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Investigation of

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

(iv) O 2 4 aminophenazone/phenol Red complex


Determination of HDL
cholesterol
LDL , VLDL and chlyomicrons are precipitated
by phosphotungstic acid in the presence of
magnesium ions leaving HDL in solution.
The cholesterol content of the supernatant
can be determined by a suitable method.

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

(ii)Colorimetric method for TG estimation


Lipoprotein lipase
Triglycerides + H2O Glycerol
+Fatty acids
Glycerokinase
Glycerol+ATP Glycerol-3-
phosphate+ADP
Glycerol-3-phosphate oxidase
Glycerol-3-phosphate+O2
H2O2+
Dihydroxy acetone phosphate
H2O2+4-Aminoantipyrine+ESPAS(N-ethyl-N-
sulpho-propyl-m-anisidine).
(B)Non enzymatic methods
At present it is difficult to recommended a
method for triglyceride measurement.
A number of improvements in methodology
have appeared only recently and consequently
there is no up-to-date report of comparisons that
include the lasted methods.
Most of the methods with modifications appear
among the kits of commercial manufactures and
some manufactures include more than one
method in their catalogue emphasising perhaps
Determination of phospholipids
Quantiative measurement of phospholipids is
rare in routine clinical practice. Phospholipids are
sometimes).
The choline-containing phospholipids lecithin,
lysolecithin and sphingomyelin which account for
at least 95% of total phospholipids in serum can
be measured by an enzymatic reaction sequence
using phosphplipase D, choline-oxidase and
horseradish peroxidase.
Kit methods with this enzymatic sequence are
available commercially.
Before the availability of enzymatic reagents
Certain choline-containing phospholipids and in
some applications their ratio have been
determined in the clinical laboratory.
For example fetal lung maturity has been
evaluated from characteristic patterns of
phospholipids in amniotic fluid.
In this instance phospholipids may be recovered
by solvent extraction applied to a silica gel plate
for separation by thin layer chromatography and
quantified after visualization by treating with
iodine vapor.
The ratio of lecithin to sphingomylin has been