03- Estimation of Blood Glucose Using Glucose Oxidase

8/3/2019 03- Estimation of Blood Glucose Using Glucose Oxidase

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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.

TUBESCONCENTRATION

(mg/dL)

GLUCOSE

STANDARD (mL)WATER (mL)

TOTAL mL

PER TUBE

1 3.0

M1V1 : M2V2

(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

M1V1 : M2V2

(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

M1V1 : M2V2

(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

M1V1 : M2V2

(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 100 ml phosphate buffer, pH 7.0.

TUBESMINUTE ADDED

ORTHO-TOLIDINEORTHO-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:

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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 + H2OGlucose Oxidase Gluconic Acid + H2O2

H2O2 + Reduced chromogenPeroxidase Oxidized chromogen + H2O

Glucose oxidase is the most specific enzyme reacting with only D-glucose and glucose

oxidase converts D-glucose to gluconic acid. Added Mutarose to the reaction can facilitate the

conversion of D-glucose to 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 expectedas 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 0.22512.0 0.225

The problems that can be come across are shown in table below;

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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.

MATERIAL1. 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

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1. Glucose oxidase reacts specifically to 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 + H2OGlucose Oxidase Gluconic Acid + H2O2

H2O2 + Reduced chromogenPeroxidase 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.

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03- Estimation of Blood Glucose Using Glucose Oxidase

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