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BIO 306.

01/02
EXPERIMENT 3
CHOLESTEROL
DETERMINATION IN SERUM
A. NAZLI BAAK

CHOLESTEROL DETERMINATION IN SERUM

Every living organism has been found to


contain sterols.
Cholesterol is the main sterol of the body
and virtually found in all cells and body
fluids.
It is also the initial starting point in many
metabolic pathways including vitamin D
synthesis, steroid hormone synthesis and
bile acid metabolism.

CHOLESTEROL DETERMINATION IN SERUM

Although cholesterol is essential for health


and growth, excess levels of serum
cholesterol is related with increased risk of
cardiovascular diseases.

Atherosclerosis results from thickening of


the inner layer of the arterial wall,
especially caused by cellular material and
deposits of lipids.

CHOLESTEROL DETERMINATION IN SERUM

Determination of serum cholesterol and


other lipids may be the initial step in the
early diagnosis of atherosclerosis and
coronary heart disease.

Chemical labs have been routinely


measuring cholesterol in patients serum
specimens for >40 years.

CHOLESTEROL DETERMINATION IN SERUM

On the basis of long experience, labs


should be capable of performing
cholesterol analysis with a high degree of
reliability.
Serum cholesterol levels may be
quantitatively determined by either
chemical or enzymatical methods.
The most widely performed method is the
chemical method of Zak, although
enzymatic kits are replacing this
conventional method nowadays.

CHOLESTEROL DETERMINATION IN SERUM

CHOLESTEROL DETERMINATION IN SERUM

CHOLESTEROL DETERMINATION IN SERUM

Cholesterol is the most hydrophobic


membrane lipid.
its

structure is quite different from the other


membrane lipids: instead of two long
hydrocarbon tails, it has a stiff carbocyclic
structure, the steroid ring system

instead

of a big hydrophilic head group, there


is only a hydroxyl group at one end of the
molecule.

CHOLESTEROL DETERMINATION IN SERUM

With this structure cholesterol is by far the


least water-soluble membrane lipid.
Cholesterol accounts for 10 % or more of
the total lipid in the plasma membrane and
the Golgi membrane, but it is less
abundant in more internal membranes.
It is prominent only in animals, including
humans. Plants have different membrane
steroids, the phytosterols, and most
bacteria have no steroids at all.

CHOLESTEROL DETERMINATION IN SERUM

Steroid

hormones are derived from


cholesterol: progestagen, cortisol,
testosterone, estrogens etc.
LDL:

major carrier of cholesterol in blood


HDL: picks up cholesterol released into the
plasma from dying cells or membranes which
undergo turnover.

CHOLESTEROL DETERMINATION IN SERUM

Preparation of Serum
Allow the blood sample to stand for at
least 1 hr. or centrifuge at 5000 rpm for 15
min.
Spin in a clinical centrifuge for 10 min.
Carefully remove the clean serum with
Pasteur pipette (do not disturb the clut).
Transfer 0,1 ml serum to a tube. The rest
can be frozen and saved for later
analyses.

CHOLESTEROL DETERMINATION IN SERUM

Procedure for Total Cholesterol


Determination
Transfer 0,1 ml of serum to test tubes. Add
10 ml of absolute ethanol and mix rapidly
on a vortex mixer for 10 sec.
Cent. the tubes for 5 at full speed in a
cilinical cent. and transfer the extracts
carefully to clean test tubes. Measure 2 ml
of extract into each of three new test
tubes. It is important to have a clean
solution; if some precipitate is carried over,
spin the tube again.

CHOLESTEROL DETERMINATION IN SERUM

The standart is prepared by adding 2 ml of


a 0.02 mg/ml solution of cholesterol to a
fourt tube.
Prepare a blank by delivering 2ml of
ethanol to a fifth tube.
Slowly add 2 ml of color reagent to each
tube and mix by gentle swirling.
(VERY STRONG ACID-HANDLE WITH
CARE!)

CHOLESTEROL DETERMINATION IN SERUM

Cover the tubes with parafilm, and allow them to


stand at RT for 30. The color that develops is
stable for about 1 hour only.
Read the absorbance of the standard and the
unknowns at 550nm against the blank, and
calculate the average mg/dl value for total
cholesterol in serum. Again set up the ratio

to calculate the conc. of unkown which is relative


to that of the standard under the same
conditions.
designed and prepared by
alburse