Estimation of Serum Cholesterol and HDL

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 ring Cyclopentano perhydro phenanthrene ring

5. Sterol: Cyclopentanoperhydrophenanthrene ring Cyclopentano perhydro phenanthrene 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

10. Lipoproteins

11. Lipoprotein Apo-lipoprotein+ Lipid  Lipoprotein

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. Dietary Fats and Cholesterol Lipoprotein Lipase Apo C2 Apo E

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

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

16. Separation of Lipoproteins Ultra-centrifugation Electrophoresis

17. Why is hyperlipidemia dangerous?

18. Hyperlipidemias

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

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

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

23. Familial Dysbetalipoproteinemia (Type III Hyperlipoproteinemia )

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. 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. Hypolipidemias

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

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

29. Desirable Levels

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 high LDL 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. Laboratory Estimation

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

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. H 2 SO4. 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 ) Fe 3+

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. Protocol for Total Cholesterol Estimation

36. Take 0.1 mL serum, add 9.9 mL of FeCl 3 -CH 3 COOH mixture and mix thoroughly with glass rod. Centrifuge at 2000 rpm for 10 min Take 3 test tubes and mark them as B, S and T 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 FeCl 3 -CH 3 COOH 5 mL 4.9 mL ------ Standard (200 mg/dL) ------ 0.1 mL ------ Conc.H 2 SO 4 3 mL 3 mL 3 mL

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. Protocol for HDL-Cholesterol Estimation

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 μ L MgCl 2 solution. Centrifuge at 2500 rpm for 10 minutes. Collect the supernatant and estimate cholesterol by the total cholesterol method

40. Precautions

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 H 2 SO 4 H 2 SO 4 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. Question

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. THANK YOU!