TOTAL CHOLESTEROL DETERMINATION ENZYMATIC COLORIMETRIC – CHOLESTEROL
TOTAL CHOLESTEROL DETERMINATION ENZYMATIC COLORIMETRIC – CHOLESTEROL OXIDASE – PAP METHOD (TRINDER REACTION) Cholesterol and triglycerides consist most of the most commonly ordered lipid tests in the laboratory used in the diagnosis and management of lipoprotein disorders as well as in cardiovascular disease. Cholesterol accounts for most of the sterol in plasma. Total cholesterol consists both the free form (unesterified, 30-40%) and the esterified (60-70%). Literature says it is not necessary to distinguish the two forms except when the contribution of the fatty acid moiety to cholestryl ester mass must be accounted for, or when the cholesterol/cholesteryl ester mass ratio is of interest (Henry Clinical Diagnosis and Management by Laboratory Methods, 19th ed.) Chemical colorimetric methods have developed from a simple direct method to a complicated four-step method. The Liebermann-Burchard colorimetric reaction remains very applicable. The Abel-Kendall (three-step) method continues as the basis for the Centers for Disease Control and Prevention (CDC) reference method for cholesterol, and due to high accuracy, the method has been transferred to the National Reference Method Laboratory Network. Enzymatic methods for cholesterol analysis were begun in the 1970’s by Flegg and Richmond. Allain and Roeschlau began using cholesterol esterase and oxidase in a single reagent to determine total cholesterol in serum. Trinder’s color system peroxidase/phenol 4-aminoantipyrine has been used successfully until now. It is important to note that certain physiologic variation and errors can alter the accuracy of lipid measurements. Techniques in blood collection, processing of samples, prolonged standing or storage of specimen before analysis, patient’s posture affect cholesterol assay. Questions for write up: 1. Describe the cholesterol metabolism. 2. What are the two pathways of esterification of cholesterol? Give the physiologic importance of each. 3. Give the functions of cholesterol in the body. 4. In the procedure, what is the importance of cholesterol esterase (cholesteryl ester hydrolase) reaction? 5. Describe the principles of: a) Lieberman-Burchard reaction b) Salkowski reaction c) Abell-Kendall method 6. Describe the general principles of the step-wise methods for cholesterol and give examples for each. 7. Give several sources of interferences of variations in cholesterol assays (in terms of specimen collection and processing and storage, quality of samples and the CHOD method itself). 8. Describe the clinical significance associated with abnormal levels of cholesterol. 9. How does liver disease affect the cholesterol levels in blood? Notes: Please kindly make the answer accurate, informative and precise. Include the references.
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