ESTIMATION OF BLOOD SUGAR (GLUCOSE)BY ORTHO-TOLUIDINE METHOD

ESTIMATION OF BLOOD SUGAR (GLUCOSE)BY ORTHO-TOLUIDINE METHOD

AIM:

To estimate the amount of blood sugar by ortho toluidine method.

CLINICAL SIGNIFICANCE:

The requirement for serum glucose level analysis is made incase of suspected diabetes mellitus (DM) .Increased serum glucose level is hyperglycemia with glycosurea is a diagnosis for diabetes mellitus. In this case of glucose tolerance test (GTT) and increased level of glucose is also noted with increased endocrine activity. A decreased level of serum glucose in hypoglycemia is often associated with starvation and hyperinsulinism, and decreased endocrine activity.

Several methods have been for glucose determination such as:

1. Benedict's quantitative method.
   
2. Folin.wu method
   
3. O-toluidine method
   
4. Glucose oxidize method
   
5. Alkaline copper reduction method
   

Among o-toluidine method is more convenient for its simplicity and closure to the true glucose level than the other method. This method dose requires protein free filtrate, which make it easy to perform. True glucose is determined only by enzymatic method. A normal range by o-toluidine is eventually with the same as that of glucose oxidizes method.

PRINCIPLE:

The aldehyde group of glucose condenses with o-toluidine in glacial acetic acid (aldehyde free colorless). On heating, giving an embral green color which is measured calorimetrically or photo metrically at 630nm. The intensity of color is proportion to the glucose concentration. The presence of thiourea stabilizes the o-toluidine reaction. This method is discouraged because of the carcinogenic effect of toluidine. It is a still popular because of its simplicity, accuracy and rapidity.

 

CH₃

 

 

O-Toluidine Glycosylamine Schiff's Base

PRECAUTION:

The chemical o-tlouidine must be handled carefully, should not touch the skin and acetic acid fumes should not be inhaled.

NORMAL VALUE OF SERUM GLUCOSE:

Normal value of serum glucose is determined as reduced substance equal to 70-110mg/dl or 80-120mg/dl.

REAGENTS:

STOCKS STANDARD SOLUTION:

Dissolve 1gm of glucose in 100ml of distilled water(1gm/100ml)

GLUCOSE WORKING STANDARD SOLUTION (100MG%):

Dilute 10ml of glucose stock standard solution to 100ml of distilled water. This solution gives100mg% of glucose.

O-TOLUIDINE REAGENT:

It contains:

• O-toluidine solution - 60ml.
  
• Thiourea - 1.5gm
  
• glacial acetic acid(aldehyde free) 1000ml
  
• Thiourea stabilizes the o-toluidine reagent.
  
• 3% TCA
  
• 3gm of trichloro acetic acid in 100ml of distilled water
  

 

PROCEDURE:

Take 3 test tubes and marked them as blank(B),standard(S),test(T).To each of 3 test tube add 10ml of o-toluidine reagent. Add 0.1ml of working standard solution which is marked as (S).To the another test tube marked as (T), add 0.1ml of serum test solution and add 0.1ml distilled water which is marked as(B).Put all the test tubes in boiling water bath. Take care to ensure that the water level in boiling water bath should cover the contents of the test tubes for heating. Boil the test tubes exactly for 5-8minutes then remove the test tubes and cool to room temperature, then determine optical density at 630nmor by using red filter.

PROCEDURE FOR BLOOD:

To 1.9ml of 3%TCA in a centrifuge tube, add 0.1ml of blood, mix well and centrifuge for 10minutes .Pipette 1ml of clear supernatant liquid into a test tube. Add 5ml of o-toluidine to the above solution and then determine the optical density value.

RESULT:

The amount of glucose presents in the given sample and blood was tested and found to be:

1. Given sample(T₁)=
   
2. Blood sample(T₂)=
   

 

CALCULATION:

1. Milligram percentage of glucose in the given test sample= optical density of test x concentration of standard Optical density of standard

2. Milligram percentage of glucose in blood=

Optical density of blood x concentration of standard

Optical density of standard

SNO	Reagent	Blank	Standard	Test	Blood
1	o-toluidine	10ml	10ml	10ml	10ml
2	Working standard solution	-	0.2ml	-	-
3	Distilled water	0.2ml	-	-	-
4	Serum or test solution	-	-	0.2ml	-
5	Supernatant of blood	-	-	-	0.2ml

 

 

SNO	Test tubes	Optical density(OD) values
1	Blank
2	Standard
3	Test
4	Blood sample