Determination of Total Cholesterol

Mr. Arvind Kumar

AIM: Determination of Total Cholesterol

 

Principle of an enzymatic method

  1. Cholesterol ester + H2O ————>Cholesterol esterase  —————> Cholesterol + Fatty acids
  2. Cholesterol + O2 —————->Cholesterol oxidase —————–> Cholesterol-4-en-3-one + H2O2
  3. 2H2O2+ 4-aminoantipyrine + Phenol ———-> Peroxidase ————> Quinoneimine dye + 4H2O

 

Sample

  1. Serum or plasma (collected after 12–14 hours of fasting for accurate results).
  2. Preparation of PFF (Protein filtrate) from serum.
    1. Take 9.9 ml ferric chloride
    2. Acetic acid reagent in a dry test tube
    3. 0.1 ml serum (Dilution of blood ≡ 1in 100).
  3. Mix by inversion using paraffin film, keep for 5 minutes, centrifuge, and use clear supernatant as PFF.

 

Reagents Composition

R1
Good’s Buffer 50 mmol/l
Phenol 5 mmol/l
4-aminoantopyrine 0.3 mmol/l
Cholesterol esterase > 200 U/l
Cholesterol oxidase > 50U/l
Peroxidase > 3 kU/l
R2 Standard —

 

Procedure

Test Standard Blank
Reagents 1 1.00 ml 1.00 ml 1.00 ml
Sample 0.01 ml – –
Standard – 0.01 ml –
Distilled water – – 0.01 ml

 

Principle Zak’s Method

  • The serum is treated with a ferric chloride-acetic acid reagent to precipitate the proteins.
  • The protein-free filtrate is treated with sulfuric acid and acetic acid.
  • The cholesterol in the protein-free filtrate is oxidized and dehydrated by ferric chloride, acetic acid and sulfuric acid to a red-coloured compound.
  • The intensity of the colour is proportional to the cholesterol content in the serum. It is read at 540 nm (green filter).

 

Reagents

  1. 05 g ferric chloride hexahydrate in acetic acid,
  2. H2SO4.
  3. Standard cholesterol solution: (5 ml ≡ 0.2 mg cholesterol), prepared fresh in ferric chloride—acetic acid reagent from stock cholesterol solution (100 mg/100 ml acetic acid).

 

 

Procedure

  Test Standard Blank
PFF 5 ml – –
Standard cholesterol solution – 5 ml –
Ferric chloride-acetic acid reagent – – 5 ml
Conc. H2SO4 3 ml 3 ml 3 ml

 

Calculation

The concentration of total cholesterol in the sample is calculated using the formula:

Cholesterol concentration (mg/dL) = Sample Absorbance/Standard Absorbance×Standard Concentration (mg/dl)

 

Normal Values

The normal range of total cholesterol depends on age, sex, and other factors. General reference ranges are:

  • Desirable: < 200 mg/dL (5.2 mmol/L)
  • Borderline High: 200–239 mg/dL (5.2–6.2 mmol/L)
  • High: ≥ 240 mg/dL (6.2 mmol/L)

Age-Specific Reference Ranges:

  • Children: 120–170 mg/dL
  • Adults: 140–200 mg/dL
  • Elderly (>65 years): Up to 220 mg/dL may be normal.

 

Clinical Significance

Elevated Total Cholesterol

High cholesterol levels (hypercholesterolemia) are a key risk factor for cardiovascular diseases. Causes include:

  • Primary hyperlipidemia (genetic):
    • Familial hypercholesterolemia.
    • Familial combined hyperlipidemia.
  • Secondary hyperlipidemia (acquired):
    • A diet high in saturated fats and cholesterol.
    • Hypothyroidism.
    • Diabetes mellitus.
    • Nephrotic syndrome.
    • Obstructive liver disease.
    • Chronic kidney disease.

Clinical Implications:

  • Atherosclerosis: Plaque buildup in arteries leading to reduced blood flow.
  • Coronary artery disease: Increased risk of heart attacks.
  • Stroke: Higher chances of ischemic stroke due to arterial blockages.

Decreased Total Cholesterol

Low cholesterol levels (hypocholesterolemia) are less common but may indicate:

  • Malnutrition or malabsorption.
  • Hyperthyroidism.
  • Liver diseases (e.g., cirrhosis or liver failure).
  • Chronic infections or inflammation.
  • Severe anaemia.

Clinical Implications:

  • Reduced steroid hormone synthesis.
  • Weak immunity due to insufficient cell membrane production.
  • Neurological disorders (rare cases linked to low cholesterol).