TITLE:

Estimation of Blood Glucose Using Glucose Oxidase

AIMS AND OBJECTIVES:
• To understand the importance of measuring blood glucose level.
• To understand the principles of enzymatic estimation of glucose.
INTRODUCTION
Enzymatic method yields maximum specificity for glucose estimation. Glucose can be
measured by its reaction with glucose oxidase, in which gluconic acid and hydrogen peroxide
are formed. Hydrogen peroxide than reacts with an oxygen acceptor, such as ortho-dianisidine,
phenylamine-phenazone or any other chromogenic oxygen acceptors, in a reaction catalysed by
peroxidase to form a colour.
One of the advantages of enzymatic method is its specificity. This enables the estimation
of glucose even in a complex mixture. Thus, this method is widely used in the field of
clinical chemistry and for food analysis. In clinical chemistry, the enzymatic analysis of
glucose in blood and urine has been modified as dipstick test. However in the present
practical, the conventional enzymatic method shall be used.
METHOD:
1. SAMPLES TEST:
a. The following substances are added in the tube.
i. 1.8 ml sodium sulphate-zink sulphate
ii. 0.1 ml blood
iii. 0.1 ml NaOH (0.1M)
b. Mix carefully.
c. Centrifuge at 3000 RPM for 5 min.
d. Transfer the supernatant into new test tube.
Note: The glucose concentration in the supernatant is 1/20 of the concentration in the blood
sample.

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 2. BLANK & STANDARD:
a. Glucose standards 12 mg/dl are provided.
b. Preparing concentration glucose standard point.

CONCENTRATION GLUCOSE TOTAL mL

TUBES WATER (mL)
(mg/dL) STANDARD (mL) PER TUBE
M1V1 : M2V2 H2O = 2.0 ml -
1 3.0 (12)V1: 3.0 (2.0 ) 0.5 mL 2.0
V1 : 0.5 mL = 1.5 mL
M1V1 : M2V2 H2O = 2.0 ml –
2 6.0 (12)V1: 6.0 (2.0 ) 1.0 mL 2.0
V1 : 1.0 mL = 1.0 mL
M1V1 : M2V2 H2O = 2.0 ml –
3 9.0 (12)V1: 9.0 (2.0 ) 1.5 mL 2.0
V1 : 1.5 mL = 0.5 mL
M1V1 : M2V2 H2O = 2.0 ml –
4 12.0 (12)V1: 12.0 (2.0 ) 2.0 mL 2.0
V1 : 2.0 mL = 0 mL

BLANK - - 2.0 mL 2.0

c. Then, add 5 ml ortho-tolidine mixture where consisting;

i. 1% ortho-tolidine
ii. 4mg (100 units) peroxidase
iii. 12.5 mg (500 units) glucose oxidase in 100 ml phosphate buffer, pH 7.0.

MINUTE ADDED
TUBES ORTHO-TOLIDINE mL TOTAL mL PER TUBE
ORTHO-TOLIDINE

BLANK 2’ 5.0 7.0

1 4’ 5.0 7.0
2 6’ 5.0 7.0
3 8’ 5.0 7.0
4 10’ 5.0 7.0
SAMPLE 12’ 5.0 7.0

iv. Mix quickly.

v. The time is staggered every 2 minutes when adding the ortho-tolidine solution
as shown table above.

vi. Incubate the tubes at room temperature for exactly 10 min.

vii. Then, measure at absorbance 625nm.
Notes:
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 • Ensure that each tubes absorbance is read exactly after 10 min.
• If the absorbance reading for the sample is too high, dilute the
supernatant which was obtained earlier, (2x) with water and repeat the
subsequent steps.

RESULTS:
GLUCOSE CONCENTRATION (mg/dL) ABSORBANCE READING (nm)
0 0.000
3.0 0.181
6.0 0.120
9.0 0.006
12.0 0.005
SAMPLE 0.045

DISCUSSION:
On this experiment, blood glucose is estimated by using enzymes glucose oxidase.
Where Ortho-toluidine is use as chemical for exploited to quantitative carbohydrates molecules to form
Schiff bases with aromatic amines. Ortho-tolidine in a hot acidic solution will yield a colored compound
with absorbance maxima at 625 nm.

• GLUCOSE OXIDASE

Glucose + O2 + H2O Glucose Oxidase

Gluconic Acid + H2O2

H2O2 + Reduced chromogen Peroxidase

Oxidized chromogen + H2O

Glucose oxidase is the most specific enzyme reacting with only β–D-glucose and glucose
oxidase converts β–D-glucose to gluconic acid. Added Mutarose to the reaction can facilitate the
conversion of α– D-glucose to β–D-Glucose. Oxygen is consumed and hydrogen peroxide is
produced. The reaction is measured based on rate of disappearance of oxygen. Spectrophotometer is
used for read absorbance, where it proportional to the amount of glucose presents in the sample.
However, on this experiment, the absorbance reading for concentration reading is not expected
as well and it fully incorrect. Because the absorbance reading supposedly from the lower thru high
value. But, on another hand for comparison with other group shown that the results reading are
correct and accordingly as table below.

GLUCOSE CONCENTRATION (mg/dL) ABSORBANCE READING (nm)

0 0.000
3.0 0.056
6.0 0.160
9.0 0.225
12.0 0.225

The problems that can be come across are shown in table below;
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 CAUSES DESCRPTION
1. Pipetting sample or chemical solution
inappropriate.
HUMAN ERROR 2. Taken wrong sample reading.
3. Unclean Cuvettes.
4. Mislabeling or not label sample.
1. Chemical are already expired.
MATERIAL
2. Improper making the chemical solution.
1. Increase levels of uric acid, bilirubin, and
ascorbic acid can cause falsely decreased
SUBSTANCES values as a result of these substances’ being
oxidized by peroxidase, which prevents the
oxidation and detection of the chromogen.
1. Galactose, an aldohexose, and mannose, an
aldopentose, will also react with Ortho-
CHEMICAL
toluidine and produce a colored compound
that can interfere with the reaction.

CALCULATION:
• Calculation concentration of blood glucose is not valid because the absorbance reading is to low to plot at the
graph.
• But, as for example, the calculation shown;
o 8.8 mg/dL (From plot graph) X 20 (times dilution)
= 176 mg/dl
• If converted to unit mmol/L
o 176 mg / dL X 1 dL/100 mL X 1000 mL/ L X 1 mmol/180 mg (gmw glucose :180)
= 9.7 mmol/L

QUESTION:
1.What are the advantages of glucose oxidase method in comparison to various other methods of
glucose analysis?
ADVANTAGES OF GLUCOSE OXIDASE
1. This method most specific enzymatic than other method such as; reduction of cupric to
cuprous salts, and reduction of ferricyanide to ferrocyanide method.
2. The reaction can be monitored polarographically either by measuring the rate of
disappearance of oxygen.

2.Discuss the principles of the enzymatic assay method.

PRINCIPLES OF THE ENZYMATIC ASSAY

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 1. Glucose oxidase reacts specifically to β–D-glucose, where it converts to gluconic acid.
2. The hydrogen peroxide is used to oxidize a dye compound.
3. Two commonly used chromogens are 3-methyl-2-benzothiazolinone hydrazone and N,N-
dimethylaniline.
4. The principle reaction as shown below;
Glucose + O2 + H2O Glucose Oxidase
Gluconic Acid + H2O2

H2O2 + Reduced chromogen Peroxidase

Oxidized chromogen + H2O

CONCLUSION:
1. The blood glucose level is not applicable on this test because value is too small.
2. However, on the experiment the glucose oxidase method is more specific test for determination the
glucose than other method.
3. Glucose test is useful in monitoring of hyperglycemia or hypoglycemia thru patient.