cholestero estimation practical

1. Estimation of Serum Total Cholesterol
by CHOD-PAP method

2. • Cholesterol:
– Most abundant animal sterol.
– Crystalline yellow solid.
– Distributed in all animal cells.
– Major component of cell memb.
• Maintain memb. Fluidity
• Determinant of membrane permeability.
– Solid alcohol from bile

3. • Occur in two form:
– Free cholesterol ( ~ 30 %)
– Cholesterol ester ( ~ 70%)
• Cholesterol esterified with FA at C3.

4. – Structure:
– cppp ( phenanthrene nucleus and cyclopentano)
• One –OH group at C3. [esterifies with FA]
• One double bond between C5 & C6.
• An 8-carbon aliphatic side chain attached to C17.
• Contain 5 methyl groups. [C20,C13,C10,C25]

5. • Properties and reaction
– Yellow crystalline solid
– Under microscope, show a notched appearance
– Insoluble in water and soluble in organic solvent such
as chloroform, benzene , etheretc
– several reaction given by cholesterol are
– SALKOWSKI’S TEST
– LIEBERMANN-BURCHARD REACTION
– ZAK’S TEST.

6. • FUNCTION
– Cell membrane: modulating effect on fluid state of the
membrane
– Nerve conduction: insulate nerve tissue
– Bile salts and acids
– Steroid hormones
– Vitamin D

7. • Principle
– Cholesterol esterase hydrolyzes Cholesterol esters in
serum to give Free cholesterol & Fatty acid.
– Cholesterol oxidase oxidises 3-OH group of Free
cholesterol to liberate Cholest-4-ene-3-one & H2O2.
– H2O2 is then converted to H2O & [O] by Peroxidase.
– 4-Amino Antipyrine takes up the [O] & together with
phenol forms a pink coloured quinoneimine dye, which
is measured at 520nm.
– Absorbance ∞ Total Cholesterol in sample.

8. Cholesterol ester Cholesterol + Fatty Acid
Cholesterol Cholest-4-en-3-one + H2O2
Cholesterol Esterase
Cholesterol Oxidase
quinoneimine

9. • Specimen
– Serum sample is used.
– Fasting blood sample is preferred.

10. • Procedure
• Mix well. Incubate at 37°C in a water bath for 10 minutes
or at RT (25- 35°C) for 15 minutes.
• Remove from water-bath & cool to RT.
• Set colorimeter to zero using blank at 520 nm & measure
the absorbance of standard, test.
Blank (B) Standard (S) Test (T)
Working cholesterol reagent 1.0 ml 1.0 ml 1.0 ml
Serum - - 10 μml
Standard (200 mg/dl) - 10 μml -

11. • Hazardous materials
– This procedure uses phenol, which is caustic.
• Avoid mouth pipetting.
• Avoid contact with skin & mucous memb.

12. • HDL cholesterol estimation
– LDL,VLDL, Chylomicron (ApoB containing lipoprotein)
are removed by precipitating them using polyanion-
divalent cation.
– Example of polyanion-divalent cation:
• Heparin-Mn2+
• Dextran sulphate-Mg2+
• Sodium phosphotungstate-Mg2+

13. • Polyanions react with +vely charged groups on
lipoproteins (facilitated in +nce of divalent cations)
causing aggregation & a cloudy precipitate.
• Precipitation is usually complete within 10-15 min at RT.

14. • Precipitate is then sedimented by centrifugation.
• Centrifugation at higher forces, accelerates sedimentation
& improve complete precipitation of apo-B containing
particles.
• HDl-cholesterol in clear supernatant is estimated by
CHOD- PAP method.

15. • LDL cholesterol estimation
– Estimated by indirect method using friedwald equation
Total cholesterol = HDLc + LDLc + VLDL
LDL = T. Cholesterol – (HDLc + VLDL)
[VLDL = TG/5]
• Thus equation becomes,
LDL = T. Cholesterol – (HDLc + TG/5)

16. • Limitations of friedwald equation
– It can’t be used when:
I. Serum Triglyceride > 400 mg/dl
II. Type III hyprelipoproteinemia
– [elevated VLDL, CM, IDL thus high TG level]
– False high value of VLDL is obtained.
– Thus false low value of LDLc is obtained.

17. Clinical significance

18. Tests included in Lipid profile test
1. Serum Total cholesterol
2. Serum Triglyceride
3. VLDL
4. LDLc
5. HDLc

19. • Normal range:
– Total cholesterol
• Desirable: <200 mg/dl
• Borderline: 200-239 mg/dl
• High risk: ≥ 240 mg/dl
– HDLc
• Low risk: ≥ 60 mg/dl
• High risk: ≤ 40 mg/dl
– LDLc
• Desirable: < 130 mg/dl
• Borderline: 130-159 mg/dl
• High risk: ≥ 160 mg/dl

20. • Causes of Hypercholesterolemia
» Nephrotic syndrome
» Diabetes Mellitus
» Obstructive Jaundice
» Hypothyroidism
» Chronic alcoholism
» Type IIa Hyperlipoproteinemia

21. Biochemical basis of hypercholesterolemia in Nephrotic syndrome
Massive proteinuria [> 3.5 gm per day]
↓
Loss of albumin
↓
Hypoalbuminemia
↓
As compensation liver synthesizes globulins [apoB100]
↓
↑ed synthesis of VLDL & LDL
↓
Hypercholesterolemia

22. Biochemical basis of hypercholesterolemia in Diabetes Mellitus
↓ Insulin : Glucagon ratio
↓
↑ed activity of Hormone sensitive Lipase
↓
↑ed Fat mobilisation
↓
↑ed FFA
↓
↑ed β- oxidation
↓
↑ed Acetyl CoA
↓
Channeled to Cholesterol Biosynthesis
↓
Hypercholesterolemia

23. Biochemical basis of hypercholesterolemia in Obstructive Jaundice
Obstruction in bile duct
↓
↓ed bile flow
↓
↓ed bile acid & bile salt excretion
↓
↓ed bile acid (salt) excretion
↓
↓ed cholesterol utilization for bile acid (salt) synthesis
↓
Hypercholesterolemia

24. Biochemical basis of hypercholesterolemia in Hypothyroidism
↓ T3 & T4
↓
↓ed expression (downregulation) of gene for LDL receptor
↓
↓ed LDL receptor
↓
↓ed uptake of LDL
↓
↑ed LDL in blood
↓
Hypercholesterolemia

25. Biochemical basis of hypercholesterolemia in Chronic Alcoholism
Over consumption of Alcohol
↓
Ethanol → Acetaldehyde → Acetate → Acetyl CoA
↓
↑ed NADH : NAD+ ratio
↓
↓ed oxidation of Acetyl CoA via TCA cycle
↓
↑ed Acetyl CoA
↓
Channeled to Cholesterol Biosynthesis
↓
Hypercholesterolemia

26. • Type IIa Hyperlipoproteinemia
[Primary Familial hypercholesterolemia]
– Defect in LDL receptor:
• LDL receptor deficiency
• Defective binding of apoB100 to LDL receptor
• [LDL-Receptor] complex isn’t internalized
– Thus elevated plasma LDL level leading to
hypercholesterolemia.

27. • Causes of Hypocholesterolemia
I. Malabsorption
II. Pernicious anemia
III. Hyperthyroidism
IV. Drugs like Nicotinamide, Clofibrate