A possible role of chenodeoxycholic acid and glycine-conjugated bile acids in fibrotic steatohepatitis in a dietary rat model.

BACKGROUND AND AIMS: Our previous study indicated that hepatic bile acids (BAs) may have deposited and stimulated the pathogenesis of a high fat-cholesterol (HFC) diet-induced fibrotic steatohepatitis in stroke-prone spontaneously hypertensive 5/Dmcr rats, based on dysregulated BA homeostasis pathways. We aimed to further characterize BA profiles in liver and evaluate their relationships to liver injury using this model. METHODS: Hepatic 21 BA levels were determined by ultra-performance liquid chromatography-tandem mass spectrometry, and their correlations with macrovesicular steatosis score, serum alanine aminotransferase (ALT) level and quantified fibrotic area were assessed using Spearman and Pearson correlations. RESULTS: Compared to control, BAs highly accumulated in HFC-fed rat liver at 2 weeks: cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) were major species, thereafter, levels of CA and DCA declined, but CDCA species persistently increased, which induced a decrease in total CA/total CDCA ratio at 8 and 14 weeks. CDCA species positively, while total CA/total CDCA negatively, correlated with macrovesicular steatosis score, serum ALT and quantified fibrotic area. Unlike control, total ursodeoxycholic acid was minor in HFC-fed rat liver, and inversely correlated to aforementioned indicators of liver injury; total glyco-BAs, rather than tauro-BAs, were predominant in HFC-fed rat liver, and positively correlated with macrovesicular steatosis score. Moreover, its ratio to total tauro-BAs positively correlated with each parameter of liver injury, while inverse associations were detected for total tauro-BAs. CONCLUSIONS: Hepatic BA accumulation may potentiate liver disease. CDCA and glyco-BAs play a more important role in the pathogenesis of fibrotic steatohepatitis.

Simple and rapid quantitation of 21 bile acids in rat serum and liver by UPLC-MS-MS: effect of high fat diet on glycine conjugates of rat bile acids.

In this report, we present a simple and rapid method for analysis of 21 kinds of bile acids and the conjugates in rat serum and liver samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) in the negative ionization mode, using cholic-2, 2, 4, 4-d4 acid as internal standard. After liquid-liguid extraction from serum and liver samples, specimens were analyzed by UPLC equipped with an Acquity TQD tandem quadrupole mass spectrometer. All of the 21 bile acids were sufficiently separated within 5 min. For most bile acids, calibration curves showed good linearities in the range of 0.25 to 5000 ng/mL for serum samples, 2.5 ng/g to 50 microg/g for liver samples. The limits of detection (LOD) were estimated to be less than 0.25 to 7.5 ng/mL in serum, less than 2.5 to 10 ng/g in liver samples. The present method was validated with respect to repeatability; the coefficient of variation (CV) values were less than 26.7% in the serum and 25.9% in the liver. In the animal study, we compared 21 bile acids in the serum and liver samples of the stroke-prone spontaneously hypertensive (SHRSP) rats fed with control (SP) diet or high-fat and high-cholesterol-containing (HFC) diet. By feeding with HFC diet, the glycine conjugates of some bile acids significantly increased and the taurine conjugate of ulsodeoxicolate (TUDC) decreased in serum and liver samples. Our results suggest that the change of bile acid profiles could be applied for the diagnosis of non-alcoholic fatty liver disease (NAFLD).