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BIOCHEMISTRY UNIT

Practical and Information Manual

For Medical Sciences Students

ACADEMIC YEAR

2012/2013

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BIOCHEMISTRY UNIT LABORATORY PRACTICALS FOR MEDICAL SCIENCES PHASE I

2012/2013

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CONTENTS PREFACE 4

INTRODUCTORY REMARKS 5

SAFETY AND LABORATORY RULES 7

EXPERIMENT 1 LABORATORY TECHNIQUES 11

EXPERIMENT 2 REACTIONS OF AMINO ACIDS AND PROTEINS 1

EXPERIMENT 3 (A) THIN LAYER CHROMATOGRAPHY OF SERUM LIPIDS 18 (B) SERUM PROTEIN ELCTROPHORESIS (DEMO) 22

EXPERIMENT 4

(A) DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE OXIDASE AND PEROXIDASE AND THE DETERMINATION OF BLOOD GLUCOSE 26

(B) THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF

GLUCOSE IN URINE 30 EXPERIMENT 5 CHOLESTEROL DETERMINATION 32

THE BIOCHEMISTRY SYLLABUS

THE BIOCHEMISTRY BOOKLIST

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P R E F A C E

Biochemistry will be taught by PBL as part of an integrated science programme which is

conducted over a period of two and a half 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: The Unit will give approximately eighty-five lectures over the course of the

first two years. The number of lectures 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 session every four weeks. Group assignments and the list of

experiments to be done will be posted on the class bulletin board at the beginning of each

semester.

When no practicals are being conducted the Unit may utilize these sessions for

small group Biochemistry tutorials.

EXAMINATIONS

All Biochemistry examinations will be conducted jointly with other disciplines of Basic

Sciences as part of the Problem Based Learning Program. Biochemistry questions may be

included in spotter examinations.

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INTRODUCTORY REMARKS

This booklet contains the protocols for the practical course, the revised course outline and the Biochemistry booklist. PRACTICALS STUDENTS WOULD NOT BE ALLOWED IN THE LABORATORY IF THEY DO NOT HAVE A LAB COAT OR PROPER FOOTWEAR! 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 Exemptions) are expected to perform all of the experiments as designated by the Biochemistry Unit Coordinator. NOTE BOOKS 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 Unit’s 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 or to answer all questions set as the case may be.

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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 at the end of each procedure. Reports should be no longer than 2 – 3 pages consisting of answers to the questions outlined at the end of each practical. All the reports must contain a copy of your results sheet bearing the Unit’s stamp.

PREPARATION Get into the habit of reading the procedures completely before you come to the laboratory. Try to understand what you are doing, and the rationale for the experiment. You will be required to present flow charts and tables for recording your results for each part of the practicals where relevant at the beginning of the laboratory session. DEMONSTRATORS Demonstrators are in the lab to help you - so ask them questions when you have problems. CODE OF BEHAVIOUR Some of the equipment in the laboratory is sophisticated and delicate. Also some pieces of equipment e.g. centrifuges are potentially lethal if not handled properly. So STUDENTS MUST NEVER: (i) Enter the LAB in the absence of a lecturer 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 AROUND IN THE LAB. (v) Smoke in the LAB. (vi) Eat in the LAB. (vii) Use their mobile devices in the LAB 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 A LAB COAT WHEN IN THE LAB. (v) Wear safety glasses while working in the laboratory (not provided).

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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.

4. Sandals, opened toe shoes are not allowed in the laboratory. 5. Some experiments will 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.

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.

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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.

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.

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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

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)

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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.

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 (750Χg) for 10 min. Carefully remove the tubes at

the end of the spin and immediately pour off the supernatants (S1) into 2

new centrifuge tubes. Do not transfer any of the loose fluffy layer on top

of the pellet (P1).

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 (4000Χg) 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.

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iv. Add 8.0 ml of cold sucrose to each of the P2 pellets and resuspend with a

glass rod. Balance the tubes and centrifuge at 5400rpm (4000Χg) 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 re-centrifugation.

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 (λmax).

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 Xs below using

graduated pipettes and repeat the dilutions using automatic pipettes.

Prepare between 2 ml to 5 ml of each dilution.

a. Χ2: a one in two dilution i.e. -1 part O: 1 part 0.02M NaOH

b. Χ3: one in three dilution i.e. -1 part O: 2 parts 0.02M NaOH

c. Χ4: one in four dilution i.e. - 1 part O: 3 parts 0.02M NaOH

d. Χ5: 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.

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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.

pH = pK + log [conjugate base]

[conjugate acid]

PROCEDURE

i. Use the 4 vials supplied as follows:

Tubes 1 and 2 containing 10 ml each of distilled H2O and tubes 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.

Draw a table as shown and use it to record your results.

Tube pH Before pH After

1

2

3

4

ii. Measure the pH of tubes 1 and 2. Record. Add 0.1 ml of 0.2M HCl (to

tube 1), mix well, and record the pH. Add 0.1 ml of 0.2M NaOH to tube 2,

mix well, and record the pH.

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iii. Measure the pH of tube 3 and 4. Record. Add 0.1 ml of 0.2 M HCl (to

tube 3), mix well, and record the pH. Add 0.1 mL of 0.2M NaOH to tube

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.

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EXPERIMENT 2

REACTIONS OF AMINO ACIDS AND PROTEINS

This experiment illustrates some of the general reactions of amino acids and proteins

which are important in the study of protein chemistry.

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 α –amino acids, to decarboxylate the amino acid, producing a blue-purple

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 flourescent products.

PROCEDURE

To 1 ml of neutral glycine solution (1g/L) in a test tube add five drops of Ninhydrin

solution #1. Place in a boiling water bath for 5 minutes to develop the blue-purple

reaction complex. Do x6 50% serial dilutions of the stock (1g/L) glycine solution and

repeat the test to 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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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-Ciocalteu’s 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.

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 H2O. Into separate tubes pipette 1.0ml each of the two

unknowns.

II. Mix (just before using) 50ml of Reagent X (2% Na2CO3 in 0.1M NaOH) with

1.0ml of Reagent Y (0.5% CuSO4 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-Ciocalteu’s 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.

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.

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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TREATMENT AND WRITE UP OF RESULTS

1) What is the role of 0.25M sucrose as the medium for the fractionation process?

2) List the major components that are present in (a) pellet P1, and (b) supernatant S2.

3) Define the Beer-Lambert Law and briefly explain why Absorbance has no units.

4) 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). Which dilution appears to be more accurate? Comment

on the spread of values.

5) Tabulate the data for the pH measurements for all solutions. State whether the test

solutions did or did not exhibit buffering capacity. Explain your observations based

on the composition of the solutions.

6) List the major buffer systems in the blood of mammals and show the equations for

each system?

7) Mention the importance of the Nihydrin test in clinical or research laboratory?

8) Plot a graph of your standard curve for the Folin-Lowry assay. Determine the

concentration of your two unknowns and report your answer in mg/ml.

9) Name the important chemical method available to determine total protein in the body

fluids

10) What is denaturation of proteins? Mention a few denaturing agents used in the

laboratory

11) What happens to the structural organization of protein when it is denatured?

12) Mention the importance of the denaturing process in the research laboratory

REFERENCE:

1. Tietz Textbook of Clinical Chemistry, Third Edition. Carl A. Burtis and Edward R.

Ashwood, eds. Philadelphia, PA: WB Saunders,

2. Manipal Manual of Clinical Biochemistry, Third edition. S Nayak, Jaypee medical

Publishers,

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EXPERIMENT 3

A. THIN LAYER CHROMATOGRAPHY OF SERUM LIPIDS

Along with proteins and nucleic acids, lipids form the other major macromolecule of

biological systems. There are many classes of lipids and lipids perform a diversity of

functions. One group of lipids, the triacylglycerides, constitute the major energy stores of

many organisms, while another group, the phospholipids, form the main component of

plasma membranes. Cholesterol, another important lipid, is also a component of membranes

and is the precursor of all of the steroid hormones.

The Lipids of serum can readily be separated into classes by thin layer chromatography. The

solvent used in this experiment separates fatty acids, triglycerides and cholesterol esters into

three distinct spots, but individual members of these three groups are not separated. Free

cholesterol runs separately; phospholipids remain on the base-line.

Serum is treated with a mixture of ether and ethanol, which strips the lipids from the

proteins to which they are bound in serum. The proteins are precipitated and the lipids

remain in the solvent. After filtration, the solution is evaporated to dryness, and the lipids

dissolved in a small volume of chloroform. This solution is used for thin layer

chromatography.

The spots are located with iodine vapour which dissolves in the lipid material giving a

brown colouration.

PROCEDURE

(1) Preparation of Serum: Obtain about 10 mL of whole blood from two members of

the class. Allow to clot for about 15 minutes in a centrifuge tube. Burst the fibrin clot

and centrifuge at 6000 r.p.m. for five minutes, then remove the serum (supernatant)

with a Pasteur pipette.

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NOTE: Each side of the bench will do the following preparation and both groups

on that side will spot from the resulting lipid solution.

(2) Preparation of Serum Lipids: Pour 20 mL of a diethyl ether-ethanol mixture

(ethanol: diethyl ether - 3:1) into a 100 mL conical flask. Transfer in 1.5 mL serum

from a pipette. Cork and shake rapidly for 1 min. Allow to stand for 30 mins and

then filter through a fluted filter paper into a 50 mL conical flask or beaker.

NOTE: You can proceed to spot the marker solutions during this thirty- minute

wait.

Evaporate just to dryness in the fume cupboard on an electric hot plate or water bath

(no naked flames). Allow to cool and add 0.5 mL chloroform and swirl to dissolve

the lipids. Spot on the thin layer plate within a few minutes or the chloroform will

have evaporated. (See spotting instructions below).

(3) Preparation of the Tank: (one per 2 pairs of students). Make sure the lid is

properly greased and well fitted. Pour in the necessary quantity of solvent (hexane:

diethyl ether: glacial acetic acid, 80:20:1 by volume). Put two filter-paper liners into

the tank against one large wall, replace the lid, and tip the tank so that the lining

papers become well soaked in solvent. Return the tank to the vertical and leave to

equilibrate. The paper liners are necessary with this extremely volatile solvent, to

ensure saturation of the vapour phase.

(4) Spotting: Marker solutions of lipids in chloroform are provided as follows:

Cholesterol 4%, cholesterol oleate 2%, phospholipids 2%, olive oil 1% oleic acid

1%. The 20 cm square plates will have been coated with silica gel and dried at

110OC. Be very careful not to damage the solid layer, which is extremely fragile.

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Place a plate flat on the bench. Prop up a ruler so that it passes over the plate without

touching it, and is parallel to and about 2 cm away from one of the edges, which is

fully coated with solid. Make a small scratch at each edge of the plate to mark the

position of the ruler, which will be the base line. Then make 8 small scratches at the

very bottom of the plate, so that they will be submerged in solvent during the run, but

will serve to indicate the position of the spots across the plate. These marks should be

2 cm apart, with the outer marks not less than 2 cm from the edges of the solid layer.

Keeping the ruler in position, apply the spots to the plate. Use a capillary pipette held

against the ruler, in the positions indicated by the scratches at the bottom of the plate.

Take great care to touch the plate very lightly with the capillary, or the silica will be

rubbed away. (A small hole in the solid at the point of application can be tolerated).

Keep the diameters of the spots to less than 5 mm. When applying several drops to

one spot, allow to dry between applications.

Apply spots as follows:

(1) Olive oil (0.5%) 50 µL

(2) Oleic acid (0.5%) 50 µL

(3) Serum extract 50 µL

(4) Serum extract 100 µL

(5) Serum extract 200 µL

(6) Cholesterol oleate (1%) 50 µL

(7) Cholesterol (2%) 50 µL

(8) Phospholipid (1%) 50 µL

(5) Chromatographic Development: Arrange one paper liner against each large wall

of the tank, and insert two plates, facing inward, with the glass of each plate resting

against the liner. Replace the lid quickly and allow to develop for 20 min. by which

time the solvent should have traveled about three quarters of the way up the plates.

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(6) Staining of Spots: Remove the plate, place horizontally and allow to dry. Stand the

plate in a dry tank containing iodine crystals, in the fume cupboard, for 15 min.

Remove the plate, place flat on the bench and at once mark the positions of the

spots by scratching their outlines with a pin. The spots fade rapidly as the iodine

evaporates, so work quickly and start with the fainter spots.

TREATMENT OF RESULTS AND WRITE UP

1. Calculate Rf values for the different lipid classes under the conditions of your

experiment.

2. Which groups of lipids are detectable in your serum?

3. Taking into consideration the apparent intensities of the spots, and the

concentrations of the marker solutions used, comment on the approximate amounts

of the different lipid classes found in serum.

4. What are some of the other biological functions of lipids?

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B. SEPARATION OF SERUM PROTEINS BY AGAR GEL ELECTRO PHORESIS This is a very common technique used in clinical and research laboratories and is used for

the separation of charged particles. Biological materials such as aminoacids, peptides,

proteins, nucleic acids posses ionisable groups and hence exist as charged molecules in

solutions, either as cations (+vely charged) or anions (– vely charged) depending upon

the pH of the medium. Even typical non-polar substances such as carbohydrates can be

given charges by derivatisation, for example as borates or phosphates.

These charged particles move in an electric field. i.e. cations towards cathode (– vely

charged electrode) and anions towards anode (+vely charged electrode). So it is obvious

that the molecules having similar charges move in the same direction. But because of the

difference in their molecular mass the extent to which they move differs. Hence the

difference in Charge: Mass ratio (C/M) forms the basis for the differential migration of

particles in an applied electric field. And this forms the general principle of

electrophoresis.

Procedure: i) Slide preparation: About 1.3ml of warm (60°C) Agar solution (100mg/10ml of

barbitone buffer) is delivered through pipette on a slide uniformly at room

temperature and allowed to solidify.

ii) Chamber saturation: Twenty minutes before starting the experiment, both the buffer

tanks of the electrophoretic chamber (figure 2.5.1) are filled with equal volume of

barbitone buffer (pH-8.6) and kept closed to saturate the chamber with solvent

vapours.

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iii) Slide placement and Wick connection: The slide prepared is kept in the chamber and

connected to buffer by means of filter paper strips (Wicks) as shown in the figure

2.5.1.

iv) Sample application: A small filter paper strip (Whatman No.3, 1x5 mm) soaked in

the serum sample is kept on the mounted slide perpendicular to the length of the

slide close towards the cathode and the chamber is closed.

v) Application of Electric field:

The electrophoretic chamber is connected to a power pack (equipment used to alter

or adjust the required current or voltage values). The power pack is switched on and

current is adjusted so that 3 mAmp current flows through each slide. The process

has to be carried out for about 90-120 minutes.

vi) Fixing of proteins: The slides are kept immersed in the absolute alcohol at 4°C for

about 30 minutes to prevent the diffusion of separated proteins.

vii) Staining: The slides are dried and kept immersed in the Amidoschwartz 10B dye for

about 2-3 minutes.

viii) Destaining: The stained slides are washed with 3% acetic acid to clear the

background and to see the protein bands clearly.

Finally the slides have to be dried. These slides can be preserved for a long time.

Since the electrophoretic pattern of serum proteins in certain diseases vary markedly

from a normal pattern it is of great diagnostic significance in several conditions like

nephrosis, liver disease, multiple myeloma, agamma-globulinemia and others.

24

25

RESULTS WRITE UP:

1. Give the reasons for: decreased serum albumin and increased alpha-2 macroglobulin in

nephrotic syndrome

2. What is multiple myeloma? Name the protein band appears during electrophoresis

3. Mention the different types of electrophoresis available to separate and identify proteins

4. Can we use this technique to separate hemoglobin? If yes in which conditions we can use

Reference: 1. Tietz Textbook of Clinical Chemistry,Carl A. Burtis and Edward R. Ashwood,

eds. Philadelphia, PA: WB Saunders,

2. Manipal Manual of Clinical Biochemistry, Third edition, Shivananda Nayak,

Jaypee Medical publishers, New Delhi,

26

EXPERIMENT 4

AIM:

I. DEMONSTRATION OF ENZYME SPECIFICITY WITH GLUCOSE OXIDASE

AND PEROXIDASE AND THE DETERMINATION OF BLOOD GLUCOSE LEVELS.

II. THE CLASSIFICATION OF SUGARS AND THE DETERMINATION OF

GLUCOSE LEVELS IN URINE.

Experiment I: Demonstration of Enzyme Specificity with Glucose Oxidase and Peroxidase.

PRINCIPLE :

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

the presence of oxygen.

Glucose oxidase

β-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 oxidises a weakly

coloured hydrogen donor DH2 (O-dianisidine) to a coloured derivative, D.

H2O2 + DH2 Peroxidase

2H2O + D

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

27

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 the concentration of glucose in blood will be determined. Also the

specificity of the enzyme will be demonstrated.

METHOD A : Determination of blood glucose using standard graph

REAGENTS:

The enzyme cocktail consist of:

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

PROCEDURE

Treatment of 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.

Pipette 0.3 ml of supernatant to a clean dry semi-micro test tube add 0.2 ml distilled water

and finally 2.0 ml enzyme cocktail (prepare in duplicate T1 and T2). Incubate at 37°C along

with your standard curve samples.

Prepare a standard curve by pipetting 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the 0.3mM

glucose solution provided (do not use the 0.1M glucose solution) into clean dry semi-

micro test tubes. Add distilled water to make the final volume in each tube 0.5 ml. Add 2.0

ml enzyme cocktail to each tube and incubate at 37°C for 45 minutes. Along with your

samples to follow (see table provided).

28

Perform the experiment as follows: Reagents Tube (Volume in ml) B S1 S2 S3 S4 S5 S6 T1 T2 L F Glucose solution(0.3mM) 0.0 0.1 0.2 0.3 0.4 0.5

Supernatant 0.3 0.3

Lactose(0.3mM) 0.5

Fructose(0.3mM) 0.5

Reagent blank 0.3

Distilled water (ml) 0.2 0.5 0.4 0.3 0.2 0.1 0.2 0.2

Enzyme Cocktail 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 at 37. C

Mix and Read the absorbance at 450 nm B= Blank T= Test, S= standard, L= Lactose, F=Fructose

Plot the standard curve of absorbance (Y axis) versus concentration of glucose in mmol

(x-axis).

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

CLINICAL SIGNIFICANCE:

Normal ranges (plasma): Fasting glucose: 60-110mg%

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 hyperfunctions of anterior pituitary and adrenal

cortex.

29

• 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

TREATMENT OF RESULTS AND WRITE UP

1. Plot the standard curve of absorbance (Y axis) versus concentration of glucose in mmol (x-axis).

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

3. Explain the term hyperglycemia with a specific example 4. List the chemical and enzymatic method(s) that are available for determination of

glucose in blood. 5. Why is an enzymatic method preferred for blood glucose estimation? 6. List any two enzymatic methods which are commonly used in laboratories of Trinidad

and Tobago to determine blood glucose 7. What happens if vacutainer does not contain fluoride? Mention the action of fluoride 8. Which hormone deficiency leads to diabetes mellitus? 9. How do you differentiate diabetes mellitus from diabetes insipidus? 10. What are the two types of diabetes mellitus? 11. List the three major clinical complications of diabetes mellitus 12. What is glycated hemoglobin and mention its clinical importance

30

Experiment II : The Classification of Sugars and the Determination of Glucose in Urine.

INTRODUCTION :

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

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.

31

TREATMENT OF RESULTS AND WRITE UP

1. Record and briefly explain your results. 2. Name the nonreducing sugar which does not give a positive Benedict’s test and

why? 3. List the metabolic actions of insulin in regulating the blood glucose? 4. From the sensitivity test, can you estimate the threshold concentration of glucose

required to give a positive Benedicts test 5. What is the renal glycosuria and mention its causes 6. Give the renal threshold value for glucose? 7. What other substances (other than carbohydrates), which may be present in urine,

are able to reduce Benedict's reagent? 8. Explain the following with specific examples a) Gestational diabetes b) Spot test 9. What is the glucose tolerance test and list the significance of the test? 10. Write the brief procedure of oral glucose tolerance test (GTT) 11. Mention the conditions in which the oral GTT is replaced with intravenous 12. How do you differentiate normal GTT from abnormal GTT?

REFERENCES

1. Clinical Chemistry, 3rd Edition, Marshall, W.J., J.B. Lippincott and Company,

London. pp 167-179. 1993

2. Manipal Manual of Clinical Biochemistry, Third edition, Shivananda Nayak,

Jaypee Medical publishers,New Delhi

32

EEXXPPEERRII MM EENNTT 55

AAIIMM:: DDeetteerrmmiinnaattiioonn ooff SSeerruumm Cholesterol [Zak’s ferric chloride method]

NOTE: For this lab two volunteers are required to donate 5mL of blood each. Volunteers

are required to come to the lab for 1:40 pm

INTRODUCTION

Cholesterol is a very low water soluble lipid found in blood. It is one of the major

constituents of plasma lipoproteins and it exists in two forms; as free cholesterol and

esterified to some long-chain fatty acids, which enhances its hydrophobicity.

Cholesterol can be obtained from the diet or manufactured in the liver from acetyl CoA

by endogenous synthesis. It plays a number of important roles such as, being a major

structural component of cell surfaces and intracellular membranes and serving as a

precursor of the bile acids and steroid hormones.

PRINCIPLE Cholesterol in acetic acid reacts with ferric chloride and sulfuric acid to produce a red

color. The absorbance of the red color is read in a photoelectric colorimeter at 540 nm

(green filter).

The proteins in serum are precipitated with ferric chloride-acetic acid reagent. Equal

volumes of protein-free filtrate containing cholesterol, a standard and blank containing

ferric chloride-acetic acid reagent are separately treated with sulphuric acid and optical

densities are read.

Specimen: serum

33

REAGENTS:

1. Glacial acetic acid

2. Ferric chloride (0.05 %): Dissolve 50 mg of ferric chloride in 100 ml acetic acid.

Store in a brown bottle and it is stable for one month.

3. Sulfuric acid, AR grade

4. Stock cholesterol standard solution: 100 mg / 100 ml acetic acid, keep in a cool,

dark place and stable for one month.

5. Working cholesterol standard solution (0.1 mg/ml): dilute 10 ml stock solution to

100 ml with 0.05% ferric chloride reagent

PREPARATION OF SERUM

Obtain 5mL 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

will be carried out by a demonstrator.

PROCEDURE Treatment of Serum:

Pipette 0.1 ml of serum and 3.9 ml of ferric chloride-acetic acid reagent into a centrifuge

tube. Cover the mouth of the tube with a piece of Parafilm and mix well with a vortex

mixer. Let stand for 10 minutes for the proteins to flocculate. Centrifuge at 3000rpm for

10 minutes.

34

Perform the experiment as follows: Reagents Blank (B) Standard (S) Test ( T1) Test (T2) Supernatant (ml) 1.0 1.0 Ferric chloride-acetic acid 1.0 ml

Working standard (0.1 mg/ml) 1.0

Sulphuric acid (ml) 1.0 1.0 1.0 1.0

Mix and incubate for 20 minutes at room temperature Read the absorbance at 540 nm Note: Take necessary precautions when pipetting concentrated sulphuric acid CALCULATION:

dardSofAbsorbance

TestofAbsorbance

tan X 0.1 x 4 = mg of cholesterol in 0.1 ml serum

X 1000 = mg of cholesterol / 100 ml serum (mg/dl) CLINICAL SIGNIFICANCE: � Normal serum cholesterol ranges from 150- 220 mg%

� The increased level of cholesterol in serum is called hypercholesterolemia. This is

seen in: nephrotic syndrome, obstructive jaundice, myxoedema, glomerulonephritis,

coronary artery thrombosis and angina pectoris.

� Decreased level is called as hypocholesterolemia and seen in hyperthyroidism,

pernicious anemia, malabsorption syndrome, acute infections and hemolytic jaundice.

35

TREATMENT OF RESULTS AND WRITE UP

1. From the results, how much cholesterol was there in 100 ml of serum?

2. Express the results for cholesterol in molar terms.

3. Name the enzyme which helps in the esterification of cholesterol

4. List the tests under lipid profile and mention one clinical significance of its usage

5. Which sample is suitable for cholesterol determination?

6. What is the normal range of values for cholesterol concentration in serum or

plasma?

7. What fraction of cholesterol is esterified in blood?

8. Draw the formula of cholesterol and name the steroid ring present in it.

9. Apart from plasma, where is cholesterol found in the animal body?

10. List the clinical conditions in which we find high blood cholesterol

11. Which lipoprotein has high cholesterol content?

12. Name the bad and good cholesterol of the human body and give the reason for it

13. Mention the four conjugated bile acids which you find in the human bile

14. List the compounds formed from cholesterol in the human body?

15. Name the precursor and the regulatory enzyme of cholesterol biosynthesis

16. Mention the technique through which you can separate lipoproteins and which

lipoprotein contains high cholesterol

REFERENCES:

2. Manipal manual of Clinical Biochemistry, Third edition, Shivananda Nayak,

Jaypee Medical publishers.

3. Tietz Textbook of Clinical Chemistry, Carl A. Burtis and Edward R. Ashwood, eds.

Philadelphia, PA: WB Saunders,

36

THE BIOCHEMISTRY BOOKLIST NAME/AUTHOR PUBLISHERS Students Should Own 1. Harper's Biochemistry Prentice Hall Inter. Granner, Mayes, Murray, Rodwell (Latest Ed.) Recommended For Reference 1. Textbook of Biochemistry John Wiley & Sons Inc. with Clinical Correlations T M Devlin (latest edition) 2. Biochemistry by Diagrams Canoe Press E. Morrison 3. Biochemistry A Case Oriented Approach C V Mosby Conway, Montgomery & Spector (5th Ed.) (St Louis, 1990) 4. Biochemistry for the Medical Sciences John Wiley & Sons Inc E A Newsholme & A R Leach (1983) 5. Biochemistry W H Freeman L Stryer (5th Ed.) (1995)

6. Essentials of Biochemistry, Jaypee Medical publishers,

S Nayak New Delhi, India

7. Biochemistry Saunders College Publishers R.H. Garrett & C.M. Grisham (1999) Harcourt Brace College (2nd Ed.) 8. Lippincott’s Illustrated Reviews Biochemistry J.B. Lippincott Company Champe & R.A. Harvey (Latest Ed. Philadelphia) 9. Elements of Medical Genetics Churchill Livingstone A E Emery & R F Mueller (7th Ed.) (1988)

37

10. Biochemistry Illustrated Churchill Livingstone P N Campbell and A L Smith (1988) (2nd Ed.) 11. Membranes and Their Cellular Blackwell Scientific Function Publication J B Finean, R Coleman and R H Michel (3rd Ed. or later) 12. Biochemistry (Board Review Series) Harwal Publishing D B Marks (1994) 13. Immune Recognition IRL Press M J Owen (1988) 14. Metabolic and Nutritional Blackwell Scientific Diseases of Cattle Payne 15. Animal Nutrition Longman McDonald, Edwards & Greenhalgh (4th Ed.) 16. Recombinant DNA Scientific J Watson, M Gilman, American Books J Witkowski, M Zoller (2nd Ed.)(1993) 17. Genes VII Oxford University Press B Lewin 7th edition (January 2000) 18. Principles of Gene Manipulation Blackwell Publishers RW Old, SB Primrose 6th Edition 2002

DENTAL 16. Basic and Applied Dental Biochemistry Churchill Livingstone Williams & Elliot (4th Ed.) (1989)