Homogeneous bioluminescence assay for galactosuria: interference and kinetic analysis

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Elevated urinary galactose is a key indicator of galactosemia and other metabolic disorders.
• Accurate quantification of galactose is crucial for timely diagnosis and management.
• Existing assays may have limitations in speed, specificity, or complexity.

Purpose of the Study:

• To develop and validate a novel quantitative assay for galactose using firefly luciferase bioluminescence.
• To assess the assay's performance, including specificity, linearity, and potential interferences.
• To compare the bioluminescence assay with conventional spectrophotometric methods.

Main Methods:

• Coupling of galactokinase and firefly luciferase reactions to monitor ATP depletion.
• Kinetic analysis of bioluminescence signal decrease correlated with galactose concentration.
• Investigation of optimal pH, interference from other urinary components, and specificity.
• Construction of calibration curves and comparison with spectrophotometric assays.

Main Results:

• The bioluminescence assay demonstrates a relatively fast and specific measurement of galactose.
• A linear detection range from 1 to 20 mM galactose was established.
• The assay's performance was evaluated concerning potential interferences and optimal conditions.
• Comparison with spectrophotometric methods validated the assay's accuracy.

Conclusions:

• The firefly luciferase bioluminescence assay provides a sensitive and specific method for quantifying urinary galactose.
• This assay offers a valuable tool for the clinical diagnosis of galactosemia and related metabolic disorders.
• The method's speed and specificity make it a promising alternative to existing diagnostic techniques.