TOTAL CHOLESTEROL (CHOD/POD METHOD)
INTERPRETATION:
Enzymatic method for in-vitro quantitative determination of cholesterol.
PRINCIPLE:
Cholesterol esters are hydrolyzed by cholesterol esterase to free cholesterol.
Cholesterol is then oxidized by cholesterol oxidase to form hydrogen peroxide.
Hydrogen peroxide reacts with 4-aminoantipyrine and phenol in presence of peroxidase to form a red quinoneimine dye.
The intensity of the color is proportional to cholesterol concentration.
REACTION:
Cholesterol esters + H₂O → Cholesterol + Fatty acids
Cholesterol + O₂ → Cholest-4-en-3-one + H₂O₂
H₂O₂ + 4-AAP + Phenol → Quinoneimine dye (red)
MATERIAL REQUIRED BUT NOT PROVIDED:
-
Test tubes
-
Pipettes
-
Analyzer
Use serum.
-
.Reagent:-
-
R1 Buffer / Enzymes / Phenol
-
R2 Cholesterol Standard 200 mg/dL
-
PROCEDURE:
| Test | Blank | Standard | Sample |
|---|---|---|---|
| Working Reagent | 1.0 ml | 1.0 ml | 1.0 ml |
| Distilled Water | 0.01 ml | – | – |
| Standard | – | 0.01 ml | – |
| Sample | – | – | 0.01 ml |
Mix and incubate for 5 minutes at 37°C.
Read absorbance of Standard (S) and Sample (T) against Blank (B) at 505 nm.
CALCULATION:
Cholesterol (mg/dL)=TS×200\text{Cholesterol (mg/dL)} = \frac{T}{S} \times 200
NORMAL VALUES:
140–250 mg/dL
(Values may vary by age, sex, population.)PRESERVATION / STABILITY:
• Do not use hemolyzed samples.
• 7 days at 2–8°C.GENERAL SYSTEM PARAMETERS:
(Values approx. may vary depending on instrumentation)
Method: End Point
Wavelength: 505 nm
Temperature: 37°C
Sample Volume: 0.01 ml
Reagent Volume: 1 ml
Linearity: Upto 1000 mg/dL
Incubation: 5 min
LIMITATIONS:
Grossly lipemic or icteric samples may interfere.
Avoid hemolyzed samples.
HDL CHOLESTEROL
INTERPRETATION:
Enzymatic method for HDL-Cholesterol estimation.
Based on PEG-modified enzymes.
MATERIAL REQUIRED BUT NOT PROVIDED:
Test tubes
Analyzer
Micropipettes
• Serum or EDTA plasma (0.3 ml)
PROCEDURE:
| Test | Blank | Standard | Serum |
|---|---|---|---|
| Enzyme Reagent | 1 ml | 1 ml | 1 ml |
| Distilled Water | 0.01 ml | – | – |
| Standard | – | 0.01 ml | – |
| Sample | – | – | 0.01 ml |
Mix well and incubate for 5 minutes at 37°C.
Read absorbance of Standard (S) and Test (T) at 600 nm.
CALCULATION:
HDL Cholesterol (mg/dL)=TS×50\text{HDL Cholesterol (mg/dL)} = \frac{T}{S} \times 50
NORMAL VALUES:
Male: 45–65 mg/dL
Female: 35–80 mg/dL
NOTES:
• Do not use hemolyzed or icteric samples.
• HDL precipitate may interfere.
• Run control serum daily.