3. Outline of Today’s lecture 1. Introduction of Lipoproteins 2. Discuss the different types of Lipoproteins 3. Identify healthy HDL & LDL levels 4. Principle’s of HDL and LDL determination

4. Objectives To explain the role of lipoproteins in the transport of lipids Master how to determine the HDL and LDL lipoproteins Understand the clinical significance of HDL and LDL

5. What is lipoprotein? A lipoprotein is a bbbb iiii oooo cccc hhhh eeee mmmm iiii cccc aaaa llll assembly that contains both pppp rrrr oooo tttt eeee iiii nnnn ssss and llll iiii pppp iiii dddd ssss, bound to the proteins, which allow fats to move through the water inside and outside cells.

6. FUNCTION Essential for the transport of insoluble lipids in plasma Provides cholesterol, phospholipids and triglycerides to tissues for: –Energy –Membrane synthesis –Hormone synthesis

7. Classification of Lipoproteins There are many different types of lipoproteins each of these particles perform different functions circulating in the body, including : Chylomicrons Very Low Density Lipoprotein (VLDL) Low Density Lipoprotein (LDL) High Density Lipoprotein (HDL)

9. Function Major Lipid Lipoprotein groups Transport of triglyceride from intestine to liver. TriglycerideChylomicron Transport of triglyceride from liver to tissues Triglyceride Very Low Density (VLDL) End product of VLDL catabolism. Transport of cholesterol to tissues Cholesterol Low Density (LDL) Transport of cholesterol from tissues. Cholesterol High Density (HDL)

10. Estimation of Lipoproteins 1. Electrophoresis ( based on the migration in an electric field) 2. Ultracentrifugation 3. Modified Ultracentrifugation 4. Precipitation

11. HDL CHOLESTEROL The “GOOD” Cholesterol ? The “GOOD” Cholesterol ? Remember: “H” is for healthy lower the risk of coronary artery disease The higher the level of HDL, the lower the risk of coronary artery disease.

12. LDL CHOLESTEROL The BAD cholesterol Remember: “L” is for LETHAL LDL carries cholesterol to tissues to be deposited The higher the LDL, the greater the risk for CAD

13. Determination of HDL-Cholesterol

15. LDL and VLDL are precipitated from serum by the action of a polysaccharide, in the presence of the divalent cations. Then, high density lipoproteins cholesterol (HDL) present in the supernatant, is determined. HDL, VLDL, LDL & Chylos

16. HDL METHODS Precipitation of LDL, VLDL, and Chylomicrons from serum specimen by the addition: –Dextran sulfate –Magnesium acetate

17. Procedure Precipitation reaction: Mix and let stand for 15 minute at room temp. Centrifuge at 2,000 × g/15 min. or 10,000 × g/2 minute. Mix and let stand for 15 minute at room temp. Centrifuge at 2,000 × g/15 min. or 10,000 × g/2 minute. Sample ( Sample (  l)300 Precipitant solution 1 DROP

18. After centrifugation, HDL is the only lipoprotein remaining in the supernatant A cholesterol method is then performed on the supernatant to determine the HDL VLDL,LDL & Chylos HDL

19. Calculations: Concentration of HDL in the supernatant = Absorbance of the Supernatant X Conc. of the Std. Absorbance of the Supernatant X Conc. of the Std. Absorbance of the Standard Absorbance of the Standard

20. 35 – 50 mg/dl Male 45 – 60 mg/dl Female

21. CLINICAL INTERPRETATION > 55 mg/dl = Low risk for CAD < 35 mg/dl = High risk for CAD

22. Determination of LDL-Cholesterol

24. LDL Cholesterol can be determined as the difference between Total Cholesterol and the Cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the presence of Polyethylene- glycol monomethyl ether. LDL, VLDL, HDL & Chylos

25. LDL METHODS Precipitation of LDL from serum specimen by the addition: ̶ Polyvinyl sulphate ̶ EDTA Na2 ̶ Polyethyleneglycol monomethyl ether ̶ Polyethyleneglycol monomethyl ether ̶ Stabiliser

26. Procedure 1. Precipitation reaction Mix and let stand for 15 minute at room temperature. centrifuge at 2,000 × g/15 min. or 10,000 × g/2 min. Mix and let stand for 15 minute at room temperature. centrifuge at 2,000 × g/15 min. or 10,000 × g/2 min. After centrifugation, HDL, VLDL & Chylos lipoproteins remaining in the supernatant After centrifugation, HDL, VLDL & Chylos lipoproteins remaining in the supernatant. Sample 200 200  l Precipitant solution 100 100  l

27. A Cholesterol method is then performed on the supernatant to determine the LDL 2. Cholesterol assay Determine the concentration of the serum Total Cholesterol according to the QCA CHOD-PAP method Determine the concentration of the serum Total Cholesterol according to the QCA CHOD-PAP method LDL HDL, VLDL, Chylos

28. Procedure QCA CHOD-PAP method ReagentsBlankSampleStandard Sample- 10 10  l- Standard-- Working reagent (ml) 111

29. Calculations: Concentration of the total Cholesterol = Absorbance of the Sample X Conc. of the Std. Absorbance of the Sample X Conc. of the Std. Absorbance of the Standard Absorbance of the Standard

30. Calculation LDL Cholesterol = Total Cholesterol – 1.5× Supernatant Cholesterol (mg/dl)

31. CLINICAL INTERPRETATION < 150 mg/dl = Low risk for CAD 135 – 159 = Possible risk for CAD > 190 mg/dl= High Risk for CAD