Lec 15: Lipid Methodologies Flashcards
Most common indications for ordering a complete lipid profile includes:
- screening for a familial lipid disorder
- establishing the risk of cardiovascular disease in an individual without prior disease
- the first lipid test in any patient being tested for a lipid disorder
- [TRUE or FALSE] Cholesterol levels remain constant with age.
- What hormone in women has cardiovascular protective effects wherein levels are lower before adolescence and after menopause?
- FALSE
• cholesterol levels rise with age - Estrogen
- Cholesterol screening should be repeated every ____ years.
- [TRUE or FALSE] Lipids are easily altered by recent dietary intake.
- Dietary intake of ___ & ___ significantly influences plasma lipid levels
- 5 years
- TRUE
- Saturated fat, cholesterol
- For lipid panel testing, how many weeks should the patient be on their usual diet?
- What medications alter lipid levels? (Include their effect)
- Lipoprotein measurements are made:
a. ___ after infection/trauma
b. ___ after childbirth
- Two weeks, neither gaining nor losing weight.
-
Oral contraceptives & postmenopausal estrogens
- false increase in cholesterol
• false increase in lipid levels -
a. 8 weeks
b. 3 to 4 months
Give the effects (increase/decrease) of the following:
- Thyroid, kidney, liver disease
- High fat diet
- Diet rich in sucrose
- Ingesting monounsaturated & polyunsaturated FA
- In the middle of a menstrual cycle
- increase in lipids & lipoproteins
- Increases serum triglycerides
- Increases triglycerides
- Reduce cholesterol
- Increase cholesterol & triglycerides, maximum estrogen secretion
- Patients should fast at least ___ before Venipuncture.
- Why is water intake permitted during fasting?
- 10 to 12 hours
- Lipids are nonpolar, so water does not affect the lipids nor interfere with the test.
Storage conditions for serum/plasma when analysis is delayed.
Refrigerated at 4°C for several days to one week
- Before taking the sample, the patient should be seated for ___ minutes to prevent ___.
- Effect of hemoconcentration.
- Effect of a recumbent position
- For 5 minutes, to prevent hemoconcentration
- Increases cholesterol by 10-15%
- 10% decrease in concentrations of TC , LDL-C, HDL-C
- Specimen of choice
- Preferred sample for electrophoretic/ultracentrifugation methods
- Anticoagulant of choice
- Anticoagulants avoided (include effects)
- Serum, Plasma
- Plasma
- EDTA
-
Citrate
• false decreased lipoprotein concentration
Heparin
• affects electrophoresis results
Liebermann Burchard Reaction
- Principle
- Components of Liebermann Burchard reagent
- Wavelength used
- End Product (color & name)
- What chemical mixture was used for precipitation & extraction?
- Acetic anhydride reacts with Cholesterol in a Chloroform solution to form a blue green color.
- Sulfuric Acid, Acetic anhydride
- 620 mm
- Cholestahexaene sulfonic acid (Blue green)
- Alcohol acetone mixture
• Alcohol precipitates proteins
• Acetone extracts lipids
Bloor Method
- Principle
- End Product (color)
- What chemical mixture was used for precipitation & extraction?
- Function of Chloroform
- Color developer
- Cholesterol is extracted with alcohol ether mixture, and it is measured with LB reaction
- Green
- Alcohol ether mixture
• Alcohol precipitates proteins
• Ether extracts lipids - Extracts cholesterol residue
- Conc. Sulfuric Acid & Acetic anhydride (LB reagent)
Abell-Kendall Method
- Chemical used to hydrolyze cholesterol
- Chemical used to extract cholesterol
- End product (color)
- Alcoholic KOH
- Petroleum jelly
- Blue green
Cholesterol Oxidase Method
(routine method)
- Enzymes in the first reaction
- Enzyme in the 2nd reaction
- Chromogens
- Cholesteryl esterase, Cholesterol oxidase
- Peroxidase
- Phenol, 4-aminoantipyrine
Cholesterol FS
- Reagent used (except the ones mentioned in COD)
- Anticoagulants used
- Anticoagulant avoided
- Wavelength used
- Interferences
- Is this method specific for cholesterol?
- Good’s buffer
- Heparin, EDTA
- Citrate
- 500 nm, Hg 546 nm
- Ascorbic acid, bilirubin
- No
This method is the current reference method and it is used to specifically measure cholesterol. This method shows agreement with the gold standard.
Gas chromatography - mass spectrometry
The reference method used by CDC for measurement of triglycerides.
Van Handel and Zilversmith method
Van Handel and Zilversmith Method
- Chemical used for extraction
- The adsorbent used and function.
- Chemical used for hydrolysis
- End product (color)
- Floch’s reagent
• Chloroform ethanol mixture - Aluminum
• removal of phospholipids - Alcoholic KOH
• hydrolyzed TAG —> glycerol, fatty acids - Blue
PRECAUTIONS
- Why are cholesterol levels slightly higher in the plasma than serum?
- Anticoagulants avoided (increase/decrease)
- TG determination interferences (increase/decrease)
- Chylomicrons & lipoproteins are trapped in the clot. Triglycerides might sink with the clot.
- Flourides, Oxalate
• false increase - Ascorbic acid, bilirubin, hemolysis
• hemolysis releases hemoglobin which leads to false decrease.
Testing Considerations: LIPOPROTEIN SEPARATION METHOD
- A method that separates lipoproteins by class on the basis of density
- not used in the clinical laboratory
- for research purposes
Ultracentrifugation
Testing Considerations: LIPOPROTEIN SEPARATION METHOD
- Separation of lipoproteins on the basis of charge
- Arrange the lipoproteins from highest to lowest charge, and state their electrophoretic mobility.
- In the reaction, what produces a red color?
- Electrophoresis
- highest to lowest:
• HDL - alpha region
• LDL - beta region
• VLDL - pre-beta region
• Chylomicron - origin - Ester bonds
Testing Considerations: LIPOPROTEIN SEPARATION METHOD
• most of the time used to separate HDL
• APO-B lipoprotein will sink in the precipitate, and HDL will be left in the supernatant to be measured.
Polyanion precipitation
Polyanion Precipitation
What are the polyanions used?
- phosphotungstate
- heparin sulfate
- dextran sulfate
• reference method for LDL determination
• combines ultracentrifugation & chemical precipitation
BETA QUANTIFICATION
- Ultracentrifugation removes VLDL and Chylomicrons.
- Chemical precipitation precipitates HDL
• reference method for HDL determination
• mixes ultracentrifugation & precipitation
• analysis of supernatant cholesterol by the Abell-Kendall assay
CDC-RMP
- Ultracentrifugation removes VLDL
- Heparin manganese removes LDL
- supernatant contains HDL
Friedewald Calculation
- indirect measurement of LDL
Formula for mg/dL:
LDL = Total Chol – HDL - (TG) ÷ 5
Formula for mmol/L:
LDL = Total Chol – HDL - (TG) ÷ 2.175
