Characterization of urine metabolic profiles of farnesoid X receptor-null mice exhibiting spontaneous hepatocarcinogenesis using mass spectrometry-based metabolomics

1218 INTRODUCTION: The farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily, controls the synthesis and transport of bile acids (BA). Previous studies with Fxr-null mice revealed a high incidence of spontaneous liver tumors by twelve months of age, perhaps resulting from BA-related inflammation. The mechanism by which these mice develop liver tumors is unknown. The purpose of this study was to characterize the urine metabolite profiles of Fxr-null versus wild-type (WT) mice to gain insight into metabolic pathways that are altered in the absence of FXR. METHODS: Twelve male WT and twelve age- and strain-matched Fxr-null mice were used for collection of 24-hour urine samples at two, six, eight, ten, eleven, and twelve months of age. Sample volumes and body weights were recorded for comparison according to genotype, and the animals were necropsied at twelve months after urine collection concluded. Urine samples were analyzed by ultra-performance® liquid chromatography-time of flight mass spectrometry. Data matrices were first normalized by urine creatinine levels and then analyzed by principal components analysis. Urine ions that differed in relative normalized concentration by genotype were identified from contributions and loadings plots. The chemical identities of the ions were predicted from their exact masses and confirmed by tandem mass spectrogram comparisons using authentic standards. RESULTS: Mean body weight of Fxr-null mice are statistically significantly lower by six months as compared with WT mice. Principal components analysis reveals dramatic separation in the first component of the scores space according to genotype as early as two months of age. This separation is indicative of distinct urine metabolite profiles exhibiting ions that are both elevated and attenuated in urine from Fxr-null versus WT mice. Moreover, these metabolite profile differences change over time. All Fxr-null mice had liver tumors by twelve months of age, whereas the no liver tumors were observed in WT mice. CONCLUSIONS: Urine metabolic profiles of male Fxr-null mice differ from age- and gender-matched WT mice in the first twelve months of life. Several large (>500 mass/charge) metabolites are substantially elevated in the urine of Fxr-null mice at all timepoints tested. In addition, the relative concentrations of a subset of urine metabolites differ according to genotype in an age- and/or disease-dependent manner. These results provide insight into the perturbations in metabolic pathways that prevail in the absence of FXR during spontaneous liver tumorigenesis and mark the first metabolomics study into FXR function.