Biochemistry: Serum Cholesterol Determination PDF

Almaaqal University Biochemistry DETERMINATION OF SERUM CHOLESTEROL Dr/ Wael Sobhy Darwish Biochemistry PhD  CHOLESTEROL Cholesterol is a substance that is essential for the formation of cell membranes, hormones a...

Almaaqal University Biochemistry DETERMINATION OF SERUM CHOLESTEROL Dr/ Wael Sobhy Darwish Biochemistry PhD  CHOLESTEROL Cholesterol is a substance that is essential for the formation of cell membranes, hormones and bile acids. Cholesterol comes both from the diet and the production of cholesterol in the liver. Cholesterol and other lipids are carried in the blood attached to proteins, forming particles known as lipoproteins. Two different types of cholesterol are: LDL cholesterol is part of low density lipoprotein.This is often called bad cholesterol, HDL cholesterol is part of high density lipoprotein. This is often called good cholesterol because it helps prevent disease by carrying cholesterol away from the cells, back to the liver, where it can be broken down and removed from the body. Out two-thirds of the plasma cholesterol is esterified with fatty acids to form cholesterol esters. Why is it used? To estimate your risk of cardiovascular disease. The determination of serum cholesterol is one of the important tools in the diagnosis and classification of lipemia. When is it requested? If you have risk factors for cardiovascular disease such as: Family history of high cholesterol or heart disease in close relatives. Being overweight or obese. Diabetes mellitus High blood pressure (hypertension) Smoking cigarettes PRINCIPLE OF THE METHOD Total serum cholesterol level was determined based on an enzymatic colorimetric method as follows: Cholesterol ester + H O Cholesterol + fatty acids 2 Cholesterol + O Cholestenone + H O 2 2 2 2 H2O2 + 4-aminoantipyrine + Phenol Quinoneimine + H2O The intensity of rosy-colored Quinoneimine in the sample which is proportional to the concentration of cholesterol in the sample was measured spectrophotometrically at λ=500 nm and compared to the color of a similarly treated standard of a known cholesterol concentration (200 mg/dL) Test Preparation Needed? No fasting is needed for a cholesterol test. Sometimes a fasting sample is required for a full lipid profile including cholesterol.  Sample (Serum or plasma)  Stability of the sample for 7 days at 2-8ºC freezing at –20ºC will keep samples stable for a 3 months PROCEDURE 1. Assay conditions: Wavelength: 505 nm (500-550) Cuvette: 1 cm light path Temperature: 37ºC /15-25ºC  PROCEDURE Blank Standard Sample WR(mL) 1.0 1.0 1.0 Standard (µL) -- 10 -- Sample (µL) -- -- 10 Mix and incubate for 5 min. at 37ºC or 10 min. at room temperature Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable for at least 60 minutes  Calculation Conversion factor: mg/dL x 0,0258= mmol/L The ranges for total cholesterol in adults: Normal: Less than 200 mg/dL. Borderline high: 200 to 239 mg/dL. High: More than 240 mg/dL  What causes high cholesterol? 1- High cholesterol may be the result of an inherited disease 2- A diet high in saturated fats. 3- For many people it is caused by a combination of both a high fat diet and an inherited tendency towards high cholesterol. In women, cholesterol is high during pregnancy. Women should wait at least six weeks after the baby is born to have cholesterol measured. Some drugs that are known to increase cholesterol levels include oral corticosteroids, beta- blockers, oral contraceptives, thiazide diuretics, oral retinoids, and phenytoin.  Measuring range: From a detection limit of 0 mg/dL to a linearity limit of 900 mg/dL. If the results obtained were greater than the linearity limit, dilute the sample 1:1 with NaCl 9 g/L and multiply the result by 2. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.