Plasma metabolomics identifies trimethylamine N-oxide as an accelerator of gallstone disease accompanied by early neoplastic alterations in mice.

India accounts for 10% of the global gallstone disease (GSD) and gallbladder cancer (CaGB) burden. CaGB's poor prognosis necessitates early detection of biomarkers. We used targeted/untargeted plasma metabolomics in human and mouse models to identify metabolite biomarkers for gallbladder disease. LC-MS-based metabolomics was performed on plasma samples in the discovery cohort (14 healthy controls, 30 GSD, and 56 CaGB). The identified metabolite signature was validated in 45 samples, and the candidate metabolite was further examined using targeted LC-MS in an independent cohort (n = 30). Trimethylamine N-oxide (TMAO) emerged as a significant metabolite that was evaluated in female C57BL/6 mice fed a lithogenic diet. Untargeted metabolomics from the discovery cohort identified 172 significantly altered metabolites (P < 0.05), with a 20-metabolite signature discriminating groups with high accuracy [area under the curve (AUC): GSD 0.98, CaGB 0.93; validation: GSD 0.87, CaGB 0.95]. TMAO was significantly elevated in GSD and CaGB (P < 0.001), with predictive values (AUC: GSD 0.96, CaGB 0.85). Targeted analysis confirmed twofold higher plasma TMAO in patients (P < 0.001). Female C57BL/6 mice fed on lithogenic diet (LD) with 0.3% TMAO (LD + TMAO) showed higher prevalence with early onset at 12 wk. TMAO induced epithelial metaplasia, hyperplasia, and altered mRNA expression of bile acid regulators [ATP-binding cassette subfamily C member 3 (ABCC3), 3 farnesoid X receptor (FXR), and Takeda G protein-coupled receptor 5 (TGR5)] and IL-10. Immunohistochemistry in mouse gallbladder tissue showed increased FXR and reduced TGR5 expression, following TMAO treatment. TMAO is a key metabolite associated with GSD and CaGB. TMAO supplementation with lithogenic diet accelerated gallstone formation, accompanied by preneoplastic changes, and disrupted bile acid regulation.NEW & NOTEWORTHY TMAO as a predictor of gallstone disease and accelerator of gallstone formation.