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.

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 beta-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 beta-muricholic acid were compared with values of N(n) 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 beta-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 micro M, 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 beta-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 beta-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.