Optimisation of an enzymatic method for β-galactosidase

Background: The enzyme β-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. Methods: Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. β-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm. Results: Reaction conditions in a citrate–phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6–5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 μl) in 80 μl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 °C. Reaction was terminated by addition of 200 μl of glycine–NaOH, pH 12.8. The assay is linear to 3000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2012 U/ml was 4.7, 3.1, and 3.4, respectively (n=10). Inter-assay CV% at 51, 496, and 1986 U/ml was 7.0, 4.0, and 3.9, respectively (n=10). Conclusions: The assay has greater practical utility and demonstrated significant differences in the perfusate β-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.