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Cat. No.: XHAKW8-100 (for 100T)

Description

Xanthine/Hypoxanthine Assay Kit with WST-8 is a colorimetric assay based on the WST-8 reagent, designed for simple, rapid, and highly sensitive detection of xanthine and hypoxanthine levels in biological fluids such as serum, plasma, urine, as well as in tissues, cells, and cell culture supernatants. Typically, 0.2-2 μl of serum or plasma sample is sufficient for this assay.

Xanthine, also known as 2,6-dihydroxypurine, with the molecular formula C5_55​H4_44​N4_44​O2_22​ and a molecular weight of 152.11, is a purine widely present in body fluids, tissues, and plant cells. Xanthine and its derivatives are a group of alkaloids commonly used as mild stimulants and bronchodilators, particularly for treating asthma or flu symptoms. Compared to other more effective stimulants like sympathomimetic amines, xanthine mainly counters adenosine and enhances central nervous system alertness. Under normal conditions, hypoxanthine is converted into xanthine by xanthine oxidase and further metabolized into uric acid and superoxide, which are then excreted in the urine. A deficiency in xanthine oxidase can lead to the rare genetic disorder xanthinuria, causing accumulation of xanthine in urine and blood and potentially leading to renal failure. Recent studies suggest that elevated xanthine levels can cause ischemic damage, making xanthine an effective marker for detecting tissue hypoxia. Early detection of xanthine changes in biological fluids is crucial for metabolic research, diagnosis, and treatment monitoring. Additionally, xanthine can be found in patients with urinary stones. During the degradation of AMP to uric acid, hypoxanthine is oxidized to form xanthine. The measurement of xanthine and hypoxanthine in plasma and urine has started to be used for monitoring the efficacy of allopurinol therapy. In hydrocephalus treatment, xanthine and hypoxanthine levels in cerebrospinal fluid (CSF) have been used as treatment guidance indicators and markers of disease progression.

Components

• Buffer A for Metabolic Assay
• Xanthine Assay Buffer
• XO (100X)
• Xanthine Indicator (100X)
• Xanthine Standard (50mM)

Principle

Xanthine/Hypoxanthine are converted into superoxide anions (O2_22​⁻) by the action of xanthine oxidase (XO). These superoxide anions then react with the WST-type indicator to produce a water-soluble formazan dye. The formazan dye has a maximum absorbance peak around 450 nm. The amount of formazan dye generated is directly proportional to the content of xanthine/hypoxanthine in the sample. By measuring the absorbance at 450 nm, the content of xanthine/hypoxanthine in the sample can be detected with high sensitivity.

Features

• This kit offers high sensitivity, a wide linear range, and requires a small sample volume. When using a sample volume of 20 µl, the kit can detect xanthine/hypoxanthine concentrations as low as 5 µM, with a good linear relationship across a concentration range of 5 µM to 1000 µM (0.1-20 nmol). The kit provides Xanthine Standard (50 mM) for calibration, allowing you to create a standard curve and calculate the xanthine/hypoxanthine content in your samples. If the sample volume is small or the xanthine/hypoxanthine content is low, you can use the Amplex Red Xanthine/Hypoxanthine Assay Kit for higher detection sensitivity.
• This kit offers versatile lysis buffer for detection. The Buffer A for Metabolic Assay provided with this kit can be used to lyse cells or tissue samples, which can then be analyzed using other metabolic assay kits from our range that also use Buffer A for Metabolic Assay. This provides strong compatibility and can also be used for protein concentration measurement, SDS-PAGE, or Western blotting for easily soluble proteins.
• The kit is flexible, fast, and widely applicable. It can be used to analyze biological fluids such as serum, plasma, and urine from mice, rats, humans, as well as cell culture supernatants, tissue, or cell samples. The entire process takes approximately 0.5-1 hour to complete. The kit is suitable for both small sample analyses and high-throughput screening automation systems.

Storage

Store at -20ºC. Valid for one year. Xanthine Indicator should be protected from light.

Precautions

• Buffer A for Metabolic Assay, Xanthine Assay Buffer, and Xanthine Indicator need to be completely thawed and equilibrated to room temperature before use to avoid affecting the test results. XO should be handled on ice, and all reagents should be stored immediately according to the conditions specified in the kit after use.
• Serum and other samples should not be stored at 4ºC for more than 2 weeks, as this may affect the accuracy of the results. Serum samples are generally best stored at -20ºC, with -80ºC being preferable.
• To minimize background interference from the diluents, dilution solutions for samples and standards should be chosen based on the type of sample. For samples lysed using Buffer A for Metabolic Assay, use Buffer A for Metabolic Assay for dilution; for blood or other types of samples, use Xanthine Assay Buffer for dilution.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, nor for use in food or drug applications, and should not be stored in ordinary residential areas.
• For your safety and health, please wear lab coats and disposable gloves while handling.