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Title: Glucose Tolerance Test

Objectives:
To determine the glucose in the urine by using the benedict solution.
To determine the blood glucose by using the glucose oxidase reagent.
Materials and Methods: Refer to the lab manual.
Result:
Part A: Determination of glucose in Urine
Type of Sample Observation
Fasting Urine Sample The solution remain blue in color
2 Hours After Glucose Intake Sample Trace amount of brick red precipitate is
deposited at the bottom of test tube.
Table 1: The following table recorded the observation of the urine color after
conducting the Benedict test.
Part B: Determination of Blood Glucose
Sample Absorbance (A) Concentration of
Glucose (mmol/L)
Concentration of
Glucose (mg/dL)
Standard
Solution
1.023 28 504.00
T
0
0.262 7.17 200.76
T
1
0.520 14.23 256.14
2

T
2
0.526 14.40 259.20
T
3
0.586 16.04 288.72
Table 2: The following table showed that the absorbance reading in different
tubes and their corresponding glucose concentration.
Calculation:
A) Calculate the Concentration of Glucose in T
0
to T
3
Given:
Absorbance of T
0
= 0.262A; Absorbance of Standard solution = 1.023A;
Concentration of Glucose Standard = 28mM/L
Conc. of Glucose =


x Conc. of Glucose Standard
=

x 28mmol/L
= 7.17mmol/L
ANS: Therefore, the concentration of glucose in Tube 0 is 7.17mmol/L.
To calculate the remaining glucose concentration in T
1
to T
3
, we can apply the
same method which shown above to obtain their respective values. The values of
concentration of glucose from T
1
to T
3
are 14.23mmol/L, 14.40mmol/L, and
16.04mmol/L respectively.
B) Conversion the Unit of Glucose Concentration in mmol/L into mg/dL
Molecular mass of glucose, C
6
H
12
O
6
= (6x12) + (12x1) + (6x16) =180
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The molecular weight of the glucose which is 180g/mol. 180g/mol is also equal to
180mg/mmol. Besides, 1 litre is equal to 10 decilitre.
1 mmol/l = 1 x 180 mg/l
= 180 mg/l
= 180 / 10 mg/dL
= 18 mg/dL (Glucose Concentration)
To convert 28mmol/L of glucose standard into mg/dL, we can use the comparison
method to solve it which shows as follows:
1mmol/L: 18mg/dL = 28mmol/L: Xmg/dL
X = 18 x 28
= 504mg/dL
ANS: Therefore, 28mmol/L of glucose standard can be converted into 504mg/dL.
To convert the rest of concentration unit from mmol/L to mg/dL in T
0
to T
1
, we
can apply the same method. Hence, the final values are 200.76mg/dL,
256.14mg/dL, 259.20mg/dL, and 288.72mg/dL respectively.
Discussion:
For the glucose urine test, we applied the Benedicts reagent to determine
the existence of glucose in the urine. Benedict's solution is a deep-blue alkaline
solution used to test for the presence of the aldehyde functional group in a
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solution. According to the principle of Benedicts solution, we can trace of the
concentration of glucose present in the urine by observe the colour change from
green to red .In other word, colour of precipitate proportional to amount of
glucose presence. From the result of the glucose urine test, there is a trace amount
of brick red precipitate at the bottom of test tube after applying Benedicts
solution. This phenomenon of presence precipitate indicating that glucose is
excreted in the urine and high probability of the person suffering from diabetes
mellitus (David C. Dugdale, 2011). Basically, from healthy individual, the
glucose will not appear in the urine because all the glucose which being excreted
into the renal tubule, eventually will reabsorb back to the vasa recta in order to
prevent the loss of glucose. In contrast, the glucose levels in diabetic patient
which go beyond the renal threshold usually 10mmol/L, the kidney will not be
able to absorb more glucose into the blood and thus excreted it into the urine.
For healthy individual, the fasting glucose concentration in the venous
plasma is below 6.1mmol/L (). The result from the patient serum sample showed
that the fasting glucose concentration is 7.17mmol/L while the 2 hours post
glucose concentration is 14.23mmol/L. The information provided from serum
glucose testing, we can determine that the patient is suffering from diabetes
mellitus. This is because his or her fasting glucose concentration is fallen outside
the normal range which is between 6.1mmol/L and 7.0mmol/L while the 2 hours
post glucose concentration is above 11.1mmol/L. According to the glucose
tolerance graph, we can observe that from 0 minute to 60 minute, both diabetes
mellitus and normal subject curve for the glucose concentration rises sharply (but
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the normal curve always below the diabetes mellitus curve). From 60 minutes
until 120 minutes, the glucose concentration in the patient increase gradually until
16.04mmol/L. This indicated that the excess glucose is not being reabsorbed from
the kidney where it is either lack of insulin due to breakdown of islet cells in the
pancreas or due to insulin resistance. The presence of insulin is to bring down the
high glucose levels in the blood after having a meal to prevent the occurrence of
hyperglycemia.
Glucose oxidase reagent is the one of the useful technique in laboratory to
determine the blood glucose levels. Glucose oxidase is an enzyme highly specific
for glucose and is not react with blood saccharides. Glucose is oxidized to
gluconic acid by the enzyme glucose oxidase (GO) in the presence of molecular
oxygen and the hydrogen peroxide is the final product of the reaction. The
hydrogen peroxide will directly react with p-Hydroxybenzene Sulfonate and 4-
aminoantipyrine in the presence of the enzyme peroxidase to form a pink colored
Quinoneimine, which is a chromogen (Roon, 1986).
The term diabetes is a chronic disease, which occurs when the pancreas
does not produce enough insulin, or when the body cannot effectively use the
insulin it produces. This leads to an increased concentration of glucose in the
blood (hyperglycaemia). Type 1 diabetes is characterized by a lack of insulin
production. Type 2 diabetes is caused by the bodys ineffective use of insulin. It
often results from excess body weight and physical inactivity. Diabetes mellitus
may present with characteristic symptoms such as thirst, polyuria, blurring of
vision and weight loss. In its more severe forms, ketoacidosis or a non-ketotic
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hyperosmolar state may develop and lead to stupor, coma, and in the absence of
treatment, death (World Health Organization, 1999).
The precaution steps are the micropipette tips must change frequently after
drawing the different types of solution to prevent any contamination to occur.
Since the GO solution is sensitive to the light, the direct exposure of the GO
solution to the broad light will alter their chemical property. After adding the
glucose oxidase (GO) solution with the patients serum in the test tube, we must
use the aluminum foil to wrap around the whole test tube. Otherwise, at the end of
the experiment, the desire result will not be obtained.
Conclusion:
At last, the glucose urine test for the patient reveals that there is a trace
amount of glucose present in the urine. This may be due to the systemic tissue
resistance or the malfunction of the beta cells which secrete low amount of insulin.
The patient serum fasting glucose concentration is 7.17mmol/L while the 2 hours
post glucose concentration is 14.23mmol/L. From the information of glucose
concentration provided, we can conclude that the patient is suffering from
diabetes mellitus. Diabetes mellitus is that the patient either lack of insulin due to
breakdown of islet cells in the pancreas (type 1) or due to insulin resistance (type
2).
Question:
1. Comment on the concentration of glucose present in the urine sample (A)
and calculate blood glucose in the plasma samples (B).
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From the result of the glucose urine test, there is a trace amount of brick
red precipitate at the bottom of test tube after applying Benedicts solution
and this indicated that glucose is excreted in the urine and high probability
of the person suffering from diabetes mellitus. Concentration of blood
glucose in the plasma samples (B) refers to result part A.

2. For the blood (plasma) samples, express your results in mmol/L and plot a
graph (in mmmol/L) of concentration vs time. Comment on your results.
By referring to the graph attached, we can determine that the patient is
suffering from diabetes mellitus. This is because his or her fasting glucose
concentration is fallen outside the normal range which is between
6.1mmol/L and 7.0mmol/L while the 2 hours post glucose concentration is
above 11.1mmol/L.
3. Sketch the GTT profile you would expect of a non-diabetic patient.
Compare your profile with the graph above.
By referring to the graph attached, the non-diabetic patient curve always
below the diabetes mellitus curve. This indicated that the excess glucose is
not being reabsorbed from the kidney where it is either lack of insulin due
to breakdown of islet cells in the pancreas or due to insulin resistance in
the diabetic patient. This causing the glucose level increase gradually until
it reached 16.04mmol/L.

4. Explain the terms of glycaemic index and glycaemic load
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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 (University of Sydney, 2011). The
glycemic load (GL) is a relatively new way to assess the impact of
carbohydrate consumption that takes the glycemic index into account, but
gives a fuller picture than does glycemic index alone. A GI value tells you
only how rapidly a particular carbohydrate turns into sugar.
References:
1) David C. Dugdale. (2011). Glucose test urine [Online]. Available at:
http://www.nlm.nih.gov/medlineplus/ency/article/003581.htm
2) Roon, R. J. (1986). Medical Biochemistry: Principles and Experiments.
University of Minnesota.
3) World Health Organization. (2006). Definition and Diagnosis of Diabetes
Mellitus and Intermediate Hyperglycemia. [Online]. Available at:
http://www.who.int/diabetes/publications/Definition%20and%20diagnosis
%20of%20diabetes_new.pdf
4) The University of Sydney. (2011). About Glycemic Index. Retrieve from
http://www.glycemicindex.com/about.php
5) Glycemic Research Institute. (2010). Glycemic Load Defined. Retrieve
from http://www.glycemic.com/GlycemicIndex-LoadDefined.htm