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A. NAZLI BAŞAK

BIO 306.01/02 EXPERIMENT 3 CHOLESTEROL DETERMINATION IN SERUM. A. NAZLI BAŞAK. CHOLESTEROL DETERMINATION IN SERUM. Every living organism has been fo un d to contain sterols.

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A. NAZLI BAŞAK

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  1. BIO 306.01/02 EXPERIMENT 3 CHOLESTEROL DETERMINATION IN SERUM A. NAZLI BAŞAK

  2. CHOLESTEROL DETERMINATION IN SERUM • Every living organism has been found to contain sterols. • Cholesterol is the main sterol of the body and virtually found in all cells and body fluids. • It is also the initial starting point in many metabolic pathways including vitamin D synthesis, steroid hormone synthesis and bile acid metabolism.

  3. CHOLESTEROL DETERMINATION IN SERUM • Although cholesterol is essential for health and growth, excess levels of serum cholesterol is related with increased risk of cardiovascular diseases. • Atherosclerosis results from thickening of the inner layer of the arterial wall, especially caused by cellular material and deposits of lipids.

  4. CHOLESTEROL DETERMINATION IN SERUM • Determination of serum cholesterol and other lipids may be the initial step in the early diagnosis of atherosclerosis and coronary heart disease. • Chemical labs have been routinely measuring cholesterol in patients’ serum specimens for >40 years.

  5. CHOLESTEROL DETERMINATION IN SERUM • On the basis of long experience, labs should be capable of performing cholesterol analysis with a high degree of reliability. • Serum cholesterol levels may be quantitatively determined by either chemical or enzymatical methods. • The most widely performed method is the chemical method of Zak, although enzymatic kits are replacing this conventional method nowadays.

  6. CHOLESTEROL DETERMINATION IN SERUM

  7. CHOLESTEROL DETERMINATION IN SERUM

  8. CHOLESTEROL DETERMINATION IN SERUM • Cholesterol is the most hydrophobic membrane lipid. • its structure is quite different from the other membrane lipids: instead of two long hydrocarbon tails, it has a stiff carbocyclic structure, the steroid ring system • instead of a big hydrophilic head group, there is only a hydroxyl groupat one end of the molecule.

  9. CHOLESTEROL DETERMINATION IN SERUM • With this structure cholesterol is by far the least water-soluble membrane lipid. • Cholesterol accounts for 10 % or more of the total lipid in the plasma membrane and the Golgi membrane, but it is less abundant in more internal membranes. • It is prominent only in animals, including humans. Plants have different membrane steroids, the phytosterols, and most bacteria have no steroids at all.

  10. CHOLESTEROL DETERMINATION IN SERUM • Steroid hormones are derived from cholesterol: progestagen, cortisol, testosterone, estrogens etc. • LDL: major carrier of cholesterol in blood • HDL: picks up cholesterol released into the plasma from dying cells or membranes which undergo turnover.

  11. CHOLESTEROL DETERMINATION IN SERUM Preparation of Serum • Allow the blood sample to stand for at least 1 hr. or centrifuge at 5000 rpm for 15 min. • Spin in a clinical centrifuge for 10 min. • Carefully remove the clean serum with Pasteur pipette (do not disturb the clut). • Transfer 0,1 ml serum to a tube. The rest can be frozen and saved for later analyses.

  12. CHOLESTEROL DETERMINATION IN SERUM Procedure for Total Cholesterol Determination • Transfer 0,1 ml of serum to test tubes. Add 10 ml of absolute ethanol and mix rapidly on a vortex mixer for 10 sec. • Cent. the tubes for 5’ at full speed in a cilinical cent. and transfer the extracts carefully to clean test tubes. Measure 2 ml of extract into each of three new test tubes. It is important to have a clean solution; if some precipitate is carried over, spin the tube again.

  13. CHOLESTEROL DETERMINATION IN SERUM • The standart is prepared by adding 2 ml of a 0.02 mg/ml solution of cholesterol to a fourt tube. • Prepare a blank by delivering 2ml of ethanol to a fifth tube. • Slowly add 2 ml of “color reagent” to each tube and mix by gentle swirling. (VERY STRONG ACID-HANDLE WITH CARE!)

  14. CHOLESTEROL DETERMINATION IN SERUM • Cover the tubes with parafilm, and allow them to stand at RT for 30’. The color that develops is stable for about 1 hour only. • Read the absorbance of the standard and the unknowns at 550nm against the blank, and calculate the average mg/dl value for total cholesterol in serum. Again set up the ratio to calculate the conc. of unkown which is relative to that of the standard under the same conditions. designed and prepared by alburse

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