Question #550f0

1 Answer
Mar 29, 2015

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.