Direct enzymic fluorimetric method for the determination of individual bile acids in bile

We describe a new, simple, fluorimetric enzymic method for the determination of individual bile acids in bile. Bile is diluted 4000-fold with water and 3 alpha- and 7 alpha-hydroxy bile acids are determined by equilibrium methods and chenodeoxycholic acid is determined by a differential kinetic method, without any prior separation and preparatory step. The equilibrium methods are based on the reaction of 3 alpha- or 7 alpha-hydroxy bile acids with beta-NAD+ in the presence of the enzyme 3 alpha- or 7 alpha-hydroxysteroid dehydrogenase (HSD). The kinetic method is based upon the reaction of chenodeoxycholic acid with beta-NAD+ in the presence of the enzyme 7 alpha-HSD. All measurements are monitored fluorimetrically. Cholic and deoxycholic acid are calculated by difference. Recovery experiments gave satisfactory results. Gallbladder bile from patients was analysed for the three major bile acids. The proposed method is suitable for clinical use.