MLAB 2401: Clinical Chemistry
Keri Brophy-Martinez
Lipids and Lipoproteins
Assessment
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
Ultracentrifuge
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.
3
Lipid Measurement
• Can be ordered as a panel
– Includes total, LDL and HDL cholesterol and
triglycerides
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
5
Cholesterol Methodology
Cholesterol ester
Cholesterol-Esterase
Free cholesterol
H2O2 + Chromogen
Cholesterol Oxidase
Horseradish Peroxidase
Free cholesterol + Fatty Acid
H 2 O2
Colored Chromogen
Three step process using a coupled reaction with cholesterol oxidase.
6
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
7
Total Cholesterol Reference Range
• varies with age, sex, & diet
– < 200 mg/dL
8
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
–
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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
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.
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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 .