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Methods used for investigation of lipids

(A)Determination of serum 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 reagent. 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
( i ) Cholesterol ester+Cholesteryl esterase cholesterol+fatty acid
*Principle:-

( ii ) Cholesterol+O2+H2O cholesterol oxidase Cholest-4-en-3-one+H2O2


( iii ) H2O2 Peroxidase H2O+O2



( iv ) O2+4-aminophenazone/phenol Red complex

(B)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.

(C)Calculation of LDL-cholesterol concentration

*LDL-cholesterol=Total cholesterol-HDL-colesterol-0.46X
triglycerides (all values are in m mol/l).

*LDL-cholesterol= Total cholesterol-(HDL+Triglycerides/5)


(mg/dl).

(D)Determination of Triglycerides

Earlier methods for triglycerides were indirect.


Sponification liberated glycerol from triglycerides and
phospholipids and converted the fatty acid from them and
from cholesterol esters to soaps. Subsequent acidification
freed the fatty acids so that total fatty acids could be
titrated. Alternatively total esterified fatty acids were
determined by the hydroxamic acid method of stern &
Shapiro. In each case phospholipids and cholesterol esters
had to be determined and the fatty acids present in them
calculated and subtracted using factors derived from the
average mol.wt. of the fatty acids present. As several
estimations thus had to be made precision was poor
particularly at low values although in
hypertriglyceridaemia useful results could be
obtained.Later such methods were largely superseded by
techniques in which the glycerol liberated by
saponification was measured chemically. Phospholipids
were first removed by absorption on to zeolite which was
sometimes mixed with other absorbents such as lioyd's
reagent (hydrated aluminum silicate). The triglycerides
were then saponified usually by alcoholic potassium
hydroxide. Fatty acids and cholesterol were removed by a
dilute acid / organic solvent partition procedure in which
the contaminants passed into the organic phase leaving
glycerol in the aqueous phase.

Finally glycerol was oxidized by metaperiodate to


formaldehyde which measured colorimetrically following
its reaction with chromotropic acid or alternatively either
colorimetrically or fluorimetrically after treatment with
acetyl acetone and ammonia (the hantzsh reaction).

Enzymatic method
(i)Triglyceride+H2O lipase Fatty acid+Glycerol

( ii ) Glycerol+ATP Glycerokinase Glycerophosphate+ADP


(iii)Glycerophosphate is oxidized by glycerol phosphate


oxidase to dihydroxyacetone and hydrogen peroxide.

(iv) Oxygen is released from H2O2 in the presence of


Peroxidase which oxidizes p-cholorophenol chromogen to
form a coloured compound. measured in research (eg , in
studies of dietary influences

(E)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 the common
quantitative method involved extraction and acid
digestion with analysis of total lipid bound
phosphorus.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 used to predict fetal lung maturity.

(F)Determination of serum lipoproteins

Visual observation of the serum specimen received in the


laboratory may provide valuable information regarding the
lipids in the specimen. The chylomicrons and VLDL scatter
light because of the large size of their molecules. These
are responsible for the opalescence of the serum.

*Appearance of fasting plasma or serum: If a specimen of


plasma or serum collected after 12 hours fasting is
allowed to stand at 4C for 18hours its appearance can be
interpreted as follows:-

Appearance Lipid abnormality


(1)Clear. Normal or may have raised
LDL(-lipoprotein).
(2)Clear body and creamy to Hyperchlomicronaemia.
layer.
(3)Turbidity dispersed Hyperlipoproteinaemia with
through the specimen. high VLDL (pre--
lipoprotein).
(4)Turbidity throughout the Hyperlipoproteinaemia with
specimen with creamy top both chylomicrons and
layer. VLDL.

Separation and measurement of serum lipoproteins by


agar-gel electrophoresis

*Principle: Classification of hyperlipoproteinaemia


according to the system of Fredrickson requires quantation
of four classes of lipoproteins. In a routine clinical practice
this is achieved by electrophoresis preferably in agarose
gel. At pH 8.8 chylomicrons if present remain at the origin.
The other major lipoprotein migrate at rates that increases
in order of HDL>VLDL>LDL. Depending on their
electrophoretic mobility the lipoproteins are also named
as:-

*HDL-Lipoprotein which moves with the globulins.

*LDL--lipoprotein which moves the -globulins.

*VLDL-Pre-lipoprotein which moves with the 2globulins.

The separated lipoproteins can be stained with a lipid


staining dye such as fat red7B , oil red O or Sudan black B.

Ultracentrifugation of lipoproteins

The lipoprotein can also be separated by


ultracentrifugation. They have lower densities than the
other solids in plasma due to their lipid content. Secondly
each class of lipoproteins has a different density. At the
density of plasma (1.006kg/l) VLDL floats at the top while
LDL and HDL are sedimented. At the density of 1.063kg/l
both the LDL and VLDL float. At the density of 1.210kg/l all
the three lipoproteins float. Lipoproteins can thus be
separated from the plasma proteins and from each other
by ultracentrifugation at appropriate density.