Compensatory Role of P-Glycoproteins in Knockout Mice Lacking the Bile Salt Export Pump

Bile salt export pump (BSEP; ATP-binding cassette, subfamily B, member 11 ) mutations in humans result in progressive familial intrahepatic cholestasis type 2, a fatal liver disease with greatly reduced bile flow. However in mice, Bsep knockout leads only to mild cholestasis with substantial bile flow and up-regulated P-glycoprotein genes (multidrug resistance protein 1a [Mdr1a] and Mdr1b). To determine whether P- glycoprotein is responsible for the relatively mild phenotype observed in Bsep knockout mice, we have crossed mouse strains knocked out for Bsep and the two P-glycoprotein genes and generated a triple knockout mouse. We found that a knockout of the three genes leads to a significantly more severe phenotype with impaired bile formation, jaundice, flaccid gallbladder, and increased mortality. The triple knockout mouse is the most severe genetic model of intrahepatic cholestasis yet developed. Conclusion: P-glycoprotein functions as a critical compensatory mechanism, which reduces the severity of cholestasis in Bsep knockout mice

Metabolic Profiling of Bile Acids in Human and Mouse Blood by LC–MS/MS in Combination with Phospholipid-Depletion Solid-Phase Extraction

To obtain a more comprehensive profile of bile acids (BAs) in blood, we developed an ultrahigh performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC–MRM-MS) method for the separation and detection of 50 known BAs. This method utilizes phospholipid-depletion solid-phase extraction as a new high-efficiency sample preparation procedure for BA assay. UPLC/scheduled MRM-MS with negative ion electrospray ionization enabled targeted quantitation of 43 and 44 BAs, respectively, in serum samples from seven individuals with and without fasting, as well as in plasma samples from six cholestatic gene knockout mice and six age- and gender-matched wild-type (FVB/NJ) animals. Many minor BAs were identified and quantitated in the blood for the first time. Method validation indicated good quantitation precision with intraday and interday relative standard deviations of ≤9.3% and ≤10.8%, respectively. Using a pooled human serum sample and a pooled mouse plasma sample as the two representative test samples, the quantitation accuracy was measured to be 80% to 120% for most of the BAs, using two standard-substance spiking approaches. To profile other potential BAs not included in the 50 known targets from the knockout versus wild-type mouse plasma, class-specific precursor/fragment ion transitions were used to perform UPLC–MRM-MS for untargeted detection of the structural isomers of glycine- and taurine-conjugated BAs and unconjugated tetra-hydroxy BAs. As a result, as many as 36 such compounds were detected. In summary, this UPLC–MRM-MS method has enabled the quantitation of the largest number of BAs in the blood thus far, and the results presented have revealed an unexpectedly complex BA profile in mouse plasma