Isolation and biochemical analysis of vesicles from taurohyodeoxycholic acid-infused isolated perfused rat livers

Abstract

AIM: To isolate biliary lipid-carrying vesicles from isolated perfused rat livers after taurohyodeoxycholic acid (THDC) infusion. Biliary lipid vesicles have been implicated in hepatic disease and THDC was used since it increases biliary phospholipid secretion.

METHODS: Rat livers were isolated and perfused via the hepatic portal vein with THDC dissolved in Krebs Ringer Bicarbonate solution, pH 7.4, containing 1 mmol/L CaCl2, 5 mmol/L glucose, a physiological amino acid mixture, 1% bovine serum albumin and 20% (v/v) washed human erythrocytes at a rate of 2000 nmol/min for 2 h. The livers were then removed, homogenized and subjected to centrifugation, and the microsomal fraction was obtained and further centrifuged at 350000 g for 90 min to obtain subcellular fractions. These were analyzed for total phospholipid, cholesterol, protein and alkaline phosphodiesterase I (PDE).

RESULTS: No significant changes were observed in the total phospholipid, cholesterol and protein contents of the gradient fractions obtained from the microsomal preparation. However, the majority of the gradient fractions (p = 1.05-1.07 g/mL and. = 1.95-1.23 g/mL) obtained from THDC-infused livers had significantly higher PDE activity compared to the control livers. The low density gradient fraction (p= 1.05-1.07 g/mL) which was envisaged to contain the putative vesicle population isolated from THDC-perfused livers had relatively small amounts of phospholipids and protein when compared to the relevant control fractions; however, they displayed an increase in cholesterol and PDE activity. The phospholipids were also isolated by thin layer chromatography and subjected to fractionation by high performance liquid chromatography; however, no differences were observed in the pattern of the fatty acid composition of the phospholipids isolated from THDC and control perfused livers. The density gradient fractions (p= 1.10-1.23 g/mL) displayed an increase in all the parameters measured from both control and THDCinfused livers.

CONCLUSION: No significant changes in biliary lipids were observed in the fractions from THDC-infused livers; however, PDE activity was significantly increased compared to the control livers.