Question #550f0
1 Answer
The detection of cholesterol uses enzyme reactions to generate hydrogen peroxide.
The H₂O₂ then reacts with a dye to form a highly-coloured product that you can measure by colorimetry or fluorimetry.
The cholesterol in blood consists of both cholesterol and cholesteryl esters.
Step 1
The cholesteryl esters are hydrolyzed by cholesterol esterase into cholesterol.
Step 2
The cholesterol is oxidized by cholesterol oxidase into H₂O₂ and a ketone (cholest-4-en-3-one).
Step 3
In the presence of horseradish peroxidase (HRP), the H₂O₂ reacts quantitatively with a sensitive dye (Amplex ® Red) to produce a coloured product (resorufin)
Resorufin can be measured in a colorimeter by its absorption at 563 nm.
When resorufin is excited at 579 nm, it emits fluorescent radiation at 587 nm, so you can also use fluorimetry to detect cholesterol.
