Bioluminescent assay for total bile acids in serum with use of bacterial luciferase.
This simple, rapid, sensitive kinetic bioluminescent method for the assay of bile acids in serum involves use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) in combination with a new, commercially available NADH Monitoring Reagent (LKB-Wallac) containing a low activity of NADH:FMN-oxidoreductase and a high activity of bacterial luciferase. Interfering dehydrogenases in serum are inactivated in the test tube with trichloroacetic acid before the assay. The standard curve is linear for concentrations of bile acids up to about 300 mumol/L. With a sample volume of 20 microL, the detection limit is about 0.2 mumol/L. The within-run precision (CV) is about 10%, both at high and low concentrations of bile acids in serum. Correlation is good (r = 0.996) between results by this method and an enzymatic method based on spectrophotometry. However, the latter method is considerably less sensitive, and it is less precise at low concentrations of bile acids.
D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D001647:Bile Acids and Salts / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D013053:Spectrophotometry; D014238:Trichloroacetic Acid
D001647:Bile Acids and Salts; D007202:Indicators and Reagents; D014238:Trichloroacetic Acid; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D008156:Luciferases