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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
× 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