Total Cholesterol

Introduction

1. Cholesterol is a waxy, fat-like substance that plays a crucial role in the body.
2. It is a key structural component of cell membranes, a precursor for synthesizing steroid hormones (like estrogen and testosterone), vitamin D, and bile acids, aiding fat digestion.
3. While cholesterol is essential for physiological functions, its excess in the blood is a significant risk factor for cardiovascular diseases, including atherosclerosis, myocardial infarction, and stroke.
4. Total cholesterol refers to the sum of all cholesterol carried in various lipoproteins, including low-density lipoproteins (LDL), high-density lipoproteins (HDL), and very low-density lipoproteins (VLDL).
5. Measuring total cholesterol provides insights into lipid metabolism and overall cardiovascular risk.
6. One of the classical methods for determining total cholesterol is Zak’s Method, which relies on chemical reactions to produce a measurable colored compound.
7. The measurement of total cholesterol is a cornerstone in lipid profile testing, which is crucial for diagnosing and monitoring conditions like hyperlipidemia, metabolic syndrome, and other cardiovascular risk factors.

 

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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

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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	—

 

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Procedure

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

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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).

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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).

 

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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.

 

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Procedure

	T	S	B
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

 

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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.

 

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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 anemia.

Clinical Implications:

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

 

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Advantages of Zak’s Method

1. Simple and cost-effective.
2. Reliable for routine cholesterol estimation in resource-limited settings.
3. Does not require advanced equipment or automated analyzers.

Limitations of Zak’s Method

1. Lack of Specificity:
   • Zak’s method may overestimate cholesterol due to interference from other substances like bilirubin and lipids.
2. Time-Consuming:
   • The method involves multiple steps, including extraction and chemical reactions.
3. Hazardous Chemicals:
   • Requires handling of concentrated sulfuric acid and chloroform, which are hazardous and need special care.