Determination of Total Cholesterol

Dr. Arvind Kumar

AIM: Determination of Total Cholesterol

Introduction

  • Total cholesterol is the total amount of cholesterol present in blood, including LDL, HDL, and VLDL fractions.

  • It is an essential lipid required for cell membrane structure, steroid hormone synthesis, bile acid formation, and vitamin D production.

  • Determination of total cholesterol is important for assessing the risk of cardiovascular diseases and diagnosing lipid disorders.

  • It is commonly estimated by the enzymatic CHOD-PAP method, where color intensity produced is directly proportional to cholesterol concentration.

  • The normal desirable serum cholesterol level in adults is less than 200 mg/dL.

 

Principle of an enzymatic method

The enzymatic method for determination of total cholesterol is based on sequential enzymatic reactions that produce a colored compound. The intensity of the color formed is directly proportional to the cholesterol concentration in the sample.

  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 = 1 in 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

  • Cholesterol in the serum reacts with acetic anhydride and concentrated sulfuric acid in the presence of ferric chloride.
  • This reaction produces a green-colored complex due to the formation of cholestadiene derivatives.
  • The intensity of the green color formed is directly proportional to the concentration of cholesterol present in the sample.
  • The color intensity is measured colorimetrically at approximately 540–560 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).