Outline of Today’s lecture
1. Introduction of Lipoproteins
2. Discuss the different types of Lipoproteins
3. Identify healthy HDL & LDL levels
4. Principle’s of HDL and LDL determination
Objectives
To explain the role of lipoproteins in the
transport of lipids
Master how to determine the HDL and LDL
lipoproteins
Understand the clinical significance of HDL
and LDL
What is lipoprotein?
A lipoprotein is a biochemical assembly
that contains both proteins and lipids, bound to
the proteins, which allow fats to move through
the water inside and outside cells.
FUNCTION
Essential for the transport of insoluble
lipids in plasma
Provides cholesterol, phospholipids and
triglycerides to tissues for:
– Energy
– Membrane synthesis
– Hormone synthesis
Classification of Lipoproteins
There are many different types of lipoproteins each of these
particles perform different functions circulating in the
body, including:
Chylomicrons
Very Low Density Lipoprotein (VLDL)
Low Density Lipoprotein (LDL)
High Density Lipoprotein (HDL)
Lipoprotein
groups
Major Lipid
Function
Chylomicron
Triglyceride
Transport of
triglyceride from
intestine to liver.
Very Low Density
(VLDL)
Triglyceride
Transport of
triglyceride from
liver to tissues
Low Density
(LDL)
Cholesterol
End product of
VLDL catabolism.
Transport of
cholesterol to
tissues
High Density
(HDL)
Cholesterol
Transport of
cholesterol from
tissues.
Estimation of Lipoproteins
1. Electrophoresis ( based on the migration in
an electric field)
2. Ultracentrifugation
3. Modified Ultracentrifugation
4. Precipitation
HDL CHOLESTEROL
The “GOOD” Cholesterol ?
Remember: “H” is for healthy lower the
risk of coronary artery disease
The higher the level of HDL, the lower
the risk of coronary artery disease.
LDL CHOLESTEROL
The BAD cholesterol
Remember: “L” is for LETHAL
LDL carries cholesterol to tissues to be
deposited
The higher the LDL, the greater the risk
for CAD
Determination of HDL-Cholesterol
LDL and VLDL are precipitated from
serum by the action of a polysaccharide, in
the presence of the divalent cations. Then,
high density lipoproteins cholesterol (HDL)
present in the supernatant, is determined.
HDL, VLDL, LDL & Chylos
HDL METHODS
Precipitation of LDL, VLDL, and Chylomicrons
from serum specimen by the addition:
– Dextran sulfate
– Magnesium acetate
Procedure
Precipitation reaction:
Sample (ml)
Precipitant solution
300
1 DROP
Mix and let stand for 15 minute at room temp.
Centrifuge at 2,000 × g/15 min. or 10,000 × g/2
minute.
After centrifugation, HDL is the only
lipoprotein remaining in the supernatant
A cholesterol method is then performed
on the supernatant to determine the
HDL
HDL
VLDL,LDL & Chylos
Calculations:
Concentration of HDL in the supernatant =
Absorbance of the Supernatant X Conc. of the Std.
Absorbance of the Standard
Male
35 – 50 mg/dl
Female
45 – 60 mg/dl
CLINICAL INTERPRETATION
> 55 mg/dl = Low risk for CAD
< 35 mg/dl = High risk for CAD
Determination of LDL-Cholesterol
LDL Cholesterol can be determined as the
difference between Total Cholesterol and the
Cholesterol content of the supernatant after
precipitation of the LDL fraction by polyvinyl
sulphate (PVS) in the presence of Polyethyleneglycol monomethyl ether.
LDL, VLDL, HDL & Chylos
LDL METHODS
Precipitation of LDL from serum specimen by
the addition:
̶ Polyvinyl sulphate
̶ EDTA Na2
̶ Polyethyleneglycol monomethyl ether
̶ Stabiliser
Procedure
1. Precipitation reaction
Sample
200 ml
Precipitant solution
100 ml
Mix and let stand for 15 minute at room
temperature. centrifuge at 2,000 × g/15 min.
or 10,000 × g/2 min.
After centrifugation, HDL, VLDL & Chylos
lipoproteins remaining in the supernatant
A Cholesterol method is then performed on the
supernatant to determine the LDL
2. Cholesterol assay
Determine the concentration of the serum Total
Cholesterol according to the QCA CHOD-PAP
method
HDL, VLDL, Chylos
LDL
Procedure
QCA CHOD-PAP method
Reagents
Blank
Sample
Standard
Sample
-
10 ml
-
Standard
-
-
10 ml
Working reagent (ml)
1
1
1
Calculations:
Concentration of the total Cholesterol =
Absorbance of the Sample
Absorbance of the Standard
X Conc. of the Std.
Calculation
LDL Cholesterol =
Total Cholesterol – 1.5× Supernatant Cholesterol (mg/dl)
CLINICAL INTERPRETATION
< 150 mg/dl = Low risk for CAD
135 – 159 = Possible risk for CAD
> 190 mg/dl= High Risk for CAD