Optimisation of an enzymatic method for beta-galactosidase.

Background: The enzyme beta-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. beta-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 microl) in 80 microl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 degrees C. Reaction was terminated by addition of 200 microl of glycine-NaOH, pH 12.8. The assay is linear to 3,000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2,012 U/ml was 4.7, 3.1, and 3.4, respectively (n=10). Inter-assay CV% at 51, 496, and 1,986 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 beta-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.