Lipids - Laboratory Tests
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What does a lipoprotein profile measure?
It measures the level of cholesterol in the blood
What are the two types of lipid profiles?
Fasting lipid profile Random lipid profile
What tests may be included in a random lipid profile? (2)
Triglycerides Total cholesterol
What may be measured in the fasting lipid profile
HDL LDL Triglycerides Total Cholesterol
What is the common method to detect cholesterol
CHOD-PAP method
What is the CHOD PAP method? (5)
CHOD = cholesterol P -> peroxidase AP -> aminoantipyrine It’s a coupled assay It generates a coloured product, quinoneimine
What is the first reaction in the CHOD-PAP method
Cholesteryl Ester is converted to Free cholesterol and fatty acids by Cholesteryl Esterase
What converts cholesteryl esters to free cholesterol and fatty acids
Cholesteryl esterase
What is the second step in the CHOD-PAP assay
The free cholesterol + O2 is converted to 4-cholesten-3-one + H2O2 by cholesterol oxidase
What converts free cholesterol + O2 to 4-cholesten-3-one + H2O2?
Cholesterol oxidase
What is the third step of the CHOD-PAP assay?
2H2O2 + aminophenazone + phenol is converted into quinoneimine + 4H2O by peroxidase
What converts 2H2O2 + aminophenazone + phenol to quinoneimine + 4H2O?
Peroxidase
What is quinoneimine?
A red chromogen measured at 500nm
What does the cholesterol oxidase enzyme work on? (2)
It accepts all sterols which have a 3B-hydroxyl group as substrates Cholesterol is a 3B-hydroxy-cholest-5-ene
Give some examples of substrates for cholesterol oxidase
Plant sterols Some steroid hormones Cholesterol oxidation products
Why is the CHOD-PAP method considered biased?
Its biased towards slightly higher results compared to the definitive analysis method (isotope-dilution mass spectrometry)
What is the definitive analysis method for cholesterol
Isotope-dilute mass spectrometry
What are two alternative cholesterol assays other than the CHOD-PAP?
Lieberman-Burchard Reaction (CDC reference method) Amplex red
Write about the Lieberman-Burchard reaction is a chemical method for cholesterol determination
Produces a blue-green colour with intensity proportional to the concentration of cholesterol
Why is the Lieberman-Burchard reaction? (2)
Reagent is not very stable and it is best to use it fresh The reagent also contains concentrated acids and is not particularly ‘user friendly’
Write about the amplex red assay (4)
Similar to the CHOD-PAP method CHOD is used to generate H2O2 but this is detected with amplex red rather than AAP Amplex red is converted to the fluorescent resorufin in the presence of peroxidase and H2O2 More sensitive assay
List some variables in cholesterol testing (5)
Age and gender -> males greater than females, this increases with age Within day variation of between 2 and 3% Seasonal variation of 3-5% Venous stasis causing an increase between 10-15% within 5 minutes of stasis Trauma can decrease lipid levels for several weeks
How are TAGs measured? (3)
TAG ASSAY A complex coupled assay with four separate enzyme reactions It measures the concentration of free glycerol via generation of a quinoneimine product
What is the first step in the TAG ASSAT
Triglycerol + 3H2O Converted to Glycerol + 3 fatty acids By Lipase