MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF BIOCHEMISTRY

DATE OF PRACTICAL: 12TH FEB 2020

ASSAY OF GLUCOSE BY GLUCOSE OXIDASE AND

GLUCOSE TOLERANCE TEST

GROUP 17
NAME REGISTRATION COURSE
NUMBER
ADOLPHE STEPHIN MBONJO 19/X/29602/PS BDSU
MBAHO
SEMAKALU GABRIEL 19/U/0791 BMAM
AUMA ESTHER LAURYNA 19/U/18884/PS BMAM
 EXPERIMENT 1: ASSAY OF GLUCOSE BY GLUCOSE
OXIDASE

ABSTRACT

In this experiment, standard glucose solutions of concentration 27mg/L, 54mg/L, 81mg/L and
108mg/L labelled (G1-G4) respectively. With fructose solution (110µg/ml) and Lactose
solution (220µg/ml) of 0.2ml were put in test tubes and same amount of colour reagent
(1.0ml) was added to each test tube. These were incubated at 37oc for 15 minutes. A control,
P-T reagent was used as blank to re-zero the colorimeter. The absorbance of the colour
produced was measured with a colorimeter using a green filter 520 nm. Absorbance is
directly proportional to the degree of breakdown of the sugar by glucose oxidase.
From the results, the absorbance of fructose and lactose were low. This means they were not
broken down by enzyme thus glucose oxidase is highly specific to glucose only. The
Calibration curve was plotted through the origin thereby respecting the Beer’s-Lambert’s
Law.

INTRODUCTION

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Glucose can be estimated specifically, in the presence of other sugars or reducing agents by
using the enzyme glucose oxidase. This enzyme catalyses the oxidation of glucose by oxygen
to lactose with formation of hydrogen peroxidase.
This hydrogen peroxide produced is measured by reacting it with a colourless dye, which it
oxidises to a coloured product catalyzed by a second enzyme peroxidase. The quantity of this
coloured product formed is measured calorimetrically and is equivalent to amount of
hydrogen peroxide and therefore the amount of glucose.

D-glucose + O2 D-gluconolactone + H2O2

Lactone reacts with water to form gluconic acid. The hydrogen peroxide is measured by
using it to oxidize a dye (colourless in the reduced form) to a coloured oxidation product.
This reaction is catalysed by peroxidase and lactone reacts with water to form gluconic acid.
The hydrogen peroxide is measured by using it to oxidize a dye (colourless in the reduced
form) to a coloured oxidation product. This reaction is catalysed by peroxidase.
The concentration of glucose in the blood is determined and expressed in mmol/L of blood.
The urine samples are tested qualitatively for glucose. The concentration of blood glucose is
plotted against time (t). Time zero is the time the glucose was taken, and the fasting blood
glucose concentration is the concentration at time zero. In the normal human, the blood rises
from a fasting level of 4-5mmol/L to about 7mmol/L within 30 to 60 minutes and then falls
over the next 3hours. In a diabetic, the initial level is higher than normal, the increase is
greater, and the fall is slower. Glucose and ketone bodies are absent from the urine of normal
subjects. In diabetics, glucose and possibly ketone bodies may be present in fasting urine and
as such glucose and/or ketone bodies will be detected in the urine after the glucose dose.

Aim of Experiment
• To test the specificity of glucose oxidase
• To monitor how blood glucose homeostasis is maintained following glucose overload
• To detect disturbances in glucose metabolism that can be linked to human conditions
such as diabetes or metabolic syndrome.

BACKGROUND

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Glucose provides the energy for life processes. It is the main end product of carbohydrate
digestion. Oxidation of glucose by the glycolytic and tricarboxylic acid pathways provides
the chemical energy needed for cellular activity. When required to maintain the blood glucose
level, liver glycogen is converted back to glucose (glycogenolysis). Muscle glycogen
provides the glucose for muscular activity.
Excess glucose is oxidized to fatty acids and stored as fat in the tissues. If needed, glucose
can also be formed from fats and protein (gluconeogenesis).
Factors that affect amount of glucose obtained.
 Fasting: This refers to blood collected after a period of no food intake. For adults, the
fasting time is usually 10 to 16 hours. For children the fasting time is 6 hours unless a
longer time is indicated, e.g. when investigating hypoglycaemia. The drinking of plain
water is permitted. The normal range of glucose concentration during fasting is 3.6–
6.4 mmol/L for adults and 2.4–5.3 mmol/L for children.
 Post-prandial: This describes blood collected after a meal has been taken. The sample
is usually taken as a 2 hr postprandial specimen.
 Random measurement: This refers to a blood sample collected at any time, regardless
of food intake. The normal range of glucose concentration measured randomly is 3.3–
7.4 mmol/L

Glucose oxidase enzyme

The glucose oxidase enzyme also known as notatin (EC number 1.1.3.4) is an oxido-
reductase enzyme isolated from the mold Penicillium notatum which catalyses the oxidation
of β- D-glucose to D-glucono-lactone and hydrogen peroxide. This enzyme is highly specific
for the β anomer of glucose and does not affect the α-anomer. In spite of this specificity, the
reaction catalyzed by glucose oxidase is commonly used in a clinical assay for total blood
glucose; that is, for solutions consisting of a mixture of β- and α-D-glucose. Glucose oxidase
is more suitable because it allows detection of small amounts of glucose. (David L. Nelson
and Micheal M. Cox, 2004). Glucose oxidase enzyme method will give a narrower reference
range for blood glucose than a Folin-Wu technique because the enzyme method is specific for
glucose. (Monica Cheesebrough, 2005)

In the post absorptive state, the concentration of blood glucose in most mammals is
maintained between 4.5 and 5.5 mmol/L. After the ingestion of a carbohydrate meal, it may
rise to 6.5 to 7.2 mmol/L, and in starvation, it may fall to 3.3 to 3.9 mmol/L. A sudden

4
decrease in blood glucose (e.g, in response to insulin overdose) causes convulsions, because
of the dependence of the brain on a supply of glucose. However, much lower concentrations
can be tolerated if hypoglycemia develops slowly enough for adaptation to occur (Robert K.
Murry et al, 2003).

In a diabetic, the initial level is higher than normal, the increase is greater and the fall is
slower. Glucose and ketone bodies are absent in urine of the normal subjects but in diabetics,
glucose and possibly ketone bodies may be present in fasting urine. Glucose and mainly
ketone bodies will be detected in urine after the glucose dose. The normal range for glucose
concentration in blood is 60 to 90 mg/100 mL, or about 4.5mM. The concentration of glucose
in blood is hormonally regulated. Glucose appears in urine when the blood glucose level
increases above 180 mg/dL. Glycosuria (presence of glucose in urine) may be the first
indicator of diabetes mellitus.

It is important that the blood glucose concentration should not rise too high for four reasons:
(1) Glucose can exert a large amount of osmotic pressure in the extracellular fluid, and if the
glucose concentration rises to excessive values, this can cause considerable cellular
dehydration.
(2) An excessively high level of blood glucose concentration causes loss of glucose in the
urine.
(3) Loss of glucose in the urine also causes osmotic diuresis by the kidneys, which can
deplete the body of its fluids and electrolytes.
(4) Long-term increases in blood glucose may cause damage to many tissues, especially to
blood vessels. Vascular injury, associated with uncontrolled diabetes mellitus, leads to
increased risk for heart attack, stroke, end-stage renal disease, and blindness.

Insulin is the most important hormone that regulates the amount of glucose in the blood, the
rate at which glucose is taken up by the tissues, and the conversion of glucose to glycogen. It
is made and secreted by the beta-islet cells of the pancreas. Only insulin is capable of
reducing the concentration of glucose in the blood. (Monica Cheesebrough, 2005)
It is important to also note that, increasing blood glucose increases metabolic flux through
glycolysis, the citric acid cycle, and the generation of ATP.

5
Insulin lowers blood glucose immediately by enhancing glucose transport into adipose tissue
and muscle by recruitment of glucose transporters (GLUT 4) from the interior of the cell to
the plasma membrane. Although it does not affect glucose uptake into the liver directly,
insulin enhances long-term uptake as a result of its actions on the enzymes controlling
glycolysis, glycogenesis, and gluconeogenesis (Robert R Murry et al, 2003)

Diabetes mellitus is a syndrome of impaired carbohydrate, fat, and protein metabolism caused
by either lack of insulin secretion or decreased sensitivity of the tissues to insulin.
There are two general types of diabetes mellitus:
1. Type 1 Diabetes—Deficiency of Insulin Production by Beta Cells of the Pancreas
Injury to the beta cells of the pancreas or diseases that impair insulin production can lead to
type 1 diabetes. Viral infections or autoimmune disorders may be involved in the destruction
of beta cells in many patients with type 1 diabetes, although heredity also plays a major role
in determining the susceptibility of the beta cells to destruction by these insults. In some
instances, persons may have a hereditary tendency for beta cell degeneration even without
viral infections or autoimmune disorders. The usual onset of type 1 diabetes occurs at about
14 years of age in the and for this reason it is often called juvenile diabetes mellitus.
However, type 1 diabetes can occur at any age, including adulthood, following disorders that
lead to the destruction of pancreatic beta cells. Type 1 diabetes may develop abruptly, over a
period of a few days or weeks, with three principal sequelae:
o Increased blood glucose levels,
o Increased utilization of fats for energy and for formation of cholesterol by the
liver,
o Depletion of the body’s proteins. Approximately 5 to 10 percent of people
with diabetes mellitus have the type 1 form of the disease.

2. Type 2 Diabetes—Resistance to the Metabolic Effects of Insulin

Type 2 diabetes is far more common than type 1, accounting for about 90 to 95 percent of all
cases of diabetes mellitus. In most cases, the onset of type 2 diabetes occurs after age 30
years, often between the ages of 50 and 60 years, and the disease develops gradually.
Therefore, this syndrome is often referred to as adult-onset diabetes. In recent years,
however, there has been a steady increase in the number of younger individuals, some
younger than 20 years old, with type 2 diabetes.

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This trend appears to be related mainly to the increasing prevalence of obesity, the most
important risk factor for type 2 diabetes in children and adults. (John E Hall, Arthur C
Guyton, 2016).
The effects of uncontrolled diabetes mellitus are:
 Glycosuria, loss of glucose in urine
 Polyuria, abnormal production of large quantities of urine usually greater than
2.5 liters over 2 hours in adults
 Ketonuria, presence of ketone bodies in urine
 Polydipsia, excessive thirst due to dehydration
 utilization of fats leading to metabolic acidosis, ketoacidosis in type 1
diabetes mellitus
 Depletion of proteins in type 1 diabetes

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METHOD

i.) Materials used.

 7 test tubes
 ml Micro pipette
 Vortex mixer
 Stop clock
 Water bath
 Colorimeter
 Biuret
 Pipette filler

ii.) Reagents used

 Standard glucose solutions

 Colour reagent (4 aminophenazone, glucose oxidase, peroxidase, phosphate buffer)
 P-T Phenol tungstate reagent (1% sodium tungstate, 1% sodium phosphate, 0.9%
sodium chloride, 0.1% phenol, PH 3.0).
 The phenol tungsten reagent precipitates protein when it’s added to a blood sample
and phenol is necessary for full color development.

iii.) Principle
Glucose oxidase (GOD) catalyses the oxidation of glucose to give hydrogen peroxide (H2O2)
and gluconic acid. In the presence of the enzyme peroxidase (POD), the hydrogen peroxide is
broken down and the oxygen released reacts with 4-aminoantipyrine and phenol to give a
pink colour. The absorbance of the colour produced is measured in a colorimeter using a
green filter of 520 nm. (Monica Cheesbrough, 2005).

iv.) Procedure

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Testing the specificity of glucose oxidase
 Four standard solutions of glucose G1, G2, G3 and G4 and standard solutions of
fructose and lactose are provided
 The standards are all dissolved in Phenol tungstate reagent.

TABLE I: GLUCOSE CONCENTRATIONS OF G1, G2, G3 & G4 SOLUTION

Solution Glucose Glucose Glucose concentration

concentration mg/L concentration µg/Ml µmoles/mL
G1 27 27 0.15
G2 54 54 0.30
G3 81 81 0.45
G4 108 108 0.60

The glucose concentrations in µmoles/mL were obtained as follows;

 G1, = 0.15 µmoles/mL

 G2, = 0.30 µmoles/mL

 G3, = 0.45 µmoles/mL

 G4, = 0.60 µmoles/mL

1. 7 test tubes were cleaned and labelled as follows: (Blank, S1, S2, S3, S4, F, L.
2. Different Solution were pipetted into the test tubes as indicated in table II below

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TABLE II: COMPOSITION OF DIFFERENT TEST TUBES
Reagents Blank S1 S2 S3 S4 F L
(ml) (ml) (ml) (ml) (ml) (ml) (ml)
P.T 0.2
G1 0.2
G2 0.2
G3 0.2
G4 0.2
Fructose(110µg/ml) 0.2
Lactose(220µg/ml) 0.2
Colour reagent 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Total µmoles glucose _ 0.03 0.06 0.09 0.12 _ _
in tubes
Total µmoles _ _ _ _ _ 0.12 0.13
fructose/lactose in
tube

The total µmoles of glucose, fructose and lactose were obtained as follows;

S1, (0.15 0.2) = 0.03 µmoles

S2, (0.30 0.2) = 0.06 µmoles

S3, (O.45 0.2) = 0.09 µmoles

S4, (0.60 0.2) = 0.12 µmoles

F, ( ) 0.2 = 0.12 µmoles

L, ( ) 0.2 = 0.13 µmoles

3. The test tubes were mixed well using a vortex mixer.

4. Test tubes were incubated 37o C for 15 minutes and were mixed manually to ensure
good aeration.
5. A timer was used to monitor.
6. Test tubes we kept to settle for 10 minutes

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 7. The absorbance of the different solutions were measured using a colorimeter, with a
green filter of wavelength 520mn. The colorimeter was recalibrated to zero using the
blank solution before measuring the absorbance of a new solution.
8. The data obtained was put in the table given.
9. A calibration curve graph was plotted.

RESULTS

Table III: THE ABSORBANCES OF THE DIFFERENT SOLUTIONS

Test tube Blank S1 S2 S3 S4 F L
Absorbance 0.00 0.059 0.111 0.172 0.235 0.000 0.000
Colorimeter type and number: Sherwood Manual Colorimeter 260
Filter Colour: Green filter(520nm)

DISCUSSION
Comparing the results in Table III above with that of Table II, it can be observed that
solutions S1, S2, S3 and S4 had an increasing absorbance respectively. The fructose and lactose
concentration respectively in tubes F and L were higher compared to the glucose
concentration in the other tubes (S1 S2 S3 S4) and thus both the F and L tubes had zero
absorbance. This shows that the enzyme glucose oxidase is very specific to its substrate
(glucose) and that even higher concentrations of any other substrate would not affect the
specificity of the enzyme glucose oxidase.

CONCLUSION
Glucose oxidase is a highly specific catalyst to glucose compared to any other sugar. The
absorbance of the dye generated from glucose is directly proportional to its concentration.

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 EXPERIMENT II: GLUCOSE TOLERANCE TEST

ABSTRACT
In this Experiment, a patient Muwazi is a 26 years old very anxious male with a weight of
59kg. He complained of mild polyuria and polydipsia with some weight loss. 1ml of blood
samples were drawn from him at time (t) 0, 30, 90 & 180 minutes after drinking glucose at a
concentration of 44.25g (dose of glucose 0.75g/kg) in H2O. The blood was deproteinized and
then diluted 20ml. 1ml of this dilution corresponds to 0.05ml blood. The concentration of
glucose is plotted against time (t) to give a Glucose tolerance curve.
From the graph, glucose concentration increased rapidly to a maximum in the first 30 minutes
and gradually decreased in the next 90 minutes to the normal value. This indicates that the
patient is normal with not (Diabetes or Hyperglycemia).

INTRODUCTION
The ability of the body to utilize carbohydrates is measured in tolerance tests. In the glucose
tolerance tests, 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 orally. Specimens of blood and urine are collected at
30 to 60 minute intervals over the next 3 to 4 hours. The concentration of glucose in the
blood is determined and expressed in mmol/L of blood. The urine samples are tested
qualitatively for glucose. The concentration of blood glucose is plotted
against time (t). Time 0 minutes is the time the glucose was taken and the fasting blood
glucose concentration is the concentration at time (t) 0 minute. This graph is called the
Glucose Tolerance Curve.

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GLUCOSE TOLERANCE TEST (GTT)
Glucose tolerance: A GTT measures the ability of the body to tolerate, or cope with, a
standard dose of glucose. The degree of tolerance to the glucose, as shown by a change in the
blood glucose level, is mainly dependent on the rate of glucose absorption and on the insulin
response.
As the glucose is absorbed, the level of glucose in the blood rises and the normal
response is for insulin to be released from the pancreas to lower the glucose level. Tolerance
is reduced when insulin is insufficient or absent. A glucose tolerance test (GTT) is usually
requested to investigate glycosuria or when random or fasting blood glucose is suggestive but
not diagnostic of diabetes. It happens only rarely however that the result of a fasting or
random blood glucose is difficult to interpret and a GTT is necessary. If glycosuria is found,
measurement of fasting glucose should be performed before the patient is subjected to a GTT.
A GTT should not be necessary in children.
Several urine glucose strip tests are commercially available. They are based on a glucose
oxidase reaction. Glucose is oxidized to gluconolactone by the enzyme glucose oxidase
(specific for glucose) with the release of hydrogen peroxide. A chromogen is then oxidized
by the hydrogen peroxide and a peroxidase enzyme is used to convert the chromogen from a
reduced colourless state to a coloured oxidized state.
(Monica Cheesbrough, 2009)

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BACKGROUND

The glucose tolerance test was first described in 1923 by Jerome W. Conn. The test was
based on the previous work in 1913 by A.T.B Jacobson in determining that carbohydrate
ingestion results in blood glucose fluctuations, and the premise (Staub-Traugott phenomenon
after its first observers H.Staub in 1921 and K.Traugott in 1922) that a normal patient fed
glucose will rapidly return to normal levels of blood glucose after an intitial spike, and will
see improved reaction to subsequent glucose feedings.
The patient is instructed not to restrict carbohydrate intake in the days or weeks before the
test. The test should not be done during an illness as results may not reflect the patient’s
glucose metabolism when healthy. A full adult dose should not be given to a person weighing
less than 42.6kg, or the excessive glucose may produce a false positive result. Usually, Oral
Glucose Tolerance Test (OGTT) is performed in the morning as glucose tolerance can exhibit
a diurnal rhythm with a significant decrease in the afternoon. The patient is instructed to fast
(water is allowed) for 8-12 hours prior to the tests.

Results Fasting plasma glucose (measured before the OGTT begins) should be below
6.1mmol/L (110mg/dL). Fasting levels between 6.1 and 7.0mmol/L (110 and 125mg/dL) are
borderline (“impaired fasting glycemia”), and fasting levels repeatedly at or above
7.0mmol/L (126mg/L) are diagnostic of diabetes (Salford Royal NHS Trust, 2012)
A 1 hour OGTT glucose level below 10mmol/L (180mg/dL) is considered normal
A 2 hour OGTT glucose level below 7.8mmol/L (140mg/dL) is normal, whereas higher
glucose levels indicate hyperglycemia. Blood plasma glucose between 7.8mmol/L
(140mg/dL) and 11.1mmol/L (200mg/dL) indicate “impaired glucose tolerance”, and levels
above 11.1mmol/L (200mg/dL) at 2 hours confirm a diagnosis of diabetes. (Hartling, L. et
al, 2012)

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REAGENTS
 Four deproteinized and diluted blood samples (X1, X2 , X3 , X4 )
 Standard glucose G2
 Phenol-tungstate (P-T) = 1 % Na2 PHO4 , 0.9% NaCl, 0.1% Phenol, pH 3.0
 Colour reagents = 4 aminophenazone, glucose oxidase, peroxidase,
phosphate buffer.

APPARATUS
 Green filter (wavelength = 520nm)
 Test tubes for holding the different solutions
 Cuvettes with a standard 1cm length to maintain the same path length of light
 Colorimeter (Sherwood colorimeter 260)
 Vortex mixers for mixing solutions
 Burettes for measuring the volumes and adding colour reagents
 Incubator 37o C
 Timer
 Pipette

PROCEDURE
1. 4 deproteinized and diluted blood samples prepared from a patient undergoing a
glucose tolerance test were provided.
2. Six test tubes were cleaned and labelled as follows: (Blank, S, X1, X2 , X3 , X4 )
3. Different solutions were pipetted into the test tubes as indicated in table I below.

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TABLE I: SHOWING COMPOSITION OF TEST TUBES OF PATIENT SAMPLE
Solution Blank S X1 X2 X3 X4
PT Reagent 0.2
Standard 0.2
glucose G2
Deprot 0.2
Sample: X1
X2 0.2
X3 0.2
X4 0.2
Colour 1.0 1.0 1.0 1.0 1.0 1.0
reagent

RESULTS
TABLE II: THE ABSORBANCE AND AMOUNT OF GLUCOSE IN THE
DIFFERENT SOLUTIONS
Blank S X1 X2 X3 X4
Absorbance 0.00 0.116 0.097 0.190 0.106 0.081
µmoles glucose 0.00 0.31 0.26 0.50 0.28 0.21
(1.0ml sample)

Deproteinized sample 1 2 3 4
mmol in 1.0ml original glucose sample 0.0052 0.0100 0.0056 0.0042
Original glucose concentration mmol/L 5.2 10.0 5.6 4.2

Sample 1, = 0.26  0.05 = 5.2 mmol/L

Sample 2, = 0.50  0.05 = 10 mmol/L
Sample 3, = 0.28  0.05 = 5.6 mmol/L
Sample 4, = 0.21  0.05 = 4.2 mmol/L

DISCUSSION

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From the graph, it is observed that the initial fasting glucose level was high (5.2 mmoles/L) at
(t) 0. The glucose concentration increased rapidly in the first 30 minutes up to maximum at
10 mmoles/L. It then decreased gradually for the next 90 minutes up to 4.2 mmoles/L. The
rapid increase in blood glucose concentration was due to its high rate of absorption into
blood.
In the last 90 minutes of the experiment, the glucose concentration decreased gradually, up to
4.2 mmoles/L which is a normal fasting glucose level. This indicates that our patient is not
Diabetic (Hyperglycemic) hence he is Normal.

EXPLANATION
Initially the blood glucose concentration was normal at 5.2mmoles/L before taking the
glucose dose. Then in the next 30 minutes there was a rapid increased of blood glucose to
10mmoles/L due to absorption of glucose in to blood from the glucose dose given. 30
minutes is not enough time for the body to regulate its glucose level hence the increase to
10mmoles/L. In the next two hours, the gradual decrease in the glucose concentration to a
normal value of 4.2 mmoles/L was due to the body glucose regulatory mechanism that
brought the glucose level back to normal. The regulatory mechanism include the hormone,
insulin that is release when high glucose level are detected in blood, insulin converts the
glucose to other compounds like protein, glycogen and others therefore reduceing the glucose
concentration to normal.

CONCLUSION
In conclusion, it was noted that the patient was Normal after observing the Glucose Tolerance
Curve.

RECOMMENDATION
The patient should seek further medical advice and run more test since the glucose
concentration is normal in other to treat polyuria and polydipsia since the glucose level is
normal.

QUESTION AND ANSWER

 Comment on the specificity of glucose oxidase by comparing the absorbances in

17
tubes F and L. why is the concentration of lactose so much higher than that of fructose
or glucose?
Despite the same concentration of glucose, fructose and lactose in the test tubes S4, F and L
the absorbance are different. The absorbance of glucose is far much greater than those of
fructose and lactose which are almost negligible. The difference is due to the specificity of
glucose oxidase reaction only on glucose resulting into formation of hydrogen peroxide
which oxidizes the dye. There is no action on fructose and lactose hence no formation of
hydrogen peroxide thus no oxidation of colored dye making the absorbance of fructose and
lactose 0.000 and 0.000 respectively.

Explanation for concentration of lactose being higher than that of fructose and glucose:
Glucose and fructose are monosaccharides, unlike lactose which is a diasaccharide. Thus in
very dilute solutions, lactose has few moles which give negligible absorbance.
Discussion:
Glucose oxidase enzyme showed a catalytic effect on specifically glucose but not on fructose
and lactose. This is because enzymes catalyze only substrates of a given structure.
Glucose and fructose have a similar molecular structure but with very different molecular
formulae. Thus due their different ways of orientation of their functional groups at different
carbon atoms, only glucose is broken down by glucose oxidase, and fructose is not. Thus
lactose and fructose were not broken to hydrogen peroxide.
The concentration of lactose is higher than that of either glucose or fructose because lactose
has a very high molecular weight (342g) which is almost twice that of glucose (180g).Thus
with the same mass of lactose and monosaccharides, lactose would have fewer number of
moles almost half way those of either glucose or fructose.
Conclusions:
 Glucose oxidase is a highly specific catalyst for glucose and not any other
sugar.
 The absorbance of the chromophores generated from glucose is directly
proportional to the concentration as stipulated in Beer-Lambert’s Law.

 Compare the absorbance of S2 in table 2 with that of the standard (S s) in table 3.

Are they the same? Should they be? Comment on the importance of any

18
 difference. Why do you think a standard is incorporated in the assay of the
samples from the patient?

S2 and Ss should have the same absorbance because the two solutions have the same
concentration, and were treated under the same conditions, colorimeter, cuvette and filter.
In our case, the S2 had an absorbance of 0.111 and Ss had an absorbance of 0.116.
Difference may be probably because the colorimeter was unstable or using different cuvettes
with different refractive indices or also during the process of dilutions of substances.
The significance of incorporating a standard into the test samples is for relative comparison
since the standard is incorporated as part of the values that were used to establish the
calibration curve. Since the concentration and absorbance of the standard are known ,they can
be used to obtain the constant of absorbance then determine concentrations of other solutions
of known absorbance from Abs=xc

REFERENCES

1. Guyton and Hall, Textbook of Medical Physiology, 2006; Elsevier Saunders,

Philadelphia, Pennsylvania, page 975

2. Hartling, L., Dryden DM, Gutherie A et al, Screening and Diagnosing Diabetes

Mellitus, October 2012, pages 1-3

3. Lehninger et al, Principles of Biochemistry, 2005, 4TH Edition, pages 245 and 271

4. K Sembulingham, 2006, Essentials of Medical Physiology, pages 421-423

5. Salford Royal NHS Trust, Glucose Tolerance Tests in Primary Care, 2012

6. Monica Cheesbrough (2005), District laboratory practice in Tropical countries, Cambridge

University, part1 second edition, Cambridge University press Pg 27, 340, 341, 343, 345, 346

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