Estimation of Blood Glucose by Glucose Oxidase Method – Amrita University

Estimation of Blood Glucose by Glucose Oxidase Method – Amrita University

October 30, 2019 9 By William Morgan


ESTIMATION OF BLOOD GLUCOSE BY GLUCOSE OXIDASE
METHOD
Glucose is the major carbohydrate present
in blood and is produced from dietary sources.
The maintenance of blood glucose in our body
is mainly controlled by hormones insulin and
glucagon. A defect in insulin secretion, insulin
action, or both results initially an impaired
glucose tolerance and cause hyperglycemia
which further leads to diabetes mellitus.
So a simple, rapid and economical method of
determining the blood sugar level is obvious
especially in the management of diabetes mellitus.
The colorimetric method, combined with an
enzymatic reaction is most widely used for
the determination of glucose in human serum.
Estimation of glucose by the enzyme glucose
oxidase gives the true glucose concentration
due to its high specificity and sensitivity.
Glucose oxidase catalyzes the oxidation of
ß-D-glucose to D-glucono-d-lactone and hydrogen
peroxide. It is highly specific for ß-D-glucose
and does not act on a-D-glucose.
MATERIALS REQUIRED
Blood sample collected in an EDTA bottle,
0.05M Phosphate buffer,
Glucose oxidase reagent, 2N Sodium hydroxide,
Sodium Sulphate- Zinc sulphate reagent,
200mg of Standard glucose solution,
Distilled water,
Test tubes,
Micropipettes,
Eppondorf tube,
5ml glass pipette and pipette pump.
PROCEDURE
First transfer 900µl of sodium sulphate — zinc
sulphate solution to 1.5 ml Microfuge tube
which is labeled as ‘test’.
Likewise, transfer 50 µl of 2N NaOH and 50
µl of blood sample into the same Microfuge
tube.
Now centrifuge the sample at 3000rpm for 5
minutes.
After 5 minutes, take out the centrifuge tube
and transfer 500 µl of supernatant carefully
from the centrifuge tube to a test tube labeled
as “TEST”.
Next transfer 125µl of glucose standard solution
to a test tube labeled as 50mg/dl.
Similarly, transfer 250µl of glucose standard
to the second test tube labeled as 100mg/dl,
375µl of glucose standard solution to the
third test tube labeled as 150mg/dl, 500µl
of glucose standard solution to the fourth
test tube which is labeled as 200mg/dl.
Now insert a fresh tip to the micropipette
and transfer 500µl of distilled water to
a test tube labeled as ‘Blank’.
In the same way add 375µl of distilled water
to the first tube, 250µl to the second tube
and 125µl to the third tube.
Now connect the glass pipette to the pipette
pump and transfer 5 µl of glucose oxidase
reagent to all the test tubes.
Cover the test tubes with aluminum foil and
place them in the water bath at 37°c for
1 hour.
After 1 hour, take out the test tubes from
the water bath and transfer the blank solution
to the cuvette.
Insert the cuvette into the slot of the colorimeter
and set the value to zero.
Next transfer 50mg/dl solution in the first
test tube to the cuvette.
Insert the cuvette into the slot of the colorimeter
and note the reading.
After getting the reading, remove the cuvette
from the slot.
Similarly, note the reading of 100mg/dl, 150mg/dl,
200mg/dl solutions and test solution and plot
a graph by taking standard glucose concentration
on X axis and Corresponding absorbance on
Y axis.
From this standard graph, the amount of glucose
concentration in the test solution can be
obtained.
Applications:
Glucose oxidase has been used widely in the
determination of free glucose in body fluids,
food, agricultural products and baking industry.
In some applications it can be used to replace
oxidants, such as bromate and L-ascorbic acid.
Other uses of glucose oxidase include the
removal of oxygen from food packaging and
removal of D-glucose from egg white to prevent
browning.