MAKERERE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF BIOCHEMISTRY

WORK BY

GROUP 6

NAME PROGRAMME REG.NUMBER

KALEDDE DAVID BPHA 22/U/5075/PS

KIYINI ROBERT DESIRE MAM 23/U/0630

KISALITA JUMA BPHA 22/U/20544/PS

TITLE: DETERMINATION OF CONCENTRATION OF GLUCOSE

IN SERUM AND SPECIFICITY OF GLUCOSE OXIDASE.
ABSTRACT.

An experiment was carried out on Patient, Mr. Ganata a moderately obese man, 36 years old,
72kg weight, who has recently developed polydipsia and polyuria and has had episodes of
faintness on missing a meal. He was given 0.5g glucose per kg of weight. (0.5g/kgwt). blood
samples of 1.0mL were taken at 0 time, 30 minutes, 90 minutes and at 180 minutes after taking
the glucose solution. The blood sample were deproteinized and diluted to 40mL. The blood
sample's glucose content was determined using glucose oxidase, an enzyme that facilitated the
oxidation of glucose with oxygen, resulting in the formation of D-gluconolactone acid and
hydrogen peroxide. The latter was quantified through colorimetric measurement.
A standard curve was generated by employing standard glucose solutions with different
concentrations, aiding in the determination of glucose concentration in the blood sample. The
concentrations of fructose and lactose determined using glucose oxidase. The outcomes for
fructose and lactose estimation revealed a similar concentration for fructose and a slightly higher
concentration for lactose compared to glucose, indicating a reduced affinity for these sugars by
the glucose oxidase enzyme. Following this, a glucose tolerance curve was constructed to
evaluate the glucose tolerance level (diabetic status) of the patient, Ganata.

According to the glucose tolerance curve, the fasting blood glucose level was 1.44 mmol/L, rose
to 2.90 mmol/L after the initial 30 minutes of oral glucose intake, and subsequently declined to a
minimum of 2.04 mmol/L within 180 minutes. The patient's initial fasting blood sugar (1.44
mmol/L) was considerably lower than the normal fasting blood sugar level of 5.6 mmol/L.
Furthermore, the final glucose level of 2.04 mmol/L remained below the normal value of 5.6
mmol/L. These findings indicate that the patient, Ganata, exhibits hyperinsulinism. The results
also suggest that the glucose oxidase enzyme demonstrates a higher specificity for glucose
compared to fructose and lactose.

Introduction.
Experiment one,
The glucose oxidase test is a biochemical assay employed to quantify the concentration of
glucose (sugar) in various samples, typically blood or urine. The estimation of glucose
concentration in a blood sample is conducted using the glucose oxidase method, chosen for
its specificity to glucose.

For the experiment, test tubes containing standard glucose solutions with concentrations of
27 mg/L, 54 mg/L, 81 mg/L, and 108 mg/L (labeled G1-G4), along with fructose solution
(110 µg/ml) and lactose solution (220 µg/ml) of 0.2 ml each, were prepared. Additionally,
1.0 ml of color reagent was added to each test tube, followed by incubation at 37°C for 15
minutes. A control, P-T reagent, served as a blank to zero the colorimeter. The absorbance
of the resulting color was measured using a Sherwood colorimeter, model 260, with a green
filter at 520 nm. The absorbance is directly proportional to the extent of sugar breakdown
by glucose oxidase.

Subsequently, a standard glucose-absorbance graph was generated (attached graph).

Experiment two: Glucose tolerance test.

The glucose tolerance test evaluates the body's capacity to manage a standard amount of glucose.
The extent of glucose tolerance, indicated by changes in blood glucose levels, primarily relies on
the rate of glucose absorption and the response of insulin. Upon glucose absorption, blood
glucose levels elevate, prompting the pancreas to release insulin in the normal physiological
response aimed at reducing glucose levels. Tolerance diminishes when there is insufficient or
absent insulin. Hyperinsulinism, characterized by elevated insulin levels, can result from reduced
insulin receptors, insensitivity of receptors to insulin, or hyperactivity of pancreatic beta cells,
often observed in type 2 diabetes.

A glucose tolerance test (GTT) is typically requested to investigate glycosuria, insulin resistance
or when random or fasting blood glucose levels suggest but do not definitively diagnose
diabetes. Although rare, instances may occur where interpreting fasting or random blood glucose
results proves challenging, necessitating a GTT. If glycosuria is detected, fasting glucose
measurement should precede the GTT. Generally, GTT is not required for children.

In this experiment, the patient fasted for 12 hours before blood collection. Subsequently, the
patient ingested a dose of glucose (0.5g/kg) dissolved in water. Blood samples were collected at
30, 90, and 180-minute intervals post-dose administration. The concentration of glucose was
plotted against time to generate a glucose tolerance curve.

INTRODUCTION
STATEMENT OF THE PROBLEM; 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
Glucose can best estimated specifically in presence of other sugars by glucose oxidase enzyme
which catalyses oxidation of glucose by oxygen to give lactone and hydrogen peroxide
D-Glucose + O2 D-Gluconolatone + H2O2
The lactone reacts with water to form gluconic acid. The hydrogen peroxide is measured by
using it to oxidise a dye to a coloured oxidation product. This reaction is catalyzed by peroxidase
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.
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. Glucose and possibly ketone bodies are
absent from urine of normal subjects, but in a diabetics, glucose and possibly ketone bodies may
be present in fasting in fasting urine. Glucose and often ketone bodies will be detected in urine
after glucose dose
In hyperinsulinism, the fasting level is below the normal range, the increase is smaller and the
fall occurs early possibly below the fasting level. No glucose or ketone bodies are detectable in
urine.
(This information was provided in the hand out)

BACKGROUND.

Glucose serves as the primary metabolic fuel for essential life processes, acting as the principal
end product of carbohydrate digestion. The oxidation of glucose through the glycolytic and
tricarboxylic acid pathways generates the necessary chemical energy for cellular activities. In
instances where immediate energy consumption by the body is unnecessary, glucose undergoes
conversion into glycogen, stored in the liver and muscles through glycogenesis.

During the maintenance of blood glucose levels, liver glycogen undergoes conversion back into
glucose via glycogenolysis, while muscle glycogen supplies the necessary glucose for muscular
activities. Excess glucose is metabolized into fatty acids and stored as fat in tissues.
Additionally, glucose can be synthesized when needed from fats and proteins through
gluconeogenesis. 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 decrease in blood glucose (e.g, in response to insulin overdose) causes convulsions, and
loss of consciousness 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 normal human or dog, the blood glucose rises from a fasting level of 4-5mmol/L to about
7mmol/L within 30 to 60 minutes, then falls to the fasting level in next 2 hours.
In individuals with diabetes, the initial blood glucose level is elevated compared to normal
levels, and the subsequent rise is more pronounced, while the decline occurs at a slower pace.
Normal subjects typically do not excrete glucose and ketone bodies in their urine. However, in
diabetic individuals, fasting urine may contain glucose, and possibly ketone bodies. Following a
glucose dose, both glucose and predominantly ketone bodies may be detected in the urine.

In cases of hyperinsulinism, the fasting blood glucose level falls below the normal range, with a
smaller increase and an early decline, potentially returning to the fasting level. Urine analysis
reveals the absence of glucose or ketone bodies.

Regarding glycosuria, nearly all the glucose passing from the blood into the glomerular filtrate is
typically reabsorbed by the kidney tubules back into circulation. Normally, less than 0.8 mmol/L
(15 mg%) is excreted in urine. Glycosuria, or glucosuria, occurs when there is an excess of
glucose in the urine. The highest blood glucose level before glycosuria occurs is termed the
renal threshold for glucose. In healthy individuals, this threshold is approximately 9–10 mmol/L
(160–180 mg%), representing the normal maximum reabsorptive capacity of the kidney tubules.
However, this threshold is lower in individuals with renal insufficiency.

Glucose becomes detectable in urine when the blood glucose level surpasses 180 mg/dL.
Glycosuria can serve as an initial indicator of diabetes mellitus. Ketonuria, the presence of
ketone bodies in urine, is observed in various conditions such as pregnancy, fever, uncontrolled
diabetes mellitus, prolonged starvation, and glycogen storage diseases (K Sembulingam, 2012).
The normal blood glucose concentration range is 60 to 90 mg/100 mL, approximately 4.5 mM.
Hormonal regulation maintains this concentration, with fluctuations attributed to dietary intake
or intense exercise being offset by hormonally induced metabolic changes across several organs.
Maintaining an optimal blood glucose concentration is crucial 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, produced and secreted by the beta-islet cells of the pancreas, stands as the most pivotal
hormone governing the blood glucose amount, the rate at which tissues absorb glucose, and the
conversion of glucose to glycogen. Its exclusive role lies in reducing the concentration of
glucose in the blood (Monica Cheesebrough, 2005).

The meticulous adjustments occurring minute by minute to maintain blood glucose levels around
4.5 mM involve the collaborative influence of insulin, glucagon, epinephrine, and cortisol on
metabolic processes across various body tissues, particularly in the liver, muscle, and adipose
tissue. Insulin serves as a signaling agent to these tissues, indicating elevated blood glucose
levels. Its secretion is triggered by the beta cells of the islets of Langerhans in response to
hyperglycemia. Consequently, heightened blood glucose levels enhance metabolic flux through
glycolysis, the citric acid cycle, and ATP generation.

An increase in ATP inhibits ATP-sensitive K+ channels, leading to the depolarization of the beta
cell membrane. This results in increased Ca2+ influx via voltage-sensitive Ca2+ channels,
ultimately stimulating insulin exocytosis. Hence, the concentration of insulin in the blood
mirrors that of blood glucose.
Various substances can stimulate the release of insulin from the pancreas, including amino acids,
free fatty acids, ketone bodies, glucagon, secretin, and the sulfonylurea drugs tolbutamide and
glyburide. These medications are employed to induce insulin secretion in type 2 diabetes mellitus
(NIDDM or non-insulin-dependent diabetes mellitus) by inhibiting ATP-sensitive K+ channels.
Conversely, epinephrine and norepinephrine impede the release of insulin.

Insulin swiftly reduces blood glucose levels by facilitating glucose transport into adipose tissue
and muscle through the recruitment of glucose transporters (GLUT 4) from the cell interior to the
plasma membrane. Although it does not directly impact glucose uptake into the liver, insulin
enhances long-term uptake through its effects on enzymes that regulate glycolysis, glycogenesis,
and gluconeogenesis (Robert R Murry et al, 2003).
Glucagon serves as a signal indicating insufficient blood glucose levels and is produced by the
alpha cells within the pancreatic islets. Its release is prompted by hypoglycemia. In the liver,
glucagon activates phosphorylase, stimulating glycogenolysis. Unlike epinephrine, glucagon
does not influence muscle phosphorylase.
Additionally, glucagon amplifies gluconeogenesis from amino acids and lactate. In all these
functions, glucagon operates through the generation of cAMP. Both hepatic glycogenolysis and
gluconeogenesis play a role in the hyperglycemic impact of glucagon, counteracting the actions
of insulin. The liver primarily clears most endogenous glucagon (as well as insulin) from the
circulation (Robert R Murry et al, 2003).
Glucose oxidase enzyme.
The enzyme glucose oxidase, also known as notatin (EC number 1.1.3.4), is an oxidoreductase
derived from the mold Penicillium notatum. It facilitates the oxidation of β-D-glucose to D-
glucono-1,5-lactone and hydrogen peroxide. This enzyme exhibits high specificity for the β
anomer of glucose, with no impact on the α-anomer. Consequently, the reaction catalyzed by
glucose oxidase is commonly employed in clinical assays for total blood glucose, encompassing
solutions containing a mixture of β- and α-D glucose. The use of glucose oxidase is particularly
advantageous as it enables the detection of minimal glucose quantities (David L. Nelson and
Micheal M. Cox, 2004).

At pH 7, glucose primarily assumes a cyclic hemiacetal form in solution, with 63.6% being β-D
glucopyranose and 36.4% α-D-glucopyranose. The presence of the linear and furanose forms is
negligible. Glucose oxidase exhibits specific binding to β-D-glucopyranose and does not act on
α-D-glucose. It can efficiently oxidize all glucose in solution due to the reaction's consumption
driving the equilibrium toward the β side.

To function as a catalyst, glucose oxidase requires a cofactor known as Flavin adenine

dinucleotide (FAD). FAD is a common participant in redox reactions. In the redox reaction
catalyzed by glucose oxidase, FAD initially serves as the electron acceptor, becoming reduced to
FADH2. Subsequently, FADH2 is oxidized by the ultimate electron acceptor, molecular oxygen
(O2), which can do so due to its higher reduction potential. This process results in the reduction
of O2 to hydrogen peroxide
To begin with, the sample undergoes deproteinization to eliminate substances like urate that
may be present in blood samples at concentrations capable of interfering in the final phase of the
reaction. Alternative approaches include:

 The o-toluidine colorimetric method should be avoided due to the highly

corrosive and toxic nature of the reagent, with the chemical o-toluidine being
carcinogenic.
 Copper and ferricyanide blood glucose reduction methods are discouraged as
they lack sufficient selectivity for glucose (Monica Cheesebrough, 2005).
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)
Factors that affect the amount of glucose obtained.
 Fasting: This pertains to blood collected following a period of abstaining from food
intake. Typically, for adults, the fasting duration is 10 to 16 hours, while for children, it
is 6 hours unless a more extended time is warranted, such as when investigating
hypoglycemia. Drinking plain water is permissible. The normal range for glucose
 concentration during fasting is 3.6–≤ 5.6 mmol/L for adults and 2.4–5.3 mmol/L for
children.
 Post-prandial: This denotes blood collected after the consumption of a meal, usually as a
2-hour postprandial specimen.
 Random measurement: This involves a blood sample collected at any time, irrespective
of food intake. The normal range for randomly measured glucose concentration is 3.3–≤
7.8 mmol/L.Values may vary depending on where in the body the blood has been drawn.
Venous plasma values are about 15% higher than whole blood values (except in
anaemia).
There is little difference between values obtained from venous plasma and capillary plasma
when the glucose concentration is normal. When the glucose is raised however, the value
obtained from capillary plasma will be slightly higher than that from venous plasma.
(Monica Cheesebrough, 2005).
A falsely high glucose level will result if a blood sample is collected from an arm receiving a
glucose (dextrose) intravenous infusion. A falsely low value may be obtained if the plasma is
markedly icteric. (Monica Cheesebrough, 2005).
Depending on physiological or pathological conditions glucose tolerance increases, impairs and
decreases.
(a) Decreased glucose tolerance.
This phenomenon is observed in diabetes mellitus. In a non-diabetic individual who is fasting,
the ingestion of 1 gram of glucose per kilogram of body weight results in a rise in blood glucose
levels from approximately 90 mg/100 ml to a range of 120 to 140 mg/100 ml, returning to below
normal levels within about 2 hours.

In contrast, for a person with diabetes, the fasting blood glucose concentration typically exceeds
110 mg/100 ml and often surpasses 140 mg/100 ml after a 2-hour oral glucose dose (John E
Hall, Arthur C Guyton, 2016). The severity of diabetes mellitus generally influences this pattern.
In mild diabetes, the fasting blood glucose level remains below 150 mg/100ml, while in severe
diabetes, it exceeds 180 mg/100ml. Following a test dose of glucose, individuals with diabetes
exhibit a higher increase in blood glucose levels compared to normal individuals, and the blood
glucose level does not return to normal even after 2 hours, which is a characteristic feature of
diabetes.

Diabetes mellitus manifests as a syndrome involving impaired carbohydrate, fat, and protein
metabolism, stemming from either a deficiency in insulin secretion or reduced sensitivity of
tissues to insulin.
Diabetes mellitus is broadly classified into two types:
Type 1 Diabetes (Insulin-Dependent Diabetes Mellitus):
Also known as insulin-dependent diabetes mellitus, this type is characterized by insufficient
insulin secretion.
Type 2 Diabetes (Non–Insulin-Dependent Diabetes Mellitus):
Referred to as non–insulin-dependent diabetes mellitus, type 2 diabetes initially stems from
reduced sensitivity of target tissues to insulin's metabolic effects, a condition commonly termed
insulin resistance.
 Type 1 Diabetes—Insufficient Insulin Production by Beta Cells of the Pancreas:
 Damage to the beta cells of the pancreas or conditions hindering insulin
production can result in type 1 diabetes. The destruction of beta cells in many
type 1 diabetes patients may be attributed to viral infections or autoimmune
disorders, with heredity playing a significant role in determining the
susceptibility of beta cells to such insults. Some individuals may possess a
hereditary predisposition for beta cell degeneration even in the absence of viral
infections or autoimmune disorders. Typically, the onset of type 1 diabetes occurs
around the age of 14, earning it the designation of juvenile diabetes mellitus.
However, type 1 diabetes can manifest at any age, including adulthood, following
disorders leading to the destruction of pancreatic beta cells. The development of
type 1 diabetes may be abrupt, unfolding over a few days or weeks, characterized
by three primary consequences:
 Elevated blood glucose levels
 Increased utilization of fats for energy and cholesterol synthesis by the liver
 Depletion of the body's proteins. Approximately 5 to 10 percent of individuals
with diabetes mellitus present with the type 1 form of the disease.
Type 2 Diabetes—Reduced Responsiveness to Insulin's Metabolic Effects:
Type 2 diabetes is notably more prevalent than type 1, constituting approximately 90 to 95
percent of all cases of diabetes mellitus. Typically, the onset of type 2 diabetes occurs after the
age of 30, frequently between 50 and 60 years, and the condition develops gradually. Hence, this
syndrome is commonly termed adult-onset diabetes. Nevertheless, in recent times, there has
been a consistent rise in the incidence of type 2 diabetes among younger individuals, some
below 20 years old. This trend is closely linked to the escalating prevalence of obesity,
identified as the paramount risk factor for type 2 diabetes in both children and adults (John E
Hall, Arthur C Guyton, 2016).

Other Forms of Diabetes:

 Diabetes insipidus. unlike diabetes mellitus which is characterized by high blood
glucose levels due to either insufficient insulin production or body’s inability to use
insulin effectively, insipidus is characterized by excessive thirsty and urination due to
deficiency of antidiuretic hormone (ADH) or kidney inability to respond to ADH, which
hormone helps to regulate the body water balance by reducing the amount of water
 excreted in urine. Common symptoms of diabetes insipidus include; excessive urination,
excessive thirsty even when fluid intake is high. However it does not result in high
glucose levels and urine lacks glucose

 Malnutrition-Related Diabetes Mellitus (MRDM):

Also known as 'tropical pancreatic diabetes,' MRDM is observed in specific regions across
tropical countries. It is associated with pancreatic damage, featuring an early age of onset and a
history of malnutrition. Exocrine pancreatic deficiency may lead to steatorrhea, and pancreatic
calcification can occur in certain cases.
 Gestational Diabetes Mellitus (GDM):
Defined as diabetes first identified during pregnancy, GDM often sees glucose values returning
to normal postpartum. However, glucose intolerance and the potential development of type 2
diabetes may manifest later in life (Monica Cheesebrough, 2005).
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. May manifest as muscle wasting.
Decreased glucose tolerance occurs in other conditions like;
 Liver disease.
 Hyperthyroidism.
 Hyperpituitarism.
 Cushing’s syndrome (excessive formation of glucocorticoids).
 Arseniasis (Chronic arsenic poisoning).
 Pregnancy.
 Old Age.
b) Increased glucose tolerance; this occurs in the following conditions:
(i). Hypopituitarism.
(ii). Addison’s disease.
(iii). Hypothyroidism.
(iv). Intestinal malabsorption.
(v) Impaired glucose reabsorption in kidney (renal glycosuria). (Monica Cheesebrough, 2005).

Depending on the method of test dose glucose administration, it is categorized as:

(a) Oral Glucose Tolerance Test: This involves assessing blood glucose levels after the oral
administration of a test dose of glucose.
(b) Intravenous Glucose Tolerance Test: Conducted for patients with impaired glucose
absorption in the gastrointestinal tract, this test administers glucose directly into the bloodstream
through a vein.

METHODOLOGY

Materials used.

MATERIAL USE
Glucose solutions: G1-G4 Used to investigate absorbance
Test tubes Contain the solutions
Test tube rack Holds test tubes
Pipette To measure the volumes of the solutions
Pipette filler Used to draw the solutions into the pipette
Vortex mixer To shake the test tube and mix the
components to form a solution
Tissue paper To clean the cuvettes
Cuvettes Hold the solution when in the colorimeter
Light filter Blocks transmission of light beyond a
specified wavelength
Colorimeter Measures the absorbance of the glucose
samples
Color reagent: 4 aminophenazone, glucose To change color according to intensity of
oxide, peroxidase, phosphate buffer glucose oxidation in the solution
P-T Phenol-tungstate: 1% sodium tungstate, Precipitates protein when it is added to blood
1% Na2PHO4, 0.9% NaCl, 0.1% Phenol, pH sample, necessary for full color development.
3.0
Stop clock. For timing the incubation period.
Thermometer For temperature measurement during
incubation.
Biuret For measuring the colour reagent.
Principle

Glucose oxidase (GOD) catalyzes 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
520 nm. (Monica Cheesbrough, 2005)
Equation
D-glucose + O2 Gluconic acid + H2O2 (Glucose oxidase)
Hydrogen peroxide H2O + [O] (Peroxidase)

[O] + 4-aminophenazone + phenol chromogen (pink)

Procedure for testing the specificity of glucose oxidase

• Four standard solutions of glucose G1, G2, G3 and G4 and standard solutions of fructose
and lactose were provided
• The standards were all dissolved in Phenol tungstate reagent.
Procedure for Glucose tolerance test

Preparation of the patient.

 Before the test the patient should be on a diet containing not less than 150 g of
carbohydrate per day for at least 3 days. If the GTT is performed following starvation or
a diet low in carbohydrate, glucose tolerance will be reduced which will make the results
of the test difficult to interpret.
 Ideally the test should be performed in the morning. The patient should be instructed not
to eat, drink (except plain water), or smoke for 10–16 hours before the test.
 The patient should be reassured if anxious. Outpatients should be allowed to rest for
about 20 minutes before starting the test especially if they have had to walk a
considerable distance to reach the hospital. Excessive exercise, excitement, or fear may
reduce glucose tolerance.
 During the test the patient should, if possible, be sitting up. If lying down the patient
should be positioned on the right side to ensure rapid emptying of the stomach.
 Ideally the patient should have no other disease at the time a glucose test is performed. A
note should be made of any drug treatments.

TABLES OF RESULTS.
The amount of glucose progressively increased from G1- G4 as indicated in the table below.
Table 1 showing the glucose concentrations in different standard glucose solutions.
Solution Glucose concentration Glucose concentration Glucose concentration
mg/L µg/mL µmoles/mL

G1 27 27 0.15
G2 54 54 0.30
G3 81 81 0.45
G4 108 108 0.60

Calculations for conversion glucose concentrations from mg/L to µg/mL and then to µmole/mL

a) converting from mg/L to µg/mL

G1
1mg = 1000µg
27mg = (27X1000) µg
= 27000 µg
» concentration of G1 = 27000 µg/L
But 1L = 1000mL
1000mL of G1 contain 27000 µg
1mL of G1 contains (27000÷1000) µg
27 µg/mL

G2
1mg = 1000µg
54mg = (54X1000) µg
= 54000 µg
»concentration of G2 = 54000 µg/L
But 1L = 1000mL
1000mL of G2 contain 54000 µg
1mL of G2 contains (54000÷1000) µg
54 µg/mL
 G3
1mg = 1000µg
81mg = (81X1000) µg
= 81000 µg
»concentration of G3 = 81000 µg/L
But 1L = 1000mL
1000mL of G3 contain 81000 µg
1mL of G3 contains (81000÷1000) µg
81 µg/mL

G4
1mg = 1000µg
108mg = (108X1000) µg
= 108000 µg
»concentration of G4 = 108000 µg/L
But 1L = 1000mL
1000mL of G4 contain 108000 µg
1mL of G4 contains (108000÷1000) µg
108 µg/mL

b) converting from µg/mL to µmoles/mL

1 mole of glucose weighs 180g
But 1g contains 1,000,000 µg
180g of glucose contain (180X 1,000,000) µg
= 180,000,000 µg
Also 1mole contains 1,000,000 µmoles
» 180,000,000 µg of glucose contain 1,000,000 µmoles
1 µg of glucose contains (1,000,000 ÷ 180,000,000) µmoles
= (1/180) µmoles
For G1
 27 µg of glucose contain (27X1/180) µmoles
= 0.15 µmoles
Since the 27µg were in 1mL then concentration of G1 is 0.15µmoles/mL

For G2
54 µg of glucose contain (54X1/180) µmoles
= 0.30 µmoles
Since the 54µg were in 1mL then concentration of G1 is 0.30µmoles/mL

For G3
81 µg of glucose contain (81X1/180) µmoles
= 0.45 µmoles
Since the 81µg were in 1mL then concentration of G1 is 0.45µmoles/mL

For G4
108 µg of glucose contain (108X1/180) µmoles
= 0.60 µmoles
Since the 108µg were in 1mL then concentration of G1 is 0.60µmoles/mL

Table 2 showing the absorbance of different glucose standard solutions and fructose and lactose
standard solutions.
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
µmoles glucose in _ 0.03 0.06 0.09 0.12 _ _
0.2mL
Total µmoles of - - - - - 0.12 0.13
fructose/lactose in the
tube
Absorbance 0.00 0.10 0.24 0.35 0.58 0.00 0.00

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

S1 (obtained from G1)

1ml of G1 contained 0.15µmoles of glucose

0.2ml of G1 contains (0.15x0.2) µmoles

The moles of glucose in S1 = 0.03 µmoles

S2 (obtained from G2)

1ml of G2 contained 0.30µmoles of glucose

0.2ml of G2 contains (0.30x0.2) µmoles

Therefore moles of glucose in S2 = 0.06 µmoles

S3, (obtained from G3)

1ml of G3 contained 0.45µmoles of glucose

0.2ml of G1 contains (0.45x0.2) µmoles

Therefore, moles of glucose in S3 = 0.09 µmoles

S4, (obtained from G4)

1ml of G4 contained 0.60µmoles of glucose

0.2ml of G1 contains (0.60x0.2) µmoles

Therefore, the moles of glucose in S4 = 0.12 µmoles

Moles of fructose used

1mole of fructose weighs 180g

180g of fructose contains 1mole

180µg of fructose contains 1µmole

The 110µg of fructose contains ((1/180) X110) µmoles

= 0.611µmole

But 110 µg were dissolved in 1mL

1mL of fructose solution contain 0.611 µmoles

0.2mL of fructose solution contain (0.611X0.2) µmoles

= 0.12 µmoles

Moles of lactose used

1mole of lactose weighs 342.3g

342.3g of lactose contains 1mole

342.3µg of lactose contains 1µmole

220µg of lactose contains ((1/342.3) X220)) µmoles

= 0.643µmoles

But 220µg of lactose were dissolved in 1mL

1mL of lactose solution contains 0.643µmoles

0.2mL of lactose contain (0.643X0.2)µmole

= 0.13µmole
Table 3: Variation of blood glucose concentration with time after oral glucose
administration.
Blank S X1 X2 X3 X4
Absorbance 0.00 0.24 0.14 0.29 0.31 0.20

Glucose 0.00 0.0360 0.0725 0.0795 0.0510

concentration
µmoles/mL

Deprot Sample X1 X2 X3 X4
Glucose concentration(m moles/L) 1.44 2.90 3.18 2.04
Glucose concetration(x10-1) mmol/L 14.4 29.0 31.8 20.4
The concentration of glucose in the 0.2ml samples used were obtained from extrapolation on the
calibration curve for absorbance of standard glucose solution against μmoles of glucose in 1.0ml
For the standard glucose solution, the above glucose concentrations in mmol/Lwere obtained as
follows;

For the X1 solution,

1 ml contain 0.0360 μ moles.

1000μmoles = 1 mmole.
0.0360μmoles = ((1/1000)X 0.0360) mmoles.
= 0.000036mmoles

0.025 mLs of blood contain 0.000036 m moles.

1000 mLs contain of blood contain ((0.000036/0.025)X1000) m moles.
= 1.44 mmoles
Therefore the glucose concentration is 1.44 mmoles/L.

For X2 solution,
1 ml contain 0.0725 μ moles.
1000μmoles = 1 mmole.
0.0725 μmoles = ((1/1000)X 0.0725) mmoles.
= 0.0000725mmoles
0.025 mLs of blood contain 0.0000725 m moles.
1000 mLs contain of blood contain ((0.0000725/0.025)X1000) m moles.
= 2.9 mmoles
Therefore the glucose concentration is 2.9 mmoles/L

For X3 solution,

1 ml contain 0.0795 μ moles.

1000μmoles = 1 mmole.
0.0795μmoles = ((1/1000)X 0.0795) mmoles.
= 0.0000795mmoles

0.025 mLs of blood contain 0.0000795 m moles.

1000 mLs contain of blood contain ((0.0000795/0.025)X1000) m moles.
= 3.18 mmoles
Therefore the glucose concentration is 3.18 mmoles/L

For X4 solution,

1 ml contain 0.051 μ moles.

1000μmoles = 1 mmole.
0.051μmoles = ((1/1000)X 0.051) mmoles.
= 0.000051mmoles

0.025 mLs of blood contain 0.000051 m moles.

1000 mLs contain of blood contain ((0.000051/0.025)X1000) m moles.
= 2.04 mmoles
Therefore the glucose concentration is 2.04 mmoles/L

A graph of glucose concentration (mmol/L) against time (minutes) was plotted (Graph attached).
DISCUSSION.

Glucose oxidase exhibited specificity solely towards glucose, with no breakdown observed for
fructose and lactose. The results demonstrated minimal absorbance in the cases of fructose and
lactose, underscoring the enzyme's high selectivity for glucose.
Although glucose and fructose share a similar molecular formula, their molecular structures
differ significantly. The distinct arrangement of functional groups at various carbon atoms sets
glucose and fructose apart. Notably, glucose oxidase demonstrates a high specificity for the β-
anomer of glucose, with no impact on the α-anomer.
In contrast, lactose possesses a distinct structure as a disaccharide composed of glucose and
galactose. Consequently, glucose oxidase is unable to break down lactose and fructose due to
their dissimilar compositions.
The molar concentration of lactose closely mirrors that of fructose (110 µg/ml) despite lactose
having a greater mass (220 µg/ml). This phenomenon arises from the considerably higher
molecular weight of lactose (342.3 g), nearly double that of fructose (180 g). Consequently, for
an equivalent mass, the number of moles of lactose is consistently nearly half of those for
fructose and glucose (molecular mass 180 g).
The normal blood glucose levels as defined by World Health Organization are as follows:

Fasting blood glucose concentration ≤ 5.6mmol/L

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

concentration

The levels of glucose in diabetes as defined by World Health Organization are as indicated
below;

Random venous blood glucose ≥ 11.1mmol/L

concentration
Fasting blood glucose concentration ≥ 7.0mmol/L
2 hour post glucose (75g) intake glucose ≥ 11.1mmol/L
concentration

The blood glucose levels for impaired glucose tolerance according to WHO are as follows;
Impaired glucose tolerance Fasting <7.0 mmol/l
2 hour specimen 7.8–11.1 mmol/l

From the second graph, it was observed that the fasting blood glucose level was notably low,
measuring 1.44 mmol/L, which falls below the normal range of ≤ 5.6 mmol/L for fasting blood
sugar. The low fasting blood glucose level may suggest the presence of hyperinsulinism in the
patient. The glucose concentration exhibited a rapid increase in the first 30 minutes, followed by
a gradual rise to a peak at 3.18 mmol/L after 90 minutes. This elevation in blood glucose
concentration was attributed to its swift absorption after oral intake.

Although the pattern of blood glucose concentration variations resembled that of a normal
person, the values recorded were significantly lower than the typical blood glucose
concentrations. Subsequently, the glucose level gradually decreased over the next 90 minutes,
reaching a minimum of 2.04 mmol/L after 3 hours of oral glucose intake. This value not only
remained lower than the fasting blood glucose level but also fell below the normal 3-hour post-
glucose intake value of ≤ 7.8 mmol/L for a healthy individual.

In a person without underlying conditions, the glucose concentration would be expected to rise
above normal and then return to a fasting level within one to two hours. However, the observed
graph indicated that the patient remained hypoglycemic, suggesting inadequate regulation of
blood glucose levels. This further supports the indication of hyperinsulinism in the patient.
Hyperinsulinism results from the excessive production of insulin by the pancreas's beta cells,
often due to insulin receptor insensitivity or cellular resistance to insulin. These factors are
recognized as potential contributors to type 2 diabetes. Therefore, it is reasonable to suspect that
Mr. GANATA may have type 2 diabetes.

Possible sources of error

 Standard preparation of glucose; There might have been some errors in the preparation of
the standard glucose which could have affected the final outcome of the experiment.

Other methods that could be;

a) HAEMOGLOBIN A/C MEASUREMENTS. When plasma glucose levels are in the
normal range 4-6% of hemoglobin A has one or both of the N-terminal valine of their
beta chains irreversibly glycated by glucose and hence referred to hemoglobin A1C
(HbA1C). The HbA1C fraction is abnormally elevated in people with diabetes with
chronic hyperglycemia. Since RBCs have a life span of 120days, the HbA1C value
reflects plasma glucose levels over the preceding 8-12 weeks. In patients who
monitor their glucose levels, HbA1C value provides a variable check on the accuracy
of their monitoring. In patients who do not monitor their glucose levels, HbA1C value
are essential to adjusting treatment.
 HbA1C can be used to diagnose diabetes. An HbA1C of 6.5% or greater, if confirmed by
repeated testing, is diagnostic of diabetes. Less than 5.7% is normal and patients with
levels 5.7-6.4% are considered at high risk of developing diabetes.
b) URINE OR BLOOD KETONES; These once detected in urine or blood are
indicative of severe diabetes as the body resorts to metabolizing fats and proteins
instead of carbohydrates.
c) SELF MONITORING OF BLOOD GLUCOSE; several paper strip methods and a
large number of blood glucose meters are available for measuring glucose on
capillary samples

MEDICAL REREVANCE OF GLUCOSE TESTING;

 Diagnosis of diabetes; blood glucose testing is the first method for diagnosing diabetes
and prediabetes. Elevated blood glucose levels can indicate diabetes or impaired glucose
tolerance.
 Monitoring diabetes management; for diabetic individuals, regular blood glucose
monitoring helps track how well their treatment plan is working.
 Prevention of complications; keeping glucose levels within a target range helps reduce
the risk of long term complications associated with diabetes such as cardiovascular
disease, kidney damage and retinopathy.
 Hypoglycemia detection; blood glucose testing is essential for detecting and managing
hypoglycemia, a potentially dangerous condition especially for individual using insulin.

CONCLUSION

From the results of our experiments one and two, it can be concluded that;
Glucose oxidase has a high specificity for glucose.
The absorbance of solutions is directly proportional to glucose concentration.
The patient is hyperinsulinic which is suggestive of type 2 diabetes.

REFERENCES.

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

Cambridge University, part1 second edition, Cambridge University press Pg 27, 340, 341, 343,
345, 348

Robert K. Murry et al (2003). Harper’s illustrated biochemistry. Lange medical books/Mc, 26th
edition, Graw-Hill companies. Pg 181, 189

David L. Nelson and Michael M.Cox (2004), Lehninger’s principles of biochemistry,

New York, 4th edition, W. H. Freeman Pg 271
John E. Hall, Arthur C. Guyton (2016). Textbook of Medical Physiology University of
Mississippi Medical Center 13th Edition Pg 996, 997

K and Prema Sembulingam (2012). Essentials of Medical Physiology 6th Edition Jaypee
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