Sensitive method for the quantification of beta-glucuronidase activity in human urine using capillary electrophoresis with fluorescence detection.
Capillary electrophoresis (CE) with fluorescence detection was used to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by beta-glucuronidase. Enzyme substrate saturation kinetics were studied in buffer and the pH range for the enzyme reaction was optimized. A linear relationship of initial enzyme reaction velocity as a function of peak area of enzyme product was obtained for enzyme activity ranging from 1 to 100 units. The beta-glucuronidase activity in urine was next determined. Freshly collected urine samples were dialyzed, the retentate was incubated with 4-methylumbelliferyl-beta-D-glucuronide, boiled and centrifuged. The supernatant was separated by CE in an uncoated capillary with 0.1 M sodium acetate buffer by applying a voltage of 12 kV. The product of the enzymatic reaction, 4-methylumbelliferone, was detected by fluorescence, facilitating the determination of as little as one unit of beta-glucuronidase activity in a 0.5-h incubation time, with an error of less than +/-5%.
Wu
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Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242, USA.
Loganathan
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D
Linhardt
R J
RJ
D000328:Adult; D000818:Animals; D002021:Buffers; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D005966:Glucuronidase / Q000652:urine*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D008297:Male; D012680:Sensitivity and Specificity
D002021:Buffers; D005456:Fluorescent Dyes; D006923:Hymecromone; C034888:4-methylumbelliferyl glucuronide; D005966:Glucuronidase