DETERMINATION OF SULFATE-CONJUGATED 3-BETA-HYDROXY BILE-ACID IN URINEBY HPLC WITH POSTCOLUMN FLUORESCENCE DETECTION USING AN IMMOBILIZED BETA-HYDROXYSTEROID DEHYDROGENASE COLUMN

A highly sensitive and specific method was established for quantificat ion of 3 beta-hydroxy-5-cholenoic acid by highperformance liquid chrom atography using an immobilized beta-hydroxysteroid dehydrogenase colum n (P-HSD column) and a postcolumn fluorescent detection. The beta-HSD column shows maximum enzyme activity and stability at 20 degrees C, an d has a very short life at 25 degrees C. The detection limit was 7 pmo l/injection. We also developed an efficient method for extracting the sulfate conjugates at position 3 from urine and conducting their enzym atic hydrolyses. This method was used to analyze urine from patients w ith congenital biliary atresia. A high concentration of 3 beta,12 alph a-dihydroxy-5-cholenoic acid was also present in the urine from these patients.