Optimisation of an enzymatic method for beta-galactosidase.

Area of Science:

• Biochemistry
• Transplantation immunology
• Analytical chemistry

Background:

• Beta-galactosidase in Kupffer cells is a potential marker for liver dysfunction before transplantation.
• Traditional spectrophotometric methods lack the necessary sensitivity for accurate detection.

Purpose of the Study:

• To develop and optimize a sensitive fluorimetric assay for beta-galactosidase.
• To enhance the utility of beta-galactosidase as a marker for pre-transplant liver assessment.

Main Methods:

• Optimized a microtitre plate-based fluorimetric assay using 4-methylumbelliferyl-galactoside (MUG) as a substrate.
• Determined optimal reaction conditions including pH (4.4), substrate concentration (3.33 mmol/l), and incubation time (30-60 min at 37°C).
• Utilized specific excitation (355 nm) and emission (460 nm) wavelengths for detecting the product, 4-methylumbelliferone (4-MU).

Main Results:

• The assay demonstrated high sensitivity and linearity up to 3,000 U/ml.
• Achieved low intra-assay coefficients of variation (CV%) of 3.1-4.7% and inter-assay CV% of 3.9-7.0%.
• Significant differences in perfusate beta-galactosidase were observed between cold-stored and warm-perfused livers in a porcine model.

Conclusions:

• The optimized fluorimetric assay provides a practical and sensitive method for detecting beta-galactosidase.
• This assay has significant utility in assessing liver function prior to transplantation.
• Demonstrated the assay's effectiveness in differentiating liver preservation strategies in a transplantation model.