3. Outline of Today’s lecture 1. Discuss the different types of cholesterol 2. Identify healthy cholesterol levels 3. Clinical significance of cholesterol 4. Principle of cholesterol estimation

4. Objectives To determine serum cholesterol level Master how to determine the conc. of cholesterol in serum. Understand the clinical significance of cholesterol

5. What is cholesterol? Cholesterol, from the Greek chole- (bile) and Cholesterol, from the Greek chole- (bile) andGreekbileGreekbile stereos (solid) followed by the chemical suffix- chemicalsuffixchemicalsuffix ol for an alcohol, is an organic chemical organicchemicalorganicchemical substancesubstance classified as a waxy steroid of fat steroid substancesteroid

6. Types of Cholesterol LDL Cholesterol-”bad” Cholesterol HDL Cholesterol-”good” Cholesterol Triglycerides Which is the most important? –L–L–L–LDL –H–H–H–HDL & Triglycerides are a close 2nd and 3rd

7. LDL—The “bad” Cholesterol Contributes to build up of fat deposits in arteries Decreases blood flow to the heart Want this to be low? –L–L–L–Lowered by diet, exercise, and most medications

8. HDL—The “good” Cholesterol Helps carry “bad” cholesterol away from arteries to liver The higher the better! How can you raise your HDL? –Exercise –Some medications help –Eating properly may help

9. DETERMINATION OF SERUM / PLASMA CHOLESTEROL Cholesterol is distributed in all the cells of the body, especially in nerve tissue. It occurs in animal fats but not in plant fats. it is widely distributed throughout the body, especially in the blood, brain, liver, kidneys, and nerve fiber myelin sheaths, and it is essential component of cell membrane development and production of bile acids, adrenal steroids, and sex hormones.

10. Cyclopentanophenanthrene Nucleus Structure of Cholesterol

11. Cholesterol is essential for life to make strong cell membrane and hormones. The body manufacturers about 1000 mg of cholesterol daily. Too much cholesterol can cause fatty plaques on arterial walls, which narrows the artery. This condition known as atherosclerosis. The build up of fat on the vessel walls can occlude and in some cases totally block the flow of blood to organs like the heart. When atherosclerosis occur in heart vessels, also known as coronary heart disease, chest pain and heart attacks can result.

12. Acute myocardial Infarction

13. The highest source of cholesterol comes from organ meats (e.g., liver) and eggs, but other common sources are any type of meat, poultry, cheese, and butterfat. The recommended daily amount of cholesterol is 300 mg for the average person.

15. The cholesterol level in serum is dependent on age, sex, geographical /cultural region and nutrition. At birth, the cholesterol level is usually below 100 mg/dl(2.6 mmol/l). Levels increase slowly throughout childhood and generally throughout life. This increase is greater in men than in women during the reproductive years.

16. A- Raised levels may occur in: Diabetes mellitus. Nephrotic syndrome. Myxoedema. Late pregnancy. Liver disease. Cholesterol is sometimes deposited in the skin as yellow nodules or xanthemas and in arteries (artheroma). However,

17. B- Low levels occur in: Severe Infection Severe Anemia. Massive Liver Cell Damage.

18. Xanthomas

19. Types of Samples Plasma Plasma Water + solids (e.g. glucose, urea, albumin, fibrinogen) No cells Serum Serum Serum = plasma – clotting factors

20. Requirements Requirements:  Automatic pipettes  Tips  Cuvettes  Spectrophotometer Specimen: Specimen: Serum or plasma Serum or plasma

21. Working Reagent Mes buffer pH 6.5 75 mM Mes buffer pH 6.5 75 mM Phenol 6 mM 3,5-Dichlorophenol 0.2 mM 4-Aminoantipyrine 0.5 mM Cholesterol Esterase ≥500 kU/L Cholesterol Oxidase ≥300 kU/L Peroxidase ≥1200 kU/L Standard soln. of Cholesterol = 200 mg/dl

22. Preparation of Cuvettes Procedure Procedure SampleStandard Blank

23. Cholesterol Esterase Cholesterol esters + H 2 O Cholesterol esters + H 2 O Cholesterol + Fatty acid Cholesterol + Fatty acid Cholesterol + O 2 + H 2 O Cholesterol Oxidase Cholesterol + O 2 + H 2 O Cholesterol Oxidase Cholestenone + H 2 O 2 Cholestenone + H 2 O 2 Peroxidase Peroxidase 2H 2 O 2 + 4-Aminophenazone + phenol 2H 2 O 2 + 4-Aminophenazone + phenol Coloured quinonic derivative + 4H 2 O Coloured quinonic derivative + 4H 2 O

24. Procedure. ReagentsBlankSampleStandard Sample ( Sample (  l)- 10 10- Standard ( Standard (  l)--10 Working reagent (ml) 111

25. Procedure Add the reagents and sample as directed Mix and let stand at room temperature for 10 min. Read the absorbance at 505 nm (495-550). Zero the spectrophotometer with the blank reagent. Read the absorbance's of the standard and samples within 1 hr Enter the absorbance's readings and calculated the concentration of the sample.

26. Calculations: Concentration of cholesterol in the sample(s) = Absorbance of the Sample X Conc. of the Std. Absorbance of the Standard

27. 50 – 200 mg/dl

28. Abnormal ??? Clinical interpretation Level mmol/LmmolLLevel mg/dLmgdL Desirable level corresponding to lower risk for heart disease < 5.2 < 200 Borderline high risk 5.2–6.2024–020 High risk > 6.2> 240