MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF BIOMEDICAL SCIENCES
DEPARTMENT OF BIOCHEMISTRY

Title of the practical: AN ASSAY OF GLUCOSE BY GLUCOSE OXIDASE AND

THE GLUCOSE TOLERANCE CURVE.

Date of Practical: WEDNESDAY 13TH FEBRUARY, 2019

NAME PROG REG. NO. SIGN

MUDDE FRANCIS BDS 18/U/662
FAUZI IBRAHIM ALI BPHA 18/U/042

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ABSTRACT

The experiment was about testing the specificity of glucose oxidase enzyme and measurement
of glucose tolerance in medical and veterinary practice. The absorbances of standard glucose
solutions were measured by colorimetry with the aid of a color reagent (colourless in reduced
form) which is oxidised in presence of hydrogen peroxide (to coloured product) produced from
the oxidation of glucose by glucose oxidase enzyme. Solutions with highest glucose concentration
had a more intense colour after incubation. A standard curve was generated from the absorbance
of samples of standard glucose concentration-this was a straight-line graph with a positive
gradient passing through the origin hence obeying Beer-Lambert’s law. Standard solutions of
fructose and lactose gave very low absorbances showing that there is almost no oxidation of the
colour reagent-hence proving enzyme specificity of glucose oxidase enzyme. Absorbances of
deproteinized samples were measured by colorimetry and used to determine corresponding
glucose concentrations from the standard curve. These concentrations were used to plot a
glucose tolerance curve. This curve showed that the patient was diabetic with mild insulin
sensitivity.
INTRODUCTION

STATEMENT OF THE PROBLEM; To determine glucose tolerance of a diabetic patient.

PURPOSE: To diagnose diabetes mellitus.

OBJECTIVES;
1. To check the specificity of glucose oxidase
2. To prepare a calibration curve for glucose
3. To draw a glucose tolerance curve for the Patient
The ability of the body to utilize carbohydrates is measured in ‘tolerance’ tests. In the glucose
tolerance test the patient is fasted from food for 12 hours, and then a sample of blood and urine
is collected. The patient is then given a dose of glucose (0.75- 1.5g/kg body weight), usually
dissolved in water and taken by mouth. Specimens of blood and urine are collected at 30 to 60
minutes intervals over the next 3 to 4 hours.
The concentration of glucose in the blood is determined and expressed in mmoles/L of blood.
The urine samples are tested qualitatively for glucose. The concentration of blood glucose is
plotted against time, time zero is the time the glucose was taken, and the fasting blood glucose
concentration is the concentration at time zero.
This graph is the glucose tolerance curve. In the normal human, the blood glucose rises from a
fasting level of 4-5mmoles/L to about 7mmoles/L within 30 to 60 minutes but then falls to the
fasting level over the next 3 hours.

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In a diabetic the initial fasting glucose level is higher than normal, the increase in blood glucose
concentration is greater and falls much more slowly.
(This information was provided in the hand out)
The glucose tolerance test is used as a tool for diagnosis of Diabetes mellitus. The fasting blood
glucose level in the early morning is normally 80 to 90mg/100ml, and 110mg/100ml is
considered to be the upper limit of normal. A fasting blood glucose level above this value often
indicates diabetes mellitus or a least marked insulin resistance. In a person with diabetes, the
fasting blood glucose concentration is almost always above 110mg/100ml and often above
140mg/100ml and the glucose tolerance test is almost always abnormal. On ingestion of
glucose, these people exhibit a much greater than normal rise in blood glucose level.
Urinary glucose can also be used as a diagnostic test. A normal person loses undetectable
amounts of glucose, whereas a person with diabetes loses glucose in small to large amounts in
proportion to the severity of disease and the intake of carbohydrate.
(Guyton and Hall, 2006, page 975)

BACKGROUND
Glucose provides the energy for life processes. Insulin is the most important hormone that
regulates the blood glucose concentration by controlling the rate at which glucose is taken up
by tissues and the conversion of glucose to glycogen in liver and muscle cells. Only insulin is
capable of reducing concentration of glucose in blood.
(Monica Cheesebrough, 1984)
This experiment is aimed at simulating a glucose tolerance test which is a routine clinical test to
investigate metabolic syndromes. The glucose tolerance test is done by first obtaining a
calibration curve which denotes the respective absorbance readings of solutions with different
glucose concentrations. This calibration curve is then used to extrapolate the unknown
concentration of glucose contained in a sample using its absorbance reading. In the case of the
glucose assay test, the sample given is the patient’s de-proteinized blood serum containing the
patient’s blood glucose. Once the patient’s blood glucose is determined at different time
intervals, these values are plotted to give the glucose tolerance curve. This curve can be
compared to the normal glucose tolerance curve and a glucose tolerance curve of a diabetic
patient. This comparison of the patient’s glucose tolerance curve and the normal glucose
tolerance curve can give an investigative conclusion regarding the existence of a metabolic
syndrome.

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MATERIALS AND METHOD
Materials used
> P-T Phenol-tungstate > Burette
> Pipette > Pipette filler

> Test tubes > Test tubes racks

> Vortex Mixer > Sherwood colorimeter 254
> Water bath
> Color Reagent
The color reagent used in this experiment is a mixture including the following
1. 4 aminophenazone
2. Glucose oxidase
3. Peroxidase
4. Phosphate buffer
The glucose oxidase is present in order to catalyze the reaction between D-Glucose with oxygen.
This reaction gives D-Glucolactone and hydrogen peroxide. The hydrogen peroxide produced
oxidizes a dye under action of the enzyme peroxidase. This dye is originally colorless and is in the
deoxidized state in the reagent. The reaction of hydrogen peroxide and dye forms a colored
oxidized product. This product is produced in a directly proportional manner with the
concentration of glucose; therefore the measurement of the concentration of the colored
product is proportional to the concentration of glucose present in a given sample. Therefore the
absorbance of the solution (caused by the colored oxidized product) is proportional to the
glucose concentration of the solution.
D-Glucose + O₂ → D-Glucolactone + H₂O₂
(Monica Cheesebrough, 1984)
Method
Using the four standard glucose solution G1, G2, G3 and G4 and the standard solutions of
fructose and lactose provided, 0.2mls were pipetted into 7 test tubes and 1.0ml of the colour
reagent, phenol tungstate added as indicated in table 1.
The solutions were mixed using a vortex mixer and incubated at 370C in a water bath for 15
minutes. After cooling the test tubes in tap water for 10 minutes, the absorbance was
measured at 520nm (green filter) using a Sherwood colorimeter zeroing the colorimeter each
time with the blank.
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 Table 1

Blank S1 S2 S3 S4 F L

P. T 0.2 - - - - - -

G1 - 0.2 - - - - -

G2 - - 0.2 - - - -

G3 - - - 0.2 - - -

G4 - - - - 0.2 - -

Fructose(110ug/ml) - - - - - 0.2 -

Lactose(220ug/ml) - - - - - - 0.2

Colour reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Total µmoles glucose in 0.00 0.15 0.30 0.45 0.60 - -
tube

Total µmoles - - - - - - -
fructose/lactose in tube

Measurement of glucose tolerance

Before the test is done, the patient is first fasted from food for 12 hours, then a sample of
blood and urine is collected. This is to draw the base line or the fasting glucose concentration in
blood. Then the patient is administered with a dose of glucose (0.75 – 1.5g/kg of body weight)
orally. Several blood samples are then drawn at different time intervals.
Four de-proteinized and diluted blood samples prepared from a patient undergoing a glucose
tolerance test were provided. In this experiment, the samples were obtained at 0, 30, 60, and
120 minutes after the oral administration of glucose.
Six test tubes were set up as shown in Table 2.
The solutions were mixed using a vortex mixer and incubated for 15 minutes at 370C in the water
bath.
The tubes were then cooled in a beaker containing tap water for 10 minutes. The absorbance
was measured by a Sherwood colorimeter 254 using a 520nm filter (green filter).

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 Table 2

Blank S X1 X2 X3 X4

P-T 0.2 - - - - -
reagent

Standard - 0.2 - - - -
glucose G2

Deprot - - 0.2 - - -
sample X 1

X2 - - - 0.2 - -

X3 - - - - 0.2 -

X4 - - - - 0.2

Colour 1.0 1.0 1.0 1.0 1.0 1.0

reagent

RESULTS AND CALCULATIONS

Table 3
Solution Glucose concentration mg/L Glucose concentration µg/ml Glucose concentration
µmoles/mL

G1 27 27 0.15

G2 54 54 0.30

G3 81 81 0.45

G4 108 108 0.60

The absorbance of the standard glucose, Lactose and fructose solutions measured using the
green filter 520nm are shown in table 3 below;
Table 4

Blank S1 S2 S3 S4 F L

Total µmoles of 0.00 0.15 0.30 0.45 0.60 - -

glucose in tube

Absorbance 0.00 0.14 0.27 0.34 0.54 0.03 0.02

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The absorbance of the de-proteinized samples from the patient at 520nm (green filter) are shown
in table 4 below;
Table 5

Blank S X1 X2 X3 X4

Absorbance 0.00 0.27 0.16 0.24 0.22 0.19

µ moles of glucose(in 1.0ml sample) 0.000 0.300 0.180 0.270 0.245 0.210

1. Changing concentration of standard glucose from mg/L to µg/mL

1mg 1000 µg
And
1L 1000 ml
27×1000
Therefore 27mg/L
1000

= 27µg/mL
The same was done to change the concentrations of other standard glucose solutions from
mg/L to µg/ml
54×1000
- = 54 µg/mL
1000

81×1000
- = 81 µg/mL
1000

108×1000
- = 108 µg/mL
1000

2. Changing concentration of standard glucose from µg/mL to µmoles/mL

Molecular weight of glucose is 180
1g 106 µg
180g 180× 106 µg

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Therefore 180× 106 µg of glucose 1 mol
But 1 mol 106 µmoles
Therefore
27
27µg × 106
180×106

= 0.15 µmoles/ mL
The same calculation was repeated to change the concentrations of other standard glucose
solutions to µmoles/mL;
54
- × 106 = 0.30 µmoles/ mL
180×106

81
- × 106 = 0.45 µmoles/ mL
180×106

108
- × 106 = 0.60 µmoles/ mL
180×106

3. Calculating amount of glucose in m moles/L in the original 1.0 ml sample of blood.

1.0ml of blood was diluted to 40mls, hence 1.0 ml of the dilution contains 0.025mls of blood.
Using the concentrations of unknown samples X1 to X4 obtained from the standard curve (Table
4)
Tube S (standard glucose) contains 0.29 µmoles/ml
1000 µmoles 1 mmole
0.300
0.300µmoles mmoles
1000

But 1 ml of the blood was diluted to 40mls hence 1 ml of the dilution contains 0.025mls
0.300
Therefore the mmoles are contained in the 0.025mls
1000
0.300
0.025mls mmoles
1000
1000 0.300
1000mls ×
0.025 1000

= 12.0 mmoles/L

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The same calculation was repeated to obtain glucose concentrations in the unknown samples
X1 to X4
1000×0.180
Tube X1 - = 7.2 mmoles/L
0.025×1000

1000×0.270
Tube X2 - = 10.8 mmoles/L
0.025×1000

1000×0.245
Tube X3 - = 9.8 mmoles/L
0.025×1000

1000×0.210
Tube X4- = 8.4 mmoles/L
0.025×1000

Table 6

Time/min Blood glucose concentration mmoles/L

0 7,2

30 10.8

60 9.8

120 8.4

Answers to questions 2 and 3

Specificity of glucose oxidase to glucose, fructose, and Lactose
The absorbance of test tube S4 is 0.54Abs while the absorbance for tubes F and L (samples
containing fructose and lactose) were 0.03 and 0.02 respectively. Therefore there is almost no
oxidation of the coloring reagent in the test tubes containing lactose or fructose.
This shows that the glucose oxidase enzyme is specific to D-glucose and not any other types of
carbohydrate. This is because D-Glucose at pH7.0 exists in cyclic hemiacetal form as 63.6% β-D-
glucopyranose and 36.4% α-D-glucopyranose, and the equilibrium is favored to β-D-

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glucopyranose. Glucose oxidase is specific to only β-D-glucopyranose which only occurs in D-
Glucose. β-D-glucopyranose would not be found in any enantiomers or any other forms
carbohydrate such as fructose and lactose. Thus glucose oxidase only catalyzes the reaction of
D-Glucose and oxygen (more specifically β-D-glucopyranose and oxygen). This explains the
reason why lactose and fructose solutions yields almost no absorbance reading when reacted
with the colored reagent.
(Stefano Ferri, 2011)
Absorbance of S2 in table 1 and S in table 2
The absorbance reading of S2 in table 4 is 0.27 and S in table 5 is 0.27. These 2 values are the
same. Any significant difference in the two values may suggest a random error in conducting
the experiment. When these two values are different then the experiment should be
considered void. This is because the standard sample absorbance (in table 5) and the S2
absorbance (in table 4) have the same glucose concentration therefore the absorbance must be
the same. If the absorbance is different, this will lead to a misinterpretation of the amount of
glucose in the patient’s serum and thus lead to a misdiagnosis.

DISCUSSION
The graph 2 shows the patients’ blood glucose concentration plotted against time. The blood glucose
concentration of the patient and the blood glucose concentrations of a diabetic and normal person
(defined by the World Health Organization) are compared. This comparison allows a conclusion to be
made regarding patient’s diabetic status.

The normal levels of blood glucose as defined by the world health organization are as follows:

Fasting blood glucose concentration ≤ 5.6mmol/L

2hour post glucose (75g) intake glucose ≤ 7.8mmol/L

concentration

The levels of blood glucose in diabetes as defined by the world health organization are as
follows:

Random venous blood glucose concentration ≥ 11.1mmol/L

Fasting blood glucose concentration ≥ 7.0mmol/L

2hour post glucose (75g) intake glucose ≥ 11.1mmol/L

concentration

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The results from the experiment states that the fasting blood glucose concentration (at time 0)
of the patient is 7.2mmols/L. This is above the described fasting glucose concentration in a
diabetic which is ≥ 7.0mmol/L, and it is also above 5.6mmol/L which is the fasting blood glucose
concentration of a non-diabetic patient. This shows that the patient is indeed a diabetic patient.
Furthermore, the glucose concentration of the patient after 2 hours of the experiment was
8.4mmoles/L which is above the described 2 hour post-glucose intake glucose concentration in a
normal person but below the described 2 hour post-glucose intake glucose concentration in a
diabetic person. This indicates hyperglycemia hence the patient is indeed diabetic however
maybe sensitive to glucose.
CONCLUSION
These two observations made, leads to the conclusion that the patient is indeed a diabetic
however may indicate some sensitivity to insulin.
Therefore the glucose tolerance test confirms the glycosuria that was observed in the patient
was as a result of diabetes.

References

1. Arthur C Guyton, John E Hall,2006, Text book of medical physiology 11 th edition,

Elsevier, Philadelphia, page 975.
2. Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycemia. 2006.
“http://www.who.int/diabetes/publications/Definition%20and%20diagnosis%20of%20d
iabetes_new.pdf”
3. Monica Cheesbrough, District Laboratory Practice in tropical countries part 1, Great
Britain Publisher 1984 (Pages 344-345, 348)
4. Stefano Ferri, Katsushiro Kojima, and Koji Sode, 2011, Journal of Diabetes Science and
Technology. Volume 5, Issue 5, September 2011. “www.jdst.org/September201”

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