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BIOCHEMISTRY DEPARTMENT
Needed for supporting the D/ of some
diseases, including DM.
When to measure: fasting state or 2 hours
after taking a meal.
Sometimes at a present time detecting a
present increased or decreased of blood
glucose level (hyperglycemia or
hypoglycemia).
1. Reduction method, which is based on the
ability of glucose to reduce Cu++ to Cu+
less sensitive, substances that could
reduce Cu++ : fructose, galactose,vitamin C,
creatinin, uric acid, glutathion, etc.
2. Enzymatic method (more specific and
precise result) : Glucose is oxidized by
glucose oxidase gluconic acid + H2O2
red dye.
Glucose oxidase (GOD) catalyzes the oxidation of glucose according to the fol owing
equation :
GOD
Glucose + O2 Gluconic acid + H2O2
The hydrogen peroxide (H2O2) which is formed, in the presence of peroxidase (POD) reacts
with 4-aminophenazone and phenol, and gives rise to 4-benzoquinon monoiminophenazon
(a red dye)
POD
H2O2 + 4-aminophenazone + phenol 4 H2O + 4 p-benzoquinon
monoiminophenazone
(red color)
Color reagent glucose oxidase (GOD-PAP) :
Glucose oxidase (GOD) > 15 KU/l
Peroxidase (POD) > 1.5 KU/l
4-aminophenazone 0.25 mmol/l
Phenol 0.75 mmol/l, mutarotase > 2.0 KU/l
Phosphate buffer pH 7.5, 0.1 mol/l.
Glucose standard solution 100 mg/dl (5.55 mmol/l).
Serum or plasma. Aquadest.
Micropipette 10 l, 1.0 ml Reaction tubes
Waterbath 37C Centrifuge
Spectrophotometer (wavelength 492-546 nm)
To make the GOD color reagent : dissolve the
content of one bottle of GOD enzyme with
its solvent available. This solution is stable
for 30 days at 2 – 8 oC temperature.
The absorbancy of the blank’s reagent must
be about 0.0 – 0.4 AU if it is read at 505 nm
wavelength compared with aquadest.
For every series of measurement, only one
standard and one blank are needed.
1. Centrifuge 3 ml EDTA blood, 2000 rpm for
10’. The plasma will be separated from the
blood cells. Use plasma for sample
2. Pipette into each of the three reaction
tubes according to the following table :
Blank Standard Sample
Aquadest 0.01 ml - -
Standard - 0.01 ml -
Plasma - - 0.01 ml
GOD color reagent 1.0 ml 1.0 ml 1.0 ml
3. Mix the content of each tube well, then
incubate them at 37oC for 10 minutes or let
stand at room temperature for 25 minutes.
Avoid direct sunlight
4. Using cuvette tube, read the sample’s and
standard’s absorbancy against the blank at
505 nm.
Calculation :
A sample A sample
C= x 100 mg/dl or C= x 55,5 mmol/l
A standard A standard