Spectrophotometric measurement of hydroperoxides at increased sensitivity by oxidation of Fe²⁺ in the presence of xylenol orange

Abstract

The method, developed by modifying the FOX methods described by Wolff (Methods Enzymol. 233, 182–189, 1994), involves the oxidation of Fe²⁺ by peroxides at low pH in the presence of both the ferric-complexing dye xylenol orange and sucrose, the amplifier of the reaction. The method proved to be a convenient, simple and efficient assay for the direct measurement of both water and lipid soluble peroxides. In fact it improves by about 60% the sensitivity of the FOX1 method for water soluble peroxides, and by 7–8 times that of the FOX2 method for lipid soluble peroxides. It allows the detection of 0.1 μM peroxide in the test solution. The method is suitable to measure the lipid hydroperoxides present in phosphatidylcholine liposomes and in human LDL. The data obtained allowed us to define a mathematical expression to calculate the lipid hydroperoxide content of liposomes knowing their oxidation index.

Keywords:

• Hydrogen peroxide
• lipid hydroperoxide
• LDL
• spectrophotometric method
• xylenol orange