Exploring the Principle of 5'-Nucleotidase (5'-NT) Detection Using Peroxidase Method

In biomedical research, the measurement of enzyme activity is crucial for the diagnosis, treatment, and drug development of various diseases. One such representative enzyme is 5'-Nucleotidase (5'-NT), which plays an indispensable role in various physiological and pathological processes. 5'-NT is an enzyme that catalyzes the hydrolysis of 5'-nucleotides to produce nucleosides and phosphate. It is widely present in various tissues and cells of the human body, with particularly high activity in the liver, biliary tract, and skeletal systems. Since the activity of 5'-NT is closely related to various hepatobiliary diseases, accurate measurement of its activity holds significant clinical importance.

The peroxidase method is a commonly used technique for detecting 5'-NT activity. It relies on the catalytic action of peroxidase and involves a series of enzymatic reactions to measure the activity of 5'-NT in a sample. This method is favored for its simplicity, high sensitivity, and specificity, making it widely used in clinical diagnostics and research.

In the peroxidase method, the sample to be tested is first mixed with a reaction solution containing the substrate 5'-inosine monophosphate (5'-IMP). Under the action of 5'-NT, 5'-IMP is hydrolyzed to produce inosine and phosphate. Subsequently, inosine is further converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then oxidized by xanthine oxidase (XOD) to produce uric acid and hydrogen peroxide (H2O2).

In this process, hydrogen peroxide is a key intermediate. Under the catalysis of peroxidase, hydrogen peroxide reacts with chromogenic agents (such as 4-aminoantipyrine and phenol derivatives) to form a colored compound. The amount of this colored compound is proportional to the concentration of hydrogen peroxide, which in turn depends on the amount of hypoxanthine produced by the catalytic action of 5'-NT. Therefore, by measuring the change in absorbance of the colored compound, the activity of 5'-NT in the sample can be indirectly calculated.

By detecting the activity of 5'-NT using the peroxidase method, we can accurately determine the level of 5'-NT in a sample, providing important insights for the diagnosis and treatment of diseases. With the continuous advancement of biomedical research and technology, it is anticipated that more advanced detection methods will be developed in the future, making greater contributions to human health.