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Determination of total cholesterol

in serum
objective
 Comprehend the method of determination
of total cholesterol in serum .
 Associate with the important of cholesterol
in vivo and its clinical significance .
Principle
 The cholesterol is determined after
enzymatic hydrolysis and oxidation
.cholesterol esters in the serum are
hydrolyzed to free cholesterol and fatty
acids in a reaction catalyzed by cholesterol
esterase.
cholesterol esterase.
cholesterol ester + H2o Cholesterol + Fatty acid
 Then the free cholesterol are oxidized by
cholesterol oxidase that generates hydrogen
peroxide .
Cholesterol oxidase
Cholesterol + o2 Cholestene-3-one + H2o2
 The indicator quinoneimine is formed from
hydrogen peroxide and 4-aminoantipyrine
in presence of phenol and peroxidase .
peroxide
4-aminoantipyrine+ Phenol + H2o2 Quinoneimine + H2o
 The amount of total cholesterol in the
sample is determined by measurement of
the absorbance of the red color at 520 nm
procedure
 1.accurately pipette into each tube:
•Blank (ul) Test •Standard (ul)
(ul)
•1.distilled H2o 30

2.Standard 30

3.sample 30

4.reaction reagent 1500 1500 1500
2.Mix the tubes and incubate for 10 min at 20℃
to 25℃ or 5 min at 37℃ .
3.Measure the absorbance at 520 nm of the test
sample (A ) and the Standard (A ) against the
T s

reagent blank using Cuvette with 0.5 cm light
path .
calculation
 Concentration of total cholesterol =
A -A
T O

× 200 (mg/dL)
•A -A
S O
Clinical significance
 Value
 <5.17 mmol/L (200mg/dl)
 Desirable blood cholesterol
 5.17-6.18 mmol/L (200-239mg/dl)
 Border-line high blood cholesterol
 ≧6.20 mmol/L (240mg/dl)
 High blood cholesterol