Metabolic signatures across the full spectrum of non-alcoholic fatty liver disease

JHEP Rep. 2022 Mar 26;4(5):100477. doi: 10.1016/j.jhepr.2022.100477. eCollection 2022 May.


Background & aims: Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease with potentially severe complications including cirrhosis and hepatocellular carcinoma. Previously, we have identified circulating lipid signatures associating with liver fat content and non-alcoholic steatohepatitis (NASH). Here, we develop a metabolomic map across the NAFLD spectrum, defining interconnected metabolic signatures of steatosis (non-alcoholic fatty liver, NASH, and fibrosis).

Methods: We performed mass spectrometry analysis of molecular lipids and polar metabolites in serum samples from the European NAFLD Registry patients (n = 627), representing the full spectrum of NAFLD. Using various univariate, multivariate, and machine learning statistical approaches, we interrogated metabolites across 3 clinical perspectives: steatosis, NASH, and fibrosis.

Results: Following generation of the NAFLD metabolic network, we identify 15 metabolites unique to steatosis, 18 to NASH, and 15 to fibrosis, with 27 common to all. We identified that progression from F2 to F3 fibrosis coincides with a key pathophysiological transition point in disease natural history, with n = 73 metabolites altered.

Conclusions: Analysis of circulating metabolites provides important insights into the metabolic changes during NAFLD progression, revealing metabolic signatures across the NAFLD spectrum and features that are specific to NAFL, NASH, and fibrosis. The F2-F3 transition marks a critical metabolic transition point in NAFLD pathogenesis, with the data pointing to the pathophysiological importance of metabolic stress and specifically oxidative stress.

Clinical trials registration: The study is registered at (NCT04442334).

Lay summary: Non-alcoholic fatty liver disease is characterised by the build-up of fat in the liver, which progresses to liver dysfunction, scarring, and irreversible liver failure, and is markedly increasing in its prevalence worldwide. Here, we measured lipids and other small molecules (metabolites) in the blood with the aim of providing a comprehensive molecular overview of fat build-up, liver fibrosis, and diagnosed severity. We identify a key metabolic 'watershed' in the progression of liver damage, separating severe disease from mild, and show that specific lipid and metabolite profiles can help distinguish and/or define these cases.

Keywords: 2-HB, 2-hydroxybutanoic acid; 3-HB, 3-hydroxybutanoic acid; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CE, cholesterol ester; Cer, ceramide; FFA, free fatty acid; FLIP, Fatty Liver Inhibition of Progression; Fibrosis; GC, gas chromatography; HCC, hepatocellular carcinoma; HSD, honest significant difference; LC, lipid cluster; LDL, low-density lipoprotein; LM, lipid and metabolite; LMC, lipid, metabolite, and clinical variable; LPC, lysophosphatidylcholine; Lipidomics; Mass spectrometry; Metabolomics; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NAS, NASH activity score; NASH, non-alcoholic steatohepatitis; NIDDK NASH-CRN, National Institute of Digestive Diseases and Kidney NASH Clinical Research Network; NRR, non-rejection rate; Non-alcoholic steatohepatitis; PC(O), ether PC; PC, phosphatidylcholine; PCA, principal component analysis; PE, phosphatidylethanolamine; QTOFMS, quadrupole-time-of-flight mass spectrometry; ROC, receiving operator characteristic; SAF, steatosis, activity, and fibrosis; SM, sphingomyelin; T2DM, type 2 diabetes mellitus; TG, triacylglycerol; UHPLC, ultrahigh-performance liquid chromatography.

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