BIOMAT Database Logo BIOMAT Database Logo

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes. 1983

J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008058 Dihydrolipoamide Dehydrogenase A flavoprotein containing oxidoreductase that catalyzes the reduction of lipoamide by NADH to yield dihydrolipoamide and NAD+. The enzyme is a component of several MULTIENZYME COMPLEXES. Lipoamide Dehydrogenase,NAD Diaphorase,NADH Diaphorase,Diaphorase (Lipoamide Dehydrogenase),Dihydrolipoyl Dehydrogenase,Glycine Decarboxylase Complex L-Protein,L-Protein, Glycine Decarboxylase Complex,Lipoamide Dehydrogenase, Valine,Lipoic Acid Dehydrogenase,Lipoyl Dehydrogenase,Valine Lipoamide Dehydrogenase,Dehydrogenase, Dihydrolipoamide,Dehydrogenase, Dihydrolipoyl,Dehydrogenase, Lipoamide,Dehydrogenase, Lipoic Acid,Dehydrogenase, Lipoyl,Dehydrogenase, Valine Lipoamide,Diaphorase, NAD,Diaphorase, NADH,Glycine Decarboxylase Complex L Protein
D008156 Luciferases Enzymes that oxidize certain LUMINESCENT AGENTS to emit light (PHYSICAL LUMINESCENCE). The luciferases from different organisms have evolved differently so have different structures and substrates. Luciferase
D008163 Luminescent Measurements Techniques used for determining the values of photometric parameters of light resulting from LUMINESCENCE. Bioluminescence Measurements,Bioluminescent Assays,Bioluminescent Measurements,Chemiluminescence Measurements,Chemiluminescent Assays,Chemiluminescent Measurements,Chemoluminescence Measurements,Luminescence Measurements,Luminescent Assays,Luminescent Techniques,Phosphorescence Measurements,Phosphorescent Assays,Phosphorescent Measurements,Assay, Bioluminescent,Assay, Chemiluminescent,Assay, Luminescent,Assay, Phosphorescent,Assays, Bioluminescent,Assays, Chemiluminescent,Assays, Luminescent,Assays, Phosphorescent,Bioluminescence Measurement,Bioluminescent Assay,Bioluminescent Measurement,Chemiluminescence Measurement,Chemiluminescent Assay,Chemiluminescent Measurement,Chemoluminescence Measurement,Luminescence Measurement,Luminescent Assay,Luminescent Measurement,Luminescent Technique,Measurement, Bioluminescence,Measurement, Bioluminescent,Measurement, Chemiluminescence,Measurement, Chemiluminescent,Measurement, Chemoluminescence,Measurement, Luminescence,Measurement, Luminescent,Measurement, Phosphorescence,Measurement, Phosphorescent,Measurements, Bioluminescence,Measurements, Bioluminescent,Measurements, Chemiluminescence,Measurements, Chemiluminescent,Measurements, Chemoluminescence,Measurements, Luminescence,Measurements, Luminescent,Measurements, Phosphorescence,Measurements, Phosphorescent,Phosphorescence Measurement,Phosphorescent Assay,Phosphorescent Measurement,Technique, Luminescent,Techniques, Luminescent
D009247 NADH, NADPH Oxidoreductases A group of oxidoreductases that act on NADH or NADPH. In general, enzymes using NADH or NADPH to reduce a substrate are classified according to the reverse reaction, in which NAD+ or NADP+ is formally regarded as an acceptor. This subclass includes only those enzymes in which some other redox carrier is the acceptor. (Enzyme Nomenclature, 1992, p100) EC 1.6. Oxidoreductases, NADH, NADPH,NADPH Oxidoreductases NADH,Oxidoreductases NADH, NADPH
D004800 Enzymes, Immobilized Enzymes which are immobilized on or in a variety of water-soluble or water-insoluble matrices with little or no loss of their catalytic activity. Since they can be reused continuously, immobilized enzymes have found wide application in the industrial, medical and research fields. Immobilized Enzymes,Enzyme, Immobilized,Immobilized Enzyme
D006913 Hydroxysteroid Dehydrogenases Enzymes of the oxidoreductase class that catalyze the dehydrogenation of hydroxysteroids. (From Enzyme Nomenclature, 1992) EC 1.1.-. Hydroxysteroid Dehydrogenase,Dehydrogenase, Hydroxysteroid,Dehydrogenases, Hydroxysteroid
D001647 Bile Acids and Salts Steroid acids and salts. The primary bile acids are derived from cholesterol in the liver and usually conjugated with glycine or taurine. The secondary bile acids are further modified by bacteria in the intestine. They play an important role in the digestion and absorption of fat. They have also been used pharmacologically, especially in the treatment of gallstones. Bile Acid,Bile Salt,Bile Salts,Bile Acids,Acid, Bile,Acids, Bile,Salt, Bile,Salts, Bile
D038181 FMN Reductase An enzyme that utilizes NADH or NADPH to reduce FLAVINS. It is involved in a number of biological processes that require reduced flavin for their functions such as bacterial bioluminescence. Formerly listed as EC 1.6.8.1 and EC 1.5.1.29. Flavin Mononucleotide Reductase,NAD(P)H-Flavin Oxidoreductase,FMN Oxidoreductase,NAD(P)H Dehydrogenase (FMN),NAD(P)H-FMN Oxidoreductase,NADH-FMN Oxidoreductase,NADH-Flavin Oxidoreductase,NADPH-Flavin Reductase,Mononucleotide Reductase, Flavin,NADH FMN Oxidoreductase,NADPH Flavin Reductase,Oxidoreductase, FMN,Oxidoreductase, NADH-FMN,Oxidoreductase, NADH-Flavin,Reductase, FMN,Reductase, Flavin Mononucleotide,Reductase, NADPH-Flavin

Related Publications

J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
A bioluminescence assay for total 3 alpha-hydroxy bile acids in serum using immobilized enzymes.
February 1984, Clinica chimica acta; international journal of clinical chemistry,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Immobilized enzymes for assaying bile acids.
January 1986, Methods in enzymology,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Bioluminescence measurement of primary bile acids using immobilized 7 alpha-hydroxysteroid dehydrogenase: application to serum bile acids.
December 1982, Journal of lipid research,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Bioluminescent assay of creatine kinase activity using immobilized firefly extract.
October 1986, Analytical biochemistry,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Bioluminescent assay for total bile acids in serum with use of bacterial luciferase.
June 1983, Clinical chemistry,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
A direct enzymic assay for 7 -hydroxy bile acids and their conjugates.
January 1973, Clinical science,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Bioluminescent assays of picomole levels of various metabolites using immobilized enzymes.
December 1982, Analytical biochemistry,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Assay procedures for immobilized enzymes.
January 1976, Methods in enzymology,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Potential bile acid metabolites. 9. 3,12-Dihydroxy- and 12 beta-hydroxy-5 alpha-cholanoic acids.
July 1985, Journal of lipid research,
J Schoelmerich, and J E Hinkley, and I A Macdonald, and A F Hofmann, and M DeLuca
Continuous-flow bioluminescent assays employing sepharose-immobilized enzymes.
January 1986, Methods in enzymology,
Copied contents to your clipboard!