Estimation of Blood Glucose level

Mr. Arif Ullah Khan Niazi

Lecturer
Bannu College of Medical Technologies Bannu
 BLOOD GLUCOSE
There are three commonly used methods for glucose
estimation, which are;-
• Copper reduction method (Folin-Wu method)
• Other reduction method such as orthotoludine
method
• Enzymatic methods such as
o Hexokinase method
o Glucose oxidase method (Recommended)
o Glucose dehydrogenase method
Estimation of Blood Glucose Level
Glucose Oxidase Method (Enzymatic method)
Apparatus:
Spectrophotometer/Colorimeter
Adjustable micropipette
Blood collection kit
Ordinary test tubes
Centrifuge with test tubes.
Estimation of Blood Glucose Level
Chemical required: Blood sample, Glucose standard, Glucose
reagent & Anti-coagulant
1. Concentration of Glucose standard= 100 mg/dl
2. Composition of Glucose reagent:
I. Glucose oxidase---------------------15 KU/L
II. Mutarotase----------------------------2 KU/L
III. Hydrogen peroxidase-------------1.5 KU/L
IV. Phenol------------------------------0.75 mol/L
V. 4-aminophenazone--------------0.25 mol/L
VI. Phosphate buffer-------------------0.1 mol/L
Estimation of Blood Glucose Level
Principal:
The enzyme Glucose oxidase catalyzes the oxidation of glucose
to Gluconic acid with the formation of Hydrogen peroxidase.
The enzyme peroxidase breaks down hydrogen peroxidase into
water and nascent oxygen. The nascent oxygen reacts with the
colorless electron acceptor 4-aminophenazone which is
oxidized to colored compound. The reaction can be followed
photo metrically. The intensity of color produced is
proportional to the amount of glucose in the sample:
Glucose+H2O+O2-------------------------------Gluconic acid+H2O2
Glucose oxidase
 Estimation of Blood Glucose Level
Procedure:
 Take 20 micro liter of the sample in the adjustable micropipette. Pour it into a small
test tube labeled as “UNKNOWN”.
 Take 20 micro liter of the glucose standard in another small test tube labeled as
“STANDARD”.
 In another small test tube labeled as “BLANK” take 20 micro liters of distilled water.
 Now add 2 ml of the glucose reagent to all the three test tubes. Wait for at least 10
minutes so that the reaction is complete at room temperature.
 Now switch on the colorimeter or spectrophotometer and do its blanking by first
placing the “BLANK” in the path of the light.
 Use the filter/monochromator of special wavelength. The routinely used is of 546
nm. Optical length of cuvette=1 cm. Temperature =20-25 or 37 degree centigrade.
Note the transmittance or the absorbance of the unknown and that of the
standard(known).
Estimation of Blood Glucose Level
Observation & Calculation:
Formula : C1/C2 = A1/A2
C1=A1/A2 × C2
Where C1= Concentration of Unknown/Test=?
C2= Concentration of Standard=1mg/ml.
A1= Color absorbance of Unknown/Test.
A2= Color absorbance of Standard.
Estimation of Blood Glucose Level

Observation & Calculation:

Formula: Concentration of T × 100

Concentration of S
Estimation of Blood Glucose Level
Normal Blood Glucose range:
Venous blood (Fasting)= 70-110 mg/dl
Venous blood (Random)= 70-180 mg/dl
CSF = 50-80 mg/dl
Urine= up to 25 mg/dl
THANK YOU