Bile Acid Metabolism in Isolated Rat Hepatocytes: Studied by Gas-Liquid Chromatography-Mass Spectrometry-Selected Ion Monitoring

Abstract

Bile acid contents in isolated rat hepatocytes were determined by gas-liquid chromatography-mass spectrometry-selected ion monitoring with the use of deuterium-labeled internal standards. This allowed us first to monitor the actual amounts of not only major but also minor bile acid components present with sufficient sensitivity and specificity and to follow the changes of individual bile acids in cultured rat hepatocytes simultaneously. In freshly isolated rat hepatocytes, cholic and beta-muricholic acids were the major components, comprising 35 and 46% of the total bile acids, respectively. These two bile acids were found to be most actively synthesized during the first 2 h of incubation and continued to increase thereafter for up to 6 h (the end of the period studied). In contrast, chenodeoxycholic and alpha-muricholic acids, which are the precursors of beta-muricholic acid, showed slight increases only in the first hour of incubation and decreased thereafter. These results suggested that the conversion to beta-muricholic acid from chenodeoxycholic acid via alpha-muricholic acid occurred rapidly in cultured rat hepatocytes. The secondary bile acids such as deoxycholic, hyodeoxycholic, and 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acids declined steadily from the start of incubation, which supported the findings that further hydroxylation of these dihydroxy bile acids occurs in rat liver.

Distinct forms of cytochrome P-450 are responsible for 6β-hydroxylation of bile acids and of neutral steroids

“…200% of the total bile acid pool [4]) are found. The predominant of these compounds, ,J-muricholic acid (3a,6,8,7fl-triOH), is thought to be formed by 6/)-hydroxylation of chenodeoxycholic acid (3a,7a-diOH) to a-muricholic acid (3af,6/,7a-triOH), followed by epimerization of the 7a-hydroxy group [5][6][7][8][9][10][11][12] (Fig. 1).…”

“…Selected steps of bile acid metabolism in the rat Cholesterol is converted in a multi-step pathway to the two primary bile acids, cholic and chenodeoxycholic acids. A part of chenodeoxycholic acid is 6,/-hydroxylated to a-muricholic acid, which in turn is converted by epimerization to fl-muricholic acid [5][6][7][8][9][10][11][12]. Bacterial 7a-dehydroxylation of chenodeoxycholic acid leads to the formation of lithocholic acid.…”

Bile Acids in the Assessment of Hepatocellular Function

“…The latter are more hydrophobic than their precursors and, potentially, more toxic. Rat hepatocytes synthesize primarily CA and r3-muricholic (3-MCA) acids with small amounts of CDCA, a-MCA, and Ô,22-r3-MCA (5,39,60). In Hep-G2 cells, CDCA is synthesized at a rate more than 7-fold greater than that for CA due to a low level of microsomal 12a-hydroxylase (66).…”