Lipids - Laboratory Tests Flashcards
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
What is the second step in the TAG assay
Glycerol + ATP
Converted to glycerol-3-phosphate + ADP
By Glycerol Kinase
What is the third step in the TAG Assay
Glycerol-3-phosphate + O2
Converted to Dihydroxyoxyacetone-P + H2O2
By Glycerol-P-Oxidase
What is the fourth step of the TAG assay
H2O2 + Aminophenazone + Chlorophenol
converted to Quinoemimine + HCL + 4H2O
By Peroxidase
What are the four enzymes in the TAG Assay
Lipase
Glycerol Kinase
Glycerol-P-Oxidase
Peroxidase
Why is investigation of lipoprotein important?
Due to the association of particular types of lipoprotein with cardiovascular disease
How are lipoproteins investigates
(4)
Number of lipoprotein analysis e.g. NMR
Overall pattern of lipoproteins e.g. electrophoresis
Lipoprotein specific lipid content e.g. gel filtration
Apolipoprotein concentration e.g. ELISA
How is HDL-Cholesterol specific lipid measurement measured?
(3)
Based on the use of reagents which cause LDL-Cholesterol to precipitate
In a fasting sample should only leave HDL-Cholesterol
Thus can use the normal assays on the supernatant and calculate the LDL as the difference between the Total Cholesterol and the HDL-C (Friedewald Formula)
What is the Friedewald formula?
LDL Cholesterol = Cholesterol - (HDL + VLDL)
VLDL = TAG/2.2
When can the Friedward Formula not be used to calculate LDL cholesterol
When the TAG is greater than 4.5 mmol/L
How are normal ranges for lipids determined
(3)
Range depends on the populations
e.g. 20% higher cholesterol in Western populations vs Asian population
Levels change with age - gradual increase
What are the normal recommendations for lipids?
(3)
Cholesterol should be less than 5 mmol/L
LDL-C should be less than 3.4 mM
TAG should be less than 1.5 mmol/L
What does a risk assessment involve
(3)
Evaluate the risk of heart disease based on measurement of blood lipid levels
Algorithms of varying complexity evaluate the 10 year risk of an event
These risk assessments may inform the decision to start treatment
Give six examples of factors measured in a risk assessment
Total cholesterol
HDL-C
LDL-C
TAG
Smoker
Blood pressure
Define dyslipidemia
(2)
A consequence of abnormal lipoprotein metabolism
Lipid levels above the recommended levels
What are the four types of dyslipidemia
Elevated Total Cholesterol
Elevated Low-density lipoproteins
Elevated triglycerides
Decreased High-density lipoproteins
What are the two types of dyslipidaemia
Primary and secondary
What is primary dyslipidaemia
(2)
Single or multiple gene mutation
Results in disturbances of LDL, HDL and TAG Production or clearance
What is secondary dyslipidaemia?
(2)
Sedentary lifestyle
Excessive consumption of cholesterol - saturated fats and trans-fatty acids
What is the common type of dyslipidaemia in Ireland
Secondary
Give some examples of lipid abnormalities
Hypercholesterolaemia
Why is hypercholesterolaemia dangerous?
Its a major risk factor for coronary heart disease
How does hypercholesterolemia cause heart disease?
Cholesterol is deposited along walls of blood vessels
LDL is a key player in this process
Plaque ruptures and clot forms -> this impedes blood flow, tissues starved of oxygen and die which can cause angina or heart attack
What are the main major risk factors for coronary heart disease?
(4)
Smoking
High blood pressure
High blood cholesterol
Physical inactivity
There are also some genetic components
What is the most common classification system used for primary Hyperlipidemia?
Based on Fredrickson
Defined categories based on the spectrum of changes in blood lipids
What is Fredrickson Type I primary hyperlipidemia
(4)
Chylomicron
Deficiency of lipoprotein lipase of apolipoprotein CII (which activates LPL)
An inability to clear chylomicrons from the circulation
Since chylomicrons are TAG rich it leads to profound hypertriglyceridemia
What is a tell tale sign of Chylomicron Syndrome (Fredrickson Type I) Primary hyperlipidemia
High levels of chylomicron TAGs leads to hepatomegaly and eruptive xanthomata
The lipids essentially burst out of the skin
How is Chylomicron Syndrom (Fredrickson Type I) Primary Hyperlipidemia syndrome treated?
Treated with a low fat diet
In ApoCII deficient forms plasma infusion may be used to temporarily replace ApoCII levels and thus LPL activity
What is Fredrickson Type IIa Primary Hyperlipidemia also called?
Familial Hypercholesterolemia
What causes FT IIa Primary Hyperlipidemia/ Familial Hypercholesterolemia
(6)
Deficiency of LDL-receptor function
Typically a mutation in the receptor
Can also have deficits in cytoplasmic adaptor proteins
This prevents LDL from being internalised i.e. it remains in plasma
1;500 people carry the mutation
LDL cannot be removed from the plasma
What are some characteristic traits of FT IIa Primary Hyperlipidemia/ Familial Hypercholesterolemia?
Cholesterol levels elevated
TAG levels generally show minor changes
What are some symptoms of FT IIa Primary Hyperlipidemia/ Familial Hypercholesterolemia?
Tendon Xanthoma
Corneal Arci
How is FT IIa Primary Hyperlipidemia/ Familial Hypercholesterolemia treated
In heterozygous FH LDL-R is upregulated -> more receptors with residual function will cleave LDL from the blood
In homozygous FH may need plasma apheresis
What is Fredrickson Type III called?
Hyperlipoproteinemia
What causes hyperlipoproteinemia
Its associated with the E2 variant of APO-E
What does FS Type III result in?
(4)
Impaired catabolism of IDL
IDL is not cleared as well from the plasma
Causes accumulation of VLDL remnants
It is often precipitated by conditions which increase VLDL
What exactly happens in secondary hyperlipidemia
Diseases lead to increases in either cholesterol or TAG as a secondary effect
Give some examples of secondary hyperlipidemias
Predominant hypercholesterolaemia
Predominant hypertriglyceridaemia
What may cause prominant hypercholesterolaemia
(2)
Cholestasis
Primary hypothyroidism
What may cause predominant hypertriglyceridemia?
(3)
Obesity
Diabetes
Metabolic Syndrome
How is hypercholesterolemia treated
(3)
Therapies target cholesterol absorption or synthesis
Dietary intervention is essential - cut out saturated fats, eggs etc (absorption), use plant stanol enriched spreads (absorption)
Theres not much you can do from a dietary perspective to stop cholesterol synthesis -> drugs
What drugs are used to treat hypercholesterolemia?
(4)
Drug therapies block either synthesis or absorption
Statin class of drugs competitively inhibit cholesterol synthesis -> lipitor, zocor
Eztimibe blocks cholesterol absorption (bile acid binding resin)
Can be used in combination
