lipids disorders
Precaution of collection
1. Patients should have been taking their
normal diet for at least 7 days before the
investigation.
2. They should have fasted for at least 10hrs
before blood specimens are collected.
3. Smoking.
4. Drugs.
5. Alcohol intake.
6. Stress.
7. The conditions adopted for the collection of
specimens (posture of patient , amount of
venous stasis).
8. Choice of anticoagulant.
Visual inspection
Collect 4 ml of serum and keep at 4C for
12hrs then observe the change:-
1. Creamy layer in surface:-Indicate to increase
of chylomicron.
2. Turbid serum:-Indicate to increase of very
low density lipoprotein.
3. Clear serum:-
.Normal serum lipid.
.Increase in low density lipoprotein or high
density lipoprotein.
Lipid electrophoresis
Using fasting serum sample separate to:-
1. Chylomicron seen in the origin.
2. VLDL seen in pre beta
position.
3. LDL seen in beta
position.
4. HDL seen in alpha
position
Estimation of lipid in
blood
(Lipid profile)
1. Cholesterol.
2. Triglycerides.
3. HDL.
4. LDL.
5. VLDL.
6. Phospholipids.
Cholesterol
1. Salkowski reaction:-
.Principle:-
.In the presence of excess acid such as
phosphoric acid and ferric (Fe+++) ions
cholesterol is oxidized to disulphonic acid
which is reddish purple in colour.
.It is read colorimertrically at 560nm(green
yellow filter).
2. Libermann-Burchard reaction(Sackett's
method):-
.Principle:-
.When dehydrated with acetic anhydride and
oxidized with sulphuric acid cholesterol forms
a green coloured complex which is read
colorimetrically at 560nm(green-yellow filter).
3. Zak's method:-
.Principle:-
.In this method proteins in serum are
precipitated with ferric chloride-acetic acid
The protein-free filtrate containing cholesterol is
treated with concentrated sulphuric acid.
Cholesterol in presence of sulphuric acid
undergoes dehydration to form3,5-cholestadiene.
This is inturn oxidized and sulphonated to form red
colored cholestapolyene sulphonic acid in the
presence of Fe+++ ions.
The intensity of red colour formed is proportional
to the amount of cholesterol present in the serum.
The colour intensity is measured by using a green
filter(540nm).
4. Enzymatic method:-
.Principle:-
(i)Cholestertol ester
Cholesterol esterase
Cholesterol fatty acid
(ii)Cholesterol O 2 H 2O 2 Cholest 4 en 3one H 2o 2
cholesteroloxidase
(iii)H 2 O 2 H 2 O O 2
Peroxidase
Calculation of LDL-
cholesterol concentration
LDL-cholesterol=Total cholesterol-HDL-colesterol-
0.46X triglycerides (all values are in mmol/l).
LDL-cholesterol= Total cholesterol -
(HDL+Triglycerides/5) (mg/dl).
Determination of Triglycerides
(A)Enzymatic method
(i)Spectrophotometric method for TG
estimation
The method employs lipase for the hydrolysis of
the triglycerides coupled with enzymatic
procedures for measuring the glycerol released.
Glycerol measurement can be made by using
glycerol kinase.
Glycerol kinaase
Glycerol + ATP Glycerol
phosphate+ADP
Pyruvate kinase
ADP+phosphoenolpyruvate ATP
Lactic dehydrogenase
Pyruvate+ NADH Lactate+NAD