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EXPERIMENT 1.

4
P13- P20

DETERMINATION of
CHOLESTEROL and
TRIGLYCERIDE IN SERUM
TC—
(CHO-PAP Enzymatic Endpoint Method, Leadman Kit)

TG—
(GPO Enzymatic Endpoint Method, Leadman Kit)
Objective
 Comprehend the principle and the
method of determination of TC and TG in
serum
 Associate with the clinical significance of
TC and TG .
1. Determination of
Total Cholesterol (TC) in Serum

Chol esterase
Chol E + H2O Chol + FA
Chol oxidase
Chol + O2 Cholestene-3-one +H2O2
POD
2 H2O2 + Phenol + 4-aminoantipyrene Quinoneimine +
Red color H2O
determine A520 nm
2. Determination of
Triglycerides (TC) in Serum
Lipases
TG + H2O Glycerol + FA
Glycerol Kinase
Glycerol + ATP Glycerol-1-phosphate + ADP
GPO
Glycerol-1-phosphate +O2 Dihydroxyacetone phosphate +
H2O2
POD
2 H2O2 + 4-aminoantipyrine + 4 Chlorophenol

Quinoneimine + HCl + 4 H2O
Reagents:
 Sample :Serum or plasma
 Standard solution
 Reaction reagent (enzymatic mixture ) –Kit
 dH2O
Procedure :
 Pipette every solution into each tube according
the tables, pay attention the volumes
 Mix the tubes and incubate for 5 min at 37℃
 Measure A520 against the blank using cuvette or
microplate reader
 Calculation
Clinical significance
 Lipid profile is a measure of the lipid contents of
the blood. It includes measurement of TG and
cholesterol (TC, HDL-C, LDL-C, VLDL chol).
 Chol and TG are insoluble in water and are
transported in the blood in lipoproteins,
complexes of lipids with apolipoproteins.
 There are four major classes of lipoprotein:
chylomicrons, VLDL, LDL and HDL. It assumes
major significance in diagnosis and management
of cases of dyslipoproteinemia.
Clinical significance
 Dyslipoproteinemia associated with Clinic
Genetic dyslipoproteinemia
Secondary dyslipoproteinemia
nephrotic syndrome, obesity, alcoholism, hypothyroidism,
Diabetes mellitus and certain drugs.
Premature atherosclerosis.
Deposition of lipid into various tissues including dermis
(xanthomas).
Elevated triglycerides presents with a risk of pancreatitis or
liver obstruction .
Clinical significance
 Lipid and Atherosclerosis or
CHD (Coronary Heart Disease)
Elevated cholesterol (LDL-C) levels, there is an increased
incidence of atherosclerosis and its complication
HDL-C is considered as the ‘good cholesterol’
Experts agree on TC/ HDL-C ratio as a better assessment
of CHD risk.
Increased TG concentrations to be positively correlated
with increased risk for CHD, however, TG concentration
represents an independent risk factor is not clear.