Bioluminescence measurement of primary bile acids using immobilized 7 alpha-hydroxysteroid dehydrogenase: application to serum bile acids.
A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. The method is based on enzymatic dehydrogenation of bile acids using a bacterial 7 alpha-hydroxysteroid dehydrogenase that is co-immobilized on Sepharose 4B beads with NADH:FMN oxidoreductase and a bacterial luciferase. The assay is specific for 7 alpha-hydroxy bile acids and has a detection limit of 0.5 pmol/tube, with a linear range of 0.5-50 pmol/tube. The assay shows good precision (6-8% intra-assay; 8-10% inter-assay). The values obtained with the bioluminescence assay showed good agreement with those obtained by gas-liquid chromatography, radioimmunoassay, or endpoint enzymatic assays. When applied to the measurement of serum bile acids, there was no interference from serum albumin, and the effect of other dehydrogenase activity in serum could be eliminated by heating the sample prior to assay. Since the method is rapid (1 minute), extremely sensitive (requires only 10 microliters of serum), and specific, it appears to be the best method currently available for the measurement of serum primary bile acids.
D001647:Bile Acids and Salts / Q000097:blood*; D002849:Chromatography, Gas; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D008163:Luminescent Measurements; D008722:Methods; D011863:Radioimmunoassay / Q000379:methods
D001647:Bile Acids and Salts; D004800:Enzymes, Immobilized; D006913:Hydroxysteroid Dehydrogenases; C025376:7 alpha-hydroxysteroid dehydrogenase