Attenuation of Slc27a5 Gene Expression Followed by LC-MS Measurement of Bile Acid Reconjugation Using Metabolomics and a Stable Isotope Tracer Strategy

The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D-4-cholic acid (D-4-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of similar to 90% gene expression was achieved followed by reduction in the reconjugation of D-4-CA to D-4-taurocholic acid (D-4-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many 1:14 and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.