We aimed to identify metabolic biomarkers and investigate the metabolic alterations in relation to primary open-angle glaucoma (POAG) and cataract in human aqueous humor. Sixteen POAG patients undergoing surgical treatments and 24 patients undergoing cataract surgeries were included in this case-control study. We performed the metabolomic analysis of aqueous humor samples using a non-targeted gas chromatography coupled to time-of-flight mass spectrometer. The area under the receiver operating

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... aracteristic curve (AUC) was computed to assess the discrimination capacities of each metabolite marker. Databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaboAnalyst were utilized to search for the potential pathways of metabolites. Aqueous humor metabolomic profiles could well distinguish POAG from controls. Fourteen metabolic biomarkers were identified as potential aqueous humor biomarkers for POAG, yielding AUC values from 0.62 to 0.86. In pathway analysis, Biotin metabolism was highly impacted, implying that these metabolic markers play important roles in the regulation of this pathway. This study identified valuable metabolic biomarkers and pathways that may facilitate an improved understanding of the POAG pathogenesis. The finding holds translational value in the development of new therapeutic measures for POAG.