MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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MLAB 2401 Clinical ChemistryKeri Brophy-Martinez

• Lipids and Lipoproteins
• Assessment

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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

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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.
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Lipid Measurement

• Can be ordered as a panel
• Includes total, LDL and HDL cholesterol and
  triglycerides

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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

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Cholesterol Methodology
Cholesterol ester
Free cholesterol Fatty Acid Free
cholesterol
H2O2 H2O2 Chromogen
Colored
Chromogen
Cholesterol-Esterase
Cholesterol Oxidase
Horseradish Peroxidase
Three step process using a coupled reaction with
cholesterol oxidase.
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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

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Total Cholesterol Reference Range

• varies with age, sex, diet
• lt 200 mg/dL

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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

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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

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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

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HDL Reference Range

• Desirable range
• gt 60 mg/dL
• Gray area
• 40-59 mg/dL
• High risk
• lt 40 mg/dL

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Triglyceride Measurement

• Methods
• Enzymatic
• Colorimetric
• Involve the liberation of glycerol by lipase

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Triglyceride Reference Range

• Recommended
• lt 150 mg/dL
• Borderline high
• 150-199 mg/dL
• High
• 200-499 mg/dL
• Very high
• gt 500 mg/dL

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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

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Example of Friedewald Formula

• Susans 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- (2524)
• LDL 181 mg/dL

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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

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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
  .