Experiment No-07

Name of Experiment: Estimation of total cholesterol in blood serum/plasma

Theory:
Cholesterol (chole-bile, stereos solid and ol- alcohol) is an organic molecule (lipid). It is an
essential structural component of animal cell membranes and serves as a precursor for the
biosynthesis of steroid hormones, bile and vitamin D. In blood, cholesterol present in the form of
cholesterol ester which gives to cholesterol and fatty acid in presence of cholesterol esterase.
This free cholesterol reacts in presence of oxygen and cholesterol oxidase to give Cholest-4-en3-one and hydrogen peroxide. This generated hydrogen peroxide breaks down to water and
nascent oxygen in presence of peroxidase. This nascent oxygen binds with chromogen and gives
Quinoneimine a pink color product. The intensity of pink color is directly proportional to total
cholesterol of blood serum/plasma. This total cholesterol is summation of HDL (high density
lipoprotein- good cholesterol), LDL (Low density lipoprotein-bad cholesterol) and triglycerides.
Hence total cholesterol = HDL + LDL + 20% Triglycerides.
Cholesterol esterase

Cholesterol ester + H2O ------------ Cholesterol + Fatty acids

Cholesterol oxidase

Cholesterol + O2-------------- Cholest-4-en-3-one + H2O2

Peroxidase

H2O2--------------------H20+ [0]
[0] + 4-amino antipyrine+ phenol------------Quinoneimine (Pink color) + H20
Requirement:
Reagent-1: it has component of Goods buffer (phosphate buffer pH 7.0), cholesterol
esterase, cholesterol oxidase, peroxidase, chromogen (4-amino antipyrine + phenol),
stabilizer and preservative
Reagent-2/Standard: it is blood serum with known concentration of total cholesterol of
200mg/dl
Blood serum/plasma

I.

II.
III.

Method:
[A] Isolation of Blood plasma/serum: liquid part of uncoagulated blood is called plasma and
coagulated blood is called serum. Isolation of Blood plasma is carried out by centrifugation.

2ml of blood is withdraw from healthy volunteer (before meal and after meal condition)
It is kept in vaccumtainer tube having some pinch of EDTA (Acts as anticoagulant)
Now it is centrifuged at 4000 rpm for 10 minutes at room temperature and pellets is
discarded and supernatant is collected which is plasma.

[B] Development of Color:

Three test tubes is taken and named as blank, positive control and unknown respectively.
In blank test tube:2000l of reagent-1 is added
In positive control test tube:2000l of reagent-1 and 20 l of reagent-2 are added
In unknown test tube:2000l of reagent-1 and 20 l of blood plasma/serum (in which
total cholesterol will be estimated) are added
All test tubes are incubated for 10 minutes at 37C in dark.
Afterwards, color is developed and absorbance/ OD are read at 490nm in
spectrophotometer/colorimeter against blank.

Tabulation:
Test tubes

Blood
serum/plasma

Reagent1

Reagent2

Blank
Positive
control
Unknown
Calculation:

----------

2000l
2000l

-----20l

20l

2000l

Incubation
at 37C for
10 minutes
in dark
,,
,,

OD
490nm

Amount of
total
cholesterol

---200mg/dl

,,

???

Amount of total cholesterol in unknown sample (mg/dl):

Amount of total cholesterol in positive control X OD of Unknown
------------------------------------------------------------------------------------OD of positive control
Result:
Case

Before meal

After meal
Remarks:
Mark Obtained:
Signature:

Total cholesterol
Standard range
Calculated
(mg/dl)
value
(mg/dl)
<200:normal
110-160:very good
170-200: good

Condition
(Normal/good/very
good range)