2.

BLANK & STANDARD:

a.Glucose standards 12 mg/dl are provided.

b.Preparing concentration glucose standard point.

TUBES
CONCENTRATION
(mg/dL)
GLUCOSE
STANDARD (mL)
WATER (mL)
TOTAL mL
PER TUBE
1
3.0
M 1V 1 : M 2V 2
(12)V1: 3.0 (2.0 )
V1 : 0.5 mL

H2O = 2.0 ml -

0.5 mL

= 1.5 mL
2.0
2
6.0
M 1V 1 : M 2V 2
(12)V1: 6.0 (2.0 )
V1 : 1.0 mL

H2O = 2.0 ml –

1.0 mL

= 1.0 mL
2.0
3
9.0
M 1V 1 : M 2V 2
(12)V1: 9.0 (2.0 )
V1 : 1.5 mL

H2O = 2.0 ml –

1.5 mL

= 0.5 mL
2.0
4
12.0
M 1V 1 : M 2V 2
(12)V1: 12.0 (2.0 )
V1 : 2.0 mL

H2O = 2.0 ml –

2.0 mL

= 0 mL
2.0
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 100ml phosphate buffer, pH 7.0.
TUBES
MINUTE ADDED
ORTHO-TOLIDINE
ORTHO-TOLIDINE mL
TOTAL mL PER TUBE
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:
2
•
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;
3
CAUSES
DESCRPTION
HUMAN ERROR
1.Pipetting sample or chemical solution
inappropriate.
2. Taken wrong sample reading.
3. Unclean Cuvettes.
4. Mislabeling or not label sample.
MATERIAL
1. Chemical are already expired.
2. Improper making the chemical solution.
SUBSTANCES
1.Increase levels of uric acid, bilirubin, and
 ascorbic acid can cause falsely decreased

values as a result of these substances’ being

oxidized by peroxidase, which prevents the

oxidation and detection of the chromogen.

CHEMICAL

1. Galactose, an aldohexose, and mannose, an

aldopentose, will also react with Ortho-

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
4

1.Glucoseoxidase 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.
5

Estimation of blood glucose using glucose

oxidase (Biomedical practical for
biochemistry II)
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RaSyidah GhaZallileft a comment

nur_fi91@yahoo.com
09 / 14 / 2010
mukundalleft a comment
Need more clinical test protocols. Very nice
09 / 17 / 2008