DEPARTMENT OF BIOCHEMISTRY

AND MOLECULAR BIOLOGY

UNIVERSITY OF MEDICAL SCIENCES















BIOCHEMISTRY
COURSE MANUAL

6-year M.D.






Edited by
P. Jagodziski Ph.D.
Head of Department







Pozna
2013/2014
Department of Biochemistry and Molecular Biology
Karol Marcinkowski University of Medical Sciences
6 wicickiego St. 60-781 Pozna (Poland)
phone (+48 61) 85 46 513, (+48 61) 85 46 519
fax (+48 61) 85 46 510
www.biolmol.ump.edu.pl


Director of the course:
Pawe Jagodziski, Ph.D.
Head of Department

Lecturer:
Pawe Jagodziski, Ph.D.
Head of Department
pjagodzi@ump.edu.pl

Instructors:
Adrianna Mostowska, Ph.D.
Adjunct
amostowska@wp.pl
Marcin Hoysz, Ph.D.
Lecturer
mholysz@ump.edu.pl
Tomasz Lehmann, Ph.D.
Assistant
tlehmann@ump.edu.pl
Agata Rycka, Ph.D.
Assistant
arozycka@ump.edu.pl
Agnieszka Rawuszko-Wieczorek, M.Sc.
Assistant
arawluszko@ump.edu.pl
Hanna Drzewiecka, M.Sc.
Assistant
epi6@o2.pl
Bartosz Frycz, M.Sc.
Assistant
bartekfrycz@gmail.com
Daria Galas, M.Sc.
Assistant
Mariusz Nawrocki, M.Sc.
Assistant
Agata Tomaszewska, M.Sc.
Assistant

THE BIOCHEMISTRY COURSE

Rules and Regulations for the 6-year M.D Program, Academic Year 2013/2014

OBJECTIVES
The main objectives of the course are to provide an understanding of biochemical processes and
to gain relevant basic laboratory skills according to the educational requirements defined in the
program of teaching biochemistry for medical students.

FORMAT
The program (160 h) consists of lectures (40 h), seminars (48 h) and practical classes (72 h). The
course is divided into four modules: Proteins, Nucleic Acids, Carbohydrates and Lipids. Each module
comprises lectures, introduction and laboratory classes, seminars, clinical correlations and review.

LABORATORY CLASSES
Prior to entering the laboratory classes within each module, during introduction classes,
a student can take the introductory review to get a bonus points (10 points for each module),
comprising biochemical background covering the respective module.
The performance during each laboratory class will be evaluated by the quality of theoretical
preparation, laboratory skills and a written protocol from the experiments, which must be delivered in
less than 3 days, and will be graded from 0 to 5 points (up to 20 points for 4 laboratory classes). For
each absence in the class, two points will be subtracted.

SEMINARS AND REVIEWS
The performance during seminar classes will be evaluated by partial test, covering the topics of
each seminar within the single module (10 one-choice questions, graded 1 point for a correct answers).
At the end of each module, the students will be subjected to a closing test during reviews, covering the
topics of all seminars and lectures within the module (30 one-choice questions, graded 1 point for a
correct answer).

CONDITIONS FOR EARNING CREDIT
During the entire course, a student can accumulate jointly for the laboratory classes, seminars and
closing tests up to 260 points (100%) plus up to 40 bonus points extra (bonus points will be added to
the final score). To receive a credit and be admitted to the final examination, a student must earn a
minimum of 182 points (70%). A student, who accumulated from 78 to 181 points (3070%), to
receive the credit and be admitted to the final examination, must pass an integrative test (50 questions)
during final review, covering topics of the four modules, and get 60% of points. A student, who did
not pass the integrative test, is entitled to two retakes, according to the schedule. A student, who
accumulated less than 78 points (<30%) is not be allowed to take the integrative test and will not
receive a credit for the course. Numbers of the points accumulated during the entire academic year will
be converted to a grade, according to the following system:

7074% satisfactory 8084% good >90% very good
7579% fairly good 8589% better than good

FINAL EXAMINATION
Students who have earned credit must take the Final Exam. The passing mark and grading system
will be set on the basis of the exam scores. If a grade obtained on the Final Exam is lower than grade
from entire academic year, the final mark can be upgraded and will be a mean of these two grades. The
final mark cannot be upgraded when the student does not pass the Final Exam.
If a failing grade is obtained, the final examination can be retaken twice according to the
schedule. The passing mark and grading system will be set on the basis of the results of retake exams.
THE BIOCHEMISTRY COURSE
Rules and Regulations for the 6-year M.D Program, Academic Year 2013/2014



1. Cheating is not allowed. Students who do not obey this rule will be subjected to
disciplinary action according to School Regulations on cheating.

2. Students are required to conduct themselves in a professional manner - see School
Regulations.

3. Using mobile-phones, mp3, radios and other electronic equipment during classes and
exams is not allowed.

4. Eating, drinking, and having any food or beverages during classes is not allowed. Food
and drinks will be immediately discarded by the tutors.

5. Filming and other recording of the lectures and seminars is not allowed. Students who do
not obey this rule will also be subjected to disciplinary action.

6. Making copies or photos of exams, tests and other quizzes is not allowed.

7. Booking travel ticket is not considered an excuse for missing any compulsory University
activity.

8. The student has the right to see each of his/her written papers or answer sheet within
7 working days following the announcement of the results.

9. In cases concerning procedures not included in the present Biochemistry Course
Regulations, School Regulations and General School Regulations apply.


I acknowledge that I have read and understood these Biochemistry Course Regulations.


Signed:.


Name (Please print):.



Safety Notes
for Students working in Biochemistry Laboratories
General
Smoking, drinking and eating are forbidden in laboratories. During classes laboratory
coats must be worn and each student should have a lab book.
Possible hazards
1. Glassware: Always inspect glassware before use for chips and cracks. The most
common laboratory accident is cut hands from damaged glassware.
2. Solvents: When handling ether, ethanol, acetone and other organic flammable liquids
risk of fire must be considered at all times. When handling corrosive materials such as e.g.
sodium hydroxide, concentrated acids or phenol, safety glasses and gloves must be worn.
3. Homogenizers and blenders: Great care should be taken to ensure that the instrument is
not switched on in the absence of the appropriate shielded glass container. The instrument
must be switched off and the rotating blades at rest before disconnecting the shielded
container.
4. Centrifuges: When using centrifuges care should be taken to ensure that the tubes are
properly balanced. Check that the rubber cushions are in the tube holders.
5. Electrical apparatus: It is forbidden to disconnect plugs from apparatus, reconnect plugs
or replace fuses. If any piece of electrical apparatus appears to be defective, it must be
checked by a qualified electrician.
6. Toxic chemicals: All handling of toxic chemicals such as e.g. cyanide, organic solvents
etc. should be conducted with great care and when necessary protective gloves should be
worn. Pipetting toxic chemicals by mouth is forbidden. Any spillage of toxic chemicals
must be cleaned up immediately.
7. Biological hazards: All samples of human blood must be regarded as major biological
hazard and handled wearing disposable gloves. When human blood is used automatic
pipettes must be employed and the material disposed in the specially designated containers.
Laboratory cleanliness:
1. All spillages of liquids and chemicals, especially onto any instrument or piece of
equipment must be cleaned up immediately.
2. Bottle stoppers must be replaced immediately after use of the reagent.
3. The weighing must be done in suitable containers.
4. All laboratory ware must be rinsed or washed after use.
5. Bench surfaces must be wiped clean and equipment and bottles arranged tidyly.
6. Biological material must be placed in special containers.
7. Cuvettes must be rinsed after use and returned to their box.

I hereby confirm that I read the safety notes:
Name .......................................... Signature ..............................................



Seminar and Laboratory Program

For students of the 6-year M.D. program Academic Year 2013/2014




I. Proteins II. Nucleic Acids
Introduction I Introduction II
Lab. 1 Plasma proteins

Lab. 2 Preparation and restriction
analysis of plasmid DNA
Sem. I Structure and functions of proteins
Sem. II Haemoglobin
Sem. III Enzymes
Sem. IV DNA structure and replication
Sem. V DNA transcription. RNA
structure and function
Sem. VI Protein biosynthesis
Sem. CC-I Clinical correlations I
Review I

Sem. CC-II Clinical correlations II
Review II
III. Carbohydrates IV. Lipids
Introduction III Introduction IV
Lab. 3 Blood glucose Lab. 4 Properties and analysis of lipids
Sem. VII Metabolism of monosaccharides
Sem. VIII Metabolism of polysaccharides
Sem. IX Glucose homeostasis in humans
Sem. X Metabolism of fatty acids
Sem. XI Biosynthesis and degradation
of lipids
Sem. XII Interorgan transport of lipids
Sem. CC-III Clinical correlations III
Review III
Sem. CC-IV Clinical correlations IV
Review IV























PROTEINS












P R O T E I N S








INTRODUCTION I


LABORATORY


Laboratory 1. Plasma proteins


SEMINARS


Seminar I. Structure and functions of proteins

Seminar II. Haemoglobin

Seminar III. Enzymes

Seminar CC-I Clinical correlations I


REVIEW I
INTRODUCTION TOPICS

PROTEINS


Structure of amino acids (structural formulas). Classification of amino acids
based on: structure of side-chain groups, essential and nonessential amino acids,
apolar and polar amino acids, glucogenic and ketogenic amino acids. Properties
of amino acids: isoelectric pH (pI), isomers, formation of peptide bonds.
Biologically active peptides: carnosine, anserine, glutathione, oxytocin,
vasopressin and bradykinin. Structure and properties of proteins based on:
amino acids contents, shape of protein molecules, solubility in water, function.
Conformation of proteins: bonds stabilising protein structures, precipitation of
proteins (dehydration and denaturation). Enzymes: nomenclature, classification,
specificity, classification and function of some coenzymes.




TEXTBOOK AND READING

Harpers Illustrated Biochemistry
27
th
edition
Murray R. K. et al.
Lange Medical Books/McGraw-Hill, 2006
ISBN: 0-07-147885-x

Harpers Illustrated Biochemistry
28
th
edition
Murray R. K. et al.
Lange Medical Books/McGraw-Hill, 2009
ISBN: 978-0-07-162591-7

Biochemistry
6
th
edition
J. M. Berg, J.L. Tymoczko, L. Stryer
W.H. Freeman, 2006
ISBN: 0716787245

Textbook of Biochemistry
6
th
edition
Devlin T. M.
Wiley, 2006
ISBN: 0-470-10989-0
LABORATORY 1
Plasma proteins
TOPICS
Structure, properties and functions of plasma proteins. Separation of plasma proteins by
electrophoresis. Determination of protein concentration by spectrophotometric method.

ELECTROPHORESIS OF SERUM PROTEINS IN AGAROSE GEL

Principles:
The electrophoretic separation can be achieved in buffer solutions of pH far from isoelectric
point of the protein (free electrophoresis), or with the aid of solid support, such as cellulose,
agarose or polyacrylamide gels. The speed of migration of the molecules depends on voltage
differences, and the charge and shape of the molecules. Electrophoresis can be used for the
separation of serum proteins. We can detect the following fractions of serum proteins:
albumins,
1
,
2
, and -globulins.

Materials:
Electrophoretic apparatus with water thermostated cell, Pt electrodes, plastic comb, power
supply, trays for staining and fixing gel.
Agarose, electrophoretic grade.
Electrophoresis buffer: 10 times concentrated Tris-borate EDTA pH 8.0 (10x TBE)
Indicator solution: 1% bromophenol blue
Fixing solution: 10% sulfosalicylic acid (w/v) and 40% ethanol (v/v) in water
Staining solution: (must be prepared immediately before use)
A. 2% phosphoric acid (w/v), 0.1% Coomassie-R-250, 6% (NH
4
)
2
SO
4
(w/v),
25% ethanol (v/v)
B. 5% Coomassie-R-250 (w/v)
C. 20% ethanol (v/v)
D. 96% ethanol (v/v)

Method:
1. Casting the gel: weigh out 1 g of agarose and transfer it into a conical flask. Add 100 ml of
ten times diluted TBE buffer (1x TBE), bring to boiling in a microwave to dissolve. Cool
solution to about 50C. Insert a comb and pour the agarose into a casting tray. Allow the
agarose to solidify and carefully remove the comb.
2. Sample application: fill the chamber with the buffer to about 5 mm above the gel surface.
Prepare samples by mixing equal volumes (e.g. 50 l) of serum and 50% sucrose solution.
Add 510 l of 1% bromophenol blue to 100 l of serum/sucrose solution and use this
sample as marker. This dye will make the albumin fraction visible during the run. Other
samples will be run without the dye. Apply 50 l of serum sample to separate wells.
3. Electrophoresis: connect the electrodes and turn the power supply on. The electrophoresis
is carried out at 20 V/cm of the gels length, or as long as the blue-stained albumin fraction
migrates about 5.5 cm (takes 45-60 min.). Turn the power supply off.
4. Fixing of gel: remove gently the gel from the chamber and insert it in the tray. Keep the
tray with gel for 10 min in fixing solution mixing from time to time.
5. Wash the gel two times for 5 min in 50 ml 20% ethanol (solution C).
6. Staining: add 98 ml solution A, 2 ml solution B and 25 ml 96% ethanol (solution D) to
conical flask (300 ml capacity). Mix vigorously to prepare colloidal solution. Cover the gel
by the above solution and mix gently for 30 min. To remove excess of the dye, wash the
gel three times in distilled water. Analyse the visible bands of proteins.

SPECTROPHOTOMETRIC DETERMINATION OF TOTAL PROTEIN
CONCENTRATION IN SERUM BY USING THE BIURET METHOD.

Principles:
Proteins and peptides that have more than one peptide bond in their structure, produces purple
colour complex with copper (II) ions in an alkaline solution (so called biuret reaction). To
avoid precipitation of Cu(OH)
2
under alkaline conditions, copper ions are chelated by sodium
potassium tartrate.

Materials:
0.9% solution of NaCl
Biuret reagent: 5 g CuSO
4
x 5H
2
O in alkaline solution with potassium sodium tartrate (45 g
potassium sodium tartrate plus 5 g potassium iodide in 1000 ml 0.2 M NaOH).

Method:
1. Preparation of standard curve: standard curve is the plot of absorbancy versus the protein
concentration. To prepare standard curve, we use serum solution of known protein
concentration. Standard serum contains 7.2 g protein per 100 ml.
2. Preparation of standard protein solutions: dilute the standard solution of serum in 50 ml
conical flask with 0.9% NaCl according to the table below:

Number of conical flask
(50 ml)
1 2 3 4 5 6 7
Volume of standard serum
(ml)
0.50 0.75 1.00 1.25 1.50 2.00 2.50
Protein concentration
(g/100 ml)
0.072 0.108 0.144 0.18 0.216 0.288 0.36

Fill up the conical flask to 50 ml using 0,9% solution of NaCl.
Simultaneously prepare samples of unknown concentration according to point 5.
3. Colour reaction: prepare two rows of tubes (7 tubes in each row) and one control tube.
Using the pipette, transfer 5 ml of each standard serum solution to parallel tubes.
Add 5 ml of 0.9% NaCl solution to last tube (control sample), then add 5 ml of biuret
reagent to each sample. Mix well the tubes and keep 30 min at room temp. Measure the
absorbancy of each sample against the blank at 570 nm wavelength.
Note: do not remove the blank, and keep it for determination of serum protein concentration
in all samples.
4. Calculation and the presentation of results: calculate mean absorbancy of parallel samples.
Draw the absorbancy (on the ordinate) versus protein concentration (on the abscissa). The
standard curve should be a straight line. Calculate the coefficient by dividing the value of
the concentration of the protein by the absorbancy.
5. Determination of protein concentration in serum: pipette 0.15 ml of serum of unknown
protein concentration to three tubes (2-4), use tube 1 as a blank. Add the biuret reagents to
samples according to the table below:

Number of tubes 1 2 3 4
0.9% NaCl 5.0 4.8 4.8 4.8
(ml)
Serum
(ml)
0.0 0.2 0.2 0.2
Biuret reagents
(ml)
5.0 5.0 5.0 5.0

6. Mix well and keep 30 min at room temp. Measure the absorbancy at 570 nm. Calculate
mean absorbancy of parallel samples. Read protein concentration from the standard curve.
Calculate final protein concentration using the following formulas:

C = F x A x D

C = D x c

Where: C final protein concentration; F coefficient; A absorbancy; D dilution of the
sample; c protein concentration of sample read from the standard curve.

Draw conclusions from the results.

SEMINAR I
Structure and function of proteins
1. The structure of polypeptide chain.

2. Levels of protein organisation:
a. primary, secondary, tertiary, quaternary structure
b. domains of protein
c. -helix, -sheet
d. triple helix of collagen type I

3. Relationship between amino acid sequence and conformation of a protein.

4. Techniques of protein separation and detection.

5. Biological relevance and function of proteins.

6. Plasma proteins:
a. constituents of plasma proteins
b. role of albumin in the maintenance of osmotic pressure and the binding of various
ligands
c. transport function of globulins
d. role of haptoglobin in haemoglobin binding
e. participation of transferrin and ceruloplasmin in iron and copper metabolism
f. role of fibrinogen in blood clotting
g. properties of human immunoglobulins
h. acute phase proteins
i. disorders in plasma protein contents Wilsons disease and
1
-antitrypsin deficiency


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 14-40, 588-605.
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 31-42, 566-582.
Stryer L. Biochemistry 6
th
edition pp. 25-64.
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 75-132, 320-327

SEMINAR II
Haemoglobin
1. Haemoglobin as an allosteric protein:
a. conformation of haemoglobin molecules, bonds stabilising its structure
b. myoglobin structure
c. normal haemoglobin

2. Participation of haemoglobin in oxygen and carbon dioxide transport:
a. haemoglobin and myoglobin oxygen affinity curves
b. influence of allosteric effectors on haemoglobin and myoglobin affinity to oxygen (2,3-
bisphosphoglycerate, CO
2
, pH, homotropic and heterotropic effects)

3. Conformation changes accompanying oxidation of haemoglobin.

4. Mechanism of carbon dioxide transport from peripheral tissues to lungs:
a. role carbonic anhydrase

5. Haeme biosynthesis and regulation:
a. disorders in globulins synthesis
b. pathological haemoglobin (thalassemias, HbS, HbM, HbC) and mechanism of haemo-
globinopathies
c. porphyrias

6. Hem catabolism and releasing of hem metabolites:
a. bilirubin synthesis
b. plasma bilirubin transport
c. mechanism of bilirubin binding in liver and releasing complex haemoglobin with bile
pigments
d. conversion of bilirubin in the intestine
e. intestine-liver circulation of bile pigments
f. jaundice


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 41-48, 279-293.
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 43-50, 271-284.
Stryer L. Biochemistry 6
th
edition pp. 183-204
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 338-351, 833-844
SEMINAR III
Enzymes
1. Structure of enzymes: catalytic site, substrate binding site, allosteric site, multifunction
enzymes and enzymatic complexes, prosthetic group, cofactors.

2. Classification of enzymes.

3. Kinetics of enzymatic reaction:
a. relation between substrate concentration and reaction velocity Michaelis-Menten
equation
b. linear form of the Michaelis-Menten equation Lineweaver-Burk plot. Hills equation
c. influence of temperature, pH and enzyme concentration on reaction velocity

4. Activators and inhibitors of enzymes, role of metal ions, competitive and non-competitive
reversible and irreversible inhibitors.

5. Methods of enzyme activity determination, units of enzyme activity.

6. Regulation of enzymatic activity by allosteric or covalent modification.

7. Intracellular compartmentalisation of enzymes.

8. Clinical applications of enzymes: isoenzymes, secretary enzymes and disease indicator
enzymes.


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 49-81
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 51-83
Stryer L. Biochemistry pp. 205-302
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 328-337, 365-412






















NUCLEIC ACIDS











N U C L E I C A C I D S








INTRODUCTION II


LABORATORY


Laboratory 2. Preparation and restriction analysis
of plasmid DNA


SEMINARS


Seminar IV. DNA structure and replication

Seminar V. RNA structure and function. DNA transcription.

Seminar VI. Protein biosynthesis

Seminar CC-II Clinical correlations II


REVIEW II
INTRODUCTION TOPICS

NUCLEIC ACIDS


Chemical formulas and nomenclature of purine and pyrimidine nucleotides.
Nucleosides and nucleosidomono-, di- and triphoshates. Nucleosides (S-
adenosylmethionine) and (nucleotides, NMN, NAD, NADP, FMN, FAD, CoA,
PAPS) as coenzymes. Deoxyribonuleic acid: structure of single strand, double
helix, higher order structures, denaturation and renaturation of DNA (melting
temperature, hiper and hipochromic effects). Ribonuleic acids (rRNA; tRNA;
mRNA; snRNA) structure and function. Hydrolysis of nucleic acids: (a) acid
hydrolysis of DNA and RNA (b) alkaline hydrolysis of RNA (c) products of
hydrolysis (d) enzymatic hydrolysis of DNA and RNA: endo- and exonucleases,
restriction enzymes.

LABORATORY 2
Preparation and restriction analysis of plasmid DNA
PREPARATION OF PLASMID DNA

Adapted from Sambrook J. et al., Molecular Cloning. A laboratory Manual 1.21-1.31, Cold
Spring Harbour Laboratory Press, 1989.

A. Harvesting of bacteria
Method:
1. Transfer a single bacterial colony into 2 ml of LB medium containing the appropriate
antibiotic in loosely capped 15 ml tube. Incubate the culture overnight at 37C with
vigorous shaking.
LB medium
To 950 ml of deionized water add: yeast extract 5 g, NaCl 10 g and dissolve.
Adjust pH to 7.0 with 5N NaOH. Make up to 1 L with deionized water.
Sterilise by autoclaving.
2. Pour 1.5 ml of culture into a microfuge tube. Centrifuge at 12 000 g for 30 sec. at 4C.
3. Remove the medium by aspiration, leaving pellet as dry as possible.
Note: the bacterial pellet will be prepared by an instructor.

B. Lysis by alkali
Method:
1. Resuspend the bacterial pellet in 100 l of ice-cold Solution I by vigorous vortexing (the
technique will be demonstrated by an instructor).
Solution I
50 mM glucose
25 mM Tris HCl pH 8.0
10 mM EDTA pH 8.0
Question: What happens at this step?
Why is it recommended not to vortex the contents of the tube?
2. Add 200 l of freshly prepared Solution II. Close the tube tightly and mix the contents by
inverting the tube rapidly five times. Do not vortex. Store the tube on ice.
Solution II
0.2N NaOH (diluted from a 10N stock)
1% sodium dodecyl sulphate (SDS)
3. Add 150 l of Solution III. Close the tube and vortex it gently in an inverted position for
10 sec. to disperse the viscous bacterial lysate. Store the tube on ice for 35 min.
Solution III
5 M potassium acetate 60.0 ml
glacial acetic acid 11.5 ml
water 28.5 ml
The solution is 3 M with respect to potassium and 5 M with respect to acetate.
Question: Why is bacterial lysate viscous?
Why is it recommended to vortex gently in an inverted position?
4. Centrifuge at 12 000 g for 5 min. at 4C. Transfer the supernatant to a fresh tube.
Question: What compounds were left in the pellet?
What compounds are in the supernatant?
5. Precipitate the double-stranded DNA with 2 vol of 95% ethanol at room temperature. Mix
by vortexing. Leave on bench for 2 min.
Question: Why can DNA be precipitated with absolute ethanol?
Does RNA co-precipitate with DNA?
6. Centrifuge at 12 000 g for 5 min in a microfuge.
7. Remove the supernatant by gentle aspiration (will be demonstrated by an instructor). Leave
the tube in an inverted position on a paper towel to drain away the fluid. Remove any drops
adhering to the walls.
8. Rinse the pellet with 1 ml of 70% ethanol at 4 C. Remove the supernatant and allow the
pellet to dry in air for 10 min.
Question: Why 70% ethanol is used for rinsing the pellet?
9. Redissolve the pellet in 20 l of TE buffer pH 8.0 containing DNase-free pancreatic RNase
(20 mg/ml).
Tris EDTA buffer (TE)
10 mM Tris HCl pH 8.0
1 mM EDTA pH 8.0
Questions: What is the role of EDTA in the buffer?
Why is it recommended to add RNase to the DNA preparation?
Note: the usual yield is about 35 l DNA/ml of culture.

RESTRICTION ANALYSIS OF PLASMID DNA

Adapted from Sambrook J. et al., Molecular Cloning. A laboratory Manual 1.21-1.31, Cold
Spring Harbour Laboratory Press, 1989.

A. Digestion of DNA with restriction enzymes
Method:
1. To analyse the DNA by cleavage with restriction enzymes to 20 l of the DNA prep add:
2 l of the buffer R
0,5 l restriction enzyme Xho I
0,5 l restriction enzyme Hind III
mix by tapping the tube
Note: restriction enzymes will be added by an instructor
2. Incubate the reaction mixture for 1 h at 37C.
Question: What happens during the incubation?
3. Analyse the DNA fragments of the restriction digest by agarose gel electrophoresis.

B. Preparation of agarose gel
Method:
1. Seal the open ends of the plastic tray within the electrophoresis apparatus as to form a
mold.
2. Add 1 g of agarose to 100 ml of 0.5x TBE buffer (containing 10 ml of 5x TBE buffer
and 90 ml of deionized water). Heat the slurry in a microwave until the agarose dissolves.
5x Tris-borate-EDTA buffer:
Tris base 54.0 g
boric acid 27.5 g
0.5 M EDTA pH 8.0 20 ml
dissolve in 1 L of deionized water
3. Cool the solution to 60C, add ethidium bromide (EtBr) to a final concentration of
0.5 mg/ml, mix thoroughly.
Ethidium bromide stock solution:
Dissolve 10 mg of EtBr in 1 ml of water, store in light-tight container (will be provided by
an instructor).
Caution: Ethidium bromide is a mutagen. Gloves should be worn when handling EtBr.
4. Position the comb 0.5-10 mm above the plate so that a well is formed when agarose is
poured.
5. Pour the warm agarose solution into the mould, let the gel set for 30 min. During this time
dilute 10-fold the TBE 5x buffer pH 8.0 with deionized water.
6. Carefully remove the comb, mount the gel in electrophoresis tank and add diluted buffer to
cover the gel to a depth of about 1 mm.

C. Agarose gel electrophoresis
Method:
1. Mix 5 volume of the sample with 1 volume of the 6x gel-loading buffer. Slowly load the
samples into the slots using a micropipette and a yellow tip (it will be demonstrated by an
instructor). The sample containing molecular size markers will also be loaded.
Gel loading buffer (6x):
Bromophenol blue 0.25%
Xylene cyanol FF 0.25%
Ficol type 400 0.15%
Question: Why is it recommended to add gel loading buffer?
What is the advantage of running the molecular size markers?
2. Cover the tank with the lid and attach the electrical leads so that DNA will migrate towards
the anode (+). Apply the voltage of 1-5 V/cm (measured from kathode to anode). Run until
the bromophenol blue has migrated about 2/3 of the distance of the gel.
Question: Why does DNA migrate towards the anode?
What is the principle of separation of DNA fragments?
3. Turn off the current, open the lid and carefully remove the tray with the gel. Examine the
gel under UV light to detect the insert that was cut out with restriction enzymes. Take
a photograph with a polaroid camera (will be demonstrated by an instructor).
Caution: UV light can damage your eyes and skin.
Always wear protective goggles.
Note: the minimum amount of DNA that can be detected by photography (it is more
sensitive than your eyes!) in about 2 ng in 0.5 cm wide band. If there is more than 500 ng
in a single band, trailing and smearing appears. However, if bands are numerous it is
possible to load over 10 mg per slot without significant loss of resolution.
Question: What is the significance of DNA analysis in medicine?

SEMINAR IV
DNA structure and replication
1. Primary structure of DNA:
a. phosphodiester bond
b. hydrogen bonds
c. single copy, moderately and highly reiterated sequences

2. Secondary structure of DNA:
a. periodic structures
b. forces that determine polynucleotide conformation
c. double helix
d. various geometric and renaturation parameters of the DNA double helix
e. denaturation and renaturation of DNA (hybridisation, probes, heteroduplexes)
f. topology of DNA molecules (linear, circular, relaxed and supercoiled DNA)
g. the nucleotide sequence of the human genome
h. function of histones in DNA packaging

3. DNA replication in Prokaryotes and Eukaryotes:
a. semiconservative mechanism of replication
b. substrates for DNA replication
c. role of template DNA
d. enzymes involved in replication (DNA polymerases, topoisomerases, ligase)
e. other proteins involved in replication
f. synthesis of primer for replication
g. chain elongation
h. control of DNA replication


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 311-314, 322-342
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 302-305, 312-330
Stryer L. Biochemistry 6
th
edition pp. 107-118, 783-804
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 23-61, 133-152
SEMINAR V
RNA structure and function. DNA transcription.
1. Structure of RNA:
a. primary
b. secondary
c. tertiary
d. modified nucleotides found in tRNA

2. Characteristics of different types of cellular RNAs:
a. mRNA
b. rRNAs
c. tRNAs
d. snRNAs

3. Mechanisms of transcription in Prokaryotes and Eukaryotes:
a. substrates for DNA transcription
b. role of template DNA (the structure of promoters and enhancers/silencers)
c. enzymes involved in transcription (properties of RNA polymerases)
d. protein factors involved in transcription (trans elements)
e. response elements (cis elements)
f. initiation of transcription
g. chain elongation
h. termination
i. regulation of gene transcription

4. Post-transcriptional processing of RNA:
a. 45S rRNA (role of RNases)
b. hnRNA (capping, addition of poly-A tail, splicing)
c. tRNA (cleavage, additions, modification of nucleotides)

5. Clinical correlations:
a. inhibitors of Prokaryotic RNA polymerase
b. inhibitors of Eukaryotic RNA polymerases
c. involvement of transcription factors in carcinogenesis


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 314-321, 348-364, 380-401
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 305-311, 335-352, 369-387
Stryer L. Biochemistry 6
th
edition pp. 118-131, 821-856
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 62-70, 175-195, 291-318
SEMINAR VI
Protein biosynthesis
1. Components of the translation apparatus:
a. mRNA as carrier of information (genetic code)
b. structure and role of ribosomes
c. tRNA as an adaptor molecule (amino acid activation and aminoacylation of tRNA)

2. Initiation of translation in Prokaryotes and Eukaryotes:
a. initiation codon
b. initiating amino acid (formylation of methionine)
c. sequence of events in the initiation process
d. role of initiation factors
e. regulation of initiation
f. antibiotics-inhibitors of chain initiation

3. Elongation of the polypeptide chain:
a. elongation process in Prokaryotes
b. elongation process in Eukaryotes
c. peptidyl transferase
d. antibiotics-inhibitors of elongation

4. Termination of translation:
a. termination codons

5. Post-translational modification of proteins:
a. glycosylation
b. phosphorylation
c. mitochondrial proteolytic processing


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 365-379
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 353-368
Stryer L. Biochemistry 6
th
edition pp. 857-891
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 201-244






















CARBOHYDRATES











C A R B O H Y D R A T E S








INTRODUCTION III


LABORATORY


Laboratory 3. Blood glucose


SEMINARS


Seminar VII. Metabolism of monosaccharides

Seminar VIII. Metabolism of monosaccharides and
polysaccharides

Seminar IX. Glucose homeostasis in humans

Seminar CC-III Clinical correlations III


REVIEW III
INTRODUCTION TOPICS

CARBOHYDRATES


Classification and nomenclature of carbohydrates. Isomerism of carbohydrates.
Structural formulas of most common monosaccharides. Physical and chemical
properties of carbohydrates (solubility, optical properties, oxidation and
reduction products). Biologically important derivatives of monosaccharides
(deoxy-, amino- sugars, phosphate esters). Glycosidic bonds. Structure and
function of oligo- and polysaccharides (disaccharides, starch, glycogen,
cellulose). Glucosaminoglycans and glycoproteins.


LABORATORY 3
Blood glucose
ENZYMATIC DETERMINATION OF BLOOD GLUCOSE BY MEANS
OF A BACTERIAL ENZYME: GLUCOSE OXIDASE

Principle:
The aldehyde group of -D-glucose is oxidised by glucose oxidase to gluconic acid and
hydrogen peroxide. The intermediate compound is D-glucono-1,5-lactone (GO). Peroxidase
(PO) and 4-amino-antypyrine are present in the reaction mixture, so that oxygen is liberated
from the hydrogen peroxide and reacts with the 4-amino-antypyrine to produce changes in the
intensity of the pink colour. The amount of formed dye is a measure of the glucose that has
been oxidised.

-D-glucopyranose + FAD D-glucono-1,5-lactone + FADH
2

D-glucono-1,5-lactone + H
2
O D-gluconic acid
FADH
2
+ O
2
H
2
O
2
+ FAD
_________________________________________________________________

GO

-D-glucopyranose + H
2
O + O
2
D-gluconic acid + H
2
O
2



PO

H
2
O
2
+ 4-amino-antypyrine oxidised 4-amino-antypyrine
(pink)

The intensity of the pink colour measured at 500 nm is proportional to the original glucose
concentration.

Materials:
5% TCA
R1 buffers + enzymes
R2 chlorophenol
R3 standard glucose 100mg/dL (5.55 mmol/L)

ENZYMATIC DETERMINATION OF GLUCOSE IN BLOOD

Method:
1. Add 0.5 ml of TCA and 50 L of blood into the centrifuge tube.
2. Mix the contents thoroughly.
3. Centrifuge the tubes for 15 min. at 3000 rpm.
4. Add 0.5 mL of 5% TCA and 50 L of glucose standard into the centrifuge tube.
5. Mix the contents thoroughly.
6. Label the tubes with 1, 2, 3.





7. Prepare solutions as shown in the table:
unknown sample
1
standard sample
2
control sample
3
supernatant 50 L
glucose standard 50 L
Water 50 L
Solution 1 mL 1 mL 1 mL
8. Mix the contents of tubes and incubate at room temperature for 20 min.
9. Read the absorbancy (A) for unknown and standard sample against control sample at
500 nm.
10.Calculate test values as follows:
A
test

Plasma glucose [mg/dl] = x 100
A
standard



DETERMINATION OF THE AMYLASE ACTIVITY IN THE BLOOD PLASMA

Principle:
Starch and amylodextrins molecule containing more than 30 glucose residues turn the
iodine solution to blue. Amylase hydrolytic activity causes the appearance of the shorten
dextrin molecules which do not generate blue colour with iodine solution. Decrease in blue
colour intensity corresponds to the amylase activity.

Method:
1. Prepare 4 glass tubes. Add 1 mL of starch substrate to each tube and incubate samples
5 min in 37C.
2. Add 20 L of blood plasma to tubes 1, 2 and 3.
3. Add 20 L of water to tube 4 (control sample).
4. Stop the reactions exactly after 7 minutes 30 seconds by adding 1 mL of iodine
solution to each sample and mix vigorously.
5. Add 5 mL of water to each tube and mix.
6. Measure the sample absorbance at the wavelength 660 nm referring to distilled water.
7. Calculate the amylase activity according to formula:

A
control
A
sample
Units of enzymatic activity (U)/100 mL of blood plasma = 800
A
control


A
control
absorbance of sample numbered 4
A
sample
calculated mean absorbance of sample numbered 1, 2 and 3

The one unit of amylase activity is defined as the amount of the enzyme hydrolysing
10 mg of starch in 30 minutes at room temperature to the stage which is not detected by
iodine solution.
In this method, 1mL of starch substrate containing 0.4 mg of starch is incubated with
0.02 mL of blood plasma for 7 minutes and 30 second at room temperature. It corresponds to
8000 mg of starch incubated with 100 mL of blood plasma for 30 minutes. Hydrolysis of
starch 8000 mg is completed by 800 U of amylase present in 100 mL of blood plasma.
SEMINAR VII
Metabolism of monosaccharides
1. Digestion of carbohydrates:
a. sites and enzymes involved in carbohydrate digestion
b. deficiencies of intestinal disaccharidases (lactase, isomaltase-sucrase)
c. intestinal absorption of monosaccharides (simple diffusion, facilitated transport, active
transport)

2. Glycolysis and gluconeogenesis.

3. Pentose phosphate pathway.






Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 151-158, 167-180, 482-483
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 149-156, 165-177, 459-460
Stryer L. Biochemistry 6
th
edition pp. 433-474, 577-591
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 581-617, 637-642, 1056-1058

SEMINAR VIII
Metabolism of monosaccharides and polysaccharides
1. Fructose metabolism

2. Galactose metabolism

3. Disorders of fructose and galactose metabolism

4. Glycogen metabolism:
a. glycogen storage diseases

5. Biosynthesis of glucuronic acid

6. Biosynthesis of aminosugars

7. Biosynthesis of complex carbohydrates:
a. glycoproteins
b. proteoglycans


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 159-166, 180-186, 523-544,
551-558
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 157-164, 177-183, 506-526,
533-539
Stryer L. Biochemistry 6
th
edition pp. 312-323, 449-452, 592-616
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 618-636, 643-658
SEMINAR IX
Glucose homeostasis in humans
1. Concentration of glucose in blood (hypo and hyperglycaemia)

2. Sources of glucose in blood

3. Control of blood glucose concentration:
a. hormonal regulation of glucose levels in blood (including insulin synthesis and
degradation as a regulatory means, glucagon, epinephrine, glucocorticoids and other
hormones involved in carbohydrate metabolism)
b. hepatic control of blood glucose levels (glucokinase, regulatory enzymes of glycolysis,
glycogenesis and glycogenolysis, regulation of gluconeogenesis)
c. other tissues involved in regulation of blood glucose concentration:
muscles (Cori and alanine cycles)
kidney (renal treshold for glucose)

4. Overview of glycogen metabolism in liver and muscle

5. Diabetes


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 167-176
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 165-173
Stryer L. Biochemistry 6
th
edition pp. 458-474
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 608-636, 863-886






















LIPIDS











L I P I D S








INTRODUCTION IV


LABORATORY


Laboratory 4. Properties and analysis of lipids


SEMINARS


Seminar X. Metabolism of fatty acids

Seminar XI. Biosynthesis and degradation of lipids

Seminar XII. Interorgan transport of lipids

Seminar CC-IV Clinical correlations IV


REVIEW IV
INTRODUCTION TOPICS

LIPIDS


Classification and nomenclature of lipids (simple lipids, complex lipids).
Major components of lipids: fatty acids, alcohols (glycerol, sphingol, inositol,
cholesterol), phosphates, organic bases, carbohydrates. Nomenclature of
saturated and unsaturated fatty acids. Physical and chemical properties of simple
and complex lipids. Digestion and absorption of lipids from the intestine. Forms
of lipid transport in blood. Principle sites of lipid synthesis and degradation.
Role of lipids as structural components of the cell. Bile composition and role in
lipid digestion.

LABORATORY 4
Properties and analysis of lipids
ISOLATION AND SEPARATION OF PLASMA AND EGG YOLK LIPIDS

Principle:
Lipids in hydrophobic, associated form may be extracted with relatively non-polar solvents,
such as ethyl ether, chloroform or petrol-ether. Membrane associated or complex lipids
however, require polar solvents, such as ethanol or methanol to disrupt the hydrogen bonding
or electrostatic forces between lipids and proteins. Covalently bound lipids, by contrast,
cannot be extracted directly by any solvent, but must first be cleaved from the complex by
acid or alkaline hydrolysis.
Another factor, which must also be considered, is enzymatic degradation of lipids during the
extraction process. In general, the use of alcohol containing solvent mixtures is sufficient to
inactivate most lipases and phosphatidases. With more stable enzymes, immersion of the
extraction mixture for 12 min. in a boiling water bath will usually inactivate them and also
enhance precipitation of the denatured protein. From the above considerations, it follows that
alcohol is an essential component of the extracting solvent and is required for disruption of
lipid-protein complexes, dissolution of the lipids, inactivation of the degradative enzymes, as
well as for the precipitation of the denatured proteins and mixing with the aqueous phase.
However, there is a drawback introduced by the use of alcoholic solvents for lipid extraction,
namely, the co-extraction of contaminants such as sugars, amino acids, salts etc. It is therefore
essential, that the crude lipid extract obtained be treated to remove these water-soluble
contaminants. The most commonly used procedures are either to wash the primary extract
with water, or to evaporate the solvent (preferably under low pressure or in a stream of
nitrogen) together with the water, and then to dry residue with a non-polar solvent, to separate
water soluble contaminations. Thus obtained lipid mixture may then be further separated,
using various methods into individual lipid classes, which can be then identified.

EXTRACTION OF PLASMA LIPIDS

Method:
Take a graduated, glass-stoppered test tube and fill it with 9.5 ml of Bloors mixture (ethyl
alcohol:ethyl ether 3:1 v/v), then add to this (dropwise) 0.5 ml of blood plasma. Stopper the
tube, mix the contents gently and open the tube again. Next, heat the contents on a hot water
bath for about 1 min. with constant swirling of the tube. Decant the supernatant into an
evaporation dish and evaporate the solvent to dryness on a water bath. Cool down the dish
with its contents. Re-extract the dry residue with about 1 ml of hexane and transfer the
re-extracted lipids into a small vial. Stopper the vial and preserve the extract for separation by
means of TLC (thin layer chromatography).

EXTRACTION OF EGG YOLK LIPIDS

Method:
Take one half of a chicken egg yolk and place it in a beaker, extract the lipids with an
approximately 20-fold volume of Bloors mixture under occasional stirring for about 10 min.
After the denatured proteins have sedimented, decant the supernatant into an evaporation dish
and evaporate the solvent to dryness on a boiling water bath. Cool down the dish with its
contents (the dry residue contains the total lipids of the egg-yolk).

A. Separation of the neutral lipid fraction from polar lipids
Principle:
Neutral lipids are readily solubilised by cold acetone, while polar ones are acetone insoluble.
By taking advantage of this difference, it is possible to separate these lipids from each other.
The so obtained sediment comprises the acetone insoluble lipid fraction and those in
solution the neutral lipid fraction.

Method:
Extract the residue obtained in the preceding procedure with cold acetone (about 10 ml) and
decant the supernatant from the sediment into another evaporation dish. Evaporate the solvent
and dissolve the residue in about 3 ml of hexane (petroleum ether). Transfer the solution into
a vial and keep for further experiments (separation by means of TLC).
Transfer a small lump of this fraction into a small vial, dissolve in about 2 ml of petroleum
ether, stopper the vial and keep for further experiments (TLC separation).

PHYSICOCHEMICAL PROPERTIES OF COMPOUND LIPIDS

A. Solubility; demonstration of the amphipathic nature of compound lipids
Method:
With the aid of a glass rod, transfer small lumps of the acetone insoluble lipids onto the
bottom of 3 dry test tubes, number them and add: 5 ml of water into tube 1, 5 ml of ethanol
into tube 2 and 5 ml of chloroform into tube 3. Shake vigorously the contents and observe the
results.
Compare the results of this experiment with those obtained in a similar experiment in which
vegetable oil was used (I.). Draw conclusions. Draw an image of the structure acquired by
these lipids when solubilised in water.

B. Detergent properties of the water solution of compound lipids
Method:
Add one drop of vegetable oil to tube 1 from the former experiment, shake vigorously and
observe the result. Draw appropriate conclusions.
Draw an image of the mixed micelles formed under these circumstances.

C. Demonstration of nitrogen bases
Principle:
Strong alkali acting at high temperature are capable of hydrolysing ester bonds formed
between the nitrogen containing alcohols (serine, ethanolamine and choline) and to
decompose these alcoholamines into free volatile aliphatic amines and ammonia. These
amines, as well as the ammonia, may be detected both by their characteristic smell and by
their alkaline reaction, which may be evidenced with the aid of suitable indicators.

Method: (Will-Varrentrap)
The experiment should be done under the fume hood.
Place a small lump of the acetone insoluble lipid fraction onto the bottom of a test tube, add a
few crystals of soda lime [NaOH-Ca(OH)
2
], and heat the contents with a lighter till dense
fumes will form. Place a damp indicator paper onto the outlet of the tube. You will soon
discover the characteristic smell and the indicator paper will change its colour indicating the
alkaline character of these fumes.
Question: Present the formulas of nitrogen bases present in phospholipids.

D. Test for glycolipids
Principle:
Sugars, when treated with concentrated sulphuric acid are transformed into cyclic aldehydes
(furfural or oxymethylene-furfural) which under anhydrous conditions form coloured
condensation products with aromatic phenols or amines (Molishs method).

Method:
The experiment should be done under the hood.
Heat the contents of tube 2 from the experiment on solubility of compound lipids (alcohol
solution) on a water bath heated to boiling temp., add to it 23 drops of -naphtol. Blend the
contents and then add carefully after tipping of the tube, 1 ml of conc. sulphuric acid, along
the tube wall. Observe the appearance at the contact surface of these two liquids a purple
coloured ring. A positive reaction is indicative of the presence of glycolipids in the tested
lipid sample.
Questions: Give a concise description of the individual glycolipid classes and of their
carbohydrate moieties.
What can be inferred from all the performed experiments with the acetone
insoluble lipid fraction?

SEPARATION OF EGG YOLK AND PLASMA LIPIDS BY MEANS OF THIN-
LAYER CHROMATOGRAPHY (TLC)

Principle:
Having extracted and partially analysed the tissue or cellular lipids (as described above) one
has some idea of the classes of compounds present in the mixture. The next stage of
investigation of lipid composition involves fractionation of the mixture into various classes of
lipids and then into pure individual components. The exact fractionation procedure to be used
at this stage will depend largely on the particular classes of lipids present. These methods may
include: solvent fractionation (as in the acetone precipitation of compound lipids); solvent
partition (counter-current distribution), column-adsorption, partition- and ion-exchange
chromatography, surface chromatography on silic acid-impregnated paper or thin-layer
chromatography (TLC).

Method:
The lipid mixtures to be separated are:
a. plasma total lipids
b. acetone soluble lipid fraction from egg-yolk

A. Preparation and conditioning of chromatographic chambers
Method:
Chamber N (neutral lipids)
Chromatographic solvent: hexane : diethyl ether : acetic acid, (84 : 16 : 0.8 V/V).
Pour the solvent into the separation chamber, just enough to cover the bottom of the jar and
screw tightly the lid onto the opening of the jar. Leave enough time to saturate the chamber
with solvent vapors (not less than 15 min.).
Chamber P (polar lipids)
Chromatographic solvent: chloroform : methanol : water (65 : 25 : 4 V/V).
Proceed as described for chamber N except that solvent P has to be used.

B. Application of lipid extracts onto pre-coated TLC micro-slides
Into a Hamilton-micro-syringe dispenser aspirate 100 l of the respective lipid extract
(acetone soluble, acetone insoluble lipids of egg-yolk and the total plasma lipids). Spot the
lipid extract drop by drop onto the starting line of a silica-gel coated micro-slide, about
0.5 cm beyond the lower edge of the slide, and along 2/3 of the slides width, so that a
continuous line of applied lipids, about 1 cm long will form. Let the solvent evaporate before
repeatedly spotting. Mark the plates on upper right corner so as to identify the sample.

C. Running and developing of chromatograms (proper separation process
and visualisation of separated spots)
Method:
Insert the slides with the applied lipids in the respective separation chamber (N and P),
close the jars tightly and allow the solvent to ascend to about 0.3 cm from the top edge.
(Caution! Dont let the solvent run off the plate).
After the solvent has reached the desired height, remove the plates from the jars and place
them horizontally onto a sheet of filter paper, let the solvent evaporate in the open air or in a
fume hood, and when dry, place the plates into an iodine containing chamber. The lipid spots
will acquire a yellow-brown colour.
Circumscribe the spots by means of a thin needle (be careful not to destroy the layer, it is
easily broken), and record an image of these chromatograms.

SERUM EGG-YOLK



N P N P

Try to identify the individual lipids on the basis of the given R
f
-values.
(R
f
-values for neutral lipids separated by means of adsorption TLC with hexane : ethyl-ether :
acetic acid are: hydrocarbons and waxes: 0.9-1.0; sterol esters: 0.9; TAG: 0.3-0.4; FFA: 0.18;
free sterols: 0.10; DAG: 0.08; MAG 0.0; polar lipids 0.0)
(R
f
-values for phospholipids separated by means of partition TLC with chloroform : methanol
: water: cerebrosides: 0.7-0.76; phosphatidic acid: 0.74; cardiolipin: 0.71; phosphatidyl-
ethanolamine: 0.62; sphingomyelin: 0.16; phosphatidylserine: 0.15; lyso-compounds: <0.1)
Questions: Compare the lipid spectra from the egg-yolk with those from plasma and
comment on the differences.
Present the normal plasma lipid composition for adults.
Which of these lipids are subject to wide diurnal variations?
What intra-vascular process enables to utilise the circulating TAG as fuel
sources by peripheral tissues?
Present by means of appropriate formulas, the reaction which is responsible for
the fact that about 75% of the total plasma cholesterol is in the esterified form?

SEMINAR X
Metabolism of fatty acids
1. Biosynthesis of FA.
Cytosolic pathway for de novo synthesis of saturated FA:
a. substrates, enzymes and cofactor requirements for the synthesis of priming units
(acetyl-CoA carboxylase)
b. FA synthase complex and sequence of reaction catalysed by this enzyme complex
c. sources of acetyl-CoA and NADPH
d. elongation of the FA chain (microsomal and mitochondrial)
e. desaturation of the FA chain
f. biosynthesis of hydroxylated FA
g. regulation of lipogenesis: nutritional state, regulation of key enzyme activity
(acetyl-CoA carboxylase, pyruvate dehydrogenase), hormonal regulation.

2. Degradation of FA:
a. -oxidation of FA: activation of FA, formation of acyl-CoA, transport of FA into
mitochondria, the site of -oxidation (enzyme and cofactor requirements); individual
steps of -oxidation (enzymes and cofactors); -oxidation of unsaturated FA; energy
balance of -oxidation
b. other oxidative pathways: - and -oxidation of FA, peroxisomal oxidation of FA;
oxidation of FA and thermogenesis, metabolism of brown adipose tissue
c. ketogenesis (under physiological and pathological conditions): ketone bodies as the
immediate fuel for extrahepatic tissues under conditions of glucose deficiency; reactions
involved in the utilisation of ketone bodies in extrahepatic tissues; regulation of
ketogenesis; ketoacidosis as a result of a metabolic imbalance between ketogenesis and
utilisation capacity.

3. Metabolism of unsaturated FA:
a. eicosanoids: cyclooxygenase pathway: prostaglandins, prostacyclins and tromboxanes
(biosynthesis, degradation and function); lipoxygenase pathway: leukotrines
(biosynthesis, degradation and function)

4. Clinical correlations:
a. genetic deficiencies in carnitine transport or carnitine palmitoyltransferase
b. genetic deficiencies in the acyl-CoA dehydrogenases
c. Refsums disease
d. diabetic ketoacidosis

Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 187-208
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 184-204
Stryer L. Biochemistry 6
th
edition pp. 617-648
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 668-675, 680-691, 730-737
SEMINAR XI
Biosynthesis and degradation of lipids
1. Metabolism of triacylglycerols (TAG):
a. degradation: main sites of TAG degradation (lipolysis): digestive tract, blood plasma
and adipose tissue. Regulatory mechanisms controlling the rate of TAG lipolysis
b. biosynthesis: main sites and routes of TAG biosynthesis: adipose tissue, liver, intestine.
Regulatory mechanisms controlling the rate of TAG synthesis

2. Metabolism and biological role of compound lipids:
a. biosynthesis of phosphoglycerides: phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, phosphatidylinositol, phosphatidylglycerols, plasmalogens (syn-
thesis de novo and remodelling routes)
b. biosynthesis of sphingomyelin
c. degradation of phospholipids: role of various phospholipases and role of degradation
products (release of polyunsaturated FA, DAG, phosphoinositol)
d. roles of various phospholipids:
lecithin in blood plasma (aiding in the transport of non-polar lipids, substrate for
LCAT activity, reaction catalysed by LCAT)
specific role of pulmonary phospholipids as surfactants: dipalmitoyl-phosphatidyl-
choline as the primary surfactant, routes of biosynthesis (RDS)
l-alkyl-2-acetyl-glycerol-3-phosphocholine: the platelet activating factor (route and
site of synthesis)
specific role of phosphatidylinositol and of other phospholipids in generating second
messengers
e. biosynthesis and degradation of glycosphingolipids (cerebrosides, sulfatides and
gangliosides)
f. sphingolipidoses as a failure in degradation of various sphingolipids

3. Clinical correlations:
a. obesity


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 209-216
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 205-211, 460-462
Stryer L. Biochemistry 6
th
edition pp.732-738
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 663-664, 676-691, 695-706, 720-
729, 1059-1064
SEMINAR XII
Interorgan transport of lipids
1. Lipoproteins: composition, properties, synthesis and function.

2. Metabolism of chylomicrons.

3. Metabolism of VLDL.

4. Metabolism of HDL.

5. Transport and uptake of FA.

6. Role of the liver and adipose tissue in the metabolism of TAG.

7. Role of the liver in the disposal of cholesterol.

8. Hypolipoproteinaemias and hyperlipoproteinaemias as inborn metabolic disorders.


Literature:
Murray R. K. et al. Harpers Biochemistry 27
th
edition pp. 217-229
Murray R. K. et al. Harpers Biochemistry 28
th
edition pp. 212-223
Stryer L. Biochemistry 6
th
edition pp.742-748
Devlin T. M. Textbook of Biochemistry 6
th
edition pp. 665-667