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Related Experiment Videos

Interference by ascorbic acid in test systems involving peroxidase. II. Redox-coupled indicator systems

R H White-Stevens, L R Stover

    Clinical Chemistry
    |April 1, 1982
    PubMed
    Summary

    Ascorbic acid causes a lag in peroxidase-based tests using redox indicators. This study identifies specific couplers, like chromotropic acid, that resist this interference, improving test accuracy.

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    Area of Science:

    • Biochemistry
    • Analytical Chemistry

    Background:

    • Peroxidase-based assays are crucial for detecting various analytes.
    • Ascorbic acid (vitamin C) can interfere with these assays, causing inaccurate results by inhibiting color development.
    • Understanding this interference is key to developing more robust diagnostic tools.

    Purpose of the Study:

    • To investigate the mechanism of ascorbic acid interference in peroxidase-based redox indicator systems.
    • To identify and select suitable coupling agents that minimize ascorbic acid interference.
    • To optimize assay conditions for improved reliability.

    Main Methods:

    • Utilized a peroxidase (EC 1.11.1.7) assay with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and various couplers.
    • Varied reaction pH, MBTH concentration, and coupler concentration (e.g., chromotropic acid).

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  • Measured color development kinetics to quantify ascorbic acid's effect and coupler resistance.

    • Main Results:

    • Ascorbic acid induces a lag phase in color development, the duration of which depends on pH and coupler type.
    • The lag phase is inversely proportional to MBTH concentration and largely unaffected by coupler concentration.
    • Fast couplers, such as chromotropic acid, Chicago acid, and H acid, show high resistance to ascorbic acid interference.

    Conclusions:

    • Ascorbic acid competes with the coupler for the oxidized MBTH intermediate (MBTHox) during the lag phase.
    • Aromatic ring systems with activating substituents are effective in developing ascorbic acid-resistant couplers.
    • The findings enable the selection of superior couplers for more accurate peroxidase-based diagnostic assays.