Lipid measurement Flashcards
Cholesteryl esters are hydrolyzed with alcoholic KOH
Unesterified cholesterol is extracted with hexane
Measured using sdfff reaction
Non-Enzymatic Methods (Abell Kendall)
Liebermann – Burchard
Non-Enzymatic Methods (Abell Kendall)
reaction
Cholesterol + sulfuric acid + acetic anhydride Green Sol.
Enzymatic Method (Cholesteryl oxidase)
Total Cholesterol (cholesteryl ester and cholesterol) + H2O –Cholesteryl esterase Cholesterol + Fatty Acid
Cholesterol + O2 –Cholesterol oxidase
4-cholestenone + H2O2
H2O2 + Dye –Peroxidase Color
Triglyceride Measurement
Non-enzymatic Method
Van Handel and Zilversmith Hantzch Condensation (Fluorometric Method)
Non-enzymatic Method
Triglyceride –Alcoholic KOH Glycerol + Fatty Acid
Glycerol + Periodic acid Formaldehyde
Van Handel and Zilversmith
Formaldehyde + Chromotropic acid Blue Solution
Hantzch Condensation (Fluorometric Method)
Formaldehyde + Diacetyl acetone + NH3 Yellow Sol’n
Triglyceride Measurement Enzymatic Methods (Glycerol Kinase)
Pyruvate Kinase
Glycerol-Phosphate Oxidase
Enzymatic Methods (Glycerol Kinase)
Triglyceride –Lipase Glycerol + Fatty Acid
Glycerol + ATP –Glycerokinase Glycerophosphate +ADP
Pyruvate Kinase
ADP + PEP –Pyruvate kinase ATP + Pyruvate
Pyruvate + NADH + H+ –LD lactate + NAD+ (340 nm)
Glycerol-Phosphate Oxidase
Glycerophosphate + O2 –GO Dihydroxyacetone + H2O2
H2O2 + Dye –Peroxidase Color
Based on the molecular density
Ultracentrifugation
Based on migration in an electric field
Electrophoresis
Lipid stains
(Oil red O, Fat Red 7B and Sudan Black)
4 bands with fat stain
Alpha-lipoprotein –fastest
Pre-β – VLDL, LP(a)
Beta – LDL
Chylomicrons – stationary at origin
High-Density Lipoprotein (HDL) Methods
Polyanion Precipitation
Lipoproteins (LDL, VLDL) are precipitated with polyanions (e.g. Heparin, dextran sulfate or Na phosphotungstate) in the presence of divalent cations (e.g. Mg or Mn), which are sediment by centrifugation (10-30 min. for 10,000g or 3 min. for 15,000g
Polyanion Precipitation
Low-Density Lipoprotein (LDL) Methods
LDL = Total cholesterol – (HDL + VLDL)
Ultracentrifugation of serum
Friedewald:
VLDL
= Triglyceride / 5 = mg/dL
= Triglyceride / 2.175 = mmol/L
Apolipoprotein Methods
Apo A-1 – for determination of HDL conc.
Apo B – for determination of LDL and VLDL conc.
