In the colorimetric determination of cholesterol using the enzyme cholesterol oxidase, which agent oxidizes the colorless organic compound 4-aminoantipyrine to a pink complex?

• A. Hydrogen peroxide
• B. Cholest-4-ene-3-one
• C. NAD
• D. Phenol

Answer: (A)

In this colorimetric cholesterol assay, the essential step is the production of hydrogen peroxide when cholesterol is oxidized by cholesterol oxidase. That hydrogen peroxide then acts as the oxidant in a peroxidase-catalyzed reaction that uses 4-aminoantipyrine and phenol to form a pink chromogen. The pink color arises from the oxidized form of 4-aminoantipyrine, produced specifically by the action of hydrogen peroxide in the presence of peroxidase. Cholest-4-en-3-one is simply the oxidation product of cholesterol, not the dye-forming agent; NAD is not involved in this pathway; phenol participates in the dye formation but does not provide the oxidizing power. Therefore, hydrogen peroxide is the agent that oxidizes 4-aminoantipyrine to the pink complex.