Specimen Collection for Lipid Analysis Serum Fasting – 12 hour fast preferred – Avoid lipemia Presence of fat droplets suspended in a solution. Lipemia affects assays by affecting absorbance due to refraction of light
3 High plasma lipid concentrations can cause excessive plasma turbidity and interfere with spectrophotometric methods. Lipoproteins can be spun down in this special centrifuge. Plasma is placed inside the “donut.” Lipids spin to the outside of the donut. Ultracentrifuge
Lipid Measurement Can be ordered as a panel – Includes total, LDL and HDL cholesterol and triglycerides
5 Historical Total Cholesterol Methodologies Colorimetric measurement procedures are less costly but are subject to interfering substances and may require extraction steps and strong acids Classical method = Liebermann-Burchard – Involved extraction & hydrolysis. Uses sulfuric & acetic acids – Results in formation of a green color, proportional to the cholesterol concentration
6 Cholesterol Methodology Cholesterol ester Free cholesterol + Fatty Acid Free cholesterol H 2 O 2 H 2 O 2 + Chromogen Colored Chromogen Cholesterol-Esterase Cholesterol Oxidase Horseradish Peroxidase Three step process using a coupled reaction with cholesterol oxidase.
7 Cholesterol Measurement Factors affecting cholesterol levels – Anything that affects HDL & LDL levels will affect cholesterol concentration-because these lipoprotein contain increased cholesterol – Thyroxine level inversely affects cholesterol level hypothyroid associated with hyper cholesterol hyper thyroid associated with hypo cholesterol – Estrogens documented that post-menopausal women have increased LDL cholesterol – Pregnancy altered endocrine function resulting in increased cholesterol – Others include hepatitis, nephrotic syndrome, emotional stress, and diabetes mellitus
8 Total Cholesterol Reference Range varies with age, sex, & diet – < 200 mg/dL
HDL Methodology Methods – Precipitation Reaction Dextran sulfate or phosphotungsate acid with magnesium chloride precipitates LDL and VLDL lipoproteins HDL left in the supernatant is tested using cholesterol assay. The answer represents the amount of HDL in the sample
Precipitation Method Drawbacks – Elevated triglyceride levels Results in overestimation of HDL Many labs will not perform HDL testing when triglyceride concentrations exceed 400 mg/dL
HDL Methodology Homogeneous Reaction Detergents or enzymes binds sites of VLDL and LDL particles. HDL is then left to react with colored products and can be measured Disadvantages- lacks specificity for HDL
HDL Reference Range Desirable range – > 60 mg/dL Gray area – 40-59 mg/dL High risk – < 40 mg/dL
Triglyceride Measurement Methods – Enzymatic – Colorimetric Involve the liberation of glycerol by lipase – 13
Triglyceride Reference Range Recommended – < 150 mg/dL Borderline high – 150-199 mg/dL High – 200-499 mg/dL Very high – > 500 mg/dL
LDL Measurement Direct measurement of LDL (VAP test) is uncommon Friedewald estimation ( calculation ) Test: Total Cholesterol, Total Triglycerides and HDL with routine procedure Estimate the LDL with the following : LDL= Chol- (HDL + VLDL) VLDL= Triglycerides/5 15
Example of Friedewald Formula Susan’s Lipid Panel results are: – Total cholesterol= 230 mg/dL – Triglyceride= 120 mg/dL – HDL= 25 mg/dL First: calculate the VLDL – Trig/5= 120/5= 24 Second: Plug results into formula – LDL= 230- (25+24) – LDL= 181 mg/dL
More on VAP VAP= vertical auto profile Advantages – Directly measures LDL, and other classes of lipoproteins – Can be performed on non-fasting samples – Identifies those at high risk for CAD Disadvantages – Not routinely ordered by physicians
18 Lipids in Stool – Normal adult with normal diet will not have more than 6 grams /day in feces. – Increased levels seen in children with malabsorption and adults with pancreatic insufficiency. – Testing done on 72 hour collection of stool. Lipid content determined as percentage of entire mass.
References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. http://chemi-girls.blog.com/page/2/ http://www.synermedinc.com/revolution.php http://www.vetlab.com/Lipoclear.htm Shokrani, M. (2014, January). Emerging approaches to the examination of lipoprotiens for cardio-metabolic risk stratification. MLO, 46(1), 16-17. Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson.