Which enzyme in the enzymatic cholesterol method is responsible for producing hydrogen peroxide in the reaction sequence?

• A. Cholesterol Esterase
• B. Cholesterol Oxidase
• C. Peroxidase
• D. Catalase

Answer: (B)

In this assay, hydrogen peroxide comes from the oxidation of cholesterol itself. Cholesterol oxidase oxidizes cholesterol to cholest-4-en-3-one and, in the process, releases hydrogen peroxide. That hydrogen peroxide is then used by the peroxidase step to convert a colorless substrate into a colored product, which is measured to determine cholesterol levels. The other enzymes have different roles: cholesterol esterase just frees cholesterol from esters, peroxidase uses hydrogen peroxide to generate color, and catalase would break down hydrogen peroxide rather than produce it. So the enzyme responsible for producing hydrogen peroxide is cholesterol oxidase.