Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver

Modifications of the analytical method to determine L-gulono-γ-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-γ-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-γ-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-γ-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-γ-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-γ-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-γ-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.