EXPERIMENT 6

6.1 TITLE: Glucose Tolerance Test

6.2 OBJECTIVES: 1. To be able to detect the presence of glucose in urine sample by using biochemical

test

2. To determine the concentration of blood glucose in the given sample

6.3 MATERIALS AND METHODS: Refer to the lab manual (page 14-15)

6.4 RESULTS:

(a) Determination of Glucose in Urine

Tube (hour) Observation

0 The blue colour of Benedict’s reagent did not
change

2 The blue colour of Benedict’s reagent changed to

brick red with precipitate

Table 1: The observation after the incubation of sample with Benedict’s reagent in boiling water bath

(b) Determination of Blood Glucose

Tube Absorbance (A)

Blank 0.041
Standard 1.121
T0 0.357
T1 0.423
T2 0.522
T3 0.546
Table 2: Absorbance (A505) for blood (serum) samples reacted with GO reagent for 20 min
6.5 CALCULATIONS:

(a) Comment on the concentration of glucose present in the urine sample and calculate the blood
glucose in the (serum) sample.

The urine sample contains glucose which is observed by the colour change of the Benedict’s reagent.
However, the concentration of glucose in urine through this method cannot be identified as the method
does not apply any quantification measurements.

Based on the absorbance values, the concentration of glucose present in the blood sample from 0 hour to
3 hours shows that the values are below the concentration of the standard. The standard used has a very
high glucose concentration.

Absorbance of sample
Concentration = × Standard
Absorbance of Sta ndard

The standard glucose concentration is 28 mmol/l. Therefore, the concentration of glucose:

T0 = (0.357 / 1.121)28 = 8.917 mmol/l

T1 = (0.423 / 1.121)28 = 10.566 mmol/l

T2 = (0.522 / 1.121)28 = 13.038 mmol/l

T3 = (0.546 / 1.121)28 = 13.638 mmol/l

(b) For the blood serum sample, express the results in mg/dl as well as mmol.l and plot a graph of
concentration vs time. Comment on the results.

Glucose concentration in mmol/l

Glucose concentration in mmol/l is calculated with the formula below:

Absorbance of sample
Concentration = × Standard
Absorbance of Sta ndard

(The calculation has been done for the previous question)

Glucose concentration in mg/dl

To convert the glucose concentration in mmol/l to mg/dl, the values in mmol/l must be multiplied with
18. Glucose concentration of:

Standard = 28mmol/l x 18 = 504 mg/dl

T0 = 8.917 mmol/l x 18 = 160.5 mg/dl

T1 = 10.566 mmol/l x 18 = 190.2 mg/dl

T2 = 13.038 mmol/l x 18 = 234.7 mg/dl

T3 = 13.638 mmol/l x 18 = 245.5 mg.dl

Test tube Glucose concentration (mg/dl) Glucose concentration (mmol/l)

Standard 504 28
T0 160.5 8.917
T1 190.2 10.566
T2 234.7 13.038
T3 245.5 13.638
Table 3: Calculated glucose concentration for the blood (serum) samples.

Based from the graph, the glucose concentration in plasma increases from 0 hour to 3 hours. The
steepness of the graph from 0 hour to 2 hours shows that the glucose is being released in the plasma. The
steepness of the graph declined after 2 hours to reach 3 hours. This indicates that the glucose introduction
into the blood has decreased.

(c ) GTT profile:
 The GTT profile of a non-diabetic shows a bell shaped graph. The glucose concentration
increases in the first 2 hours and then decreases rapidly when it reaches 3 hours. The rapid decline in the
glucose concentration in blood plasma is due to the homeostasis effect by the body in which the body
produces insulin normally to regulate blood glucose concentration in the body by converting the excess
blood glucose into glycogen and restore the normal plasma glucose level. Compared to the experimental
graph, the concentration of glucose in plasma did not rapidly fall when reaching 3 hours. This may be due
to the failure of the pancreatic cells to produce enough insulin for glucose conversion.

(d) i. Glycemic index

The Glycemic Index (GI) is a numerical scale used to indicate how fast and how high a particular food
can raise our blood glucose (blood sugar) level. A food with a low GI will typically prompt a moderate
rise in blood glucose, while a food with a high GI may cause our blood glucose level to increase above
the optimal level.

ii. Glycemic Load

The glycemic load (GL) is a relatively new way to measure how the intake of carbohydrates affects blood
sugar levels. It is often used in tandem with glycemic index (GI), which helps determine how quickly
food is digested and absorbed into your system. The quicker it is absorbed, the faster blood sugar levels
rise. The glycemic load can also tell the content of carbohydrates of a particular food. The glycemic load
considers the serving size of the food and calculates the number of carbohydrates in that particular
serving of food. As a result, a more accurate means of predicting how the carbohydrates of a given food
will affect the blood sugar can be obtained.
6.6 DISCUSSION:

Two tests were done in this experiment. The first test which uses Benedict’s reagent is a test to
determine the presence of glucose in the urine sample. The change of colour from blue to brick red with
precipitate indicates that the urine sample contains glucose. The production of brick red colour with
precipitate is due to the chemical reaction between glucose and Benedict’s reagent. Benedict's reagent is a
solution of copper sulfate, sodium hydroxide, and tartaric acid. Aqueous glucose is mixed with Benedict's
reagent and heated. The reaction reduces the blue copper (II) ion to form a brick red precipitate of copper
(I) oxide. Because of this, glucose is classified as a reducing sugar. This method can’t be used to calculate
the concentration of glucose in the sample as it is only based on observation alone without the aid of any
measuring machines.

The second test uses blood plasma in which the detection and the measurement of plasma blood
glucose was done. The blood sample was taken from a patient that has fasted overnight after oral glucose
dosage intake. The fasting state eliminates the presence of glucose from other sources such as from the
diet that will leave out only the presence of glucose from the oral dose to be measured by using clinical
tests. The blood serum from the blood drawn at 0, 1, 2 and 3 hours were added with Glucose Oxidase
reagent to catalyze the oxidation of glucose to hydrogen peroxide and Gluconic acid.

D-Glucose + H2O + O2  H2O2 + D-Gluconate

The hydrogen peroxide will then reacts with oxygen acceptor such as ortho-dianisidine,
phenylalanine-phenazone or other chromogenic oxygen acceptors to form a colour. The reaction is
catalyzed by peroxidases. This enzymatic method provides a specific estimation of glucose even in a
complex mixture. The resulting color is measured for the absorbance by using spectrophotometer for the
determination of serum glucose concentration.

A standard was use as the control for the experiment. The concentration of glucose in the
standard in this experiment is very high, in which it exceeds the normal range. Normal blood glucose
level in a healthy individual should range from 3.6 to 5.8 mmol/l. The result shows that the blood glucose
concentration value is higher than the normal range. This indicates that the patient is suffering from
Diabetes Mellitus. The graph curve shape indicates that the patient may suffer from Diabetes Mellitus
Type 2. However the type of diabetes could not be confirmed as there was no observation of any decline
in the blood glucose concentration from the graph. Based from the graph, the detection of blood glucose
in blood after 2 hours is lower than that detected after 0 and 1 hour. The difference might be due to the
consumption of oral glucose dose that has been totally broken down. It is also because the patient did not
uptake any new glucose dosage into their system.
 Theoretically, a person who has type 1 Diabetes Mellitus would have the blood glucose
concentration under 9mmol/l for adults and under 10mmol/l for children after the oral dosage whereas a
person who has type 2 Diabetes Mellitus would normally have blood glucose concentration below
8.5mmol/l after the consumption of the oral glucose dosage. However, the blood glucose level obtained
from the experiment shows values greater than 9mmol/l. The abnormally high level of serum glucose
might be due to the sample being used.

High glucose levels in blood impose many health risks for individuals who suffer from Diabetes.
The glucose that is introduced into the body could not be broken down efficiently for energy or storage.
Thus, the glucose would have to be excreted out from the body through urine which explains the presence
of glucose in the urine for the first test. Since glucose can’t be broken down for energy, the cells within
the body would use protein, nucleic acids and fats to be broken down and release energy. Fats break down
would produce ketone bodies. Accumulation of ketone bodies will lower the pH of the blood and causes
Diabetic Ketoacidosis. Imbalance in pH regulation can cause dysregulation of many biochemical
processes within the body.

6.7 CONCLUSION:

Glucose tolerance test can be used to identify the body function mechanism towards introduced glucose.
The test is also reliable as it is done after a fasting state as well as because it uses Glucose oxidase
enzyme which acts specifically in oxidizing the glucose to enable glucose concentration quantification in
the serum sample. The obtained results which shows glucose concentration in T 0, T1, T2 and T3 in mmol/l
is 8.917, 10.566, 13.038 and 13.638 respectively. The results shows that the patient has very high blood
glucose value that is greater than 7.0mmol/l which suggests that the patient has diabetes mellitus.
6.8 REFERENCES:

1. Ellis Toussier (1999). Assess The State of Health of Your Pancreas. “How To Estimate How Far You
Have Already Travelled on the Road to Diabetes.” Retrieved March 1, 2011 from
http://www.rajeun.net/assesspancreas.html

2. The George Mateljan Foundation (2011). “What is the Glycemic Index?” Retrieved March 1, 2011
from http://www.whfoods.com/genpage.php?tname=faq&dbid=32

3. J.-M. Ekoé, Marian Rewers, Rhys Williams. “The epidemiology of diabetes mellitus” (2008).
Diagnosis and classification (page 11-15). Wiley: New York

4. William E. Winter, Maria Rita Signorino. Diabetes mellitus: pathophysiology, etiologies,

complications, management and laboratory evaluation (2002). American association for clinical
chemistry.
 UNIVERSITY TUNKU ABDUL RAHMAN

FACULTY OF SCIENCE

Subject code : UDEE 3144

Subject title : CLINICAL BIOCHEMISTRY

Name : Kalai Selvi a/p Ganesan

Student ID : 08 ANB 05006

Year/Trimester : Year 3/ Trimester 2

Experiment Title : Glucose Tolerance Test

Date of Submission : 3rd March 2011

Instructor : Ms Tan Ming Hooi

Group members : Kaviyarasi a/p Kannimuthu Sudhashini John

Saritha a/p Manikan Katherine Usha a/p Goval (Group 1)

DECLARATION

I, KALAI SELVI A/P GANESAN hereby declare that this assignment is our original work and all

materials used from other sources have been properly acknowledged.

Signature,

______________