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
2015/2016

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.

pjagodzi@ump.edu.pl

Head of Department

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

Hanna Drzewiecka, M.Sc.

Assistant

epi6@o2.pl

Bartosz Frycz, M.Sc.

Assistant

bartekfrycz@gmail.com

Mariusz Nawrocki, M.Sc.

Assistant

THE BIOCHEMISTRY COURSE

Rules and Regulations for the 6-year M.D Program, Academic Year 2015/2016
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 (70 h) consists of lectures (6 h), seminars (12 h) and practical classes (52 h). The
course is divided into two modules: 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 10 points for 2 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 130 points (100%) plus up to 20 bonus points extra (bonus points will be added to
the final score). To receive a credit student must earn a minimum of 91 points (70%). A student, who
accumulated from 39 to 90 points (3070%), to receive the credit must pass an integrative test
(30 questions) during final review, covering topics of the two 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 39 points (<30%) is not be allowed to take the integrative test and
will not receive a credit for the course.
During the entire Biochemistry course (Biochemistry I and II) a student can accumulate jointly up
to 260 points (100%) plus up to 40 bonus points extra (bonus points will be added to the final score).
Numbers of the points accumulated during the entire academic year will be converted to a grade,
according to the following system:
7074% satisfactory
7579% fairly good

8084% good
8589% better than good

>90% very good

FINAL EXAMINATION
Students who have earned credit must take the final exam prepared by the National Board of
Medical Examiners (NBME). NBME exam covers material of entire Biochemistry course
(Biochemistry I and II). If a passing grade on NBME is obtained, the final mark can be upgraded and
will be a mean of the grade obtained during the entire Biochemistry course and the grade obtained on
the NBME exam. If a failing grade on NBME is obtained, the examination can be retaken twice
(according to the schedule) before the beginning of the next academic year (MCQ test, 100 questions
graded 1 point for a correct answer). However, in this case, the final mark cannot be upgraded.

THE BIOCHEMISTRY COURSE

Rules and Regulations for the 6-year M.D Program, Academic Year 2015/2016

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 2015/2016

III. Carbohydrates

IV. Lipids

Introduction III

Introduction IV

Lab. 3

Lab. 4

Properties and analysis of lipids

Sem. VII Metabolism of monosaccharides

Sem. X

Metabolism of fatty acids

Sem. VIII Metabolism of polysaccharides

Sem. XI

Biosynthesis and degradation

of lipids

Sem. IX

Blood glucose

Glucose homeostasis in humans

Sem. XII Interorgan transport of lipids

CC-IIIA Clinical correlations IIIA

CC-IVA Clinical correlations IVA

CC-IIIB Clinical correlations IIIB

CC-IVB Clinical correlations IVB

Review III

Review IV

CARBOHYDRATES

CARBOHYDRATES

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

CC-III A

Clinical correlations IIIA

CC-III B

Clinical correlations IIIB

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 + FADH2
D-glucono-1,5-lactone + H2O D-gluconic acid
FADH2 + O2 H2O2 + FAD
_________________________________________________________________
GO

-D-glucopyranose + H2O + O2 D-gluconic acid + H2O2

PO

H2O2 + 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:

supernatant
glucose standard
Water
Solution

unknown sample
1
50 L

standard sample
2

control sample
3

50 L
1 mL

1 mL

50 L
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:
Atest
Plasma glucose [mg/dl] = x 100
Astandard

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:
Acontrol Asample
Units of enzymatic activity (U)/100 mL of blood plasma = 800
Acontrol
Acontrol absorbance of sample numbered 4
Asample 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 27th edition pp. 151-158, 167-180, 482-483
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 149-156, 165-177, 459-460
Stryer L. Biochemistry 6th edition pp. 433-474, 577-591
Devlin T. M. Textbook of Biochemistry 6th 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 27th edition pp. 159-166, 180-186, 523-544,
551-558
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 157-164, 177-183, 506-526,
533-539
Stryer L. Biochemistry 6th edition pp. 312-323, 449-452, 592-616
Devlin T. M. Textbook of Biochemistry 6th 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 27th edition pp. 167-176
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 165-173
Stryer L. Biochemistry 6th edition pp. 458-474
Devlin T. M. Textbook of Biochemistry 6th edition pp. 608-636, 863-886

LIPIDS

LIPIDS

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

CC-IVA

Clinical correlations IVA

CC-IVB

Clinical correlations IVB

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 THINLAYER 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

Try to identify the individual lipids on the basis of the given Rf-values.
(Rf-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)
(Rf-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; phosphatidylethanolamine: 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 27th edition pp. 187-208
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 184-204
Stryer L. Biochemistry 6th edition pp. 617-648
Devlin T. M. Textbook of Biochemistry 6th 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 (synthesis 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-phosphatidylcholine 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 27th edition pp. 209-216
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 205-211, 460-462
Stryer L. Biochemistry 6th edition pp.732-738
Devlin T. M. Textbook of Biochemistry 6th edition pp. 663-664, 676-691, 695-706, 720729, 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 27th edition pp. 217-229
Murray R. K. et al. Harpers Biochemistry 28th edition pp. 212-223
Stryer L. Biochemistry 6th edition pp.742-748
Devlin T. M. Textbook of Biochemistry 6th edition pp. 665-667