Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7β-hydroxysteroid dehydrogenase

Abstract

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7β-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7β-hydroxyl groups were quantified using 7β-hydroxysteroid dehydrogenase and 3α-hydroxyl groups (total bile acids) were quantified using 3α-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7β-hydroxyl groups]/[3α-hydroxyl groups] × 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7β-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8–24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.