Xanthine Oxidase (XO), a form of xanthine oxidoreductase which generates reactive oxygen species (ROS), produces uric acid through the catabolism of purine bases. XO is highly abundant in endothelial cells, where its metabolic byproducts regulate nitric oxide and acts as a physiological inflammatory signal. Excess XO activity drives vascular oxidative stress by its byproduct, superoxide, reacting with nitric oxide to form peroxynitrite, ultimately leading to endothelial dysfunction. This impairs vasodilation contributing to hypertension and ischemia-reperfusion injury. In the liver, elevated activity of XO can indicate acute cellular damage, systemic hyperuricemia, and promote fibrosis and necrosis associated with MASLD/NAFLD progression. Monitoring XO levels is important for understanding liver health and evaluating chronic metabolic dysfunction.

XO Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring XO activity in plasma, serum, cell lysates, saliva, and other biological liquid samples. This assay is based on a coupled enzyme reaction. Xanthine oxidase (XO) catalyzes the sequential oxidation of hypoxanthine to xanthine and uric acid producing the byproduct hydrogen peroxide (H₂O₂). The hydrogen peroxide produced reacts with colorimetric probe to generate a pink-colored product that is measurable colorimetrically (570 nm) and fluorometrically (Ex/Em = 535/587nm). The change in absorbance is directly proportional to XO activity in the sample.

• Microplate reader capable of measuring OD 570 nm or Ex/Em = 535/587 nm
• 96-well black plate for fluorometric assay
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted H₂O₂ standards, samples, or positive control to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Immediately measure the absorbance at 570 nm or fluorescent intensity at Ex/Em 535/587nm for OD1 (or RFU1) read using a plate reader.
5. Incubate the plate for 20 minutes at room temperature with mixing in the dark then measure the absorbance at 570 nm or fluorescent intensity at Ex/Em 535/587 nm for OD2 read using a plate reader.