Lab Report 3

Maria Kamal Katha

ID:22336048

Submitted for: BTE258

Biotechnology Lab III

Section:05

Submitted to: MD Tawsif Ur Rashid

Brac University
Summer Semester 2024
Experiment no: 03 Date:30.06.2024

Name of the experiment: Determination of blood glucose under normal and diabetic conditions
using enzymatic colorimetric test.

Objective:

This experiment aimed to measure blood glucose levels from different blood samples using the
colorimetric test.

Principle:

Blood is a specialized bodily fluid that delivers oxygen and nutrients to the cells and transports
metabolic waste products away from cells in animals. It is composed of blood cells and blood
plasma. Plasma constitutes 55% of blood fluid and is mostly water (92% by volume). It
contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide, and blood cells.
Glucose is a major energy source for most body cells, including brain cells. Carbohydrates
quickly turn into glucose in the body and raise the blood glucose level—hormones such as
insulin and glucagon help in controlling blood glucose levels.

Various external and internal factors such as body composition, age, physical activity, and sex
alter the blood glucose level in the body. The level of glucose circulating in the blood at a given
time is known as the blood glucose level. It is an important parameter for diabetes, a disease
related to the abnormal metabolism of blood sugar and defective insulin production. In this case,
the body doesn’t produce enough insulin or respond to insulin properly, resulting in sugar build-
up in the bloodstream and damaging organs, blood vessels, and nerves. This condition of the
presence of excess sugar in the bloodstream is called hyperglycemia. On the contrary, the
condition where blood glucose level drops below 70 mg/dL is known as hypoglycemia.

Colorimetry is a technique in physical and analytical chemistry that is used to determine the
concentration of colored compounds in solution. A colorimeter or spectrophotometer is a device
used to measure the absorbance of solutions at a specific wavelength of light. Colorimetric
assays use reagents that undergo a measurable color change in the presence of the analyte to
detect the presence of enzymes, antibodies, hormones, and other specific compounds. This
method requires less time and apparatus and yields accurate results.

To determine blood glucose level, glucose oxidase is employed as this enzyme is highly specific
for glucose and does not react with blood saccharides. Glucose is determined after enzymatic
oxidation in the presence of glucose oxidase by the following reactions:

The components of reagents are mainly:

Components Amount

Phosphate buffer (pH 7.5) 100 mmol/L

4-aminophenazone 0.25 mmol/L

Phenol 0.75 mmol/L

Glucose oxidase 15 KU/L

Peroxidase 1.5 KU/L

Matriptase 2 KU/L

Sodium aside 0.095%

Stabilizers

Table 3.1: Reagents’ components and their amount

The blood glucose analysis detects both hyperglycemia and hypoglycemia for the diagnosis of
diabetes. For this, the following chart can be followed:
 Fasting blood glucose ≤ 6.1 mmol/L

2 hours after eating < 7.8 mmol/L (for people aged 50 and younger)
< 8.3 mmol/L (for people aged 50 - 60)
< 8.9 mmol/L (for people aged 60 and older)

Random (Usual) 4.4 - 6.6 mmol/L (before meals / when waking up)
5.5 - 7.7 mmol/L (at bedtime)
Table 3.2: Blood glucose level chart

Apparatus:

● Microcentrifuge tubes
● Sterile syringes (5 mL)
● Sterile screw cap glass tube
● Micropipette and tips
● Microcentrifuge machine
● Cuvettes
● Spectrophotometer

Reagents:

● Fresh human blood (5 mL)

● Enzyme reagent
● Standard solution (Glucose 5.55 mmol/L or 100 mg/dL)

Methods:

1. At first, four volunteers were chosen and 5 mL of blood was collected from each
individual into glass test tubes by puncturing the vein with a sterile syringe.
2. The test tubes were kept at a slanted position and incubated at 37˚C for 5 minutes.
3. Five microcentrifuge tubes were labeled and an equal amount of blood samples were
taken from each test tube into the microcentrifuge tubes using a micropipette.
4. The microcentrifuge tubes were then centrifuged at 13000 rpm for 10 minutes.
5. The clear supernatant (serum) was collected from each tube into cuvettes using a
micropipette, leaving the pellets (erythrocytes) at the bottom of the tube.
6. Then the samples were prepared accordingly adding the serum from each tube with the
reagent and standard solution:

Cuvettes solution Reagent (ml) Standard solution (µl) Sample (µl)

Sample 1 2 0 20

Sample 2 2 0 20

Sample 3 2 0 20

Sample 4 2 0 20

Sample 5 2 0 20

Standard (Positive control) 2 20 0

Negative control 2 0 0
Table 3.3: Preparation of blood samples for measuring glucose
concentration

7. The solutions were incubated for 10 minutes at 20 - 25ºC temperature and the absorbance
was measured at 500 nm using a spectrophotometer.
8. The concentration of glucose (mg/dL) for each sample was measured using the formula:
(Absorbance of sample / Absorbance of standard) x Concentration of standard
 (A) (B) (C)

Figure 10: (A) Standard Solution, (B) Blank, (C) Sample

Calculation:

For sample 1, glucose concentration = (0.123 / 0.213) x 100 = 57.746 mg/dL

= 57.746 x 0.055 = 3.176 mmol/L

For sample 2, glucose concentration = (0.183 / 0.213) x 100 = 85.915 mg/dL

=85.915 x 0.055 = 4.725 mmol/L

For sample 3, glucose concentration = (0.222 /0.213) x 100 = 104.225 mg/dL

= 104.225 x 0.055 = 5.732 mmol/L

For sample 4, glucose concentration = (0.156 /0.213) x 100 = 73.239 mg/dL

= 73.239 x 0.055 = 4.028 mmol/L

For sample 5, glucose concentration = (0.221 /0.213) x 100 = 103.755 mg/dL

=103.755 x 0.055 = 5.706 mmol/L

Result and observation:

Vial number Absorbance Glucose concentration Interpretation

Sample 1 0.123 nm 3.176 mmol/L Low level of blood glucose

Sample 2 0.183 nm 4.725 mmol/L Low level of blood glucose

 Sample 3 0.222 nm 5.732 mmol/L Normal level of blood glucose

Sample 4 0.156 nm 4.028 mmol/L Low level of blood glucose

Sample 5 0.221 nm 5.706 mmol/L Normal level of blood glucose

Positive control 0.213 nm 100 mg/dL –

Negative control 0 nm _ _

Table 3.4: Glucose concentration of the blood samples

Discussion:

In the experiment, blood samples were collected at random. Samples 3 & 5 had normal levels of
blood glucose whereas samples 1, 2, and 4 had low levels of blood glucose, indicating
hypoglycemia. This might be due to fasting for a longer period, medication, or any medical
condition influencing blood sugar levels. A slightly red-colored dye was present in the standard
and sample solutions, confirming the breakdown of glucose with the help of a reagent. No
absorbance reading was found in the negative control solution as no glucose was present to react
with the reagent.

Fresh blood samples were taken to get a more accurate result as the age and condition of blood
samples can affect the enzyme activity. The reagent was also fresh to ensure correct results as
they degrade and lose effectiveness over time. For this, the reagent container was covered with
aluminum foil to maintain its working temperature and stability by protecting it from light and
other contaminants. Moreover, before blood collection, the individuals followed their usual diet
and were in their normal state of health.

The test tubes containing the blood samples were kept in a slanted position so that the liquid
plasma could easily be collected after the blood clots. For this, the incubation time was properly
maintained to let the blood clot so that blood cells would not be collected while collecting the
blood plasma. Bubble formation was avoided while pipetting and all the samples were taken
around equal volumes. The centrifugation speed and period were also maintained to separate the
supernatant-containing serum from the erythrocyte pellets. Moreover, serum was used instead of
plasma as blood clotting factors are present in plasma. The serum is devoid of clotting factors,
making it less prone to cross-contamination due to enzymatic reactions.
Positive and negative controls helped to maintain the accuracy of the experiment. A standard
solution with the reagent was taken as the positive control to ensure that the reagent was working
properly. As the concentration of the standard solution was known, it helped in determining the
unknown concentrations of glucose in the blood samples. For negative control, only a reagent
was taken where no glucose was present. It gave the zero-absorbance reading and removed any
previous absorbance value in the spectrophotometer which might have resulted in incorrect
readings of the samples. As a result, it helped in identifying any non-specific reaction due to the
presence of contaminants and confirmed that there were no false-positive results.

After mixing the solutions, the absorbance was measured within 60 minutes for a valid result as
glucose is affected by heat. However, the accuracy of the experiment could be affected as it was
done in the open air without the use of a laminar air hood. Many people worked in the same
laboratory and the cuvettes containing the samples were kept open, which could result in
contamination and altered absorbance readings.

Precautions:

1. The working area should be sterilized before and after experimenting.

2. The syringe must be sterile.
3. Blood plasma should be collected carefully so that the blood cells are not collected.
4. The reagent should be at the correct working temperature and protected from light.
5. The centrifugation speed and period should be maintained strictly.
6. The micropipette should be calibrated properly and pipetting should be done carefully to
avoid bubble formation.
7. The pipette tubes should be discarded in the wet discard after every use.
8. The microcentrifuge tubes should be loaded opposite to each other in the centrifuge
machine.
9. The cuvettes should be placed in the spectrophotometer properly keeping the clear side
along the source of light.