Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.     

Disposition and metabolism of 2-fluoro-beta-alanine conjugates of bile acids following secretion into bile

Since 2-fluoro-beta-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439-5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholyglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugated during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.     

Effect of biliary obstruction on formation and metabolism of bile acids in rat

The effect of biliary obstruction in the rat on several hydroxylations involved in the formation and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The rate of 7α-hydroxylation of cholesterol increased two- to threefold between 24 and 48 hours after ligation of the bile duct and remained at this level the next 48 hours. During the first 24 hours of obstruction the rates of 1 2α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid decreased but returned to control levels between 24 and 48 hours after operation. The rate of 6β-hydroxylation of lithocholic acid and taurochenodeoxycholic acid increased gradually and reached a plateau between 24 and 48 hours at which time the rate was two to three times faster than in the controls. The increase in 6β-hydroxylase activity was reflected in the pattern of the bile acids excreted in urine. After 48 hours of obstruction β-muricholic acid accounted for 50% or more of the bile acids in urine.     

Bile acids and lipoprotein metabolism: a renaissance for bile acids in the post-statin era?

Based on an improved molecular understanding of how bile acid metabolism is regulated, an exciting period of research developments can be expected. By new ways of stimulating cholesterol breakdown to bile acids, novel therapeutic principles can be forseen which will further improve our potential for treating and preventing atherosclerosis.     

Effect of vanadate on the metabolism of bile acids in diabetic rats

The effect of treatment with vanadate on the metabolism of bile acids was studied in normal and diabetic rats. In the normal rats, the composition of biliary bile acids was not changed by drinking an aqueous solution of 0.2 g/l NaVO3-5 g/l NaCl ad libitum for two weeks. By contrast, the increased proportion of cholic acid, accounting for 88% of the total biliary bile acids, in the diabetic rats decreased to 46% by the treatment with vanadate without any elevation of serum insulin level. These results indicate that vanadate with an insulin-like effect on glucose metabolism in diabetic rats has such an effect also on bile acid metabolism in an insulin-deficient state.