In the current study, a change in the serum metabolome following LCA-induced liver injury was assessed in mice fed LCA-supplemented diets in order to determine the mechanism of cholestatic liver injury and to discover biomarkers for disease progression. Epacadostat in vitro Comparison of the LCA-induced metabolic changes and altered gene expression patterns in the farnesoid X receptor (Fxr)-null mouse that is resistant to LCA-induced liver injury provided further understanding of the mechanism of the LCA-induced liver toxicity. ALP, alkaline

phosphatase; ALT, alanine aminotransferase; CHK, choline kinase; CHPT1, choline phosphotransferase 1; CM, ceramide; FXR, farnesoid X receptor; LCA, lithocholic acid; LPC, lysophosphatidylcholine; LPCAT, lysophosphatidylcholine acyltransferase; OPLS, orthogonal projection to latent structures; PC, phosphatidylcholine; PCYT1, phosphate cytidylyltransferase 1; PLA, phospholipase A; PLD, phospholipase D; SGMS, sphingomyelin synthase; SM, sphingomyelin; SMPD, sphingomyelin phosphodiesterase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; UPLC-TOFMS, ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry. Female mice (C57BL/6NCr), farnesoid X receptor (Fxr)-null mice, and background-matched wildtype mice20

were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. For the LCA studies, mice were given 0.6% LCA-supplement AIN93G diet with the AIN93G diet as a control (Dyets, Bethlehem, PA). All animal studies were carried out in accordance with Institute

of Laboratory Animal Resources (ILAR) guidelines Trichostatin A datasheet and protocols approved by the National Cancer Institute Animal Care and Use Committee. Serum was prepared using serum separator tubes (Becton, Dickinson, Franklin Lakes, NJ). The serum catalytic activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) was measured with ALT and ALP assay kits, respectively (Catachem, Bridgeport, CT). Serum was prepared using serum separator tubes (Becton, Dickinson). The serum was diluted with 19 volumes of 66% acetonitrile selleck and centrifuged twice at 18,000g for 20 minutes to remove insoluble materials. UPLC-TOFMS was performed as reported.21 The eluant was introduced by electrospray ionization into the mass spectrometer (Q-TOF Premier; Waters, Milford, MA) operating in either negative or positive electrospray ionization modes. Data processing and multivariate data analysis were conducted as reported.7 Orthogonal projection to latent structures (OPLS) and contribution analyses were performed using SIMCA-P+12 (Umetrics, Kinnelon, NJ). RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and quantitative polymerase chain reaction (qPCR) was performed using complementary DNA (cDNA) generated from 1 μg total RNA with a SuperScript II Reverse Transcriptase kit and random oligonucleotides (Invitrogen). Primers were designed using qPrimerDepot.