1. Total Serum Cholesterol Test19
Total Serum Cholesterol Test
Fifth Stage Student By
Dr. Sakar Karem Abdulla
M.Sc. Clinical Biochemistry

2. Cholesterol Cholesterol is a very hydrophobic compound.
It consists of four fused hydrocarbon rings called steroid nucleus.
Present as a free cholesterol and cholesteryl ester.
Elevated levels of cholesterol are associated with atherosclerosis.
Ring D has an eight-carbon, branched hydrocarbon chain attached to carbon 17
Ring A has a hydroxyl group at carbon 3.
Ring B has a double bond between carbon 5 and carbon 6.

3. Cholesterol structure

4. Sources of Cholesterol
Cholesterol can be (exogenous) derived from food.
Cholesterol can be (endogenous) synthesized de novo in liver, intestine, adrenal cortex and reproduction tissues.

5. Synthesis of Cholesterol
Synthesis of HMG-CoA
Synthesis of mevalonic acid
Synthesis of squalene
Synthesis of cholesterol

6. Synthesis of Cholesterol

7. Synthesis of Cholesterol

8. Functions of Cholesterol
Structural component of cell membranes
Precursor for the synthesis of bile acid and bile salt
Precursor for the synthesis of steroid hormones and vitamin D

9. Transport of Cholesterol
Cholesterol is relatively insoluble in aqueous media and transported in body fluids as soluble protein complexes called lipoproteins.
Lipoproteins are composed of a lipid core (containing triglyceride and cholesterol ester) surrounded by a hydrophilic shell (containing Apo lipoproteins, phospholipids and free cholesterol).

10. Lipoproteins Structure

11. Lipoproteins Chylomicron Very low-density lipoproteins (VLDL)
Intermediate-density lipoproteins (IDL)
Low-density lipoproteins (LDL)
High-density lipoproteins (HDL)

12. Fate of cholesterol

13. Primary Hypercholesterolaemia
Familial hypercholesterolemia
Familial defective apoB
Familial combined hyper lipidaemia
Polygenic hyper cholesterolaemia
Hyper alpha lipoproteinaemia

14. Secondary Hypercholesterolaemia
Hypothyroidism
Diabetes mellitus
Nephrotic syndrome
Cholestasis
Acute intermittent porphyria
Anorexia nervosa
Certain drugs e.g. Anticonvulsants, oral contraceptives
Alcohol

15. Principle of the test
Cholesterol esters → cholesterol + free fatty acids (by cholesterol esterase)
Cholesterol + O2 → cholesten + H2O2 (by cholesterol oxydase)
2H2O2 +phenol + 4-aminoantipyrine → quinoneimine + 4 H2O (by peroxidase)
The absorbance of coloured complex proportional to the concentration of cholesterol in the sample

16. Reagents composition
R1 Buffer: phosphate buffer, chloro-4- Phenol, sodium cholate, preservative
R2 Enzyms: cholesterol oxidase (CO), cholesterol esterase (CE), Peroxidase (POD), 4 – Amino-antipyrine
R3 Standard: Cholesterol 200mg/dl
Reagent preparation: add the content of vial R2 into vial R1, mix gently until complete dissolution.

17. Test Procedure Working reagent 1000 µl standard 10 µl / serum
Assay
Working reagent
1000 µl
standard
10 µl /
serum
Mix. Let stand for 10 minutes at room temperature
Record absorbance at 500 nm
Colour is stable for 1 hour

18. Calculation
Total cholesterol=Abs.Assay/Abs.standard  x conc.standard (200mg/dl)
Normal value  < 200 mg/dl
Low risk mg/dl
High risk ≥ 240 mg/dl