
5	8.936	Pentanoic acid, 4-oxo-	94
6	9.412	Butanoic acid	83
7	11.940	Urea	95
8	12.511	Glycerol	91
9	12.590	Silanol	97
10	13.933	Butanedioic acid	97
11	14.293	Propanoic acid	94
12	14.705	Pyrimidine	93
13	14.774	Triacetin	83
14	15.657	2-Piperidinecarboxylic acid	90
15	16.070	L-threonine	87
16	16.460	N-(1-oxobutyl)-Glycine	90
17	17.270	N-(2-methyl-1-oxopropyl)-Glycine	91
18	17.530	(R*,S*)-3,4-Dihydroxybutanoic acid	94
19	19.301	Malic acid	90
20	19.492	N-(3-methyl-1-oxobutyl)-Glycine	98
21	19.566	2,3,4-oxy-Butanal	90
22	19.814	1,2,3,4-oxy-Butane	90
23	20.497	L-proline	96
24	20.909	L-threonic acid	90
25	21.475	Creatinine	96
26	25.108	Hexanedioic acid	90
27	25.632	Arabitol	91
28	25.843	Nonadecane	83
29	26.018	Xylitol	93
30	26.166	Ribitol	91
31	26.838	4-Pyrimidinecarboxylic acid	96
32	26.938	1-Propene-1,2,3-tricarboxylic acid	91
33	27.208	Phosphoric acid	90
34	28.768	Citric acid	91
35	29.032	Myo-inositol	83
36	30.328	Mannonic acid	95
37	30.540	Hydrazone	96
38	30.730	N-Phenylacetyl glycine	93
39	31.037	Silane	91
40	31.449	L-Gluconic acid	99
41	32.422	D-Gluconic acid	91
42	33.025	Dehydrocholic Acid	92
43	34.612	Hexadecanoic acid	99
44	35.691	Uric acid	98
45	38.388	Retinoic acid, methyl ester	95
46	39.065	Octadecanoic acid	99

Peaks in the total ion chromatograms are numbered according to their retention time. The identification of metabolite is based on national institute of standards and technology mass spectra database according to the match of masses (m/z) between the interested peak’s fragmentation pattern and that from the standard database.

Table 2 Marker metabolites found in normal and cancer groups

Metabolites	Retention time	P value1	A (normal)	B (cancer2)	R3
Lactic acid	7.196	2.4 × 10-5	79.24 ± 6.1	187.04 ± 71.99	1.36
Butanoic acid	9.412	0.000	16.79 ± 0.52	27.33 ± 4.98	0.63
Propanoic acid	14.293	0.000	60.58 ± 9.79	147.77 ± 15.3	1.43
Glycerol	12.511	0.000	147 ± 8.98	269.13 ± 50.31	0.83
Pyrimidine	14.705	0.000	61.68 ± 8.05	163.11 ± 12.23	1.64
Butanedioic acid	13.933	0.1 × 10-5	161.51 ± 5.85	267.89 ± 54.64	0.66
Malic acid	19.301	0.000	10.7 ± 1.91	32.15 ± 1.16	2.00
Citric acid	28.768	1.4 × 10-4	1291.89 ± 364.74	2164.74 ± 529.58	0.68
Hexadecanoic acid	34.612	4.17 × 10-4	1347.84 ± 304.67	2066.57 ± 437.28	0.53
Uric acid	35.691	0.000	172.2 ± 17.03	214.52 ± 7.74	0.25

¹P values were calculated based on Student t test (significance at P < 0.05);

²Cancer group included the non-metastasis group and the metastasis group;

³R value was calculated from the arithmetic mean values of each group. R = (B-A)/A. R with a positive value indicates a relatively higher concentration in cancer group while a negative value means a relatively lower concentration as compared with the normal group.

Table 3 Metabolic differences in the two groups

Metabolites	Retention time	P value1	A (non-metastasis)	B (metastasis)	R2
Alanine	8.105	0.000	173.75 ± 39.59	19.28 ± 10.63	-0.89
Butanoic acid	9.412	0.000	32.09 ± 1.00	22.58 ± 0.72	-0.30
Glycerol	12.511	0.003	303.23 ± 26.16	235.04 ± 45.64	-0.22
Butanedioic acid	13.933	0.1 × 10-5	216.36 ± 2.63	319.43 ± 17.89	0.48
L-proline	20.497	0.000	184.99 ± 10.26	117.78 ± 7.05	-0.36
L-threonic acid	20.909	2.28 × 10-4	284.94 ± 46.47	181.48 ± 37.25	-0.36
Myo-inositol	29.032	0.000	33.08 ± 3.58	114.8 ± 2.20	2.47

¹P values were calculated based on Student t test (significance at P < 0.05);

²R value was calculated from the arithmetic mean values of each group. R = (B-A)/A. R with a positive value indicates a relatively higher concentration in metastasis group while a negative value means a relatively lower concentration as compared with the non-metastasis group.