A bioluminescent assay for 12-α-hydroxy bile acids using immobilized enzymes

✍ Scribed by Juergen Schoelmerich; John E. Hinkley; Ian A. Macdonald; Alan F. Hofmann; Marlene DeLuca

• Book ID: 102986914
• Publisher: Elsevier Science
• Year: 1983
• Tongue: English
• Weight: 519 KB
• Volume: 133
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90250-6

✦ Synopsis

A bioluminescent assay for 12-cr-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-a-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPHPMN oxidoreductase or bacterial diaphorase. The assay was specific for 12+hydroxy bile acids and the lower limit of detection was 4 pmol/O.S 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°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-o-hydroxy bile acid concentrations in the serum of patients or experimental animals.