1. Cholesterol Ester Hydrolysis:
- Cholesterol esterase (CE) hydrolyzes cholesterol esters into free cholesterol and fatty acids.
```
Cholesterol ester + H2O → Cholesterol + Fatty acid
```
2. Cholesterol Oxidation:
- Cholesterol oxidase (CHOD) oxidizes free cholesterol to cholest-4-en-3-one and hydrogen peroxide (H2O2).
```
Cholesterol + O2 → Cholest-4-en-3-one + H2O2
```
3. Peroxidase Reaction:
- Peroxidase (POD) catalyzes the reaction between H2O2 and a chromogenic substrate, 4-aminophenazone (4-AP), in the presence of a phenol, typically phenol 4-aminoantipyrine (PAP). This results in the formation of a colored quinoneimine dye.
```
H2O2 + 4-AP + PAP → Quinoneimine dye + H2O
```
Key Points:
* Coupled Reactions: The reactions are coupled, meaning the product of one reaction serves as the substrate for the next. This ensures that the formation of the colored product is directly proportional to the initial cholesterol concentration.
* Spectrophotometric Measurement: The intensity of the colored product is measured using a spectrophotometer at a specific wavelength. The absorbance is directly proportional to the cholesterol concentration.
* Calibration: The method requires a calibration curve using known cholesterol standards to relate the absorbance readings to cholesterol concentrations.
Advantages of the CHOD-PAP method:
* High sensitivity and accuracy
* Relatively simple and rapid
* Widely available and standardized
Disadvantages of the CHOD-PAP method:
* Can be affected by lipemia (high triglyceride levels) and hemolysis
* Requires specialized reagents and equipment
Overall, the CHOD-PAP method is a reliable and accurate method for measuring cholesterol levels in blood. It is widely used in clinical laboratories and is considered the gold standard for cholesterol determination.
