Determination of ursodeoxycholic acid in serum by a new fluorometric enzymatic method using 7 beta-hydroxysteroid dehydrogenase from Clostridium absonum.
A fluorometric enzymatic method for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates in human serum has been developed. A simple and fast purification and preconcentration procedure using Sep Pak C18 cartridges was employed for the UDCA extraction from human serum. UDCA and its conjugates were determined in the extracted sample by an equilibrium method based on the enzymatic conversion of the 7 alpha-hydroxy group into 7-oxo group by beta-nicotinamide adenine dinucleotide phosphate in the presence of 7 beta-hydroxysteroid dehydrogenase (7 beta-HSD) and the produced NADPH was monitored fluorometrically. The 7 beta-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC No. 27555) and purified by affinity chromatography. The method has a limit of detection of 0.8 microM in serum and the precision varied from 6.1 to 2.0% for low and high concentrations, respectively. The recovery of UDCA from serum samples was about 99% (range 85-105%). The method was successfully applied to UDCA determination in serum samples from patients treated with UDCA for primary biliary cirrhosis.