Clinical Chemistry 1, MLAB211

University of Balamand

Faculty of Health Sciences

Clinical Chemistry 1, MLAB211

Laboratory report 1

Experiment 1

Blood Glucose Determination

 Maria F.Hajjar
Presented to:
 Maria Ch. Moujaes Dr. Hala Chamieh
 Mariam A. Hamze

 Karam N. Al Aryan 1
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 Fatima Kataya
 TABLE OF CONTENTS
1. Background information
1.1. What is glucose
1.2. Metabolism
1.3. Chemical structure
1.4. Normal general range in blood
1.5. Associated diseases

2. objectives & purposes& principle

3. Materials required
3.1. Laboratory equipments
3.2. Specimen used
3.3. Reagents used

4. Methodology
4.1. Usage of micropipette
4.2. Tubes
4.3. Incubation

5. Results
5.1. Calculating blood glucose levels
5.2. Spectrophotometer usage
5.3. Calculating the absorbance

6. Discussion and interpretation

6.1. what was the reason for the error
6.2. what is the importance of micropipettes?
6.3.

7. What other additional tests could be used

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 1. Background information
1.1.What is glucose
Glucose is the most important carbohydrate fuel in the body. In the fed state, the
majority of circulating glucose comes from the diet; in the
fasting state, gluconeogenesis and glycogenolysis maintain
glucose concentrations. Very little glucose is found in the
diet as glucose; most is found in more complex
carbohydrates that are broken down to monosaccharides
though the digestive process.

1.2. Metabolism:
Your body is designed to keep the level of glucose in your blood constant. Beta cells in your pancreas monitor
your blood sugar level every few seconds. When your blood glucose rises
after you eat, the beta cells release insulin into your bloodstream. Insulin
acts like a key, unlocking muscle, fat, and liver cells so glucose can get
inside them.

1.3. Chemical structure

Glucose is a simple sugar monosaccharide having two isoforms,
alpha and beta, with a chemical structure of C6H12O6

1.4. Normal ranges

The expected values for normal fasting blood glucose
concentration are between 70 mg/dL (3.9 mmol/L) and 100
mg/dL (5.6 mmol/L). When fasting blood glucose is between 100 to 125 mg/dL (5.6 to 6.9
mmol/L) changes in lifestyle and monitoring glycemia are recommended.

1.5. Associated diseases

In type 1 diabetes, your body doesn't have enough insulin. The immune system
attacks and destroys cells of the pancreas, where insulin is made.

In type 2 diabetes, the cells don't respond to insulin like they should. So the
pancreas needs to make more and more insulin to move glucose into the cells.
Eventually, the pancreas is damaged and can't make enough insulin to meet the
body's needs.

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 2. Purposes & objectives
2.1. Objective & Purposes

The purpose of this experiment is to determine the amount of glucose levels in human serum
with a quantitative calorimetric enzymatic method in vitro

2.2. test Principle

 Initially, glucose and glucose oxidase rapidly form an enzyme-substrate

complex, and the oxidation process takes place through a hydride transfer
from the anomeric carbon of the carbohydrate to the FAD co-factor (presented
in the glucose oxidase enzyme close to the active site) to generate FADH -.
Sequentially, this anionic form reacts with molecular oxygen to form a radical
pair (RP), which is converted into flavinhydroperoxide (FHPO) and then
releases hydrogen peroxide in the biological media.
 Secondly, an equivalent amount of phenol and 4-aminoantipyrine reacts in the
presence of four equivalents of hydrogen peroxide and peroxidase, resulting in
dye formation and four molecules of water
 The term peroxidase is related to a class of enzymes with potential to induce
oxidative processes by using hydrogen peroxide. two molecules of phenol are
transformed in the respective phenoxy radicals, first by action of π-cation
radical, which was generated from hydrogen peroxide and Por-Fe(III), and then
in the regeneration of the native enzymes. Subsequent reaction of the antipyril
radical, generated by action of the phenoxy radical, reacts with another
phenoxy radical to afford a pyrazolone derivative, which is converted into
quinoneimine dye by the action of hydrogen peroxide.

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 3. Materials required
3.1. Laboratory Equipments

 Test tubes
 Two Micropipettes
 Cuvettes
 Tube rack
 Spectrophotometer

3.2. Specimen used

 Serum heparinized

3.3. Reagents use

 Working reageant containing:
Enzymes:
- Glucose oxidase enzyme, GOD
- Peroxidase enzyme, POD
- 4-aminoantipyrine

Buffer reagent:

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 -Phosphate buffer reagent (ph= 7)

 Standard solution:

-Glucose solution of 100 mg/dl

4. Methodology
Steps to follow when using a micropipette:
 Select the volume and set the tip
 Press and hold the plunger at the first stop
 Place the tip in the liquid and slowly release the plunger
 Pose for a seconde and then move the tip and inserted into the delivery vessel
 Press the plunger to the second stop and pose for two seconds
 Remove the tip and release the plunger
 Inject the tip into the waste container

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 Tube 1: Blank Tube 2: standard

TO PREPARE THE TO PREPARE THE Tube 3: blood,serum

BLANK TUBE, 1000 ΜL STANDARD TUBE 1000 sample
OF WORKING REAGENT ΜL OF WORKING
WAS ADDED ONLY REAGENT+ 10ΜL OF TO PREPARE THE
STANDARD WERE SAMPLE TUBE
ADDED 1000ΜL OF
TABLE 1: Table showing all the tube content WORKING REAGENT
+10ΜL OF BLOOD,
WERE ADDED
Tube content Blank standard sample

1-working reagent 1000µl 1000µl 1000µl

2-standard - 10 µl -
3-blood,serum
- - 10µl
sample


Mix your tubes well
 Incubate all the tubes for 15
minutes at 37 degree Celsius
 After the incubation, the tubes were analyzed using a
spectrophotometer to measure the absorbance of each one of them at a
specific wavelength
 The blank absorbance was substracted from the sample
 Measure the absorbance of the sample
 Measure the absorbance of the standard
 Use the values of the absorbances calculated to calculate the blood sugar concentrations in the
sample tube, using a calibration curve based on the standard absorbance values
 Record your results

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  The results were interpreted according to the established guidelines for normal and abnormal blood
sugar levels.
 Any discrepancies or unexpected results were noted and further investigation was conducted as
needed.

!!! Procedure Spectrophotometric Technique

1. Plug in the spectrophotometer and turn it on by turning the left dial clockwise. Allow the
spectrophotometer to warm up at least 5 minutes before proceeding.

2. Set the wavelength dial to 600 nm. This is a wavelength of light that gets absorbed readily

3. With no vial in the spectrophotometer, the light path is closed. Therefore no light is transmitted, or in
other words there is an infinite absorption.

4. Now fill a cuvette (a small test tube that is 12 mm in diameter and 100 mm in length) with Blank 1.

5. Insert the cuvette containing Blank 1 into the sample chamber. As you do the light path will be opened.
This blank solution does not contain any Grape Kool-Aid, and so the absorbance should be set to zero.
Use the right knob to set the absorbance to zero.

6. Next fill a cuvette with Grape Kool-Aid test solution. Insert it into the spectrophotometer and record its
absorbance in question number one of your assignment.

5. Results
Calculations :

 Absorbance of the standard: 0,223 in 100mg/dl of glucose

 Absorbance of the sample : 0.117 in blood serum glucose ???

0.117× 100
Formula : 0.223 = 50 mg / dl of glucose in blood serum ( hypoglycemic)

 The amount of glucose obtained in the serum sample was less than the normal range : 75-
110mg/dl

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  The glucose concentration in the serum should’ve been more than 65 mg/dl, since that the serum
was obtained from man

 Our results showed that this man is hypoglycemic, which is false

 In the discussion/interpretation part we will discuss why, and what was the reason for this error
and false calculations

6. Discussion/ Interpretation of the result

Why was the amount of glucose calculated false? What is the error
that was done in the lab that has lead to a false result?
 Adjusting the volume on micropipettes

1. During extraction of the human blood serum, we might have not correctly extracted the
right amount of volume which is 10µl
2. We might have not released the thumb knob correctly.
3. We might have not released slowly the serum collected in our tube, which will lead to
bubbles formation, hence falsifying our results

Our results showed 50 mg/dl of glucose in the human serum, however the right answer should be
within the normal range.

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 Why did we use micropipettes not normal pipettes?
Micropipettes are the most important and latest technical tool for the laboratories. There are so many
different types of laboratories and in all of them people have to work with liquids and semi liquids. For
getting perfect volume of them they need something which can carry the material in perfect volume and
can release it at any required place.

Why did we use spectrophotometry for blood glucose levels?

The spectrophotometer measures absorbance. Absorbance values, by themselves, do not describe the
concentration of a substance. However, we can
determine the concentration of a substance in a
solution using a standard curve. A standard
curve translates absorbance values into
concentration.. We can construct a standard
curve by making solutions with a known
concentration of the substance we are
measuring and then measuring their
absorbance. Graphing the concentration on the
x-axis and the absorbance on the y-axis, we
can see that there is a linear relationship
between concentration and absorbance. Thus a
standard curve is not really a curve, but a
straight line. Beers Law describes this linear relationship:
Using the standard curve, we can determine the concentration of other solutions, by locating the
absorbance of that solution on the y-axis and drawing a horizontal line to the standard curve line. Then
you can draw a vertical line from that intersection to the x-axis to determine the concentration.

What is the importance of the enzymatic essay for blood glucose

determination?
This method is highly specific, the enzymatic glucose analyses are based on the two methods most
commonly used and employes in clinical chemistry, the hexokinase method, and the glucose
oxidase method

7. what other tests could be used to

determine the blood glucose levels?

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