Quantitative Determination of Total Cholesterol in Serum/Plasma
"Learn how to quantitatively determine total cholesterol levels in serum or plasma using an enzymatic color/endpoint method. Cholesterol plays a vital role in various bodily functions and is a significant risk factor for heart disease. Discover the principle, procedure, and calculations involved in this crucial diagnostic test." (299 characters)
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Presentation Transcript
Exp#4 Cholesterol Quantitative determination of total cholesterol in serum /plasma by enzymatic color/endpoint method
Cholesterol is a fatty substance found in blood, bile and brain tissues. It serves as a precursor to bile acids, steroids and vitamin D. The determination of serum cholesterol is a major aid in the diagnosis and classification of lipemias,hepatic and thyroid diseases -High blood cholesterol is one of the major risk factors for heart disease. -Major dietary sources of it cheese, egg yolks ,beef, fish, and shrimp
Principle The cholesterol present in the sample originates a colored complex, according to the following reaction: Cholesterol esters + H2O CHE Cholesterol + F.A CHOD Chol+ O2 Cholestene-3-one + H2O2 POD 2H2O2 + Phenol + 4-Aminoantipyrine Quinonimine( red color) +4H2O
CHE= cholesterol esterase CHOD= cholesterol oxidase POD= peroxidase This dye is proportional to the cholesterol concentration of Cholesterol in the sample *Specimen: Serum or heparinized sample
Procedure Blank Std Test Chol Rgt-ml 1 1 1 Pre-warm at 37 C for 3min then: Std- l --- 10 --- Sample- l --- --- 10 Mix and incubate at 37C for 10 min, then read Aat 505 nm
Calculation A test X Conc. Of Std(200 mg/dl)= Chol (mg/dl) A Std *Normal Range: Desirable ..<200 mg/dl Borderline high ..200-239 High . 240
Interfering substance Anticoagulants, such as fluoride and oxalate will result in false low values. The test is not influenced by hemoglobin values up to 200 mg/dl or by bilirubin levels up to 10 mg/dl. Interference from grossly icteric and heavily hemolysed specimens is correctable by use of a serum/ plasma blank.
Limitation This reagent is linear up to 500 mg/dl. Samples with value of above 500 mg/dl should be diluted 1:1 with isotonic saline and re- run. Multiply the final results by two (2). Grossly lipemic serums require a "sample blank". Add (10 l) of sample to 1.0 ml saline, mix and read the absorbance against water
