Determination of Sulfate-Conjugated 3β-Hydroxy Bile Acid in Urine by HPLC with Postcolumn Fluorescence Detection Using an Immobilized β-Hydroxysteroid Dehydrogenase Column

抄録

A highly sensitive and specific method was established for quantification of 3β-hydroxy-5-cholenoic acid by high- Performance liquid chromatography using an immobilized -β-hydroxysteroid dehydrogenase column (β-HSD column) and a postcolumn fluorescent detection. The β-HSD column shows maximum enzyme activity and stability at 20°C, and has a very short life at 25°C. The detection limit was 7pmol/injection. We also developed an efficient method for extracting the sulfate conjugates at position 3 from urine and conducting their enzymatic hydrolyses. This method was used to analyze urine from patients with congenital biliary atresia. A high concentration of 3β, 12α-dihydroxy-5- cholenoic acid was also present in the urine from these patients.