SITI ANNISA DEVI TRUSDA
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.
2.
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.
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 37C
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
1.0 ml
1.0 ml
1.0 ml
GOD color reagent
 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
C=
A sample
x 100 mg/dl or
C=
A standard
C : Glucose concentration of the sample
x 55,5 mmol/l
A standard
1.
2.
3.
4.
By this method the blood glucose level can be
measured linearly up to 600 mg/dl. If the blood
glucose level is higher than 600 mg/dl, dilute the
plasma three times by adding 2 volumes of
aquadest, then repeat the procedure. Multiply the
result three times.
The result is not influenced by blood creatinin,
fructose, galactose, uric acid, glutathion, ascorbic
acid or bilirubin as long as these substances’s
concentration are at normal range.
Bilirubin concentration up to 10 mg/dl does not
influence the result, but a high dose of oral
ascorbic acid (vitamin C) could decrease the result.
The color produced will be stable for 2 hours.
 Urine
Reduction Test (Benedict’s Test) :
 Principle :
 Glucose reduces the alkaline copper solution,
Cu++ Cu+ and is precipitated as Cu2O, a red
brick color substance.
 Depend on concentration of Cu2O produced,
the shaked solution will produce different
color that can be used to estimate the
glucose concentration
Urine of a diabetic patient as sample
Benedict’s qualitative solution (semi
qualitative) :
- 17 gr CuSO4
- 100 gr NaCO3 anhydride
- 170 gr tri – Na- Citrate 2 H2O
Dissolved in 1000 ml of aquadest
Test tubes
Waterbath 100 0C/ Bunsen burner
 Mix
3 ml Benedict’s solution with 3 drops of
urine in a test tube, heat it in 100oC
waterbath or just boil it for a moment, note
the color produced after shaking the tube.
 Repeat the same procedure with the urine
diluted 2, 4 and 8 times.
 The
positive result of Benedict’s test could
be given by many reductors, such as :
 - Sugars : glucose, pentose, lactose,
galactose.
 - Drugs : antipyrine, pyramidon, PAS,
xanthonin.
 - High concentration of normally urine
substances : indican, uric acid, creatinin.
 - Preservative agents :formalin, CHCl3.
 To
read the result, shake the tube and then
note the solution’s color produced :
 - Blue
: (-) → means there is
no glucose in the sample urine
 - Green
: (+) → 0.5 – 1 gr % of
glucose
 - Yellowish green : (++) → 1 – 1.5 gr % of
glucose
 - Yellow
: (+++) → 2 – 3.5 gr %
glucose
 - Red brick color : (++++) → 4 gr % of
glucose