A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes.
A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.
D001647:Bile Acids and Salts / Q000032:analysis*; D008058:Dihydrolipoamide Dehydrogenase; D004800:Enzymes, Immobilized; D038181:FMN Reductase; D006913:Hydroxysteroid Dehydrogenases; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases
D001647:Bile Acids and Salts; D004800:Enzymes, Immobilized; D006913:Hydroxysteroid Dehydrogenases; C019189:12 alpha-hydroxysteroid dehydrogenase; D008156:Luciferases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase