Sensitive method for the quantification of β-glucuronidase activity in human urine using capillary electrophoresis with fluorescence detection

Abstract

Capillary electrophoresis (CE) with fluorescence detection was used to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl–β- d-glucuronide by β-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 β-glucuronidase activity in urine was next determined. Freshly collected urine samples were dialyzed, the retentate was incubated with 4-methylumbelliferyl–β- 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 β-glucuronidase activity in a 0.5-h incubation time, with an error of less than ±5%.