Cholesterol practical pdf

cholestero estimation practical

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

2. •Cholesterol:
–Mostabundantanimalsterol.
–Crystallineyellowsolid.
–Distributedinallanimalcells.
–Majorcomponentofcellmemb.
•Maintainmemb.Fluidity
•Determinantofmembranepermeability.
–Solidalcoholfrombile

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

4. –Structure:
–cppp(phenanthrenenucleusandcyclopentano)
•One–OHgroupatC
3.[esterifieswithFA]
•OnedoublebondbetweenC
5&C
6.
•An8-carbonaliphaticsidechainattachedtoC
17.
•Contain5methylgroups.[C
20,C
13,C
10,C
25]

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
–CholesterolesterasehydrolyzesCholesterolestersin
serumtogiveFreecholesterol&Fattyacid.
–Cholesteroloxidaseoxidises3-OHgroupofFree
cholesteroltoliberateCholest-4-ene-3-one&H
2O
2.
–H
2O
2isthenconvertedtoH
2O&[O]byPeroxidase.
–4-AminoAntipyrinetakesupthe[O]&togetherwith
phenolformsapinkcolouredquinoneiminedye,which
ismeasuredat520nm.
–Absorbance∞TotalCholesterolinsample.

8. Cholesterol ester Cholesterol + Fatty Acid
Cholesterol Cholest-4-en-3-one + H
2O
2
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 reagent1.0 ml 1.0 ml 1.0 ml
Serum - - 10 μml
Standard (200mg/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(ApoBcontaininglipoprotein)
areremovedbyprecipitatingthemusingpolyanion-
divalentcation.
–Exampleofpolyanion-divalentcation:
•Heparin-Mn
2+
•Dextransulphate-Mg
2+
•Sodiumphosphotungstate-Mg
2+

13. •Polyanionsreactwith+velychargedgroupson
lipoproteins(facilitatedin+nceofdivalentcations)
causingaggregation&acloudyprecipitate.
•Precipitationisusuallycompletewithin10-15minatRT.

14. •Precipitateisthensedimentedbycentrifugation.
•Centrifugationathigherforces,acceleratessedimentation
&improvecompleteprecipitationofapo-Bcontaining
particles.
•HDl-cholesterolinclearsupernatantisestimatedby
CHOD-PAPmethod.

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 LDLcis 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
»Nephroticsyndrome
»Diabetes Mellitus
»Obstructive Jaundice
»Hypothyroidism
»Chronic alcoholism
»Type IIaHyperlipoproteinemia

21. Biochemical basis of hypercholesterolemia in Nephroticsyndrome
Massive proteinuria[> 3.5 gm per day]
↓
Loss of albumin
↓
Hypoalbuminemia
↓
As compensation liver synthesizes globulins [apoB
100]
↓
↑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
↓ T
3& T
4
↓
↓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 IIaHyperlipoproteinemia
[Primary Familial hypercholesterolemia]
–Defect in LDL receptor:
•LDL receptor deficiency
•Defective binding of apoB
100to LDL receptor
•[LDL-Receptor] complex isn’t internalized
–ThuselevatedplasmaLDLlevelleadingto
hypercholesterolemia.

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