Plasma metabolomic profile in nonalcoholic fatty liver disease

Abstract

The plasma profile of subjects with nonalcoholic fatty liver disease (NAFLD), steatosis, and steatohepatitis (NASH) was examined using an untargeted global metabolomic analysis to identify specific disease-related patterns and to identify potential noninvasive biomarkers. Plasma samples were obtained after an overnight fast from histologically confirmed nondiabetic subjects with hepatic steatosis (n = 11) or NASH (n = 24) and were compared with healthy, age- and sex-matched controls (n = 25). Subjects with NAFLD were obese, were insulin resistant, and had higher plasma concentrations of homocysteine and total cysteine and lower plasma concentrations of total glutathione. Metabolomic analysis showed markedly higher levels of glycocholate, taurocholate, and glycochenodeoxycholate in subjects with NAFLD. Plasma concentrations of long-chain fatty acids were lower and concentrations of free carnitine, butyrylcarnitine, and methylbutyrylcarnitine were higher in NASH. Several glutamyl dipeptides were higher whereas cysteine-glutathione levels were lower in NASH and steatosis. Other changes included higher branched-chain amino acids, phosphocholine, carbohydrates (glucose, mannose), lactate, pyruvate, and several unknown metabolites. Random forest analysis and recursive partitioning of the metabolomic data could separate healthy subjects from NAFLD with an error rate of approximately 8% and separate NASH from healthy controls with an error rate of 4%. Hepatic steatosis and steatohepatitis could not be separated using the metabolomic profile. Plasma metabolomic analysis revealed marked changes in bile salts and in biochemicals related to glutathione in subjects with NAFLD. Statistical analysis identified a panel of biomarkers that could effectively separate healthy controls from NAFLD and healthy controls from NASH. These biomarkers can potentially be used to follow response to therapeutic interventions.

Figure 1

Box plots of plasma levels of bile salts in healthy controls and subjects with steatosis and NASH; median scaled values are presented on the y axis; only bile salts which were significantly different (p<0.05) between controls and NASH are shown. Others are presented in supplemental tables.

Figure 2

Glutathione metabolism is upregulated in subjects with NAFLD. The plasma levels (box and whisker plots) of glutamyl amino acids. All are significantly different (p<0.05) in NASH and steatosis compared with controls except gamma-glutamylleucine, which is significantly higher in NASH only.

Figure 3

Box and whisker plots of plasma concentration of carnitine and acylcarnitines in subjects with NAFLD and healthy controls. (Carnitine, butyrylcarnitine: p<0.05 NASH vs. controls and steatosis vs. controls; propionylcarnitine and 2-methylbutyroylcarnitine: p<0.05 NASH vs. controls, NS: steatosis vs. controls.)

Figure 4

Box and whisker plots of plasma concentration of branched chain amino acids, tyrosine and glutamate in healthy controls and subjects with steatosis and NASH. (NASH vs. controls, p<0.05 for all; steatosis vs. controls, p<0.05 for glutamate, tyrosine and isoleucine.)

Figure 5

Random forest importance plot for all subjects.

Figure 6

Random forest importance plot for controls vs. NASH.