Decoding NAFLD Through Microbiome-Associated Metabolomics and Metabolic Phenotypic Expression

Background: Non-alcoholic fatty liver disease (NAFLD) is a progressive metabolic liver disorder strongly associated with gut microbiome-associated metabolic dysfunction. This study aimed to identify serum metabolomic signatures associated with NAFLD severity using untargeted metabolomics.

Methods: Serum samples were collected from healthy controls (HC, n = 19), non-alcoholic fatty liver (NAFL, n = 16), non-alcoholic steatohepatitis (NASH, n = 10), and cirrhosis patients (CH, n = 6). Untargeted metabolomic profiling was performed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS). Multivariate chemometric and pathway enrichment analyses were conducted to identify disease-associated metabolic alterations.

Results: A total of 105 metabolites were identified across all study groups. Distinct metabolic profiles were observed among HC, NAFL, NASH, and CH cohorts. Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, were significantly reduced in NAFLD patients, whereas cholic acid, palmitoylcarnitine, and L-carnitine levels were elevated in advanced disease stages. Pathway analysis revealed perturbations in butanoate metabolism, primary bile acid biosynthesis, fatty acid degradation, and energy metabolism pathways. Multivariate analyses demonstrated clear metabolic segregation among disease groups.

Conclusions: NAFLD progression is associated with profound serum metabolomic alterations involving reduced microbiome-associated SCFAs, dysregulated bile acid metabolism, and impaired mitochondrial β-oxidation. These findings provide insight into gut-liver metabolic interactions underlying NAFLD progression and identify potential circulating metabolic biomarkers associated with disease severity.

Significance: Distinct serum metabolomic signatures were identified across NAFL, NASH, and CH. Altered SCFA, bile acid, and acylcarnitine metabolism highlighted dysregulated gut-liver metabolic pathways associated with NAFLD severity.