Lab 1: ESTIMATION OF GLUSOCE LEVEL

Lab 1: ESTIMATION OF GLUSOCE LEVEL

Method: Indirect Ortho-Toluidine.

Objective: To determine the level of glucose in blood.

Introduction:

Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate. Glucose is one of the main products of photosynthesis and starts cellular respiration.

            The blood glucose test is ordered to measure the amount of glucose in the blood right at the time of sample collection. It is used to detect both hyperglycemia and hypoglycemia, to help diagnose diabetes, and to monitor glucose levels in persons with diabetes.

Blood glucose may be measured on a fasting basis (collected after an 8 to 10 hour fast), randomly (anytime), post prandial (after a meal), and/or as part of an oral glucose tolerance test (OGTT / GTT). An OGTT is a series of blood glucose tests.

Principle:

Protein in blood is deposited by TCA and separated by centrifuge. Glucose supernatant reacted towards O-Toluidine, when heated, produced a blue-green colour which is measured by 630nm spectrophotometer. This reaction involves condensation of glucose with a primary aromatic acid in glacial acid. The blue-green chromogen formation is a stable reaction of glucosamine and a Schiff Base.

Material:

1.      Test tube

2.      Pipette

3.      Centrifuge

4.      Spectrophotometer

Reagents:

·         Standard glucose solution 10g/L

·         Working glucose solution 200mg/L

·         9% Tricholoroacetic acid (TCA) solution

·         O-Toluidine reagent

Procedure:

Protein deposition phase

1.      3 test tube with label each it as BLANK, STANDARD, and TEST were prepared.

2.      As  the table below:

	BLANK	STANDARD	TEST
Distilled water (ml)	1.1	1.0	1.0
Working standard glucose solution 10mg/dL	-	0.1	-
Specimen	-	-	0.1
9% TCA solution	0.5	0.5	0.5

3.      All solution is mixed well and leaves it for 5 minutes.

4.      Leave it for 5 minutes, and then centrifuge it at 3500rpm for 5 minutes.

Reaction phase

1.      3 test tube with label each it as BLANK, STANDARD, and TEST were prepared.

2.      After that follow the table below:

	BLANK	STANDARD	TEST
Supernatant from Blank	0.5	-	-
Supernatant from Standard	-	0.5	-
Supernatant from Test	-	-	0.5
O-Toluidine reagent	3.0	3.0	3.0

3.      Covered all the test tube with cotton. Then mix all the solution well and put all the test tube into a boiling bath for 12 minutes.

4.      After that, cool the test tube under running water for 5 minutes. Then mix again gently. Finally read the absorbance at 630nm spectrophotometer. Start at Zero with Blank.

 Result:

Reading	Test	Standard
1	0.437	0.168
2	0.439	0.171
3	0.439	0.175
Average	0.438	0.171

The figure shows that TEST tube shows blue green colour, the STANDARD tube show light blue and the BLANK tube show colourless

Calculation:

Mg of glucose in 100ml blood

__Absorbance test__ x factor (16)

Absorbance standard

·         Absorbance test=0.438 nm

·         Absorbance standard=0.171 nm

0.438 nm x 16 = 40.982 mg/100ml

0.171 nm

*Reference: Fasting specimen: (FBS) 65-100mg /100mL

Discussion:

The purpose of the experiment is to determine the level of glucose in a patient’s blood. The result obtained is that the level of glucose is 40.98 mg/100ml. The solution in the test tube has shown a color change thus indicates the glucose is present in the patient’s blood. It is because the presence of heat and acetic acid makes the ortho-toluidine reacts rapidly with glucose to form a green-coloured.

Protein in blood is deposited by TCA and separated by centrifuge. Glucose supernatant reacted towards O-Toluidine, when heated, produced a blue-green colour which is measured by 630nm spectrophotometer. This reaction involves condensation of glucose with a primary aromatic acid in glacial acid. The blue-green chromogen formation is a stable reaction of glucosamine and a Schiff Base.

Based on the result, the level of the glucose is 40.98 mg/100ml. This show that the patient having a hypoglycemia. Hypoglycemia is a condition that occurs when your blood sugar (glucose) is too low. Blood sugar below 70 mg/100ml is considered low. Hypoglycemia occurs when body's sugar (glucose) is used up too quickly.

            We also must take precaution to prevent any accident occur in laboratory. We should wear glove and lab coat when handling a blood or other specimen. Furthermore, we must check the expiry date of chemical reagent frequently before proceeds any experiment. As student, we must be clean and neat attire. For example, we must washing hands after handling specimen.

Conclusion:

            At the end of the experiment, students are able to determine the level of glucose in the patient’s blood. The result is 40.98 mg/100ml. this shows the patient has hypoglycemia

Question:

1. Explain the function of :
1. Ortho-toludine: to give color to the glucose to become blue green.
2. Trichloroacetic acids(TCA): reagent to detect albumin.
2. Give 2 factors that causing diabetes mellitus?

Pancreases do not produce insulin because the beta cell are destroy or loss function and the fat cell does not response to insulin normally by certain factor.

3. Why do we need to add fluoride oxalate in the sample for glucose analysis?

It used to stabilized the enzyme system that involved in glycolysis.to stop the gltycolysis.

4. Name the other method that can be used to estimate blood glucose level?

Using the glucose meter to check the level of glucose in the blood.

5. Name the condition of high glucose?

Hyperglycemia.

References:

·         Arthur Schoenstadt, MD. (2008). What Is Diabetes. From: MedTV.com. website: http://diabetes.emedtv.com/diabetes/diabetes.html

·         Cara L. Thompson. (2002). Hyperglycemia in the Hospital. From: Diabetes Spectrum. Website: http://spectrum.diabetesjournals.org/content/18/1/20.long

·         Vasudevan A Raghavan. (2010). Hypoglycemia. From: medscape reference. Website: http://emedicine.medscape.com/article/122122-overview