30
2 INVESTIGATION OF LIPID METABOLISM &
CHOLESTEROL
Topics to be reviewed
Lipids in blood, lipoproteins. Cholesterol ­ transport and metabolisms. Decision-making analysis, critical
difference.
Pre-lab questions
1. Draw formulas of cholesterol and cholesteryl linolate.
2. Explain what the blank is. Which substances are absent in the blank for the determination of serum
cholesterol?
3. Which biological samples can be analyzed by Reflotron and Accutrend?
4. Write Friedewalďs equation and the two limitations of its usage.
5. Which lipoprotein fraction poses a risk for atherosclerosis? In which serum protein fraction is it
comprised? Give its two possible names.
Introduction
Recently, lipid metabolism disorders have come into account in connection with the prevention and
treatment of cardiovascular disease.
Basic examination of lipid metabolism involves analysis of triacylglycerols, total cholesterol, HDLcholesterol
and from these values calculated concentrations of LDL-cholesterol and non-HDL cholesterol.
The examination is completed with the chylomicrons test and occasionally with the analysis of
phospholipids and electrophoresis of lipoproteins.
Hyperlipidaemia is the condition in which the levels of lipids in the blood are too high.
With regard to the development of atherosclerosis we recognize three basic types of hyperlipidaemias*
:
I. Isolated hypercholesterolaemia (isolated increase of the total cholesterol)
II. Isolated hypertriacylglycerolaemia (isolated increase of triacylglycerols)
III. Combined hyperlipidaemia (simultaneous increase of cholesterol and triacylglycerols)
*Formerly used classification according to Frederickson, which is based on the determination of total cholesterol,
triacylglycerols a electrophoresis of lipoproteins, is no longer used.
Hyperlipoproteinaemia is the condition in which the level of one or more classes of lipoproteins in the
blood is increased. Often, the increase in blood lipids is combined with the decrease in the concentration
of HDL-cholesterol, therefore, the term dyslipoproteinaemia is also used.
Laboratory diagnostics of lipid metabolism disorders must be supported by the examination of blood
lipids from at least two blood collections in 2­8 weeks interval, at normal lifestyle. Critical differences of
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the individual parameters should be evaluated (see Exp. 1.6.3) and, if they are lower than acceptable, the
arithmetic mean is calculated for each analyte. In case that the critical difference is exceeded, the third
measurement should be carried out.
The critical differences of lipid metabolism parameters:
Analyte Critical difference (%)
Total cholesterol > 20
LDL-cholesterol > 25
HDL-cholesterol > 25
Triacylglycerols > 65
The examination of lipid metabolism is highly influenced by lifestyle (diet, physical activity, and body
weight), pharmacotherapy (hormonal contraception, hormonal substitution therapy, etc.) and by acute or
non-compensated disease. Therefore, the examination of blood lipids must not be done, when it is
expected, that the result will not describe the situation at the normal lifestyle (shortly after vacation, during
hospitalization from other reasons, acute diabetes mellitus, gravidity and half a year later, etc.).
2.1 Determination of total cholesterol in serum and blood
Cholesterol is transported in blood as a component of lipoproteins, mainly LDL, less in HDL and VLDL.
About two thirds of the cholesterol in the plasma is esterified with long chain unsaturated fatty acids, the
remaining one third is present as free (unesterified) cholesterol.
2.1.1 Enzymatic determination of serum cholesterol
Cholesterol esters are hydrolyzed by cholesterol esterase into free cholesterol and fatty acids. The free
cholesterol is then oxidized with the aid of cholesterol oxidase and consumption of oxygen giving rise to
cholestenon and hydrogen peroxide. The latter, in the peroxidase-catalyzed reaction yields a red dye from
of 4-aminophenazon and phenol.
Materials: Kit CHOL (Roche Diagnostics*
) containing: Reagent-cholesterol (containing Tris buffer 100 mmol/L, pH 7.7; Mg2+
50 mmol/L; 4-aminophenazon 1 mmol/L; sodium cholate 10 mmol/L; phenol 6 mmol/L; 3,4-dichloro-phenol
4 mmol/L, polyglycol ether 0.3 %; cholesterol esterase 6.67 kat/L; cholesterol oxidase 4.17 kat/L; peroxidase
3.33 kat/L), calibrator-cholesterol (concentration is given on the label of vial). Specimens of blood serum, set of
test tubes, micropipettor 20 L, pipettor adjusted to 2 mL. Spectrophotometer Spekol 1300 and software WinAspect
or spectrophotometer Helios Delta and VisionLite Fixed. *
Alternatively, the kit of BioVendor, Pliva-Lachema or
Human can be used; the composition of reagents may be then different.
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Procedure
Pipette the components into labelled test tubes according to the following table:
Reagens (L) Blank Test Standard
Reagent-cholesterol 2 000 2 000 2 000
Demi-water 20 - Serum
- 20 Calibrator-cholesterol
- - 20
Mix well and incubate for 10 minutes at room temperature.
Measure absorbances*
of samples (Ax) and standard (ASTD) at 500 nm against the blank within 1 h.
*Measure absorbances according to the enclosed instructions to the spectrophotometers and use relevant software.
Calculation of the concentration of serum total cholesterol: STD
STD
x
x c
A
A
c
where cSTD is concentration of cholesterol in calibrator (it is given on the label of the vial).
Evaluation
According to the recommendation for diagnostics and treatment of hyperlipoproteinaemias of adult
people, prepared by the Czech Society for Atherosclerosis, the examination of cholesterol should be
performed once in every five years in all adult people from 20 to 75 years old during preventive medical
examination. If the concentration of total cholesterol is below 5 mmol/L, next control should follow after
five years.
Serum cholesterol above 5 mmol/L should be the reason for further examination, especially the finding of
the cholesterol distribution in the lipoprotein fractions. Increased concentration of cholesterol is often
found in diabetics or in case of hypothyroidism, decreased (< 2.5 mmol/L) e.g. in case of late hepatic
cirrhoses or hyperthyroidism.
2.1.2 Determination of blood cholesterol using analyzer Reflotron
Reaction zone of the test strip contains enzymes cholesterol esterase, cholesterol oxidase, peroxidase and
chromogenous substrate dimethylbenzidine. Determination is based on the sequence of enzymatic
reactions described in Exp. 2.1.1. The resulting colouring is evaluated by the reflectance method.
Materials: Analyzer Reflotron
with strips designated for cholesterol, micropipettor 30 L, lancets or sterile needles,
disinfectant Ajatin, cellulose swabs.
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Procedure
Follow detailed enclosed instructions to the analyzer Reflotron.
Preparation of the instrument. Open flap of measurement chamber.
Preparation of the strip. Remove the foil protecting the test strip.
Capillary blood collection. Using the pipettor take up exactly 30 L of capillary blood directly from
the puncture site:
Strictly follow the rules for the proper collection of capillary blood (see Exp. 1.1.2):
wash hands in warm water
disinfect the puncture site
point the puncture using of lancet from lateral side of finger pad
wipe off the first drop of blood
support formation of the other drops by gently pressure to the surrounding tissue
after collection of blood apply a cotton wool wetted in disinfect solution on the puncture site
Application of blood on the strip. Apply blood in the centre of the red application zone on the test
strip (don't touch the application zone by tip).
Starting measurement closing flap. Within 15 seconds after sample application, insert test strip (the
magnetic band turned down) into the measuring chamber until it snaps in. Close flap immediately.
Result reading. The value CHOL is displayed (a test specific magnetic code recognizes the parameter
of interest) and time left to displayed result. Cholesterol value in mmol/L is displayed.
Evaluation
The total cholesterol value should be below 5 mmol/L, see below table ,,Border value of blood lipids in
common population". Investigation of lipids by ,,dry chemistry" methods is regarded as screening. The
diagnostic and treatment dyslipoproteinaemias must be always supported by examination of lipids from
venous blood using the certified method.
2.1.3 Determination of blood cholesterol using analyzer Accutrend
Determination is based on the sequence of enzymatic reactions described in Exp. 2.1.1. Reaction zone of
the test strip contains enzymes cholesterol esterase, cholesterol oxidase, peroxidase and chromogenous
substrate dimethylbenzidine. The resulting colouring is evaluated by the reflectance method.
Materials: Analyzer Accutrend
with strips designated for cholesterol, lancets or sterile needles, disinfectant Ajatin, cellulose
swabs.
Procedure
Follow detailed instructions of the analyzer Accutrend
:
Switch the instrument on. To start a measurement, switch the meter on. The display shows the stored
code number. If the meter is already coded skip next step.
Coding. Insert the check strip into the instrument and the code is saved.
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Insert test strip. Insert a cholesterol test strip until it snaps in. The meter recognizes the type of test
strip and confirms correct placement by an acoustic signal. A blank value of the test strip is taken
automatically.
Capillary blood collection. Strictly follow the rules for the proper collection of capillary blood (see
Exp. 1.1.2)
wash hands in warm water
disinfect place of puncture site
point the puncture using of lancet from lateral side of finger pad
wipe off the first drop of blood
support formation of the other drops by gently pressure to the surrounding tissue
after collection of blood apply a cotton wool wetted in disinfect solution on the puncture site
Application of the blood on the strip. Open the flap and apply the drop of blood directly from the
punctured finger onto the yellow mesh of the test strip. If the application field is not completely
covered with blood, repeat this step and put a drop of blood on a new test strip.
Initiation of measurement. Close the flap immediately. After having applied a drop of blood, close the
flap immediately. Only now the counting of the time starts. The cholesterol value is displayed after
180 seconds and stored automatically with time and date.
Alternatively result reading. The round colour field on the reverse side of the test strip has to be
stained uniformly. If not, repeat the measurement with a new test strip. In addition, please compare
the colour field with the colour scale on the test strip package.
Evaluation
The concentration of total cholesterol should be below 5 mmol/L, see below table "Cut-off values of
blood lipids in common population". Generally, the examination of lipids by "dry chemistry" methods is
regarded as screening. The diagnostics and treatment of dyslipoproteinaemias must be always supported
by the examination of lipids from venous blood using the certified method.
2.2 Determination of blood triacylglycerols using analyzer
Reflotron
Determination of triacylglycerols belongs among basic methods for the examination of lipid metabolism.
The Reflotron
analyzer and capillary blood are used for testing in the practical lesson.
Principle: After placing the blood sample on the test strip, the erythrocytes and other cell elements are
separated. Consecutively, a sequence of enzymatic reactions takes place in the reaction zone of the test
strip. The triacylglycerols (TG) are hydrolyzed by esterase into fatty acids and glycerol. Then glycerol is
phosphorylated by glycerol kinase to glycerol-3-phosphate, which is oxidised by glycerol-phosphate
oxidase in presence of oxygen. Arising hydrogen peroxide reacts with the redox indicator catalysed by
peroxidase to form coloured product. The resulting coloration is evaluated by the reflectance method.
Methods used in the automatic analyzers are based on the similar principle.
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Materials: Analyzer Reflotron
with strips designated for triacylglycerols, micropipettor 30 L, lancets or sterile needles,
disinfectant Ajatin, cellulose swabs.
Procedure
Follow detailed instructions of the analyzer Reflotron
.
The procedure is analogous as in Exp. 2.1.2.
Evaluation
Serum triacylglycerols in the healthy population (the cut-off level) should be less than 2 mmol/L.
Samples of serum with the concentration of TG above 3.4 mmol/L are opalescent, TG concentrations over
11.3 mmol/L indicate the presence of chylomicrons and serum has milky turbidity. Therapy is strictly
based on the diet with low content of fats and sugars, increased physical activity, sufficient amount of
antioxidants in the nutrition and the overall hypolipidemic treatment.
2.3 Determination of HDL-cholesterol and LDL-cholesterol
A high proportion of serum total cholesterol incorporated in HDL is considered as a sign of the
satisfactory ability of an organism to eliminate undesirable excess cholesterol. On the contrary, an
increased concentration of LDL-cholesterol represents the high coronary risk involved in
hypercholesterolaemia.
The determination of HDL-cholesterol (HDLChol) is based on the procedure, in which chylomicrons,
VLDL and LDL are precipitated with a suitable precipitating agent (e.g. phosphotungstic acid and MgCl2
or heparin and MnCl2). The supernatant after centrifugation contains cholesterol associated with soluble
HDL. As the concentration of cholesterol in HDL is very low, a sensitive enzymatic method must be used
for the determination (see Exp. 2.1.1).
The recent methods are able to determine HDL-cholesterol directly in the serum without the previous precipitation of the other
lipoprotein particles. Enzymes cholesterol esterase and cholesterol oxidase modified by polyethylene glycol in presence of Mg2+
ions and cyclodextrin sulphate catalyse selectively only the transformation of HDL-cholesterol.
Moreover, there is also a new turbidimetric method for the direct determination of LDLChol (without any preparatory steps and
calculations), which uses a polyanionic detergent. LDL particles form insoluble complexes with this detergent. The resulting
turbidity is measured by the turbidimetric method.
Procedure and evaluation
In spite of lack of time, the task is carried out theoretically.
Evaluate the concentrations of HDLChol provided by the assistant in accordance with the reference
values given below.
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2.4 Calculation of LDL-cholesterol and derived parameters
The concentration of LDL-cholesterol (LDLChol) is usually calculated from the difference between
concentration of total cholesterol (CholTot) and the concentration of HDL-cholesterol and triacylglycerols
(TG) (all values are in mmol/L) using Friedewalďs formula:
LDLChol = CholTot TG/2.2 HDLChol (mmol/L)
This approximately formula is based on the assumption that in most cases chylomicrons are absent after
overnight fasting. Serum triacylglycerols originate mostly from VLDL, in which the molar ratio
TG/cholesterol is usually a constant having the value approximately of 2.2. This formula may be used
only if serum TG concentration is less than 4.5 mmol/L.
Additional derived parameters of the lipid metabolism
The aim of these calculations is to make the determination of the risk, resulting from the measured
concentrations of blood lipids, more accurate.
CholTot/HDLChol index
This index takes into account that higher level of HDLChol is a "negative risk factor", which lowers the
risk of the atherosclerotic cardiovascular disease. Concurrent increase of the total and HDL-cholesterol
may not increase the risk of atherosclerotic cardiovascular disease.
Non-HDLChol
Non-HDLChol = CholTot ­ HDLChol (mmol/L)
This parameter takes into account the concentrations of all atherogenic lipoproteins. It is used in case,
when the calculation of LDL cholesterol cannot be carried out because of the increased concentration of
triacylglycerols.
Cut-off values of blood lipids in common population:
CholTot
(mmol/L)
LDLChol
(mmol/L)
TG
(mmol/L)
HDLChol
(mmol/L)
Index
CholTot/HDLChol
Non-HDLChol
(mmol/L)
< 5.0 < 3.0 < 2.0 > 1.0 < 5 < 3.8
If any of the measured concentrations exceeds the cut-off value, additional examinations must be performed to reveal the disorder
of lipid metabolism and to evaluate the risk of atherosclerotic cardiovascular disease.
Procedure and evaluation
Using Friedewalďs formula and the given values of CholTot, TG and HDLChol calculate the value of
LDLChol and compare all of them with the desired ones (see above).
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2.5 Electrophoresis of serum lipoproteins
The lipoproteins can be separated electrophoretically at an alkaline pH on agarose gel according to their
different mobility. The separated lipoproteins are stained with a lipid specific Sudan Black.
Materials: Kit Hydragel LIPO + Lp(a) (Sebia) containing: Tris-barbital buffer, pH 8.75 (0.05 mol/L, prepared by a dilution of
22.5 mL of the stock solution from the kit with demi-water to final volume of 300 mL), Sudan black solution (2 mL
of the 6% solution from the kit in 160 mL of ethanol and 140 mL of demi-water), destaining solution (45 % ethanol),
agarose gels (ready to use). Micropipettor 5 L, applicator, strips of filter paper, migration chamber CUVE K20,
source of voltage, hair dryer. Specimens of serum.
Procedure
Preparation of the gel. Unpack the HYDRAGEL agarose gel plate and place it on the filtration paper
(the gel side up). Place one thin filter paper onto the gel surface (in the application point, on the plane
with arrows) to absorb the excess of liquid. Remove the paper immediately. Do not leave the filter
paper for a too long contact with the gel to avoid its dehydration.
Application of the samples. Place the applicator with the notches on a flat surface. Apply gradually
5 L of serum samples into the applicator wells and let it seep for 5­10 minutes. Remove the nonseeped
serum by a strip of the filtration paper and take away the applicator.
Electrophoretic separation. Measure about 150 mL of Tris-barbital buffer into each compartment of
the migration chamber. Place the gel plate on the bridge with the gel side facing down and according
to the polarity indicated on the gel (the application side of the gel on the cathodic side); the gel should
be dip about 1 cm into the buffer on each side. Plug the chamber to the power supply. Migration takes
place for 90 minutes at the constant voltage of 50 V (initial current 112 mA per gel). After
migration, unplug the chamber and remove the gel plate.
Fixation and staining. Dry the gel with hot air (max. temperature 80 °C). The gel must be perfectly
dry. Immerse the dried and cooled gel in the staining solution for exactly 15 min. Destain the gel in
destaining solution for exactly 5 min. Wash gel by immersing into demi-water for several minutes.
Drying of the gel. Drain vertically excess liquid on the gel surface and dry the gel with hot air.
Evaluation
After the electrophoretic separation the chylomicrons stay at the start, LDL ( -lipoproteins) migrates at
the position of 2-globulins, VLDL (pre- -lipoproteins) at the position of -globulins. HDL particles
have the highest mobility and they appear at the position of 2-globulins.
A fraction characteristic for lipoprotein (a) ­ Lp(a) ­ a risk factor of atherosclerosis and coronary heart
disease ­ may appear between - and -lipoproteins (i.e. HDL and LDL). It may be determined
quantitatively by immunoassays or electroimmunodiffusion.
Electrophoregram can be evaluated densitometrically at 580 nm. The results are then expressed in
percentages of the optical density for the individual fractions with respect to the whole coloured area. The
measured values together with other indicators (e.g. total cholesterol, triacylglycerols) are used to
characterize hyperlipoproteinaemias.
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Reference intervals: -lipoproteins 23­46 %
pre- -lipoproteins 3­18 %
-lipoproteins 42­63 %
In practice, electrophoregram is evaluated visually. Individual serum fraction is compared with the reference one and evaluated
as normal/increase/decrease lipoprotein fraction.
The other tests of lipid metabolism
Chylomicrons test
Measurement of chylomicrons in serum is not commonly indicated. Samples collected after overnight
fasting (12 h at least) should be free of chylomicrons. Their presence in serum is indicated by severe
chylosity. The chylomicrons test may be used for their detection. Chylomicrons presence is indicated by
an opalescent or white ring after 12hours standing of serum at 4 °C. In this time, the possibly present
chylomicrons are floating to the surface of the sample.
Related problems
1. Mr. J. (45 years) had the concentration of cholesterol 6.0 mmol/L and after 8 weeks 5.2 mmol/L.
The critical difference (CD) for cholesterol is 20 %. a) What does the CD value express? b)
What conclusion could be made from both examinations? Should they be followed by the third
examination, or can we conclude, that the mean value from both examinations is 5.6 mmol/L?
2. Mr. P. (36 years old) had repeatedly the level of cholesterol about 8.1 mmol/L, LDL-cholesterol
4 mmol/L. His daily intake of cholesterol was about 600 mg. He was recommended a diet with
low cholesterol content and a change of lifestyle. After three months, the mean value of
cholesterol was 7.4 mmol/L. Then, man had prescribed Colestid, which bounds bile acids in the
small intestine and increases their excretion in urine. Taking of this drug lead to decrease of
cholesterol level to the range of 5.7­6.4 mmol/L.
a) How the first findings of cholesterolaemia should be interpreted?
b) Which types of food should be excluded and which should prevailed in the diet of Mr. P.?
c) Is the cholesterol intake in food the only source of cholesterol in the organism?
d) What is the relation between cholesterol and bile acids? Why could the taking of Colestid
lead to the decrease of the cholesterol concentration?
3. Mr. Z. (40 years old) had confirmed high serum concentrations of the total and LDL-cholesterol
(10 mmol/L and 8.6 mmol/L, respectively). Clinical examination proved characteristic
xanthomas on tendons. Mr. Z.'s father died of myocardium infarction at the age of 55. It was
concluded, that the patient suffers from familiar hypercholesterolaemia (the cause is the
defective gene for the LDL receptor).
a) What can explain the increased level of cholesterol in this patient?
b) How could we positively confirm the diagnosis?