The University of the West Indies

St. Augustine
Practical and Information Manual for courses
MDSC 1001, MDSC 1101, MDSC 1102, OPTM 1062

Academic Year:
(2016-2017)

BIOCHEMISTRY UNIT
DEPARTMENT OF PRECLINICAL SCIENCES

FACULTY OF MEDICAL SCIENCE

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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

BIOCHEMISTRY UNIT
DEPARTMENT OF PRECLINICAL SCIENCES
FACULTY OF MEDICAL SCIENCE
Date (dd/mm/yy)

Course

Student Name

Student ID number

Experiment number :

Experiment name

Marks Obtained

Comments

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CONTENTS

PREFACE

INTRODUCTORY REMARKS

SAFETY AND LABORATORY RULES

EXPERIMENT 1

LABORATORY TECHNIQUES

12

EXPERIMENT 2

REACTIONS OF AMINO ACIDS

AND PROTEINS

19

EXPERIMENT 3

DEMONSTRATION OF ENZYME SPECIFICITY

WITH GLUCOSE OXIDASE AND PEROXIDASE
AND THE DETERMINATION OF BLOOD GLUCOSE 24
THE CLASSIFICATION OF SUGARS AND THE
DETERMINATION OF GLUCOSE IN URINE

30
EXPERIMENT 4
REFERENCES

CHOLESTEROL DETERMINATION

33
38

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PREFACE
Biochemistry will be taught by PBL as part of an integrated basic science
programme which is conducted over a period of two years. There will be
one of these PBL sessions per week each one lasting about three hours. In
addition to this integrated approach, the Biochemistry Unit will conduct
teaching by the more traditional methods of lectures, practicals and
tutorials.
Lectures
There will be approximately eighty-five Biochemistry lectures over the
course of the first two years. The number varies from one to four per week
depending on the Block.
Practical
The Unit will aim to conduct four Biochemistry practicals (two per
Semester). For all practical sessions the class will be divided into groups
depending on the size of the class and each group will have a session
about once every four weeks. Group assignments and the list of
experiments to be done will be posted on the class bulletin board and on
Myelearning at the beginning of each semester.
When no practicals are being conducted the Unit may utilize these sessions
for small group Biochemistry tutorials.
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Examinations
Laboratory

practical

assignments

will

contribute

towards

students

continuous assessment marks. Biochemistry content will also be examined

jointly with other disciplines of Basic Sciences as part of the integrated
Problem Based Learning Program (continuous assessment and final exams).
Biochemistry questions may be included in spotter examinations.

INTRODUCTORY REMARKS
This booklet contains the protocols for the practical course, the revised
course outline and the Biochemistry booklist.
Practicals
The aims of the practical are threefold:
(i)

to reinforce concepts introduced in the lecture course

(ii)

to introduce biochemical concepts best taught in a practical

setting
(iii) to introduce common biochemical techniques
As a consequence all students (refer to note on Exemptions) are expected
to perform all of the experiments as designated by the Biochemistry Unit
Coordinator.
Laboratory note books

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Before the first practical session all students must possess a hard cover
laboratory notebook and a permanent marker (for labeling). Please note
that you will only be doing four experiments so you do not require a very
thick book.
All results and working must be entered directly into your laboratory
notebook in pen (no pencils). Recordings made on scraps of paper or loose
sheets will not be accepted. Please ensure that the page containing your
results receives the Biochemistry Units stamp at the end of each practical
session. (This will be done by the demonstrator; it is your responsibility to
take your book to him/her).
Students will work in pairs but for each experiment each partner is
expected to provide a full practical report and to answer all of the questions
in the treatment of results and write-up section associated with each
practical exercise.
Laboratory practical reports
Students will be required to submit short reports for marking. You will be
expected to present and process your results, and answer relevant
questions as outlined unde r treatment of results and write-up section
found at the end of the description of each procedure. Reports must
consist of (i) a cover page (page two of this manual), (ii) a copy
of your signed and stamped results and (iii) the answers to the
questions given at the end of each lab.
The deadline for submission of each report will be advertised. Plagiarism
and other forms of cheating are treated very harshly; whether the source of
materials is the Internet or a colleagues report. If you are discovered plagiarizing you
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will lose marks. For information regarding the university regulations on plagiarism
please

refer

to

http://sta.uwi.edu/resources/documents/Exam_Regulations_Plagiarism.pdf.
Preparation
Study the experimental procedures completely before your laboratory
session. You will be required to present flow charts and tables (for
recording your results), for each of the practicals, at the beginning
of the laboratory session. Try to understand what you are doing, and the
rationale for the experiment. Designing the flow charts and tables prior to
the laboratory session ensures that you study and think about the various
aspects of the experiment before you perform it.

Flow charts are

diagrammatic representations of the sequence of operations or steps that is

required to conduct the experiments.

Typically, they employ the use of

simple shapes (squares, rectangles, circle etc.) connected by arrows or lines.

Within these shapes the action steps are briefly stated. Demonstrators will
be in the lab to help you, so ask questions when you are unsure.
Code of behaviour
Some of the equipment in the laboratory is sophisticated and delicate. Also some pieces of
equipment and other materials used are potentially lethal if not handled properly.
Students must never:
(i)

Enter the lab in the absence of a lecturer, technician or demonstrator.

(ii)

Attempt to use a piece of equipment until they are familiar with its mode of
operation.

(iii)

Attempt to use any equipment when it is not directly related to the experiment being
conducted.

(iv)

Play in the lab.

(v)

Eat/drink/smoke the lab.

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Students must:
(i)

Report all accidents immediately all minor spills should be cleaned up

immediately and major spills should be reported to the laboratory supervisor.

(ii)

Report all broken or faulty equipment (including glassware).

(iii)

Dispose of broken glassware, syringes and needles into specified bins.

(iv)

Always wear long sleeved lab coats in the lab.

(v)

Wear safety glasses while working in the laboratory (not provided).

(vi)

Wear shoes that cover the entire foot.

STUDENTS WOULD NOT BE ALLOWED IN THE LABORATORY IF THEY

DO NOT HAVE A LAB COAT OR PROPER FOOTWEAR!

SAFETY AND LABORATORY RULES

One of the first things any scientist learns is that working in the laboratory can be an
exciting experience. However, the laboratory can also be quite dangerous if proper
safety rules are not followed at all times. In order to prepare yourself for a safe year in
the laboratory, read over and adhere to the following safety rules.
A.

Dress Code
1. Laboratory coats should also be worn in the laboratory at all times.
2. Tie back long hair in order to keep it away from any chemicals, burners,
and candles, or other laboratory equipment.
3. Any article of clothing or jewelry that can hang down and touch chemicals
and flames should be removed or tied back before working in the
laboratory. Sleeves should be rolled up.
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4. Sandals, opened toe shoes are not allowed in the laboratory.

5. Some experiments may require that you wear safety goggles (safety
goggles will not be provided).
B.

General Safety Rules

1. Read all directions for an experiment several times. Follow the directions
exactly as they are written. If you are in doubt about any part of the
experiment, ask the lecturer or the demonstrator for assistance.
2. Never perform activities that are not authorized by the lecturer or
demonstrator.
3. Never handle any equipment unless you have specific permission.
4. Take extreme care not to spill any material in the laboratory. If spills occur,
ask the lecturer or demonstrator immediately about the proper clean-up
procedure. Never simply pour chemicals or other substances into the sink
or trash container.
5. Never eat or drink in the laboratory. Wash your hands before and after
each experiment.
6. There should be no loud talking or horseplay in the laboratory.
7. When performing a lab, make sure the work area has been cleared of
purses, books, jackets, etc.
8. Know the location and use of all safety equipment (goggles, aprons,
eyewash, fire blanket, fire extinguishers, etc.)
9. Read your assignment before coming to class and be aware of all safety
precautions. Follow directions.
10. Never work alone in the lab.

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C.

Heating and Fire Safety

1. Again, never use any heat source such as a candle or burner without
wearing safety goggles.
2. Never heat any chemical that you are not instructed to heat. A chemical
that is harmless when cool can be dangerous when heated.
3. Always maintain a clean work area and keep all materials away from
flames. Never leave a flame unattended.
4. Never reach across a flame.
5. Make sure you know how to light a Bunsen burner. (Your lecturer will
demonstrate the proper procedure for lighting a burner.) If the flame leaps
out of a burner towards you, turn the gas off immediately. Do not touch the
burner as it may be hot.
6. Always point a test tube that is being heated away from you and others.
Chemicals can splash or boil out of a heated test tube.
7. Never heat a liquid in a closed container. The expanding gases produced
may blow the container apart, injuring you or others.
8. Never pick up any container that has been heated without first holding the
back of your hand near it. If you can feel the heat on the back of your
hand, the container may be too hot to handle. Always use a clamp or
tongs when handling hot containers.

D.

Using Chemicals Safely

1. Never mix chemicals for the "fun of it." You might produce a dangerous,
possibly explosive substance. No unauthorized experiments should be
performed.
2. Never touch, taste, or smell any chemical (unless instructed by lecturer).
Many chemicals are poisonous. If you are instructed to note the fumes in
an experiment, always gently wave your hand over the opening of a
container and direct the fumes toward your nose. Do not inhale the fumes
directly from the container.
3. Use only those chemicals needed in the activity. Keep all lids closed when
a chemical is not being used. Notify the lecturer or demonstrator when
chemicals are spilled.
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4. Dispose of all chemicals as instructed by your lecturer.

5. Be extra careful when working with acids or bases. Pour such chemicals
over the sink, not over your work bench.
6. When diluting an acid, always pour the acid into water. Never pour water
into the acid.
7. Rinse any acids off your skin or clothing with water. Immediately notify the
lecturer or demonstrator of any acid spill.
8. Never pipette by mouth.
9. Be sure you use the correct chemical. Read the label twice.
10. Do not return any excess back to the reagent bottle.
11. Do not contaminate the chemical supply.
12. Keep combustible materials away from open flames (alcohol, carbon
disulfide, and acetone are combustible).
13. Do NOT use the same spatula to remove chemicals from two different
containers. Each container should have a different spatula.
14. When you remove the stopper from a bottle, do NOT lay it down on the
desk, but place the stopper between your two fingers and hold the bottle
so the label is in the palm of your hand so drips won't ruin the label, etc.
Both the bottle and the stopper will be held in one hand. Be sure and rinse
any drips that might have gotten on the outside of the bottle.
15. Be careful not to interchange stoppers from two different containers
16. Replace all stoppers and caps on the bottle as soon as you finish using it.
17. Mercury spills must be cleaned up immediately. Alert the lecturer or
demonstrator if there is a spill. DO NOT touch the mercury.
E.

Using Glassware Safely

1. Glass tubing should never be forced into a rubber stopper. A turning
motion and lubricant will be helpful when inserting glass tubing into rubber
stoppers or rubber tubing. Your lecturer will demonstrate the proper way to
insert glass tubing.
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2. When heating glassware, use a wire or ceramic screen to protect

glassware from the flame of a Bunsen burner.
3. If you are instructed to cut glass tubing, always fire-polish the ends
immediately to remove sharp edges.
4. Never use broken or chipped glassware. If glassware breaks, notify the
lecturer or demonstrator and dispose of the glassware in the proper
container.
5. Always thoroughly clean glassware before putting it away.
F.

Using Sharp Instruments

1. Handle scalpels or razor blades with extreme care. Never cut any material
towards you: always cut away from you.
2. Notify your lecturer or demonstrator immediately if you are cut in the
laboratory.

G.

Electrical Equipment Rules

1. Batteries should never be intentionally shorted. Severe burns can be
caused by the heat generated in a bare copper wire placed directly across
the battery terminals. If a mercury type dry cell is shorted, an explosion
can result.
2. Turn off all power when setting up circuits or repairing electrical
equipment.
3. Never use such metal articles as metal rulers, metal pencils or pens, nor
wear rings, metal watchbands, bracelets, etc. when doing electrical work.
4. When disconnecting a piece of electrical equipment, pull the plug and not
the wire.
5. Use caution in handling electrical equipment which has been in use and
has been disconnected. The equipment may still be hot enough to
produce a serious burn.
6. Never connect, disconnect, or operate a piece of electrical equipment with
wet hands or while standing on a wet floor.

H.

End-of-Experiment Rules
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1. When an experiment is completed, always clean up your work area and

return all equipment to its proper place.
2. Wash your hands after every experiment.
3. Make sure all candles and burners are turned off before leaving the
laboratory. Check that the gas line leading to the burner is off as well.
I.

Other Safety Rules

1. Do not use hair spray or hair mousse during or even before coming to
laboratory class. These are highly flammable and might cause automatic
ignition when in close proximity to a heat source.

(http://www.sanbenito.k12.tx.us/teachers/science_safety/Safety_And_Lab_Rules.html)

EXPERIMENT 1
LABORATORY TECHNIQUES
The aim of this experiment is to allow students to become familiar with some basic
operations that are routinely performed in a biochemistry laboratory. The procedures
include centrifugation, volumetric measurements, dilutions, spectrophotometry, and pH
measurement. The concept of buffer action is also introduced.
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A.

Preparation of Mitochondria from Calf Liver

In biochemistry is it often necessary to isolate specific cell organelles for
investigation of particular biomolecules, enzymes, metabolic pathways etc. Such
separation or fractionation of cell components is accomplished by differential
centrifugation after the cells have been broken, by subjecting the tissue to one of
several cell-rupturing procedures.
PROCEDURE
A 10% (w/v) liver homogenate in 0.25M sucrose, 0.001M EDTA, pH7.2 has been
prepared for you.

i.

Obtain approximately 20 mL of homogenate and add equal amounts into 2

plastic tubes for centrifugation. Make sure that the tubes are balanced and counter
poised when placed in the centrifuge.

ii.

Centrifuge at 2300 rpm (750g) for 10 min. Carefully remove the tubes at
the end of the spin and immediately pour off the supernatants (S 1) into 2 new centrifuge
tubes. Do not transfer any of the loose fluffy layer on top of the pellet (P 1).

iii.

Balance the tubes as in (i), above. You may use some sucrose medium to
add weight to one of the tubes. Centrifuge at 5400rpm (4000g) for 10 min. Pour off the
supernatants (S2) into a single test tube and save. Note the appearance of the pellets
(P2) i.e. crude mitochondria.

iv.

Add 8.0 mL of cold sucrose to each of the P 2 pellets and resuspend with a
glass rod. Balance the tubes and centrifuge at 5400rpm (4000g) for 10 min. discard
the supernatants (S3). Note the appearance of the pellets (P3) i.e. mitochondria.
N.B.
Cleaner mitochondria may be obtained by further resuspensions and recentrifugation.
B. Spectrophotometry: Beer-Lambert Law.
The measurement of absorbance is the final step in many quantitative
determinations in the laboratory. The compound p-nitrophenol, in the dissociated
form i.e. alkaline conditions, absorbs in the visible range of the EM spectrum with
an absorption maximum centered at 405nm (max).
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PROCEDURE
i.
Collect approximately 10 mL of solution N, a 0.30 mM p-nitrophenol
solution and prepare a stock solution (O). To prepare O: Take 8 mL of
solution N and make up the volume to 40 mL using 0.02M NaOH.
ii.

Using your stock solution O, prepare the dilutions X2 to X5 below

using graduated cylinders and repeat the dilutions using automatic
pipettes. Prepare between 2 to 5 mL of each dilution.
a.
b.
c.
d.

2: a one in two dilution i.e. -1 part O: 1 part 0.02M NaOH

3: one in three dilution i.e. -1 part O: 2 parts 0.02M NaOH
4: one in four dilution i.e. - 1 part O: 3 parts 0.02M NaOH
5: one in five dilution i.e. - 1 part O: 4 parts 0.02M NaOH

iii.

Zero the spectrophotometer at 405nm using 0.02 M NaOH as the

blank.

iv.

Record the absorbances for the 2 sets of dilutions and for solution
O.

C. Buffer Action
In biological systems constant pH is essential for most cellular processes. The
maintenance of this narrow range of pH is accomplished by buffer systems,
which resist the changes in pH that would otherwise occur in metabolism. A
buffer solution is characterized by the pK value of the weak acid (base) and the
ratio of conjugate base to acid as shown by the Henderson-Hasselbalch
equation.
conjugate base
pH = pKa + log10 conjugate acid
PROCEDURE
i.

Use the 4 vials supplied as follows:

Vials 1 and 2 containing 10 mL each of distilled H 2O and vials 3 and 4
containing 10 mL each of any one of solutions A, B, C, or D ( these are
already measured for you) to be tested for buffering capacity.
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Draw a table as shown and use it to record your results.

Vial
1
2
3
4

pH Before

pH After

ii.

Measure the pH of vials 1 and 2. Record. Add 0.1 mL of 0.2M HCl (to vial
1), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to vial 2, mix well, and record
the pH.

iii.

Measure the pH of vial 3 and 4. Record. Add 0.1 mL of 0.2 M HCl (to vial
3), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to vial 4, mix well, and record
the pH.

iv.

Obtain results for the three other solutions from other members of the
class (eg. if your solution was A, obtain results for solutions B, C and D).
The compositions of the test solutions are:
A.
500mL of 0.1M NaH2PO4 added to 500 mL of 0.1M Na2HPO4.
B.
500mL of 0.1M HCl added to 500 mL of 0.2M Na2HPO4.
C.
50mL of 0.1M acetic acid added to 950mL of 0.2M sodium acetate.
D.
50mL of 0.1M NaH2PO4 added to 50mL of 0.1M Na2HPO4 and
diluted
to 1L with H2O.
TREATMENT OF RESULTS AND WRITE-UP
(1)

(25 marks)

What is the role of 0.25M sucrose as the medium for the fractionation process?
(2 marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
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(2)

List the major components that are present in (a) pellet P 1, and (b) supernatant
S2.

(2 marks)

(a)______________________________________________________________
(b)______________________________________________________________

(3)

(i)

Define the Beer-Lambert Law.

(2 marks)

________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

(ii)

Briefly explain why Absorbance has no units.

(1 mark)

________________________________________________________________
________________________________________________________________

(4)

The molar extinction coefficient of p-nitrophenol at 405nm is 18.810 3 Lmol1

cm-1. Use this value to calculate the concentration of solution O. (show your

calculations in the box below).

(3 marks)
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(5)

Plot a graph of absorbance vs. concentration for both sets of dilutions (use the
concentrations calculated from your dilutions, do not use the concentrations
obtained by using Beer-Lambert law). Attach the graph to your report.
(4

marks)

Which dilution appears to be more accurate? Comment on the spread of values.

(2
marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
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(6)

(i) Tabulate the data for the pH measurements for all solutions in the space
below.

(2

marks)

(ii) State whether each of the test solutions did or did not exhibit buffering
capacity.

(2 marks)

________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

(iii) Explain your observations based on the composition of the solutions.

(2 marks)
________________________________________________________________
________________________________________________________________
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________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

(7)

List the major buffer systems in the blood of mammals and show the equations
for each system?

(3 marks)

________________________________________________________________
________________________________________________________________
________________________________________________________________

EXPERIMENT 2
REACTIONS OF AMINO ACIDS AND PROTEINS
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This experiment illustrates some of the general reactions of amino acids and proteins
which are important in the study of protein chemistry.
(A)

Ninhydrin Reaction
The most common means of detecting amino acids is by reaction with Ninhydrin
(triketohydrindene hydrate). Ninhydrin, which is a very strong oxidizing agent,
reacts with amino acids, to decarboxylate the amino acid, producing a bluepurple compound, CO2, H20 and an aldehyde. Proline gives a yellow colour.
Under controlled conditions, the reaction is used for quantitative estimation of
amino acids, but is not as sensitive as newer methods that yield fluorescent
products.

PROCEDURE
Place 1 mL of neutral glycine solution (1g/L) in a test tube. Prepare six (6) 50%
serial dilutions of the neutral glycine solution (1g/L). Add five drops of Ninhydrin
solution #1 to all tubes. Place all tubes in a boiling water bath for 5 minutes to
develop the blue-purple reaction complex. Determine the approximate limit of
detection of this qualitative test.
For the quantitative test, pipette 0.70 mL each of 0.5mM solutions of glycine,
tyrosine, and proline in separate test tubes. Also set up a blank tube with 0.70mL
of distilled H2O. Add 0.8 mL of Ninhydrin solution #2, mix well, and place the
tubes in a boiling water bath for 15 minutes.
Cool the tubes and add 4mL of 50 % aqueous n-propanol to each tube. Mix and
leave at room temperature for 10 minutes. Read the absorbance of the amino
solutions against the blank at 570nm and at 440nm for proline (you will have to
blank the instrument at 570nm and 440nm).
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(B)

Quantitative Determination of Protein

The Folin-Lowry assay is very sensitive and it is one of the most commonly used
procedures for determining protein concentration. The protein solution is first
heated with alkaline copper solution. Folin-Ciocalteus reagent contains
phosphotungstic and phosphomolybdic acids and produces a blue-green colour,
the intensity of which depends on the tyrosine and tryptophan residues in the
protein.
PROCEDURE
i. Prepare a standard curve using 0.2, 0.4, 0.6, 0.8 and 1.0 mL of the
standard protein solution (20mg/100mL). Make up all volumes to
1.0mL with distilled H2O. Set up a blank tube with 1.0mL of H 2O.
Into separate tubes pipette 1.0mL each of the two unknowns.
ii. Mix (just before using) 50mL of Reagent X (2% Na 2CO3 in 0.1M
NaOH) with 1.0mL of Reagent Y (0.5% CuSO 4 in 1% sodium
citrate).
iii. Add 4 mL of the prepared solution to all tubes, mix and leave at
room temperature for exactly 10 minutes.
iv. Add 0.5 mL of Folin-Ciocalteus reagent to all tubes, mix well, and
leave standing at room temperature for 30 minutes.
v. Read the absorbances of all tubes against the blank at 750nm.

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(C)

Protein Precipitation and Denaturation

A wide variety of chemicals, including certain organic acids, concentrated neutral
salts, heavy metal ions and certain organic solvents, can precipitate proteins. In
many cases, the precipitation results in irreversible denaturation of the protein.

PROCEDURE
i.

With a small measuring cylinder, put approximately 3 mL of protein

solution (5mg/mL) into 4 test tubes. To each add only one of the following:

a.

A few drops of 0.1M CuSO4.

b.

2mL of 10% trichloroacetic acid.

c.

2mL of saturated (NH4)2SO4 solution.

d.

10mL of cold ethanol.

ii.

Place the tubes, except the one with ethanol, in a boiling water bath
for 5 minutes. Note the results.

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(20

TREATMENT AND WRITE UP OF RESULTS

marks)
(1)

Was there any significant difference in the absorbance values for

glycine and tyrosine in the quantitative Ninhydrin test?
(1 mark)
________________________________________________________________
Explain.

(3 marks)

________________________________________________________________
________________________________________________________________
________________________________________________________________

(2)

State an important use of the Ninhydrin test in a clinical or research laboratory,

other than quantification.

(2 marks)

________________________________________________________________
________________________________________________________________

(3)

Plot a graph of your standard curve for the Folin-Lowry assay. Determine the
concentration of your two unknowns in mg/mL.

Attach the graph to your

report.
(6 marks)
________________________________________________________________
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________________________________________________________________
________________________________________________________________
________________________________________________________________

(4)

Define what is meant by the denaturation of proteins, and state any two
(4 marks)

denaturing agents commonly used in the laboratory.

________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

(5)

Although (NH4)2SO4 precipitates protein, at lower concentrations of the salt

protein solubility is increased. Explain this phenomenon.

(4 marks)

________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

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EXPERIMENT 3
(A) DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE
OXIDASE AND PEROXIDASE AND THE DETERMINATION OF BLOOD
GLUCOSE LEVELS.
(B) THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF
GLUCOSE LEVELS IN URINE.
(A)

DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE

OXIDASE AND

PEROXIDASE

AND

THE

DETERMINATION

OF

BLOOD GLUCOSE LEVELS.

Glucose oxidase specifically oxidizes -D-glucopyranose to the lactone of
gluconic

acid in the presence of oxygen.

Glucose oxidase

-D-Glucose + H2O + O2

D-gluconic Acid + H2O2

When the enzyme peroxidase is included in the reaction mixture, the

peroxidase substrate can be oxidized by the "indicator reaction". The oxygen
liberated oxidizes a weakly coloured hydrogen donor DH2 (O-dianisidine) to a
coloured derivative, D.
H2O2 + DH2

Peroxidase

2H2O + D

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The conditions of this reaction are so arranged that the oxidation of

glucose go to

completion. Thus, by using these coupled enzyme reactions,

the amount of the

easily measured compound, the dye, can be used to

quantify the amount of D-

glucose oxidized, by direct proportionality. The

absorption spectrum of the dye

formed from o-dianisidine has a broad

peak centered around 450 nm. In this

experiment

glucose in blood will be determined. Also the

the

concentration

of

specificity of the enzyme will

be demonstrated.
Clinical significance of blood glucose
Normal ranges (plasma):

Fasting glucose:

60-100mg%

Post-prandial glucose:

90-140 mg%

Random glucose:

90-150 mg%

The increase in blood glucose is called as hyperglycemia.

The blood glucose level is increased in uncontrolled diabetes mellitus.
The increased levels are also seen due to hyper-functions of anterior
pituitary and

adrenal cortex.

In hyperthyroidism, fasting blood sugar level may be normal but there

is a

pronounced hyperglycemia in the fed state.

The decrease in blood glucose is referred to as hypoglycemia (< 40

mg %).
This condition is seen in insulin secreting tumors of the beta cells of
pancreas.
Occasionally it is encountered in renal diabetes.
Over dosage Insulin also causes hypoglycemia
PROCEDURE
The enzyme cocktail consist of:
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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

Glucose oxidase

12.5 mg

Peroxidase

4.0 mg

O-dianisidine dihydrochloride (1% in ethanol)

0.5 mL

Add 0.5 M sodium phosphate buffer pH 7.2 to make

100 mL

Treatment of the blood sample:

Pipette 0.1 mL of blood into a centrifuge tube containing 1.5 mL distilled
water. Add 0.2 mL Ba(OH)2 solution (4.7%), mix well and then add 0.2 mL
ZnSO4 solution (5%). Prepare a reagent blank by pipetting 1.6 mL water
into a centrifuge tube.

Add 0.2 mL Ba(OH) 2 solution (4.7%), mix well

and add 0.2 mL ZnSO4 solution (5%).

Mix thoroughly and centrifuge for 5 minutes at 500g. The supernatant
should be

clear.

Assay
Perform the assay according to the following table.
Reagents
Glucose
(0.3 mM)
Supernata
nt
Lactose
(0.3 mM)
Fructose
(0.3 mM)
Reagent
blank
Distilled
water
Enzyme
cocktail

Tube (mL)
S4
S5
S6
0.3 0.4 0.5

B
-

S1
0.0

S2
0.1

S3
0.2

T1
-

T2
-

L
-

F
-

0.3

0.3

0.5

0.5

0.3

0.2

0.5

0.4

0.3

0.2

0.1

0.2

0.2

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

Mix and incubate for 45 minutes

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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

Mix and read the absorbance at 450 nm

B = Blank T = Test, S = standard, L = Lactose,

Fructose
Pipette 0.3 mL of supernatant to a clean dry semi-micro test tube and
add 0.2 mL distilled water (prepare in duplicate; T1 and T2).
standard curve by

pipetting 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the

0.3mM glucose solution

solution) into

Prepare a

provided (do not use the 0.1M glucose

clean dry semi-micro test

tubes. Add distilled water to

make the final volume in each tube 0.5 mL. Pipette

0.5 mL of lactose and

fructose into separate semi-micro test tubes. Add 2.0 mL

enzyme

cocktail

to each of the eleven (11) tubes and incubate at 37C for 45 minutes. Mix by
vortex and read the absorbance at 450 nm.
Plot the standard curve of absorbance (y-axis) versus concentration of
glucose in mmol (x-axis).
concentration of glucose

Use the standard graph to determine the

in your sample of blood in mmol/L then express

your results in mg/dL.

(20 marks)

TREATMENT OF RESULTS AND WRITE UP

(1)

Plot the standard curve of absorbance (y-axis) versus concentration of

glucose in mmoL (x-axis). Attach the graph to your report.

(6.0

marks)

(2)

Use the standard graph to determine the concentration of glucose in your sample
of blood in mmol/l then express your results in mg/dL.

Show

your

30
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

calculations in

the box below.

(4.0 marks)

(3)

Name the two enzymatic methods which are commonly used in laboratories

to

determine blood glucose.

(1.0 mark)

________________________________________________________________
________________________________________________________________

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Biochemistry Unit
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2015-2016

(4)

What are the two advantages of enzymatic methods used to analyse

blood glucose?

(1.0

mark)
________________________________________________________________
________________________________________________________________
Briefly describe the purpose of sodium fluoride in blood collection
tubes, where

glucose measurements are to be done.

(1.0 mark)
________________________________________________________________
________________________________________________________________
________________________________________________________________

(5)

Which hormone is deficient in diabetes mellitus?

(0.5 marks)

_________________________________________________________________
List three mechanisms by which this hormone regulates blood glucose.
(1.5 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
(6)

What is glycated hemoglobin (HbA1c)?

(1

mark)
_________________________________________________________________
32
Biochemistry Unit
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2015-2016

_________________________________________________________________

Briefly describe the clinical importance of HbA1c.

(3

marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

(7)

List 4 clinical symptoms that may occur if hypoglycemia is not treated?

(1 mark)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

33
Biochemistry Unit
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2015-2016

(B)

The Classification of Sugars and the Determination of Glucose

in Urine.
Carbohydrates may be classified as either reducing or non-reducing sugars.
The reducing sugars have a free aldehyde or ketone group or a potentially free
aldehyde group as in the hemi-acetal forms present in the cyclic molecule.
These monosaccharides reduce alkaline solutions of copper (Cu2+), with the
formation of a coloured (usually brick-red) precipitate of cuprous oxide (Cu2O).
Benedict's solution contains cupric sulphate (CuSO4), sodium carbonate
(Na2CO3) and sodium citrate.
The Benedict's

test is utilized in the detection and quantitation of

monosaccharides, such as glucose, in biological fluids e.g. urine. You may recall
that patients suffering with diabetes mellitus have abnormally high blood
glucose levels, which may be detected in their urine.
PROCEDURE
Carry out Benedict's test on 0.1 M solutions of the glucose, fructose, lactose
and sucrose solutions provided. Examine the sensitivity of the test, in the case
of glucose, by diluting the 0.1 M glucose solution provided to give solutions of
concentrations 0.05 M, 0.025 M, 0.01 M, 0.001 M and 0.0001 M. Carry out
Benedict's test on each of these diluted solutions. Obtain a sample of urine and
carry out the Benedict's test. Do not dilute the urine sample.
The Benedict's test

34
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

Pipette 2.0mL of Benedict's reagent into a test tube. Add 2.0mL of the test
solution mix well and place in a boiling water bath for 5 minutes. Let the tube
cool slowly (do not place under running water). A green, red or yellow
precipitate is indicative of a positive reaction.

TREATMENT OF RESULTS AND WRITE UP

(15

marks)
(1)

Briefly explain your results.

(7.0 marks)

_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

(2)

Name the non-reducing sugar which does not give a positive Benedicts test and
explain why?

(2.0 marks)

_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
35
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

(3)

(2.0 marks)

What is renal glycosuria and what are its causes?

_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

(4)

(1.0 mark)

What is the renal threshold value for glucose?

______________________________________________________________
______________________________________________________________

(5)

What is the significance of performing oral glucose tolerance tests?

(2.0 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

36
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

(6)

(1.0 mark)

What is gestational diabetes?

_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

EXPERIMENT 4
Serum Lipids
Lipids are a very diverse group of biomolecules that carry out a wide range of
bodily function. They are insoluble in water and consequently need to be
transported in plasma in association with various lipoprotein particles.
Plasma lipoproteins can be separated into five major classes: chylomicrons,
very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL),
low-density lipoproteins (LDL), and high-density lipoproteins (HDL).

Each

lipoprotein has specific roles in lipid metabolism and contains different

amounts of cholesterol, cholesterol esters, triglycerides, phospholipids and
fatty acids.

Abnormal proportions of lipoproteins in blood are usually

indicative of disease conditions due to altered metabolism.

In this experiment total cholesterol, triglycerides and HDL cholesterol will be
measured in serum samples using commercially available enzymatic
preparations.

37
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

PROCEDURE
Reagents provided:
1.

Cholesterol standard (200 mg/dL) and enzyme cocktail

2.

Triglycerides standard(200 mg/dL) and enzyme cocktail

3.

HDL standard (50 mg/dL) and enzyme cocktail

4.

Two serum samples A, B, C or D

Preparation of serum (this will be done by a demonstrator)
Obtain 5 mL of whole blood each from 2 members of the class. Allow

the blood to

clot for about 15 minutes in a centrifuge tube. Burst the

fibrin clot and centrifuge

at 6000 r.p.m. for 5 minutes, then remove the

serum (supernatant) with a Pasteur

pipette.

This serum will then be

suitably diluted for use in the assays.

Assay
Measure total cholesterol, triglycerides and HDL-cholesterol in the two
samples given and in the serum sample using following procedure.
Reagents
Blank
Water
100L
Standard/sa -

Standard
100L

Sample X
100L

mple
Enzyme

1 mL

1 mL

1 mL

cocktail
Vortex and incubate for 10 min at room temperature
Read absorbances at 500nm against the blank
The amount (mg/dL) of total cholesterol, triglycerides and HDLcholesterol in each sample can be calculated based on the change in
absorbance and the concentration of analyte in the standard.
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Biochemistry Unit
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2015-2016

Sample calculation:

sample

A sample
A standard

standard =

TREATMENT OF RESULTS AND WRITE UP

mg/dL

(20

marks)
1.

Tabulate your results and calculate the serum concentration of

cholesterol, triglycerides and HDL-cholesterol in your samples. Show
your working in the space below.
(6 marks)

39
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

2.

State the Friedewald Formula and use it to estimate the concentrations

of VLDL

and LDL in your samples.

below.

(3 marks)

Show your working in the space

40
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

3.

List the lipoproteins which constitute a lipid profile and briefly discuss

the clinical significance of the profile.

(4 marks)

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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

4.

List any four clinical conditions in which you may find high blood

cholesterol.
(2 marks)
_________________________________________________________________
_________________________________________________________________
_________________________________________________________________

42
Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016

5.

Why is the total cholesterol/HDL ratio considered an atherogenic index?

(3 marks)

6.

Which of your samples could be considered atherogenic.

(2

marks)
________________________________________________________________
________________________________________________________________
________________________________________________________________
REFERENCES:
1.

Burtis, CA and Bruns DE. 2014. Tietz Fundamentals of Clinical

Chemistry and
2.

Molecular Diagnostics 7th edition, Saunders, USA

Nayak, S. 2013. Manipal manual of Clinical Biochemistry, 4thedition, Jaypee

Medical publishers.
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Biochemistry Unit
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2015-2016

3.

Gaw A, Murphy MJ, Srivastava R, Cowan RA and OReilly D St. J. 2013.

Clinical

Biochemistry:

An

Illustrated

Colour

Text,

Churchill Livingstone, New York

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Biochemistry Unit
Dept. of Preclinical Sciences
2015-2016