Xanthine is a key purine base and intermediate in catabolic pathway of purine metabolism. Xanthine is produced during catabolic breakdown of DNA and RNA from hypoxanthine via oxidation by xanthine oxidase, yielding uric acid, and guanine via deamination by guanine deaminase. Xanthine is distributed throughout the body in tissues, blood, and excretory fluids regulated by xanthine oxidoreductase. Xanthine oxidoreductase deficiency can cause xanthinuria leading to excessive xanthine in the blood and urine. This can result in urolithiasis, severe renal colic, and renal failure. Xanthine serves as an indicator in severe cellular stress during ischemia injury. Under these conditions, ATP breaks down to hypoxanthine and xanthine triggering the accumulation of destructive reactive oxygen species (ROS). Xanthine can be modified chemically to yield methylxanthines, which are used in therapeutic applications for managing Chronic Obstructive Pulmonary Disease, apnea of prematurity in newborns, and asthma. Monitoring Xanthine/Hypoxanthine concentration is critical for managing drug therapies and monitoring conditions associated with abnormal purine metabolism.

Xanthine/Hypoxanthine Assay Kit provides a simple, reproducible, and sensitive tool of measuring Xanthine/Hypoxanthine concentration in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. Xanthine oxidase catalyzes the conversion of hypoxanthine and xanthine to uric acid and 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 signal measured is directly proportional to the concentration of Xanthine/Hypoxanthine present 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 Xanthine Standards or samples 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. Incubate the plate for 30 minutes at room temperature with mixing in the dark.
5. Measure the absorbance at 570 nm or fluorescent intensity at Ex/Em 535/587 nm using a plate reader.