Introduction
- LDL stands for Low-Density Lipoprotein, commonly called bad cholesterol.
- LDL is the major carrier of cholesterol from liver to peripheral tissues.
- It contains a high proportion of cholesterol and apolipoprotein B.
- LDL supplies cholesterol for membrane synthesis, steroid hormone production, and cellular metabolism.
- Excess LDL cholesterol deposits in arterial walls and promotes plaque formation.
- Therefore, LDL estimation is one of the most important tests in lipid profile analysis.
- Serum LDL cholesterol is a major indicator of cardiovascular risk.
- Increased LDL level is strongly associated with atherosclerosis and coronary artery disease.
Principle
- LDL cholesterol estimation is based on direct homogeneous enzymatic method.
- In the first step, special detergents block or solubilize non-LDL lipoproteins such as HDL, VLDL, and chylomicrons.
- Only LDL cholesterol remains available for enzymatic reaction.
Reaction 1
- LDL cholesterol esters are hydrolyzed by cholesterol esterase.
LDL Cholesterol Ester + H₂O → Cholesterol + Fatty acids
Reaction 2
- Free cholesterol is oxidized by cholesterol oxidase to produce hydrogen peroxide.
Cholesterol + O₂ → Cholestenone + H₂O₂
Reaction 3
- Hydrogen peroxide reacts with 4-aminoantipyrine and chromogen in presence of peroxidase to form colored quinoneimine dye.
2H₂O₂ + 4-AA + Chromogen → Quinoneimine dye + H₂O
- The color intensity formed is directly proportional to LDL concentration.
- Absorbance is measured at 540–600 nm depending on analyzer.
Specimen
Sample Type
- Serum is preferred specimen
- Heparin plasma may also be used
Precautions
- Use fresh non-hemolyzed sample
- Fasting sample preferred
- Avoid lipemic contaminated specimen
Stability
- Stable for 24 hours at room temperature
- Stable for 7 days at 2–8°C
Reagents
Reagent 1 (R1)
- Buffer solution
- Selective detergent
- Protective agents
Reagent 2 (R2)
- Cholesterol esterase
- Cholesterol oxidase
- Peroxidase
- 4-aminoantipyrine
- Chromogen
Function of Reagents
- R1 blocks non-LDL lipoproteins
- R2 selectively measures LDL cholesterol
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Colorimeter / semi-auto analyzer
- Cuvette
- Timer
- LDL reagent kit
Procedure
| Components | Blank | Standard / Calibrator | Test |
|---|---|---|---|
| Reagent 1 | 300 µL | 300 µL | 300 µL |
| Distilled water | 4 µL | — | — |
| Calibrator | — | 4 µL | — |
| Sample | — | — | 4 µL |
First Incubation
- Mix properly
- Incubate at 37°C for 5 minutes
Then Add
| Components | Blank | Standard / Calibrator | Test |
|---|---|---|---|
| Reagent 2 | 100 µL | 100 µL | 100 µL |
Second Incubation
- Mix properly
- Incubate at 37°C for 5 minutes
Reading
- Measure absorbance against blank
- Read at 546 nm / 600 nm
Calculation
Formula
LDL Cholesterol = (Absorbance of Test / Absorbance of Calibrator) × Calibrator concentration
Alternative Formula (Friedewald Formula)
LDL = Total Cholesterol − HDL − (Triglycerides / 5)
- Friedewald formula is used when triglycerides are below 400 mg/dL.
Normal Reference Values
| Category | LDL Value |
|---|---|
| Optimal | < 100 mg/dL |
| Near Optimal | 100 – 129 mg/dL |
| Borderline High | 130 – 159 mg/dL |
| High | 160 – 189 mg/dL |
| Very High | ≥ 190 mg/dL |
Clinical Significance
Increased LDL Cholesterol
- Atherosclerosis
- Coronary artery disease
- Myocardial infarction
- Diabetes mellitus
- Hypothyroidism
- Nephrotic syndrome
- Obesity
Decreased LDL Cholesterol
- Hyperthyroidism
- Severe liver disease
- Malnutrition
Diagnostic Importance
- Assesses cardiovascular risk
- Essential part of lipid profile
- Monitors lipid lowering therapy
- Predicts atherosclerotic disease risk
