Homogeneous bioluminescence assay for galactosuria: interference and kinetic analysis.

Elevated galactose concentration in urine is an important clinical symptom of galactosemia and other metabolic disorders. A quantitative assay for galactose using firefly luciferase bioluminescence is presented. The assay couples the galactokinase and firefly luciferase reactions. A higher concentration of galactose present in the sample produces a faster decrease in ATP concentration, which is monitored by firefly luciferase bioluminescence. The kinetic assay is modeled and analyzed. The interference between the two reactions, the interference of certain sugars and other components in the urine, the specificity, and the optimal pH for galactokinase were studied. Calibration curves were constructed and compared with a conventional spectrophotometric assay for galactose. The bioluminescence assay is relatively fast and specific for galactose with a linear range from 1 to 20 mM galactose. The effect of other galactose metabolites (galactonate and galactitol) has also been studied.