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

Figure 1 Flow diagram of the study protocol. NASH: Non-alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease.

Figure 2 Characteristics of the study participants. Mean concentrations of (A) glycosylated hemoglobin (HbA1c); (B) fasting blood glucose (FBG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), total cholesterol (TC); (C) alanine aminotransferase (ALT), and aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and alpha-fetoprotein (AFP); and (D) total bilirubin (TBil) and direct bilirubin (DBil) in the non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and healthy control groups. E: Results of ultrasound examination in the three groups. Significant differences among the three groups were assessed by one-way ANOVA in A-D and by t-tests in E. ^aP < 0.05, ^bP < 0.01 vs the control group; ^cP < 0.05, ^dP < 0.01 vs the NAFLD group.

Figure 3 S-plots of PCA analysis (A) with electrospray ionization (ESI+) and (B) without electrospray ionization (ESI-) in the non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, no comma symbol and control groups. PCA: Principal component analysis; NASH: Non-alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease.

Figure 4 S-plots following (A) PCA, (B) PLS, and (C) OPLS analyses with (A1, B1 and C1) electrospray ionization (ESI+) and without (A2, B2 and C2) electrospray ionization (ESI-) in the non-alcoholic fatty liver disease and control groups. PCA: Principal component analysis; NASH: Non-alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease.

Figure 5 Receiver operating characteristic curves for 2-methylguanosine, 7-methylxanthine, gluconic acid, and indoxylsulfuric acid in the non-alcoholic fatty liver disease and control groups. NAFLD: Non-alcoholic fatty liver disease.

Figure 6 S-plots following (A) PCA, (B) PLS, and (C) OPLS analyses with (A1, B1, C1) electrospray ionization (ESI+) and without (A2, B2, C2) electrospray ionization (ESI-) in the non-alcoholic steatohepatitis and control groups. PCA: Principal component analysis; NASH: Non-alcoholic steatohepatitis.

Figure 7 Receiver operating characteristic curves for indoleacetic acid, gluconic acid, 2-methylguanosine, cAMP, indoxylsulfuric acid, and acetyl-DL-leucine in the non-alcoholic steatohepatitis and control groups. NASH: Non-alcoholic steatohepatitis.

Figure 8 S-plots following (A) PCA and (B) PLS analyses with (A1, B1) electrospray ionization (ESI+) and without (A2, B2) electrospray ionization (ESI-) in the non-alcoholic steatohepatitis and non-alcoholic fatty liver disease groups. NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.

Figure 9 Receiver operating characteristic curves for 3-indoleacetic acid, indoleacetic acid, L-carnitine, and pyroglutamic acid in the non-alcoholic steatohepatitis and non-alcoholic fatty liver disease groups. NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.

Figure 10 Venn diagram of metabolites differentially expressed in urinary samples of the non-alcoholic fatty liver disease vs control, non-alcoholic steatohepatitis vs control, and non-alcoholic fatty liver disease vs non-alcoholic steatohepatitis groups. NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.