Determination of Total Cholesterol Levels (Quantitative)

MATERIAL I DETERMINATION OF TOTAL CHOLESTEROL LEVELS (QUANTITATIVE) Basic Theory Cholesterol is a yellowish, waxy fat produced by the human body, especially in the liver. Cholesterol is an important fat, but if there is too much of it in the blood it can be dangerous to health. When viewed from a chemical perspective, cholesterol is classified into the lipid (fat) group with a steroid alcohol component. Cholesterol is a nutrient that is difficult for the body to absorb, and enters the body's organs through the lymphatic system. Cholesterol in blood plasma is mainly found bound to fatty acids and circulates in the form of cholesterol esters. Sources of cholesterol come from all food ingredients of animal origin, meat, eggs, milk and fishery products, brain tissue, nerve tissue and egg yolks. Cholesterol in the body also has important functions, including: the formation of the testosterone hormone in men and the hormone estrogen in women, the formation of vitamin D, and as a source of energy. Cholesterol is formed through acetate which is produced from nutrients and energy as well as other metabolic products besides cholesterol which also produces energy. Excessive energy sources result in excess formation of acetate as an intermediate, and fat in the body will also increase. The formation of cholesterol through acetate is a very complex process, one of which plays an important role is the reductase enzyme HMG – Co.A. Restricting cholesterol consumption will result in increased cholesterol levels in the blood if the enzyme system works abnormally. Under normal circumstances, cholesterol is synthesized in the food eaten, converted into tissue, hormones, vitamins which then circulate into the body through the blood, but there is also cholesterol that returns to the liver to be converted into bile acids and salts, the results of cholesterol synthesis are stored in body tissue. . Lipoproteins are a combination of lipid and protein molecules that are synthesized in the liver. Each type of lipoprotein differs in size, is synthesized and transports various types of lipids in different amounts. The type of lipoprotein that can trigger atherosclerosis consists of total cholesterol, LDL, HDL and triglycerides. These particles have special and different properties in the process of atherosclerosis formation, as follows: a) LDL (low density lipoprotein), which carries the most cholesterol in the blood. b) HDL (high density lipoprotein), transports less cholesterol. Its function is to remove excess LDL cholesterol in the arteries back to the liver for processing and disposal. c) VLDL (very low density lipoprotein), has the highest amount of triglycerides in the blood. Some VLDL is converted into LDL. d) Triglycerides, which is a type of fat in the blood that can affect cholesterol levels in the blood. Excess triglycerides will be hydrolyzed by the enzyme lipoprotein lipase, the remaining hydrolysis is then metabolized by the liver into LDL cholesterol. Lipid balance in the blood is regulated by several mechanisms, namely increasing or decreasing the rate of release of lipoproteins in the blood and reducing the formation of lipoproteins and the amount that enters the blood. LDL cholesterol increases the risk of heart attack because LDL can penetrate blood vessel walls and inhibit blood flow in the coronary arteries that supply it. heart. The cholesterol carried by HDL will reduce the risk of heart attack because this cholesterol carries the remaining cholesterol to the liver to be metabolized. Basic Principles for Determining Total Cholesterol Levels The basic principle of the experiment is the determination of cholesterol through enzymatic hydrolysis and oxidation. The colorimetric indicator is a quinoneimine indicator produced from 4- aminoantypyrine and phenol by hydrogen peroxide under the influence of a peroxidase catalyst (trinder reaction). cholesterol ester + H2OCHECholesterol + cholesterol + O fatty acids CHO Cholesterol-3-one + H2O2 2H2O2+ Phenol + 4 aminoantipyrinePODQuinonimine + 4H2O2 Procedure 1. Wavelength : Hg 546 nm ( 500 nm ) 2. Optical path : 1 cm 3. Incubation temperature : 20 – 25°C or 37° Material : 1. Blood Sample 10-15 ml Reactor : 1. Good buffer pH 6.7 : 50 mmol/L 2. Phenol : 5 mmol/L 3. 4-aminoantypyrine : 0.3 mmol/L 4. Cholesterol esterase: ≥ 200 U/L 5. Cholesterol oxidase : ≥ 50 U/L 6. Peroxidase : ≥ 2kU/L 7. Standards : 200 mg/dL (5.2 mmol/L Specimen (biological sample) Serum, plasma heparin or plasma EDTA Stability: • 7 days at a temperature of 20-250C, • 7 days at temperature 7-80C and • 30 months at temperature -200C Tool : 1. Test tube 2. EDTA 3. Centrifuge 4. Kits 5. Spectrophotometer 6. Myropipette Implementation: Pipetted in a reaction tube: Pipette in cuvette Blank Standard Sample Reagent 1000 µl 1000 µl 1000 µl Sample - 10 µl 10 µl aquabides 10 µl - - Mix, incubate for 10 minutes at temperature 37oC. Measure the absorbance of the standard and sample against the reagent blank within 60 minutes. (∆A