Introduction
- Cholesterol is a sterol and an essential lipid present in all body cells.
- It is an important structural component of cell membranes.
- Cholesterol is required for synthesis of steroid hormones, bile acids, and vitamin D.
- It is synthesized mainly in the liver and also obtained from dietary sources.
- Cholesterol circulates in blood bound to lipoproteins.
- Serum cholesterol estimation is one of the most important lipid profile investigations.
- It helps assess cardiovascular risk, liver function, endocrine disorders, and lipid metabolism abnormalities.
- Increased serum cholesterol is called hypercholesterolemia.
- Decreased cholesterol is called hypocholesterolemia.
Principle
- Serum cholesterol estimation is based on enzymatic CHOD-POD method.
- The reaction occurs in three enzymatic steps.
First Reaction
- Cholesterol esters are hydrolyzed by cholesterol esterase.
- Free cholesterol and fatty acids are formed.
Reaction
Cholesterol ester + H₂O → Cholesterol + Fatty acids
Second Reaction
- Free cholesterol is oxidized by cholesterol oxidase.
- Cholest-4-en-3-one and hydrogen peroxide are produced.
Reaction
Cholesterol + O₂ → Cholest-4-en-3-one + H₂O₂
Third Reaction
- Hydrogen peroxide reacts with 4-aminoantipyrine and phenol in presence of peroxidase.
- A red colored quinoneimine dye is formed.
Reaction
2H₂O₂ + 4-AAP + Phenol → Quinoneimine dye + 4H₂O
Principle of Measurement
- Intensity of color formed is directly proportional to cholesterol concentration.
- Absorbance is measured at 505 nm.
Specimen
Sample Type
- Serum is preferred specimen
- Plasma may also be used
- Heparin plasma acceptable
- EDTA plasma acceptable
Precautions
- Use fresh non-hemolyzed sample
- Avoid contaminated sample
Stability
- 7 days at 20–25°C
- 7 days at 4–8°C
- 3 months at −20°C
Reagents
Reagent 1
- Good’s buffer
- Phenol
- 4-aminoantipyrine
- Cholesterol esterase
- Cholesterol oxidase
- Peroxidase
Standard
- Cholesterol standard solution
Role of Reagents
- Cholesterol Esterase – Hydrolyzes esterified cholesterol
- Cholesterol Oxidase – Oxidizes cholesterol
- Peroxidase – Catalyzes color reaction
Materials Required
- Test tubes
- Micropipette
- Pipette tips
- Colorimeter / semi-auto analyzer
- Cuvette
- Timer
- Cholesterol reagent kit
Procedure
| Components | Blank | Standard | Test |
|---|---|---|---|
| Reagent 1 | 1.00 ml | 1.00 ml | 1.00 ml |
| Distilled water | 0.01 ml | — | — |
| Standard | — | 0.01 ml | — |
| Sample | — | — | 0.01 ml |
Incubation
- Mix properly
- Incubate at 37°C for 10 minutes
Reading
- Measure absorbance of sample and standard against blank
- Read at 500–505 nm
Calculation
Formula
Cholesterol (mg/dL) = Absorbance of Sample / Absorbance of Standard × Standard concentration
Example
- Sample absorbance = 0.45
- Standard absorbance = 0.50
- Standard concentration = 200 mg/dL
Calculation
- 0.45 / 0.50 × 200 = 180 mg/dL
Normal Reference Values
Adults
| Category | Value |
|---|---|
| Desirable | < 200 mg/dL |
| Borderline High | 200 – 239 mg/dL |
| High | > 239 mg/dL |
Children
| Category | Value |
|---|---|
| Desirable | < 170 mg/dL |
| Borderline High | 170 – 199 mg/dL |
| High | > 199 mg/dL |
Clinical Significance
- Serum cholesterol estimation is one of the most important biochemical tests used to assess lipid metabolism and cardiovascular risk.
- Cholesterol is an essential lipid required for normal body function, but abnormal levels are strongly associated with many diseases.
- It is an important structural component of cell membranes and is necessary for synthesis of steroid hormones, bile acids, and vitamin D.
- Serum cholesterol level reflects the balance between dietary intake, endogenous synthesis, utilization, and excretion.
- Abnormal cholesterol concentration is clinically significant in cardiovascular disease, liver disorders, endocrine disease, renal disorders, and metabolic syndromes.
Increased Cholesterol (Hypercholesterolemia)
- Hypercholesterolemia means increased serum cholesterol above normal range.
- Persistent elevation increases the risk of lipid deposition in blood vessels.
- This leads to progressive vascular damage and organ complications.
Atherosclerosis
- High serum cholesterol is one of the major causes of atherosclerosis.
- Excess cholesterol deposits in arterial walls.
- These deposits form atherosclerotic plaques.
Effects of Plaque Formation
- narrowing of arteries
- reduced blood flow
- loss of arterial elasticity
Clinical Importance
- major risk factor for cardiovascular disease
- early detection helps prevent vascular complications
Coronary Artery Disease
- Elevated cholesterol strongly increases risk of coronary artery disease.
- Cholesterol-rich plaques narrow coronary arteries supplying the heart.
Clinical Consequences
- angina pectoris
- myocardial infarction
- ischemic heart disease
Clinical Importance
- serum cholesterol screening helps estimate cardiac risk
Cerebrovascular Disease
- High cholesterol contributes to cerebral artery narrowing.
May Cause
- stroke
- transient ischemic attack
Peripheral Vascular Disease
- Cholesterol deposition in peripheral arteries reduces blood flow to limbs.
Clinical Features
- leg pain during walking
- poor circulation
Hypothyroidism
- Thyroid hormone deficiency reduces cholesterol metabolism.
- Hepatic cholesterol clearance decreases.
- Serum cholesterol rises significantly.
Clinical Importance
- Hypercholesterolemia is common in untreated hypothyroidism
Diabetes Mellitus
- Diabetes alters lipid metabolism.
- Serum cholesterol often rises, especially when diabetes is poorly controlled.
Clinical Importance
- increases cardiovascular risk further
- requires lipid monitoring
Nephrotic Syndrome
- Nephrotic syndrome causes marked hypercholesterolemia.
- Liver increases lipoprotein synthesis to compensate protein loss.
Clinical Importance
- very high cholesterol is characteristic finding
Obstructive Jaundice
- Cholesterol excretion through bile decreases in biliary obstruction.
- Serum cholesterol rises significantly.
Seen In
- gall stone obstruction
- biliary tract disease
Familial Hypercholesterolemia
- Genetic disorders may cause severe cholesterol elevation.
Clinical Importance
- early cardiovascular disease
- tendon xanthomas may develop
Obesity and Sedentary Lifestyle
- Obesity commonly increases cholesterol levels.
- Sedentary lifestyle reduces lipid utilization.
Clinical Importance
- lifestyle correction is essential
Dietary Causes of Increased Cholesterol
- Excess saturated fat intake increases serum cholesterol.
Foods Contributing
- animal fat
- butter
- fried foods
- egg yolk
Drug-Induced Hypercholesterolemia
Drugs Causing Increase
- corticosteroids
- oral contraceptives
- diuretics
Decreased Cholesterol (Hypocholesterolemia)
- Low cholesterol is less common but clinically important in some disorders.
Hyperthyroidism
- Excess thyroid hormone increases cholesterol metabolism.
- Serum cholesterol becomes low.
Severe Liver Disease
- Liver synthesizes cholesterol.
- Severe liver damage reduces cholesterol production.
Seen In
- severe hepatitis
- cirrhosis
- liver failure
Malnutrition
- Poor dietary intake reduces cholesterol availability.
Malabsorption Syndromes
- Intestinal absorption of fat is reduced.
- Cholesterol level decreases.
Diagnostic Importance
- Serum cholesterol is essential in lipid profile evaluation.
Clinical Uses
- assesses cardiovascular risk
- monitors lipid disorders
- evaluates endocrine disease
- assesses liver and biliary disorders
Monitoring Importance
- Follow-up of hyperlipidemia treatment
- Monitoring statin therapy
- Assessing dietary control
- Evaluating preventive cardiovascular care
Cholesterol and Lipoproteins
- Total cholesterol should always be interpreted with:
- HDL cholesterol
- LDL cholesterol
- triglycerides
Clinical Importance
- LDL indicates atherogenic risk
- HDL indicates protective effect
Prognostic Importance
- Persistent high cholesterol indicates long-term vascular risk
- Lowering cholesterol reduces risk of heart attack and stroke
