Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzyme Assays

Background:

• The Amplex Red and horseradish peroxidase (HRP) reaction is widely used to quantify hydrogen peroxide.
• This assay relies on the production of a fluorescent product, resorufin, upon oxidation of Amplex Red by H2O2 in the presence of HRP.
• Accurate measurement of hydrogen peroxide is crucial in various biological and chemical applications.

Purpose of the Study:

• To investigate the impact of dietary antioxidants on the Amplex Red/HRP assay.
• To determine if biological concentrations of antioxidants cause interference in hydrogen peroxide quantification.
• To propose a mechanism for the observed interference.

Main Methods:

• Utilized the Amplex Red/HRP assay to detect hydrogen peroxide.
• Introduced dietary antioxidants at biological concentrations into the reaction mixture.
• Monitored the fluorescence signal generated by resorufin production.
• Analyzed the dose-dependent effect of antioxidants on the assay results.

Main Results:

• Dietary antioxidants at biological concentrations caused significant interference in the Amplex Red/HRP assay.
• The interference resulted in the over-quantification of hydrogen peroxide.
• The observed interference was dose-dependent, increasing with higher antioxidant concentrations.
• A potential mechanism for antioxidant interaction with HRP in this assay was proposed.

Conclusions:

• Dietary antioxidants can interfere with the Amplex Red/HRP assay, leading to inaccurate hydrogen peroxide measurements.
• The findings highlight the importance of considering potential interferences from dietary components in enzyme-coupled assays.
• Further research is needed to fully elucidate the interaction mechanism and develop strategies to mitigate this interference.