ESTIMATION OF TOTAL

CHOLESTEROL BY ENZYMATIC
METHOD

Ravoori.Saideswar Rao
Department of Biochemistry
 Importance of cholesterol

• Cholesterol is required to build and maintain cell

membranes.

• Regulates membrane fluidity over a wide range of

temperatures

• Aids in the manufacture of bile salts.

• Precursor for the synthesis of vitamin D and steroid

hormones
 Structure
• Cholesterol is an acyclic compound which includes:

Cyclopentano perhydro phenanthrene ring structure

Methods of estimation of serum
cholesterol are as follows:

1.Colorimetric method
2.Enzymatic assays
3.Automated methods
 Colorimetric method
Cholesterol (2 molecules)Cholesterol(2 molecules)

Cholesta-3-5-diene (2 molecules) (oxidation)
↓
Oxidation by H2SO4 (hydration)
↓
Bis cholesta - 3-5-diene (1 molecule)

Sulphonation Sulphonation
(Liebermann Burchard reaction) Fe +3(Salkowski reaction)

Mono sulphonic acid(green colour) Disulphonic acid derivatives

(redcolour)
 Enzymatic assays
C.E. hydrolase
(i) Cholesterol esters cholesterol + fatty acids
or ethanolic KOH

Cholest. oxidase
(ii) Cholesterol + O2 cholest -4-en-3-one + H2O2

Peroxidase
(iii) 2H2O2 + 4 amino phenazone o-quinone-imine-dye + 4H2O
+ phenol
 Reagents
Pipes Buffer (pH6.9)
Cholesterol esterase
Phenol
Cholesterol oxidase
Sodium cholate
Peroxidase
4- aminoantipyrine
 Procedure:
Pipette into test Blank STD1 STD II STD III Test
tubes marked
as
Working 2ml 2ml 2ml 2ml 2ml
reagent
Standard - 20ul 20ul 20ul -
Test - - - - 20ul

Mix and incubate at 37oC for 10 minutes Read the

absorbance of test standard and blank at 500 nm or with
green filter. The final color is stable for 1 hr.
Calculation:
 Ref. range

Triglycerides = < 150 mg/ dl

Total cholesterol= < 200 mg/ dl
LDL cholesterol = <100 mg / dl
HDL cholesterol = >40 mg / dl
 Clinical significance
• Atherosclerosis- increased levels of
cholesterol for prolonged periods
• It is deposits in the subintimal region of
arteries and leading to hardening of
coronary arteries and cerebral blood
vessels.
Hypercholesterolimia
PRIMARY- LDL recepters defects.
SECONDARY-
Nephrotic syndrome
Diabetes mellitus
Obstructive jaundice
Hypothyroidism
Nephrotic syndrome-
Increased globulin fraction.
Increased beta fraction(lipoprotein
fraction)

Diabetes mellitus- Acetyl coA pool

is increased and more mol. are
channeled to Cholesterol synthasis.
Obstructive jaundice-
Excration of cholesterol through
bile is blocked.
Hypothyroidism-
Receptors for HDL on liver cells are
decreased and so excreation is not
effective.
 Hypocholesterolemia
severe infection,
severe anaemia
Immunocompromised diseases
liver damage
hyperthyroidism
malnutrition
 Major risk factors
• Cigarette smoking
• Hypertension
• Low HDL cholesterol
• Family history of premature
• Age above 55 years
 Interference
• Bilirubin
• Ascorbic acid
• HB