Confirming lack of metabolism of vanillin to vanillic acid by xanthine oxidase

XO is also a molybdenum hydroxylase enzyme and can potentially metabolize vanillin to vanillic acid. Vanillin was incubated with partially purified rat liver XO fraction. Briefly, incubations were done in a volume of 500 μl and at final concentrations of vanillin (25 μM), and XO fraction (0.2 mg protein/ml). Incubations were conducted for 30 min and terminated with 250 μl of 0.5 M perchloric acid containing p-nitrocatechol as internal standard (IS, 3 μg/ml). The samples were vortexed, centrifuged, and an aliquot of the supernatant (50 μl) was injected onto the HPLC.

Further, inhibition of formation of vanillic acid was also studied using allopurinol, a specific inhibitor of XO. The incubation conditions were similar to that mentioned above and contained allopurinol at a final concentration of 5 μM. Post incubation, the samples were similarly processed.

Since XO rich fractions can potentially be contaminated by small amounts of AO,[15,16] the inhibition of formation of vanillic acid by raloxifene was also studied in the XO fractions. The incubation conditions were identical to that mentioned earlier, except that they contained raloxifene at a final concentration of 10 μM. Post incubation, the samples were similarly processed.