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Updated: Dec 10, 2025
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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
13.8K
Exploration and quantification of ascorbate affecting peroxidase-catalyzed chromogenic reactions with a
Yong-Sheng Li1, Yang Zhao, Qiao-Jing Li
1School of Chemical Engineering, Sichuan University, Chengdu 6100651, China. lysgxf2005@qq.com.
Chemical Communications (Cambridge, England)
|August 29, 2020
Summary
Ascorbic acid (AsA) interferes with peroxidase (POD) assays by reacting with chromogenic products, not by inhibiting POD or depleting H2O2. Removing AsA interference is crucial for accurate POD activity and analyte determination.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Enzymology
Background:
- Peroxidase (POD)-catalyzed reactions are widely used in biochemical assays.
- Ascorbic acid (AsA) is a common biological reductant that can interfere with these assays.
- Understanding the mechanism of AsA interference is critical for accurate enzymatic activity determination.
Purpose of the Study:
- To investigate the interaction between ascorbic acid (AsA) and peroxidase (POD) in catalyzed reactions.
- To elucidate the mechanism by which AsA interferes with POD activity assays.
- To develop a method for simultaneous determination of POD activity and AsA.
Main Methods:
- Utilized a recirculating-flow catalysis detection (RFCD) system.
- Studied the competition and influence of AsA in POD-catalyzed reactions.
- Employed Trinder and enzyme-linked reaction systems.
Main Results:
- Ascorbic acid (AsA) does not inhibit peroxidase (POD) or directly deplete hydrogen peroxide (H2O2).
- AsA reacts with chromogenic products, forming colorless intermediates that are re-generated by H2O2.
- The RFCD system enabled simultaneous determination of POD activity (EPOD) and AsA.
Conclusions:
- Ascorbic acid (AsA) interference in POD assays stems from its reaction with chromogenic products, not direct enzyme inhibition.
- Accurate determination of POD activity (EPOD) and analytes requires removal of AsA interference.
- The developed RFCD method allows for simultaneous quantification of EPOD and AsA, improving assay reliability.
