Journal of Lipid Research, Vol. 24, 316-323, March 1983
Copyright © 1983 by Lipid Research, Inc.

Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B

Herman Jan Kempen , Margreet Vos-van Holstein , and Jan de Lange

Gaubius Institute, Health Research Division TNO, Herenstraat 5d, 2313 AD Leiden, The Netherlands

After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and ßbeta;-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N^a) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N^a for cholic and ßbeta;-muricholic acid were compared with values of Nⁿ or N^o, theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first hour of incubation should be totally derived from pre-existent cholesterol, whereas 50% and 25% of the cholic and ßbeta;-muricholic acid, respectively, produced during the second and third hours of incubation should be derived from newly synthesized cholesterol. The contribution of newly made cholesterol as substrate for bile acid production was also estimated by using ML-236B. In a concentration of 12 µM, it depressed cholesterol synthesis by 90% during 1 or 3 hours of incubation of hepatocytes of cholestyramine-fed rats. Mass production of cholic acid was depressed by 25% and that of ßbeta;-muricholic acid was not inhibited at all by ML-236B during the first hour of incubation, while they were depressed by 71 and 52%, respectively, during the second plus third hours of incubation. It is concluded that 1) in isolated hepatocytes newly made cholesterol can be a significant substrate for bile acid formation; 2) there are separate cholesterol substrate pools for the productions of cholic or ßbeta;-muricholic acid; 3) of the total carbon flux directed into cholesterol synthesis, the major part ends up as free cholesterol and only a minor part as bile acids, even in hepatocytes with a derepressed bile acid production.—Kempen, H. J., M. Vos-van Holstein, and J. de Lange. Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B.

Supplementary key words cholestryamine • newly synthesized cholesterol • endogenous cholesterol

Submitted on January 4, 1982
Revised on August 31, 1982

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?