Urinary metabolomics analysis identifies key biomarkers of different stages of nonalcoholic fatty liver disease

Table 1 Demographic and clinical characteristics of the study participants (mean ± SD)

Group	NASH	NAFLD	Healthy group
Number	45	33	30
Age (yr)	39 ± 10	44 ± 14	39 ± 4
Gender	Male (38) Female (7)	Male (19) Female (14)	Male (21) Female (9)
Height (cm)	169 ± 8	170 ± 6	168 ± 6
Weight (kg)	76 ± 13b	78 ± 11b	68 ± 9
BMI (kg/m2)	26.40 ± 3.49b	26.81 ± 3.43b	23.73 ± 1.95

^bP < 0.01 vs the healthy group. NASH: Non-alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease; BMI: Body mass index.

Table 2 Laboratory test results in the three groups of study of participants (mean ± SD)

Group	NASH	NAFLD	Healthy group
HbA1C (%)	5.51 ± 0.58b	5.8 ± 0.62b	4.83 ± 0.47
FBG (mmpl/L)	5.31 ± 0.69	5.4 ± 1.05	5.25 ± 0.33
HDL (mmpl/L)	1.2 ± 0.36	1.28 ± 0.3	1.33 ± 0.37
LDL (mmpl/L)	3.57 ± 0.88b	3.53 ± 0.77b	1.64 ± 0.95
TG (mmpl/L)	2.62 ± 1.4b	2.39 ± 1.64b	1.32 ± 0.41
TC (mmpl/L)	5.37 ± 0.98b	5.3 ± 0.77b	4.32 ± 0.83
FFA (mmpl/L)	0.58 ± 0.25	0.5 ± 0.24	0.44 ± 0.05
ALT (U/L)	100.66 ± 48.4bd	28.9 ± 10.76	27.36 ± 9.76
AST (U/L)	49.68 ± 23.1bd	27.17 ± 12.74	26.41 ± 13.05
GGT (U/L)	75.26 ± 53.1bd	40.25 ± 23.66	27.39 ± 12.04
ALP (U/L)	78.5 ± 33.31	73.43 ± 17.05	79.1 ± 16.02
TBil (μmol/L)	16.42 ± 6.24b	16.63 ± 7.04b	8.99 ± 1.92
DBil (μmol/L)	4.07 ± 2.18	3.7 ± 1.72	3.05 ± 1.34
B ultrasound examination	2.51 ± 0.55ac	1.67 ± 0.48a	0 ± 0

^aP < 0.05,

^bP < 0.01 vs the healthy group;

^c < 0.05,

^dP < 0.01 vs the NAFLD group. NASH: Non-alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease; HbA1c: Glycosylated hemoglobin; FBG: Fasting blood glucose; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; TG: Triglycerides; TC: Total cholesterol; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase; Tbil: Total bilirubin; Dbil: Direct bilirubin.

Table 3 Urinary metabolites differentially expressed in non-alcoholic fatty liver disease patients and healthy controls

No.	Metabolites	VIP-value (OPLS-DA)	P value (t-test)	Fold change
ESI+
1	L-Carnitine	1.488	0.002	1.531
2	Creatinine	1.195	0.015	0.257
3	L-Valine/betaine	1.195	0.015	-0.544
4	Acetylcarnitine	1.658	0.001	1.669
5	Nα-Acetyl-L-arginine	1.297	0.008	-0.350
6	Hypoxanthine	1.883	0.000	0.968
7	1-Methylguanine	1.376	0.005	0.483
8	Adipic acid	1.534	0.001	-0.627
9	Xanthosine	1.467	0.002	0.389
10	Guanosine	1.448	0.003	0.273
11	7-Methylxanthine	1.489	0.002	1.922
12	2-Methylguanosine	1.654	0.001	0.475
13	Butyryl-L-carnitine	1.499	0.002	0.560
14	Gluconic acid	1.391	0.004	-0.733
15	Xanthurenic acid	1.351	0.006	0.485
16	Kynurenic acid	1.590	0.001	0.560
17	Indole-3-carboxylic acid	1.189	0.015	0.496
18	6β-hydroxytestosterone	2.203	0.000	1.251
19	Androstenedione	1.500	0.002	0.779
20	PGA2 methyl ester	1.709	0.000	0.676
21	Cortisol	1.340	0.006	0.641
22	Deoxycorticosterone	1.770	0.000	0.841
23	Corticosterone	1.568	0.001	0.766
24	Cortisone	1.383	0.004	0.661
25	Testosterone glucuronide	1.838	0.000	0.844
26	EPA	1.208	0.014	0.714
27	Decanoyl-L-carnitine	1.592	0.001	1.101
28	Androsterone	2.276	0.000	0.918
29	Eicosapentaenoic	1.282	0.009	-0.417
	Acid ethyl ester
30	Ursodeoxycholic acid	1.599	0.001	0.474
ESI-
31	Shikimate-3-phosphate	1.195	0.020	-0.344
32	2-keto-D-gluconic acid	1.902	0.000	-0.426
33	α-D-glucose	1.647	0.001	-0.484
34	Pyroglutamic acid	1.545	0.002	0.334
35	(S)-2-hydroxyglutarate	1.299	0.011	-0.579
36	2-Deoxy-D-ribose	1.264	0.014	-0.455
37	1-Methyluric acid	1.181	0.022	0.870
38	Salicyluric acid	1.455	0.004	-0.723
39	Salicylic acid	1.170	0.023	-0.494
40	Indoxylsulfuric acid	1.829	0.000	0.646
41	Ferulic acid 4-O-glucuronide	2.046	0.000	-2.303
42	Caffeic acid 3-sulfate	1.320	0.010	-2.214
43	2,3-Dihydroxybenzoic acid	1.857	0.000	-1.004
44	3,3-Dimethylglutaric acid	1.635	0.001	-1.225
45	Ferulic acid 4-sulfate	1.695	0.001	-1.779
46	Deoxyinosine	1.315	0.010	-1.049
47	Indolelactic acid	1.251	0.015	-0.868
48	3-Methylsuberic acid	1.577	0.002	-1.825
49	L-Homocitrulline	1.619	0.001	-1.883
50	Glycocholic acid	1.187	0.021	0.408
51	Glycoursodeoxycholic acid	1.392	0.006	0.932
52	L-homotyrosine	1.475	0.004	-0.625
53	Ethisterone	1.702	0.001	-0.659

Table 4 Urinary metabolites differentially expressed in non-alcoholic steatohepatitis patients and healthy controls

No.	Metabolites	VIP-value (OPLS-DA)	P value (t-test)	Fold change
E
ESI+
1	L-Lysine	1.291	0.007	-0.747
2	Suberic acid	1.037	0.032	-0.370
3	L-Carnitine	1.015	0.036	0.804
4	Creatinine	1.052	0.030	0.206
5	L-Valine/betaine	1.668	0.000	-0.852
6	Citrulline	1.539	0.001	-0.451
7	L-Dopa	1.249	0.009	-0.322
8	Acetylcarnitine	1.213	0.012	0.971
9	Nα-Acetyl-L-arginine	1.128	0.019	-0.658
10	L-Threonine	1.009	0.037	-0.300
11	L-Tyrosine	1.217	0.011	-0.304
12	Uridine	1.106	0.022	0.114
13	Hypoxanthine	2.406	0.000	1.093
14	2'-O-Methyladenosine	1.345	0.005	0.222
15	1-Methylguanine	1.921	0.000	0.744
16	6-Hydroxynicotinic acid	1.617	0.001	-0.957
17	Adipic acid	1.635	0.001	-0.808
18	Glycerophosphocholine	1.623	0.001	0.299
19	cAMP	2.233	0.000	0.417
20	L-Proline	1.046	0.031	0.397
21	Dimethyl fumarate	1.716	0.000	-0.627
22	5-Hydroxy-L-tryptophan	1.118	0.021	0.190
23	Xanthosine	1.467	0.002	0.318
24	D-Ribose	0.994	0.040	0.141
25	2-Methylguanosine	2.145	0.000	0.528
26	Butyryl-L-carnitine	1.941	0.000	0.569
27	α-Hydroxyhippuric acid	1.134	0.019	-0.540
28	Gluconic acid	1.098	0.023	-0.537
29	N-Acetylproline	1.486	0.002	0.491
30	Kynurenic acid	1.543	0.001	0.465
31	Indoxylsulfuric acid	1.444	0.002	0.494
32	Ferulic acid	0.982	0.043	-0.948
33	5-Hydroxyindoleacetic acid	1.420	0.003	0.312
34	Acetyl-DL-leucine	1.419	0.003	-0.522
35	Indole-3-carboxylic acid	0.969	0.046	0.359
36	3-Indoleacetic acid	1.055	0.029	-0.458
37	6β-Hydroxytestosterone	2.074	0.000	0.929
38	Estrone glucuronide	1.111	0.021	-1.448
39	PGA2 methyl ester	1.305	0.006	0.389
40	Cortisol	1.864	0.000	0.658
41	Tetrahydrocortisone	1.173	0.015	0.412
42	Corticosterone	1.165	0.016	0.406
43	Deoxycorticosterone	1.502	0.002	0.551
44	Cortisone	1.444	0.002	0.633
45	Ethisterone	1.541	0.001	0.689
46	EPA	0.953	0.050	0.456
47	Decanoyl-L-carnitine	1.295	0.007	0.828
48	Androsterone	2.370	0.000	0.741
49	Lauroylcarnitine	1.455	0.002	0.700
50	Palmitic amide	1.277	0.008	0.674
51	Stearamide	1.655	0.000	1.008
52	Ursodeoxycholic acid	1.231	0.010	0.398
ESI-
53	N-Acetylneuraminic acid	0.993	0.034	-0.257
54	5-aminosalicylic acid	2.042	0.000	-0.877
55	Guanine	1.082	0.021	-0.369
56	p-Coumaric acid	1.315	0.004	-0.852
57	2-Keto-glutaramic acid	1.058	0.024	0.201
58	L-2-Aminoadipic acid	0.968	0.039	0.265
59	N-Acetyl-L-glutamic acid	1.046	0.026	0.248
60	Pyroglutamic acid	2.089	0.000	0.483
61	2-Deoxy-D-ribose	1.040	0.026	-0.672
62	N-Acetylaspartylglutamic acid	1.091	0.020	0.250
63	(S)-2-Hydroxyglutarate	1.512	0.001	-0.754
64	Vanillylmandelic acid	1.065	0.023	0.269
65	(S)-(-)-2-Hydroxyisocaproic acid	1.449	0.002	-0.487
66	Salicyluric acid	0.975	0.038	-0.792
67	2-Phenylglycine	0.943	0.045	-0.829
68	Succinylacetone	0.973	0.038	0.405
69	Veratric acid	0.944	0.045	-0.573
70	Acetyl-DL-valine	0.956	0.042	0.264
71	Salicylic acid	1.034	0.027	-0.574
72	Indoxylsulfuric acid	1.573	0.001	0.542
73	2-Isopropylmalic acid	0.934	0.047	-0.817
74	Caffeic acid 3-sulfate	0.929	0.048	-2.412
75	Dihydroferulic acid 4-sulfate	1.351	0.003	-1.229
76	Pyridoxal phosphate	0.965	0.040	-1.215
77	2,3-Dihydroxybenzoic acid	1.702	0.000	-1.477
78	L-Glutamine	1.070	0.022	0.288
79	3-Methyladipic acid	1.838	0.000	-1.158
80	Ferulic acid 4-sulfate	1.903	0.000	-1.748
81	Isoferulic acid 3-O-glucuronide	1.438	0.002	-2.353
82	2-Keto-D-gluconic acid	1.594	0.000	-0.890
83	(±)-Propionylcarnitine	1.639	0.000	0.816
84	Indolelactic acid	1.420	0.002	1.305
85	3-Methylsuberic acid	1.460	0.001	-1.358
86	L-Homocitrulline	1.484	0.001	-1.419
87	Androsterone sulfate	1.735	0.000	0.680
88	L-Homotyrosine	1.384	0.003	-0.464

Table 5 Urinary metabolites differentially expressed in patients with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

No.	Metabolites	VIP-value (OPLS-DA)	P value (t-test)	Fold change
ESI+
1	L-Carnitine	1.253	0.067	-0.727
2	L-Dopa	0.952	0.092	0.234
3	Acetylcarnitine	1.181	0.094	-0.698
4	L-Histidine	1.229	0.081	-1.339
5	Pyroglutamic acid	1.541	0.021	0.143
6	3-Methylxanthine	2.403	0.002	-1.981
7	α-D-Glucose	1.739	0.013	-1.376
8	5-Hydroxyferulate	0.895	0.058	-0.266
9	2-Oxosuberate	0.850	0.073	-0.315
10	p-Hydroxyphenylacetic acid	1.354	0.095	0.398
11	3-Indoleacetic Acid	0.915	0.052	-0.378
12	β-Estradiol	1.883	0.002	-0.635
13	Phosphorylcholine	1.731	0.032	0.716
14	17α-Hydroxypregnenolone	0.854	0.087	-0.281
15	Deoxycorticosterone	0.865	0.093	-0.312
16	Progesterone	0.866	0.084	-0.301
ESI-
17	2-Keto-glutaramic acid	1.653	0.027	0.210
18	cAMP	1.659	0.041	0.196
19	7-Methylxanthine	1.595	0.036	-0.925
20	(S)-(-)-2-Hydroxyisocaproic acid	1.344	0.089	-0.250
21	Gluconic acid	1.638	0.025	-1.111
22	N-Acetylproline	1.865	0.021	0.399
23	Acetyl-DL-valine	1.636	0.053	0.284
24	Pyridoxal phosphate	1.490	0.050	-0.990
25	N-Acetyl-DL-tryptophan	1.440	0.059	-0.951
26	2-Keto-D-gluconic acid	1.258	0.088	-0.340
27	D-(+)-3-Phenyllactic acid	2.021	0.017	-1.621
28	Indoleactic acid	1.495	0.051	0.832
29	3-Hydroxy-sebacic acid	1.270	0.092	-0.292
30	Sebacic acid	1.847	0.024	0.420
31	Deoxyguanosine	1.548