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Estimation of
Serum Cholesterol & HDL
Ashikh Seethy
Objectives:
At the end of this practical, you should be able to:
◉ Explain role of cholesterol and lipoproteins in health ...
Sterol and Steroid
Steroid:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
Sterol:
Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
Sterols
Phytosterols Zoosterols Ergosterol
◉ Sitosterol
◉ Sigmasterol
◉ Fungi
◉ Protozoa
Cholesterol
Functions of Cholesterol
In plasma membrane
◉ Membrane fluidity
◉ Lipid rafts
Precursor molecule of:
◉ Steroid hormones
◉ Vitamin D
◉ Bile Acids
Ne...
Cholesterol Synthesis
From Acetyl CoA;
HMG CoA Reductase
Mainly in Liver
ER and Cytosol
Transported
to
peripheral
tissues
...
Lipoproteins
Lipoprotein
Apo-lipoprotein+ LipidLipoprotein
Apo-B48 (Structural)
Apo-E (Binds to Apo-E receptor)
Apo-C2 (Activates LpL)
Apo-A1
(Structural)
Apo-B100
(Structural, Bind...
Dietary Fats and Cholesterol
Lipoprotein Lipase
Apo C2
Apo E
Endogenous Fats and Cholesterol
Lipoprotein
Lipase
Hepatic
Lipase
Apo C2
Apo E
Apo B-100
◉ After conversion to bile acids
◉ Directly into the bile
◉ Exfoliation of cells
Excretion of Cholesterol
Separation of Lipoproteins
Ultra-centrifugation Electrophoresis
Why is hyperlipidemia dangerous?
Hyperlipidemias
Causes of Hyperlipidemia
Secondary:
◉ Type 2 Diabetes Mellitus
◉ Hypothyroidism
◉ Nephrotic syndrome
◉ Alcoholism
◉ High c...
Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and...
↓LpL or Apo CII
↓
Familial
Hyperchylomicronemia
(Type I)
◉ Autosomal Recessive
◉ Elevated Triglycerides
◉ Eruptive Xanthom...
Defect in LDL-Receptor
↓
Familial
Hypercholesterolemia
(Type IIa)
◉ Autosomal Dominant
◉ Elevated LDL-C
◉ Tendon Xanthomas
Familial
Dysbetalipoproteinemia
(Type III
Hyperlipoproteinemia)
Trudy M. Forte et al. J. Lipid Res. 2009;50:S150-S155
GPIHBP1
Deficiency
ApoA-V
Deficiency
Familial Hypertriglyceridemia
•...
Causes of Hyperlipidemia
Primary:
Phenotype I IIa III IV V
Lipoprotein,
elevated
Chylomicrons
and VLDL
LDL Chylomicron
and...
Hypolipidemias
Defect in Microsomal Triglyceride transfer Protein
↓
Abeta-lipoproteinemia
(Bassen-Kornzweig syndrome)
Defect in ABCA1 or Apo-A1
↓
Tangiers disease
Desirable Levels
Total Cholesterol
< 200 mg/dL Desirable
200-239 mg/dL Borderline high
> 240 mg/dL High
HDL Cholesterol
< 40 mg/dL Low
≥ 60...
Laboratory Estimation
Enzymatic Method
◉Cholesterol ester Cholesterol + Fatty acid
◉Cholesterol + O2 Cholest-4-en-3-one + H2O2
◉H2O2 + 4-AP + Ph...
Zak’s Method
•The proteins present in the serum sample are first precipitated by adding Ferric
chloride- Acetic acid reage...
Lipid profile
◉Total Cholesterol, Tri-Acyl Glycerol, LDL and HDL
◉ Fasting sample for Tri-Acyl Glycerol estimation
◉ Tests...
Protocol for Total
Cholesterol Estimation
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...
Calculation:
Serum total cholesterol (mg/dL) =
(T-B)/(S-B) x Concentration of Standard x Dilution factor
Serum total chole...
Protocol for HDL-Cholesterol
Estimation
◉ LDL, VLDL and chylomicrons are precipitated by polyanions in the
presence of metal ions to leave HDL in solution.
◉ The ...
Precautions
◉ Glacial acid is extremely volatile, irritant and corrosive to mucous
membrane. Mouth pipetting of acetic acid should be ...
Question
A 32 year old woman was hospitalized with an acute myocardial
infarction. Coronary angiography indicated the presence of >...
THANK YOU!
Estimation of Serum Cholesterol and HDL
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Estimation of Serum Cholesterol and HDL

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

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Estimation of Serum Cholesterol and HDL

  1. 1. Estimation of Serum Cholesterol & HDL Ashikh Seethy
  2. 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. 3. Sterol and Steroid
  4. 4. Steroid: Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
  5. 5. Sterol: Cyclopentanoperhydrophenanthrene ringCyclopentanoperhydrophenanthrene ring
  6. 6. Sterols Phytosterols Zoosterols Ergosterol ◉ Sitosterol ◉ Sigmasterol ◉ Fungi ◉ Protozoa Cholesterol
  7. 7. Functions of Cholesterol
  8. 8. In plasma membrane ◉ Membrane fluidity ◉ Lipid rafts Precursor molecule of: ◉ Steroid hormones ◉ Vitamin D ◉ Bile Acids Nerve conduction Signal transduction
  9. 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
  10. 10. Lipoproteins
  11. 11. Lipoprotein Apo-lipoprotein+ LipidLipoprotein
  12. 12. 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
  13. 13. Dietary Fats and Cholesterol Lipoprotein Lipase Apo C2 Apo E
  14. 14. Endogenous Fats and Cholesterol Lipoprotein Lipase Hepatic Lipase Apo C2 Apo E Apo B-100
  15. 15. ◉ After conversion to bile acids ◉ Directly into the bile ◉ Exfoliation of cells Excretion of Cholesterol
  16. 16. Separation of Lipoproteins Ultra-centrifugation Electrophoresis
  17. 17. Why is hyperlipidemia dangerous?
  18. 18. Hyperlipidemias
  19. 19. Causes of Hyperlipidemia Secondary: ◉ Type 2 Diabetes Mellitus ◉ Hypothyroidism ◉ Nephrotic syndrome ◉ Alcoholism ◉ High carbohydrate intake ◉ Glycogen storage disorders ◉ Cushing syndrome
  20. 20. 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 +/– +++ +++ +/– +/–
  21. 21. ↓LpL or Apo CII ↓ Familial Hyperchylomicronemia (Type I) ◉ Autosomal Recessive ◉ Elevated Triglycerides ◉ Eruptive Xanthomas
  22. 22. Defect in LDL-Receptor ↓ Familial Hypercholesterolemia (Type IIa) ◉ Autosomal Dominant ◉ Elevated LDL-C ◉ Tendon Xanthomas
  23. 23. Familial Dysbetalipoproteinemia (Type III Hyperlipoproteinemia)
  24. 24. 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
  25. 25. 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 +/– +++ +++ +/– +/–
  26. 26. Hypolipidemias
  27. 27. Defect in Microsomal Triglyceride transfer Protein ↓ Abeta-lipoproteinemia (Bassen-Kornzweig syndrome)
  28. 28. Defect in ABCA1 or Apo-A1 ↓ Tangiers disease
  29. 29. Desirable Levels
  30. 30. 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
  31. 31. Laboratory Estimation
  32. 32. 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
  33. 33. 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+
  34. 34. 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
  35. 35. Protocol for Total Cholesterol Estimation
  36. 36. 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
  37. 37. 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
  38. 38. Protocol for HDL-Cholesterol Estimation
  39. 39. ◉ 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
  40. 40. Precautions
  41. 41. ◉ 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.
  42. 42. Question
  43. 43. 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.
  44. 44. THANK YOU!

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