The Determination of Glucuronides in Biological Fluids by Lucigenin Chemiluminescence (beta-Glucuronidase, Glucuronic Acid, Immobilized Enzyme, (Hplc))
Klopf, Lori Lynn
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Permalink
https://hdl.handle.net/2142/70292
Description
Title
The Determination of Glucuronides in Biological Fluids by Lucigenin Chemiluminescence (beta-Glucuronidase, Glucuronic Acid, Immobilized Enzyme, (Hplc))
Author(s)
Klopf, Lori Lynn
Issue Date
1985
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Abstract
The use of certain chemiluminescence reactions as detection techniques for organic compounds has been explored during the past decade. In this research project the chemiluminescence reaction between alkaline lucigenin (10,10'-dimethyl-9,9'-biacridinium nitrate) and glucuronic acid (an important organic reductant in humans) was studied. The main goal of this project was to develop an analytical method for the quantitation of conjugated glucuronic acid (i.e., glucuronides) in physiological fluids using this chemiluminescence reaction as the detection technique.
Initial investigations showed that the addition of the surfactant sodium dodecyl sulfate to lucigenin prevented the inconvenient precipitation of the products of the lucigenin chemiluminescence reaction. Hydrolysis of the glucuronide bond, which is necessary for analysis of the resulting glucuronic acid by lucigenin chemiluminescence, was accomplished enzymically. (beta)-Glucuronidase (E. coli) was immobilized on controlled-pore glass and this support was packed into a column, which was then inserted into the continuous-flow system for on-line hydrolysis of glucuronides.
This system was successful for the quantitation of individual solutions and mixtures of various glucuronides. In order to analyze the species in urine and blood samples, however, a method had to be developed to prevent other compounds (e.g., other organic reductants) in these samples from interfering with the lucigenin chemiluminescence reaction. The best technique found for elimination of such interferences was a separation of glucuronic acid from the primary interferences in an anion exchange high-performance liquid chromatography column. By using appropriate valving, only the portion of the column effluent containing glucuronic acid was allowed to pass into the continuous-flow system for subsequent reaction with the chemiluminescence reagents. The detection limit (10-15 (mu)M), the linear working range (0.01-2 mM), and the precision (2% rsd) are all quite satisfactory for the analysis of the glucuronide levels usually found in biological samples. The chemiluminescence method developed here was compared with a standard colorimetric technique in the determination of the total (conjugated plus free) glucuronic acid concentration in urine samples. The chemiluminescence method was found to be superior to the colorimetric method in terms of accuracy, precision, and time per analysis.
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