Dietary antioxidants interfere with Amplex Red-coupled-fluorescence assays.
Oxidation of Amplex Red by hydrogen peroxide in the presence of horseradish peroxidase (HRP) gives rise to an intensely colour product, resorufin. This reaction has been frequently employed for measurements based on enzyme-coupled reactions that detect hydrogen peroxide as a final reaction product. In the current study, we show that the presence of dietary antioxidants at biological concentrations in the reaction medium produced interferences in the Amplex Red/HRP catalyzed reaction that result in an over quantification of the hydrogen peroxide produced. The interference observed showed a dose-dependent manner, and a possible mechanism of interaction of dietary antioxidants with HRP in the Amplex Red-coupled-fluorescent assay is proposed.
Serrano
José
J
Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRBLleida, Lleida, Spain. jceserrano@mex.udl.cat
Jové
Mariona
M
Boada
Jordi
J
Bellmunt
María Josep
MJ
Pamplona
Reinald
R
Portero-Otín
Manuel
M
D000975:Antioxidants / Q000737:chemistry*; D004032:Diet; D036681:Fluorescence Recovery After Photobleaching / Q000592:standards*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry*; D010084:Oxidation-Reduction
D000975:Antioxidants; D010078:Oxazines; C470430:Amplex Red; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase