Bile Acids Metabonomic Study on the CCl₄- and α-Naphthylisothiocyanate-Induced Animal Models: Quantitative Analysis of 22 Bile Acids by Ultraperformance Liquid Chromatography−Mass Spectrometry

Abstract

Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver and intestinal disorders and other diseases affecting BA metabolism. A rapid, simple, and sensitive analytical method is needed to demonstrate the full metabolic profile and simultaneously determine the individual BAs in biological samples. In our present study, an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method has been established and validated for simultaneous quantitation of 22 BAs and a metabonomic study was performed based on the chemometric analysis of the serum samples from carbon tetrachloride (CCl4)- and alpha-naphthylisothiocyanate (ANIT)-induced liver failure rats. The optimal chromatographic condition was effected by UPLC (Acquity UPLC BEH column, 1.7 microm, 2.1 mm x 100 mm) using a linear gradient elution system of methanol-5 mM ammonium acetate containing 0.01% acetic acid after a simple-step deproteinization by precipitation. The separation of the 22 BAs can be finished in less than 12 min, and the concentrations of these BAs in rat serums were simultaneously determined using a selective ions monitoring mode. The method was validated with respect to repeatability (relative standard deviation < 9.78%) and accuracy (relative errors from -13.55 to 9.58%). The range of each BA was found from not detected (nd) to 8301 ng mL(-1), respectively. Furthermore, the developed method was successfully applied to the metabonomics analysis of BAs in CCl4- and ANIT-induced liver failure rats, using principle component analysis and canonical discriminant analysis. The serum samples from the two types of rat liver injury could be distinguished from each other and from the untreated animals according to the varieties of BAs. It indicated that the level of BAs could be considered as a sensitive parameter of hepatotoxicity induced by different chemical toxins. This novel metabonomics study of BAs based on the UPLC-MS profile provides not only an accurate quantitative assay of the serum concentrations of biomarkers but also a promising methodology for evaluation of liver injury.

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 ).…”

“…Physiological concentrations of circulating BAs in healthy subjects are low; a number of conditions, including hepatobiliary and intestinal diseases or drug-induced liver injury, can alter BA homeostasis, leading to increased BA levels (10)(11)(12). Accumulation of BAs is considered a hallmark of chronic cholestatic liver disease; more specifi cally, it has been shown that its deleterious toxic effects depend on the profi le of accumulated BAs ( 13 ).…”

A Perceptive of Mass Spectrometry in Clinical Chemistry: Hyphenation for Benefit of Mankind

“…LC/MS is a very important hyphenation technique used for the clinic chemistry. There are large numbers of reports on the analysis for different compounds to understand nephrotoxicity and hepratoxicity [30][31][32][33] by analysis of urine and serum samples. These studies were carried out to understand the mechanism or as biomarkers.…”