Target group: MBBS and BDS students Slides prepared in Nov 2016

1. Estimation of
Serum Cholesterol & HDL
Ashikh Seethy

2. Objectives:
At the end of this practical, you should be able to:
◉ Explain role of cholesterol and lipoproteins in health and disease
◉ State the desirable levels of total cholesterol and different
lipoproteins
◉ Describe various causes of dyslipidemias
◉ State the principle of cholesterol estimation & HDL estimation,
interpret the results obtained and correlate them with the clinical
findings

3. Sterol and Steroid

4. Steroid:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring

5. Sterol:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring

6. Sterols
Phytosterols Zoosterols Ergosterol
◉ Sitosterol
◉ Sigmasterol
◉ Fungi
◉ Protozoa
Cholesterol

7. Functions of Cholesterol

8. In plasma membrane
◉ Membrane fluidity
◉ Lipid rafts
Precursor molecule of:
◉ Steroid hormones
◉ Vitamin D
◉ Bile Acids
Nerve conduction
Signal transduction

9. Cholesterol Synthesis
From Acetyl CoA;
HMG CoA Reductase
Mainly in Liver
ER and Cytosol
Transported
to
peripheral
tissues
Before transport, cholesterol is esterified to form cholesterol esters

11. Lipoproteins

12. Lipoprotein
Apo-lipoprotein+ LipidLipoprotein

13. Apo-B48 (Structural)
Apo-E (Binds to Apo-E receptor)
Apo-C2 (Activates LpL)
Apo-A1
(Structural)
Apo-B100
(Structural, Binds to LDL-Receptor)
Apo-E
Apo-C2
Apo-B100

14. Dietary Fats and Cholesterol
Lipoprotein Lipase
Apo C2
Apo E

15. Endogenous Fats and Cholesterol
Lipoprotein
Lipase
Hepatic
Lipase
Apo C2
Apo E
Apo B-100

16. ◉ After conversion to bile acids
◉ Directly into the bile
◉ Exfoliation of cells
Excretion of Cholesterol

17. Separation of Lipoproteins
Ultra-centrifugation Electrophoresis

18. Why is hyperlipidemia dangerous?

19. Hyperlipidemias

20. Causes of Hyperlipidemia
Secondary:
◉ Type 2 Diabetes Mellitus
◉ Hypothyroidism
◉ Nephrotic syndrome
◉ Alcoholism
◉ High carbohydrate intake
◉ Glycogen storage disorders
◉ Cushing syndrome

21. Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and VLDL
remnants
VLDL Chylomicrons
and VLDL
Triglycerides +++ N ++ ++ +++
Cholesterol
(total)
+ +++ ++ N/+ ++
LDL-cholesterol - +++ - - -
Atherosclerosis +/– +++ +++ +/– +/–

22. ↓LpL or Apo CII
↓
Familial
Hyperchylomicronemia
(Type I)
◉ Autosomal Recessive
◉ Elevated Triglycerides
◉ Eruptive Xanthomas

23. Defect in LDL-Receptor
↓
Familial
Hypercholesterolemia
(Type IIa)
◉ Autosomal Dominant
◉ Elevated LDL-C
◉ Tendon Xanthomas

24. Familial
Dysbetalipoproteinemia
(Type III
Hyperlipoproteinemia)

25. Trudy M. Forte et al. J. Lipid Res. 2009;50:S150-S155
GPIHBP1
Deficiency
ApoA-V
Deficiency
Familial Hypertriglyceridemia
• Type IV hyperlipoproteinemia
• Type V hyperlipoproteinemia
Endothelial Cell

26. Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and VLDL
remnants
VLDL Chylomicrons
and VLDL
Triglycerides +++ N ++ ++ +++
Cholesterol
(total)
+ +++ ++ N/+ ++
LDL-cholesterol - +++ - - -
Atherosclerosis +/– +++ +++ +/– +/–

27. Hypolipidemias

28. Defect in Microsomal Triglyceride transfer Protein
↓
Abeta-lipoproteinemia
(Bassen-Kornzweig syndrome)

29. Defect in ABCA1 or Apo-A1
↓
Tangiers disease

30. Desirable Levels

31. Total Cholesterol
< 200 mg/dL Desirable
200-239 mg/dL Borderline high
> 240 mg/dL High
HDL Cholesterol
< 40 mg/dL Low
≥ 60 mg/dL High
Tri-Acyl Glycerol
< 150 mg/dL Normal
150-199 mg/dL High
200-499 mg/dL Hypertriglyceridemic
>500 mg/dL Very highLDL Cholesterol
< 70 mg/dL
In patients with
risk factors
< 100 mg/dL Optimal
160-189 mg/dL High
>190 mg/dL Very high NCEP-ATP III Guidelines

32. Laboratory Estimation

33. Enzymatic Method
◉Cholesterol ester Cholesterol + Fatty acid
◉Cholesterol + O2 Cholest-4-en-3-one + H2O2
◉H2O2 + 4-AP + Phenol 2H2O + Quinone-imine
◉Absorbance of Quinoneimine at 510 nm is directly proportional to the
concentration of cholesterol in serum
CE hydrolase
Cholesterol
Oxidase
Peroxidase

34. Zak’s Method
•The proteins present in the serum sample are first precipitated by adding Ferric
chloride- Acetic acid reagent. The protein free filtrate is treated with conc. H2SO4.
Cholesterol
↓dehydration
Cholesta-3-5-diene (2 molecules)
↓oxidation
Bis cholesta-3-5-diene (1 molecule)
↓sulphonation
Liberman-Burchard reaction Salkowski reaction
Monosulphonic Acid derivatives Disulphonic Acid derivatives
(Green colour) (Red colour)
Fe3+

35. Lipid profile
◉Total Cholesterol, Tri-Acyl Glycerol, LDL and HDL
◉ Fasting sample for Tri-Acyl Glycerol estimation
◉ Tests should be repeated on a different occasion
◉ Friedwald equation:
Total Cholesterol = HDL + LDL + VLDL
VLDL = TAG/5
Not valid if TAG > 400 mg/dL

36. Protocol for Total
Cholesterol Estimation

37. 1. Take 0.1 mL serum, add 9.9 mL of FeCl3-CH3COOH mixture and mix
thoroughly with glass rod.
2. Centrifuge at 2000 rpm for 10 min
3. Take 3 test tubes and mark them as B, S and T
4. Mix well and keep in water bath at 50-60°C for 10 min. Cool to room
temperature and measure the OD at 540 nm.
Blank Standard Test
Supernatant ------ ------ 5 mL
FeCl3-CH3COOH 5 mL 4.9 mL ------
Standard
(200 mg/dL)
------ 0.1 mL ------
Conc.H2SO4 3 mL 3 mL 3 mL

38. Calculation:
Serum total cholesterol (mg/dL) =
(T-B)/(S-B) x Concentration of Standard x Dilution factor
Serum total cholesterol (mg/dL) =
(T-B)/(S-B) x 200 mg/dL x Dilution factor
Dilution factor = (0.1/8)/(0.05/8)
= 2

39. Protocol for HDL-Cholesterol
Estimation

40. ◉ LDL, VLDL and chylomicrons are precipitated by polyanions in the
presence of metal ions to leave HDL in solution.
◉ The cholesterol content of the supernatant is estimated by
employing the procedure of total cholesterol estimation.
◉ To 1 mL serum, add 0.1 mL Phosphotungstate reagent and 50 μL
MgCl2 solution. Centrifuge at 2500 rpm for 10 minutes.
◉ Collect the supernatant and estimate cholesterol by the total
cholesterol method

41. Precautions

42. ◉ Glacial acid is extremely volatile, irritant and corrosive to mucous
membrane. Mouth pipetting of acetic acid should be strictly avoided
◉ No mouth pipetting of H2SO4
◉ H2SO4 containing solution should be handled carefully and any
contact with skin should be avoided
◉ Always add acid to water
◉ Standard precautions should be followed for handling serum.

43. Question

44. A 32 year old woman was hospitalized with an acute myocardial
infarction. Coronary angiography indicated the presence of >75%
stenosis in all the three coronary arteries
Family history revealed that her father and two of her five siblings also
had myocardial infarction at young age.
Laboratory investigation shows TAG-135 mg/dL.
Estimate Total Cholesterol and HDL Cholesterol. Calculate the LDL
Cholesterol level.

45. THANK YOU!