Lipid measurement

Question 1

Cholesteryl esters are hydrolyzed with alcoholic KOH
Unesterified cholesterol is extracted with hexane
Measured using sdfff reaction

Answer 1

Non-Enzymatic Methods (Abell Kendall)

Liebermann – Burchard

Question 2

Non-Enzymatic Methods (Abell Kendall)

reaction

Answer 2

Cholesterol + sulfuric acid + acetic anhydride  Green Sol.

Question 3

Enzymatic Method (Cholesteryl oxidase)

Answer 3

Total Cholesterol (cholesteryl ester and cholesterol) + H2O –Cholesteryl esterase Cholesterol + Fatty Acid

Cholesterol + O2 –Cholesterol oxidase
4-cholestenone + H2O2

H2O2 + Dye –Peroxidase Color

Question 4

Triglyceride Measurement

Answer 4

Non-enzymatic Method

Van Handel and Zilversmith
Hantzch Condensation (Fluorometric Method)

Question 5

Non-enzymatic Method

Answer 5

Triglyceride –Alcoholic KOH Glycerol + Fatty Acid

Glycerol + Periodic acid  Formaldehyde

Question 6

Van Handel and Zilversmith

Answer 6

Formaldehyde + Chromotropic acid  Blue Solution

Question 7

Hantzch Condensation (Fluorometric Method)

Answer 7

Formaldehyde + Diacetyl acetone + NH3 Yellow Sol’n

Question 8

Triglyceride Measurement
Enzymatic Methods (Glycerol Kinase)

Answer 8

Pyruvate Kinase

Glycerol-Phosphate Oxidase

Question 9

Enzymatic Methods (Glycerol Kinase)

Answer 9

Triglyceride –Lipase Glycerol + Fatty Acid

Glycerol + ATP –Glycerokinase Glycerophosphate +ADP

Question 10

Pyruvate Kinase

Answer 10

ADP + PEP –Pyruvate kinase ATP + Pyruvate

Pyruvate + NADH + H+ –LD lactate + NAD+ (340 nm)

Question 11

Glycerol-Phosphate Oxidase

Answer 11

Glycerophosphate + O2 –GO Dihydroxyacetone + H2O2

H2O2 + Dye –Peroxidase Color

Question 12

Based on the molecular density

Answer 12

Ultracentrifugation

Question 13

Based on migration in an electric field

Answer 13

Electrophoresis

Question 14

Lipid stains

Answer 14

(Oil red O, Fat Red 7B and Sudan Black)

Question 15

4 bands with fat stain

Answer 15

Alpha-lipoprotein –fastest
Pre-β – VLDL, LP(a)
Beta – LDL
Chylomicrons – stationary at origin

Question 16

High-Density Lipoprotein (HDL) Methods

Answer 16

Polyanion Precipitation

Question 17

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

Answer 17

Polyanion Precipitation

Question 18

Low-Density Lipoprotein (LDL) Methods

Answer 18

LDL = Total cholesterol – (HDL + VLDL)

Ultracentrifugation of serum

Question 19

Friedewald:

VLDL

Answer 19

= Triglyceride / 5 = mg/dL

= Triglyceride / 2.175 = mmol/L

Question 20

Apolipoprotein Methods

Answer 20

Apo A-1 – for determination of HDL conc.

Apo B – for determination of LDL and VLDL conc.