Metabolomic investigation of cholestasis in a rat model using ultra‐performance liquid chromatography/tandem mass spectrometry

Abstract

Metabolomics follows the changes in concentrations of endogenous metabolites, which may reflect various disease states as well as systemic responses to environmental, therapeutic, or genetic interventions. In this study, we applied metabolomic approaches to monitor dynamic changes in plasma and urine metabolites, and compared these metabolite profiles in Eisai hyperbilirubinemic rats (EHBR, an animal model of cholestasis) with those in the parent strain of EHBR - Sprague-Dawley (SD) rats - in order to characterize cholestasis pathophysiologically. Ultra-performance liquid chromatography/tandem mass spectrometry-based analytical methods were used to assay metabolite levels. More than 250 metabolites were detected in both plasma and urine, and metabolite profiles of EHBR differed from those of SD rats. The levels of antioxidative and cytoprotective metabolites, taurine and hypotaurine, were markedly increased in urine of EHBR. The levels of many bile acids were also elevated in plasma and urine of EHBR, but the extent of elevation depended on the particular bile acid. The levels of cytoprotective ursodeoxycholic acid and its conjugates were markedly elevated, while that of cytotoxic chenodeoxycholic acid remained unchanged, suggesting the balance of bile acids had shifted resulting in decreased toxicity. In EHBR, reduced biliary excretion leads to increased systemic exposure to harmful compounds including some endogenous metabolites. Our metabolomic data suggest that mechanisms exist in EHBR that compensate for cholestasis-related damage.

Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method

“…The method includes the simultaneous quantifi cation Serum and tissue liver sample preparation Most methods used to target BAs in serum involve the removal of proteins by precipitation using organic solvents, followed by a solid-phase extraction (SPE) ( 12,22,24,(28)(29)(30)(31)(32)(33). On the other hand, preparation of liver tissue involves a homogenization step, usually followed by an SPE step ( 17,34 ).…”

“…The patterns of BAs that we obtained in serum samples from each species are in good agreement with those reported by other authors. However, our method provides a more specifi c and comprehensive profi ling of BAs in rodents than previous reports in which ␣ -, ␤ -, and -MCA, and their taurine conjugates were not properly quantifi ed ( 12,23,31 ). The results are expressed in nM as mean ± SD (n = 8).…”

“…By considering that BAs are signaling molecules involved in controlling the expression of different genes, any alteration of the intrahepatic BA content (concentration and/or composition) could affect the metabolic homeostasis of the liver and the whole organism. Defective canalicular export, due to impaired canalicular transport or a physical obstruction to bile fl ow, leads to intrahepatic BA accumulation, which may contribute have been reported in serum from patients with cholestasic liver diseases ( 13,28 ), in plasma and urine from a genetic model of cholestasis in rats ( 31 ), in serum of rats treated with hepatotoxins ( 12 ), and in rat bile and liver samples during chemically induced hepatocarcinogenesis ( 37 ). Moreover, an in vitro study has suggested that LCA, a minor BA form, plays an important role in controlling the expression of several proteins involved in BA synthesis, metabolism, and transport ( 6 ).…”

The metabolomic window into hepatobiliary disease

“…Metabolomics revealed that cholestasis induced in Fxr -null mice by a cholic acid diet resulted in increased urinary excretion of bile salt tetrols, predominantly 3α,6,7α,12α-tetrahydroxy-5β-cholestan-26-oyltaurine, due to an adaptive upregulation of the steroid-hydroxylating cytochrome P450 CYP3A11 in these mice [99]. An adaptive response was also characterized in a rat cholestasis model, with a shift from cytotoxic to cytoprotective bile acids in plasma and urine [100]. …”

Application of ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry in identification of three isoflavone glycosides and their corresponding metabolites

“…Ultra‐performance liquid chromatography (UPLC) operates with sub‐2 μm packed columns and a fluid system capable of operating at higher pressure, providing better peak resolution, higher sensitivity and fast speed for complex mixture separation compared with conventional high‐performance liquid chromatography (HPLC) . The combination of UPLC and quadrupole time‐of‐flight mass spectrometry has been one of the predominant analytical platforms for identification of the constituents of natural products and metabolites …”