Lab 4 Estimation of Serum Cholesterol (Total & HDL) PDF

Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ z Estimation of...

Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ z Estimation of Serum Cholesterol (Total & HDL) Lipid profile: A pattern of lipids in the blood.  A lipid profile usually includes the levels of total cholesterol, high- density lipoprotein (HDL) cholesterol, triglycerides, and by using these values, can also calculate: Low-density lipoprotein (LDL) Cholesterol. Cholesterol: is a fatty waxy substance that is present in every cell of the body and is carried in the blood stream. You need some cholesterol to keep your cells and organs healthy and it is play a number of roles in maintaining vital body functions including hormone production and digestion.  The liver produces cholesterol in the body and also comes from the foods you eat, especially meat, eggs, poultry, and dairy products (about 70% synthesized in body, 30% from food).  Foods that are high in dietary fat can also make your liver produce more cholesterol. There are two types of cholesterol: 1. LDL (low density lipoprotein):  LDL: bad cholesterol "carry cholesterol from liver to blood then to organs.  It has less protein content and contains more cholesterol.  LDL cholesterol is easy to stick to the walls of blood vessels.  High LDL in blood associated with atherosclerosis, heart disease and myocardial infraction  Reducing LDL levels is a major treatment target for cholesterol-lowering medications.  Because high LDL in blood will deposited in blood artery and trigger clot formation Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ 2. HDL (high density lipoprotein):  HDL: good cholesterol, carry cholesterol from organs and blood to liver to get rid of it.  It removes excess cholesterol from tissues (it cleans blood).  High levels linked to a reduced risk of heart and blood vessel disease. The higher your HDL level, the better. Risk of high LDL and heart disease:  LDL (“bad”) cholesterol builds up in your artery walls, making them hard and narrow. HDL (“good”) cholesterol cleans up excess “bad” cholesterol and moves it away from the arteries, back to your liver.  Hyperlipidemia: is caused by having too much LDL cholesterol in your blood and not enough HDL cholesterol to clear it up. Unhealthy lifestyle choices can raise “bad” cholesterol levels and lower “good” cholesterol levels. Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------  Too much LDL cholesterol in your blood may put you at risk for heart disease and other serious conditions. High LDL levels can cause the build-up of plaque, a fatty substance that narrows the arteries and blocks blood from flowing normally. When blood flow to the heart is blocked, it can cause a heart attack. When blood flow to the brain is blocked, it can lead to stroke and peripheral artery disease. Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ Normal value: Total Cholesterol Level Category Less than 200mg/dL Desirable 200-239 mg/dL Borderline high 240mg/dL and above High LDL (Bad) Cholesterol Level LDL Cholesterol Category Less than 100mg/dL Optimal 100-129mg/dL Near optimal/above optimal 160-189 mg/dL High 190 mg/dL and above Very High HDL (Good) Cholesterol Level HDL Cholesterol Category Considered protective against heart 60 mg/dL and higher disease 40-59 mg/dL The higher, the better Less than 40 mg/dL A major risk factor for heart disease Measuring LDL- level: Total cholesterol is defined as the sum of HDL, LDL, and VLDL. Usually, only the total, HDL, and triglycerides are measured. For cost reasons, the VLDL is usually estimated as one-fifth of the triglycerides and the LDL is estimated using the Friedewald formula (or a variant): VLDL can be calculated by dividing total triglycerides by five. Direct LDL measures are used when triglycerides exceed 400 mg/dl. The estimated VLDL and LDL have more error when triglycerides are above 400 mg/dl. Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ Estimated LDL = [Total cholesterol] − [Total HDL] − [Estimated VLDL] LDL = Total cholesterol - ( HDL + TG/5 ) 1. Estimation of serum cholesterol Methods: (enzymatic colorimetric method). Principle: The enzyme cholesterol esterase is used to hydrolyze the cholesterol esters present in serum into free cholesterol and free fatty acids. The enzyme cholesterol oxidase, in the presence of oxygen, oxidizes cholesterol to cholest- 4-en-3one and hydrogen peroxide. Hydrogen peroxide oxidizes phenol and 4- aminoantipyrine to produce red color that can be measured spectrophotometrically. The series of the reactions involved in the assay system is as follows: CHE Cholesterol esters + H2O Cholesterol + free fatty acids CHOD Cholesterol + O2 Cholest-4-en-3-one + H2O2 POD 2 H2O2 + Phenol+ 4- aminoantipyrine Quinoneimine dye + 4H2O The intensity of the color formed (red dye quinoneimine) is proportional to the cholesterol concentration in the serum. Procedure: Take 3 test tubes and label them as test (T), standard (S) and blank (B). Blank Standard Sample Reagent (R) 1000 μl 1000 μl 1000 μl Standard - 10 μl - Sample - - 10 μl Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------  Mix well and incubate at 37 oC for 5 min or at 25 oC for 10 min.  Measure the absorbance of the standard and test sample at 505 nm against blank.  After incubation, the color is stable between 60 min. Calculation: (A) Sample Cholesterol (mg/dl) = x (standard conc.) (A) Standard 2. Estimation of serum HDL cholesterol Methods: (a) separation method. (b) enzymatic colorimetric method. Principle: This technique uses a separation method based on the selective precipitation of apoliprotein B-containing lipoproteins (VLDL, LDL and (a)Lpa) by phosphotungstic acid/MgCl2, sedimentation of the precipitant by centrifugation, and subsequent enzymatic analysis of high density lipoproteins (HDL) as residual cholesterol remaining in the clear supernatant. Procedure: A) Precipitation: Take 2 test tubes and label them as test (T) and standard (S). Tubes Standard Sample Precipitation Reagent 400 μl 400 μl Standard 200 μl - Sample - 200 μl  Vortex and allow to stand for 10 minutes at room temperature.  Centrifuge for 10 minutes at 4000 r.p.m (Revolutions per minute).  Separate the clear supernatant within 2 hours. Lecturer: M.Sc Hiba Muayad Medical Laboratory Techniques Clinical Biochemistry Stage: second // Lab: 4 ------------------------------------------------------------------------------------------------ B) Colorimetry: Measure HDL Cholesterol in the supernatant by using the cholesterol Reagent as fallow : Take 3 test tubes and label them as test (T), standard (S) and blank (B). Standard Sample Tubes Blank supernatant supernatant Monoreagent 1000 μl 1000 μl 1000 μl Standard - 50 μl - Supernate - - 50 μl  Mix and let the tubes stand for 10 minutes at room temperature or for 5 minutes at 37 oC.  Read the absorbance of sample (A) supernatant and standard (A) standard at 500 nm against reagent blank. Calculation: (A) Supernatant HDL Cholesterol (mg/dl) = x (standard conc.) (A) Standard