DANYLO HALYTSKYI
LVIV NATIONAL MEDICAL UNIVERSITY
DEPARTMENT OF BIOLOGICAL CHEMISTRY
BIOLOGICAL CHEMISTRY
METHODICAL INSTRUCTIONS FOR PRACTICAL
EXERCISES
for students of pharmaceutical faculty
PART I
LVIV – 2011
1
Principal rules of security of the work in biochemical laboratory.
The work in biochemical laboratory demands permanent care and attention.
Carelessness may be the cause of dangerous consequences, such as explosion,
combustion, poisoning, fire etc. Taking this into account a working person must follow
the next principal rules of security .
1. The working place must be always in order. All unnecessary things (bags, not needed
books or papers, clothes, hindering things) must be eliminated.
2. By heating a tube on an open flame don’t look into the tube or direct the tube orifice
to the own face of researcher or upon surrounding colleagues. During the heating the
tube must be rotated in a flame in order to prevent its breakdown.
3. The dilution of concentrated acids, especially sulfuric acid, is performed by pouring
acid into the water, not vice versa.
4. Measurement of the quantity of strong acids, alkalies, poisonous reagents must be
done with graduated cylinders. Measurement with pipettes and sucktion of mentioned
reagents is forbidden.
5. Smell of gases or vapours must be done with a care, beginning from some distance.
6. Evaporation of acids, ammonia, substances with unpleasant smell must be performed
under the chemical hood.
7. Don’t look inside the flask or tube, in which a reaction proceeds, it is dangerous.
8. Inflammable substances (benzene, acetone, ether, alcohol) must be stored in special
boxes and keep on a distance from an open fire. The work with these solvents is
recommended to be conducted under the hood.
9. The work with strong poisons ( benzidine, phenylhydrazine, phenol, cyanides,
alkaloids) must be conducted very attentively in a proper place, hands must be carefully
washed.
10 Testing of different substances or reagents by taste is forbidden. Only in special
cases it may be performed according to the instruction or recommendation of assistent
professor.
11. Smoking in laboratory is forbidden. The working student must have an overcoat
and hood.
12. After finishing the work all glassware must washed and stored in a proper place
according to recommendations of an assistent. The remnants of biomatherial must be
eliminated after use or at the end of the work.
13. Don’t throw out filter paper, pieces of glass, matches, cotton or similar things into
the washbowl. After finishing the work the taps of water and gas must be closed,
electric power switch off. To leave laboratory after the permission of the assistent.
2
Module 1. General principles of metabolism.
Metabolism of carbohydrates, lipids and its regulation.
Thematic plan of practical lessons in module 1
N
Theme of the lesson
Sense module 1: Introduction to biochemistry. Simple and conjugated
proteins. Enzymes.
1
Introduction to biochemistry. Methods of biochemical investigation.
Amino acid composition, structure, physico-chemical properties,
classification and functions of simple and conjugated proteins
Enzymes: structure, physico-chemical properties, classification and
2
mechanism of action of enzymes. Methods of detection of enzymes in
biological material.
Kinetics of enzymatic reactions. Regulation of enzymatic activity,
3
determination of enzymatic activity.
Regulation of enzymatic processess enzymopathias and mechanisms of
4
their development. Application of enzymes as pharmaceutical
preparations.
Role of cofacors and coenzymatic vitamins in catalytic activity of
5
enzymes.
Sense module 2: General principles of turnover of substances and energy
General principles of turnover of substances and energy.
6
Functioning of citric acid cycle.
Biological oxidation, oxidative phosphorylation and ATP synthesis.
7
Inhibitors and uncouplers of tissue respiration and oxidative
phosphorylation in respiratory chain of mitochondria
Sense module 3: Metabolism of carbohydrates and its regulation
Anaerobic and aerobic oxidation of glucose, alternative pathways of
8
carbohydrate metabolism.
Catabolism and biosynthesis of glycogen.Regulation of glycogen
9
metabolism Biosynthesis of glucose - gluconeogenesis,
10 Mechanisms of metabolic and humoral regulation of carbohydrate
metabolism. Disorders of carbohydrate metabolism.
Sense module 4: Lipid metabolism and its regulation
11 Catabolism and biosynthesis of triacylglycerols and phospholipids.
Intracellular ipolysis and molecular mechanisms of its regulation.
12 -Oxidation of fatty acids and their biosynthesis. Investigation of fatty
acids ketone bodies metabolism.
13 Biosynthesis and biotransformation of cholesterol/ Regulation of lipid
metabolism and its disorders.
Summary control of module 1: “General principles of metabolism.
14
Metabolism of carbohydrates, lipids and its regulation”
3
N
1
2
3
4
5
6
7
8
9
10
11
Thematic plan of lectures on module 1
for students of pharmaceutical faculty
Hours
Theme of the lecture
Introduction to biochemistry. Amino acid composition of proteins.
2
Structure, properties and classification of proteins.
Enzymes: structure, properties and classification. Mechanism of action and
2
regulation of enzymatic activity. Kinetics of enzymatic reactions.
General principles of turnover of biomolecules and energy. Tricarboxylic
2
acid cycle.
Molecular princip;es of bioenergetics. Biological oxidation. Oxidative
2
phosphorylation and its regulation.
Digestion of nutrients in gastrointestinal tract of human being.
2
Carbohydrates: structure, classification, functional significance. Metabolism
of monosaccharides, anaerobic and aerobic oxidation of glucose.
2
Gluconeogenesis.
Metabolism of polysaccharides
2
Regulation of carbohydrate metabolism and its disorders.
2
Lipids: structure, classification and functional significance. Transport forms
2
of lipids in blood. Metabolism of simple lipids.
Metabolism of complex lipids and its regulation. Correction of disorders of
2
lipid metabolism by pharmaceutical preparations.
General catabolic pathways of amino acids. Biological role of products of
2
amino acids catabolism.
Criteria for evaluation of students knowledge level in module 1
Minimal
Maximal
number of
number of
points
points
65
117
Current learning activity
Summary module control
50
80
Total
115
197
The current learning activity and answers on control lessons of modules are
evaluated according to traditional system of marks and in points as follows:
“5” – 9 points, “4” – 7 points, “3” – 5 points, “2” – 0 points.
Student obtains permission for participance in summary module control after the
fulfillment of educational program and in a case when he (she) achieved for current
learning activity points number not less then 65 points.
The summary module control is accepted if the student shows the knowledge of
practical methods of investigation and achieved in control of theoretical level points
number not less then 50.
4
Topic № 1. Introduction to biochemistry. Methods of biochemical investigations.
Structure of proteins, amino acid composition, physico-chemical properties,
classification and function of simple and conjugated proteins.
Objective: introduction to the subject and assignments of biological chemistry,
their practical employment; to make an acquaintance with methods of biochemical
investigation. To demonstrate some physico-chemical methods of investigation as well
to learn about devices used in biochemistry. To learn structure, classification and
physico-chemical properties of proteins; to perform qualitative reactions on amino acids
and quantitative determination of protein.
Specific objectives:
 To analyze the present status and perdpectives of biochemistry? Application of its
achievements in clinical and industrial pharmacy.
 To interpret amino acid composition, structural organization, physico-chemical
properties and methods of protein purification. To discuss a multiple functions and
biological properties of proteins and peptides.
 To discuss classification and characterization of simple and conjugated proteins,
significance of selected representatives.
Actuality of the theme. Proteins are high molecular organic compounds which are the
background of the structure of cells and tissues, take part in metabolism, are involved
into processes of growth, reproduction and constant renewal of body compounds. They
are widely used in practical medicine for treatment and diagnostics.
Theoretical questions
1. Definition of biochemistry as a science and its position among other medical and
biological disciplines. An object and assignments of biochemistry. Principal trends and
parts of biochemistry.
2. A short history of biochemistry and principal stages of its developement.
Contribution of scientists of the Dept. of Biochemistry of Lviv State Medical University
to the development of biochemistry.
3. Principal methods of biochemical investigation:
- optical methods in biochemistry( photoelectrocolorimetry, spectrometry,
spectrophotometry, fluorescent analysis).
- electrophoresis (zonal electrophoresis, electrophoresis in gels, isoelectrofocusing,
immunoelectrophoresis).
- chromatography and its types (ion-exchange chromatography, affinity
chromatography, thin layer chromatography - TLC, gel filtration).
- principles of polarographic methods. Manometric and radioisotope methods in
biochemical investigations.
4. General characterization of amino acids. Classification of amino acids (due to their
structural, electrochemical, biological properties).
5. Biologically active peptides, their significance and employment in medicine
(glutathione, hormones of hypophysis and hypothalamus, insulin etc.).
5
6. Modern concept of structural levels in organization of protein molecule and types of
chemical bonds in protein molecule. Physical and chemical properties of proteins.
Isoelectric point, proteins as amphoteric electrolytes.
7. Methods of protein purification and isolation. Criteria of protein purity. Dialysis of
proteins and its employment in medicine. Methods of quantitative determination of
proteins.
8. Classification of proteins. Characterization of simple proteins.
9. Conjugated proteins, their characteriristics.
10. Amino acids, peptides and proteins as medicinal agents.
Practical part
Experiment 1. Measurement of optical density (D) of solutions which contain
different quantities of phosphorus.
Principle. Phosphates in a solution of sulfuric acid form phosphomolybdate
complexes with molybdate, which are then reduced to molybdate blue. The amount of
phosphates is then measured colorimetrically according to a colour intensity of
molybdate blue complex.
Procedure. Add the following solutions to five tubes (as indicated in the table):
N
1
2
3
4
5
Reagents
Standard phosphate solution (10
mg/ml)
Distilled water, ml
Molybden reagent, ml
Phosphorus concentration, mg%
Results of optical density measurement
Tube number
1
2
3
4
5
-
0.5
1.0
2.0
3.0
5.0
5.0
0
4.5
5.0
50
4.0
5.0
100
3.0
5.0
200
2.0
5.0
300
After mixing a colour develops, which is measured on a photoelectrocolorimeter
with a red light filter. Optical density must be measured at distinct time after the
addition of the last reagent, usually after 5 min. According to the results obtained a plot
is constructed (a calibration curve).
Experiment 2. Biuret method of protein quantitation.
Principle. Proteins in alkaline medium interact with copper sulfate with formation
of compound with violet color.
Reagents. Blood serum, standard protein solution (50 g/l ), biuret reagent (0,15 g
copper sulfate, 0,6 g sodium, potassium tartrate are dissolved in 60 ml of water, 30 ml
of 10% NaOH solution are added, volume adjusted to 100 ml, then 0,1 g KJ is added).
Procedure. To the first tube 0,1 ml of saline is added (control), to the second tube
0,1 ml of standard protein solution is introduced, into the third and fourth tubes 0,1 ml
of tested protein solutions are added. To all tubes 5 ml of biuret reagent are added,
mixed and after 10-30 min the extinction (optical density) is measured on a
photocolorimeter using green light filter against control solution.
6
Calculation. The concentration of protein in test solution is calculated according to
formula
X= (AxB)/C, where
X –protein concentration in test solution, (g/l),
A – protein concentration in standard solution (g/l),
B – extinction of tested protein solution,
C – extinction of standard protein solution.
Clinical and pharmaceutical significance. In a healthy person the content of
protein consists from 60 g/l to 80 g/1. Below 60 g/1 is hypoproteinemia and above 80
g/1 is hyperproteinemia. A drop in blood protein concentration occurs as a result of a
decrease in protein biosynthesis, of water balance disorders, or as a result of an increase
in protein breakdown and protein loss. Hypoproteinemia is observed in nephritic
syndrome, malabsorption syndrome (enteritis, chronic pancreatitis), exudative
enteropathias, skin diseases (combustion, exematous lesions), during massive blood
loss, retention of water and mineral salts (chronic kidney diseases),
agammaglobulinemias, hypogamma-globulinemias, and during starvation or improper
nutrition.
Decrease in blood protein concentration is observed during cardiac failure as a
result of water retention causing a swelling of tissues or during kidney diseases when
proteins are excreted in the urine. Protein biosynthesis disturbances take place during
cancer cachecsia and during chronic inflammatory diseases, when accompanied by
degenerative processes.
Hyperproteinemia is observed in plasmocytoma, Waldenstrom macroglobulinemia,
rheumatoid arthritis, collagenoses, liver cirrhos as well as in diseases accompanied with
dehydration, that is during diarrhea, vomiting, diabetes insipidus. As a result of heavy
mechanical lesions (traumas) the increase in blood protein concentration may be due to
the loss of a substantial part of the intravascular fluid. In acute infections the increase in
blood protein concentration may due to the increased production of acute phase
proteins, in chronic infectious diseases it may be due to enhanced synthesis of
immunoglobulins.
Control of laboratory work.
1. What method is used most frequently for quantitative determination of protein in
biological material?
2. What compounds develop a positive biuret reaction?
3. General content of protein in blood of a person is 100 g/L. Does it corresponds to
normal values? Yow is called this status?
4. General content of protein in blood of a person is 45 g/L. How is called this status
and what is the cause of its appearance?
Examples of tests
1. A linear polymer chain of amino acids is called:
A. Polypeptide
B. Polyamine
7
C. Polynucleotide
D. Polyacrylate
E. Polyprene
2. In protein biosynthesis are involved:
A. L- amino acids only
B. D-amino acids only
C. D-amino acids predominantly
D. L-amino acids predominantly
E. Both D- and L- amino acids equally well
3. How many asymmetric carbons contains α-alanine?
A. One
B. Two
C. Three
D. Four
E. None
4. Secondary structure of proteins includes the next structural element:
A. α-Helix
B. Triple superhelix
C. Disulphide bridges
D. Acetylated amino groups
E. Attachment of oligosaccharides to a polypeptide chain
5. Chose the correct value of normal protein concentration in human blood plasma
A. 65-85 g/l
B. 25-40 g/l
C. 45-60 g/l
D. 85-100 g/l
E. 100-150 g/l
Individual independent students work
Methods of fractionation and purification of proteins
1.
2.
3.
4.
References.
Murrey R.K., Granner D.K., Mayes P.A., Rodwell V.W. Harper’s Biochemistry, 26th Ed. 2003, 927 p.
Trudy McKee, James McKee. Biochemistry. Wm C Brown Publishers. 1966, 654
p.Bhagavan N.V. Medical biochemistry. Jones and Bartlett Publishers International:
Boston-London. – 1992, 980 p.
Champe P.C., Harvey R.A. Biochemistry.2-nd Ed. Lippincott’sIllustrated reviews.
1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Part I Lviv2002, 102 p.
8
5. Mardashko O.O., Yasinenko N.Y., Biochemistry. Texts for lectures. Jdessa State
Medical University. 2003, 416 p.
Topic № 2. Enzymes: structure and physico-chemical properties, classification of
enzymes and mechanisms of enzymatic action. Studies of methods of enzymes
detection in biological material.
Objective: To study principles of organization and general properties of enzymes.
To learn the method of determination of salivary amylase activity and the specificity of
action of distinct enzymes.
Specific objectives:
 To interpret biochemical principles of structure and functioning of different classes
of enzymes.
 To explain chemical nature of enzymes and their properties as biocatalysts.
 To analyze theories of mechanisms of enzymatic action and stages of enzymatic
catalysis.
 To learn principles of determination of enzymatic activity in biological fluids.
Actuality of the theme. Enzymes are biocatalysts of almost exclusively protein
nature which play a key role in metabolism of alive cells, tissues and organs. They are
widely used as diagnostic tools, medicinals and pharmaceutical preparations. Many
pharmaceutical substances influence, mainly inhibits, activity of enzymes and are used
in treatment of different pathological states.
Theoretical questions.
1.
2.
3.
4.
5.
6.
7.
8.
Function of enzymes.
Chemical nature of enzymes and their properties as biocatalysts.
Structural organization of enzymes.
Structure of simple and complex enzymes. Prosthetic groups of complex.
enzymes, vitamins as precursors in biosynthesis of coenzymes.
General properties of enzymes and specificity of their action.
Actual classification and nomenclature of enzymes.
Mechanisms of enzymatic action. Stages of enzymatic catalysis.
Hypotheses and theories of the mechanisms of enzymatic action.
Practical part
Experiment 1.Detection of amylase in saliva.
Principle. Starch with iodine gives a blue coloured complex.
Dextrins in
presence of iodine form red-brown colour or no change in colour at all. Maltose also
does not form a coloured complex with iodine, but it can be detected with the Trommer
reaction.
Method. Place 10 tubes in the tube holder and put into each tube 2 ml of water
and 1 drop of Lugol solution. Pour into a separate tube 5 ml of 0.5 % starch and 1 ml of
diluted saliva. Mix the contents of the tube and after 60 sec 0.5 ml of the fluid is
drained and added to the tubes 1, 2, 3 etc. in the tube holder. The hydrolysis of starch is
considered to be complete when the probe will not change the colour of iodine in the
9
test tube. Then, the Trommer reaction is performed with hydrolysate. To the several ml
of test solution an equal volume of 5% NaOH is added and several drops of CuSO4
solution. The content is mixed and heated on a flame to boiling. The appearance of
yellow-red sediment indicates on the presence of reducing substances, namely, reducing
disaccharide maltose.
Explain the results and draw a conclusion.
Experiment 2. Investigation of substrate specificity of salivary amylase and yeast
sucrase
Principle. Enzymes exhibit selectivity to substrates, which is called substrate
specificity. In many instances this property is the essential characteristic that renders
enzymes markedly different from inorganic catalysts. The high specificity of enzymes is
attributable to the conformational complementarities between the molecules of enzyme and
substrate due to the unique structure of active centre of the enzyme. These properties
provide for the high affinity and selective course of a particular chemical reaction among
the vast variety of other chemical reactions simultaneously occurring in the living cells.
Depending on the degree of selectivity enzymes are distinguished as having relative
(or group specificity) or absolute specificity. Pepsin splits the proteins of animal and plant
origin, although these substrates of pepsin may differ in the chemical composition, as well as
in physical and chemical properties. However, pepsin does not split carbohydrates or fats.
This is explained by the fact that the point for pepsin attack in the substrate molecule is the
peptide bond. For lipase, which catalyzes the hydrolysis of fats to glycerol and fatty
acids, the point of attack is the ester bond. Enzymes exhibiting analogous selectivity in
hydrolysis of proteins are trypsin, chymotrypsin, peptidases Enzymes that hydrolyse
glycoside bonds in polysaccharides, are called glycosidases.. Usually, these enzymes are
involved in the digestive process and their specificity has a definite biological significance.
Relative specificity is also exhibited by certain intracellular enzymes, such as hexokinase
capable of catalysingthe phosphorylation of hexoses using ATP as donor of phosphate and
energy. Simultaneously in the cells are present enzymes, which are specific for distinct
individual hexose (e.g. glucose) and catalyse the same reaction of phosphorylation.
The absolute specificity of enzymatic action refers to the ability of an enzyme to
catalyse the conversion of only a single substrate. Any changes (modifications) in the
substrate structure render it inaccessible to enzymatic attack.
There is experimental evidence for the existence of the so-called -stereochemical
specificity associated with the L or D stereoisomeric forms or cis- and trans- isomers of
chemical compounds.
Materials and reagents: 1 % starch solution, sample of sucrase from yeasts,
1 % solution of sucrose, 0,1 n solution of J2 in KJ (Lugol solution), 10% sol. of sodium
hydroxide, 5% sol. of CuSO4. Diluted saliva, water bath, tube holder, pipettes.
Method:
1. Specificity of amylase: in each of two tubes is placed 1 ml of saliva two fold
diluted. Into tube N1 is added 2 ml of 1% solution of starch, into tube N2 – 2 ml
of 1% solution of sucrose. Tubes are incubated at 37 C in thermostate for 15 min.
Thereafter the content of both tubes is submitted to Trommer reaction.
2. Specificty of yeasts sucrase: into each of two tubes is placed 1 ml solution of
sucrase from yeasts. Into tube N 1 is added 2 ml of 1% solution of sucrose, to the
10
2-nd tube is added 2ml of 1% starch solution. Tubes are incubated at 37 C in
thermostate for 15 min. Thereafter with the content of both tubes is performed
Trommer reaction.
Results of the experiment are noted in the table.
Tube №
Enzyme
Substrate
Result of Trommer
reaction
1
2
3
4
Explain the results and draw a conclusion.
Clinical and pharmaceutical significance.
Activity of amylase is increased in blood and in urine in case of inflammation of
salivary glands (mumps) and pancreas (acute pancreatitis). Activity of amylase in saliva
has almost no diagnostic significance.
Control of experimental work
1. What reaction catalyzes salivary amylase?
2. What are products of amylase action upon starch?
3. What changes in amylase activity are observed in pathology?
4. What types of enzyme specificity
5. What type of specificity is characteristic to amylase and sucrase?
Examples of tests
1. Chose a correct statement about common feature of enzymes and inorganic catalysts.
A. Acceleration of thermodynamically permitted reactions
B. Dependence of activity from pH of reaction medium
C. High selectivity to type of catalyzed reaction
D. Specific dependence from substrate concentration
E. Dependence from the presence of cofactors
2. Prosthetic group of complex (conjugated) enzyme is defined as:
A. Coenzyme
B. Apoenzyme
C. Holoenzyme
D. Allosteric factor
E. Suppressor
3. As cofactors of enzymes the most frequently are met the next compounds:
A. Vitamins and their derivatives
B. Hormons, e.g. thyroxine
C. Carbohydrates
D. Polynucleotides
E. Hydrocarbons
4. Chose from listed below enzymes ONE, which belongs to a class of hydrolases.
A. Pepsin
B. Aldolase
11
C. Glucokinase
D. Phenol oxidase
E. ATP synthase
5. Chose from listed below enzymes ONE which exhibits specificity to peptide bonds:
A. Chymotrypsin
B. Arginase
C. Urease
D. Alcohol dehydrogenase
E. Cellulase
Topic № 3. Kinetics of enzymatic reactions. Regulation of enzymatic activity and
determination of enzymes.
Objective: To learn the main principles of regulation of metabolic pathways. On
an example of amylase and choline esterase to investigate the influence of activators
and inhibitors upon activity of enzymes.
Actuality of the theme: Enzymes are biocatalysts with a changeable activity,
which is submitted to regulation. Estimation of enzymatic activity is routinely used in
laboratory investigations with diagnostic purposes. Additionally enzymes are used as
medicinal and drugs in practical medicine.
Specific objectives:
 To explain the mechanism of enzymatic action on basis of the affinity of enzyme to
substrate and events which occur in enzyme-substrate complex.
 To interpret kinetics of enzymatic reactions.
 To explain the employment of inhibitors and activators of enzymes in cases of
metabolic disorders.
 To analyze the influence of activators and inhibitors upon activity of amylase and
choline esterase.
Theoretical questions:
1. Activation and inhibition of enzymes:
- Activation of enzymes;
- inhibition of enzyme (reversible, irreversible, competitive, noncompetitive).
2. Regulation by change in catalytic activity of enzymes.
- allosteric enzymes;
- covalent modification of enzymes;
- activation of enzymes by limited proteolysis;
- the effect of regulatory proteins;
- cyclic nucleotides in regulation of enzymatic processes.
3. Regulation by changes in quantity of enzymes: constitutive and adaptive enzymes.
4. The main principles of kinetics of enzymatic catalysis:
- dependence of enzymatic reaction rate from concentration of substrate;
- dependence of enzymatic reaction rate from concentration of enzyme;
- formation of substrate-enzyme complex and process of substrate transformation;
12
- equation of Michaelis-Menten; sense of
substrate).
5. Units of enzymatic activity.
Km value (affinity of enzyme to
Practical part
Experiment 1. Investigation of the influence of activators and inhibitors on
activity of salivary amylase.
Principle. Compounds, which enhance the activity of enzymes – called activators
– are a number of metal ions, e.g. Na+, Mg2+, Mn2+, Co2+, as well as organic substances,
especially intermediate metabolites. Amylase is activated by sodium chloride (NaCl),
inhibited – by copper sulphate (CuSO4). As indicator of the influence of mentioned
compounds on the activity of amylase is a degree of starch cleavage under the action of
amylase in the presence of NaCl or CuSO4.
Method. Saliva is diluted two fold. Then three tubes are taken. Into tube N1 is
poured 1 ml of water, tube N 2 – 0,8 ml of water and 0,2 ml of 1 % NaCl, N3 – 0,8 ml
of water and 0,2 ml of 1% solution of CuSO4. Into each tube is added 1 ml of saliva
preparation, mixed and then 2 ml of 1% starch solution. Tubes are incubated at 37C
during 15 min and then reaction with iodine is performed. (0,1% solution of iodine in
0,2% sodium iodide). Changes in color development are observed and registered in a
table (see below).
N of tube
Tube content
1
2
3
Water (ml)
1
0,8
0,8
NaCl, 1 %-solution (ml)
0,2
CuSO4, 5 %-solution (ml)
0,2
Saliva 2 fold diluted (ml)
1
1
1
Starch, 1 %-solution (ml)
2
2
2
Final color after addition of
iodine
Explain the results and draw a conclusion.
Clinical and pharmaceutical significance. Inhibitors of enzymes are widely used
in medicine as drugs and medicinals, e.g. acetylosalicylic acid (aspirin) is an inhibitor of
cyclooxygenase (prostaglandin syntase) and employed as anti-inflammatory drug.
Trasylol (inhibitor of trypsin) and contrical – inhibitor of different proteinases – (one of
them are kallikreins) are used in treatment of pancreatitis, allopurinol –inhibitor of
xanthine oxidase- is used for treatement of gout, etc.
Experiment 2. Investigation of the influence of phosphacol and calcium ions on
activity of cholinesterase.
Principle. Organic phosphocompounds are irreversible inhibitors of
cholinesterase and acetyl-cholinesterase due to irreversible binding with an active site of
enzyme and block their activity. Preparations of phosphororganics are strong poisons
13
for insects (pesticides) as well as for higher animals. Mechanism of inhibitory action
consists in covalent binding with OH group of serine in active center of enzyme.
In conduction of nerve excitation an increase of calcium ions in nerve ending
occurs and this is a signal for release of acetylcholine into the synaptic cleft and
subsequent interaction with cholinoreceptors of postsynaptic membrane and hydrolytic
cleavage by cholinesterase. Besides, calcium ions are strong activators of cholinesterase
Method of quantitative determination of cholinesterase is founded on the titration
with alkali the acetic acid, which is released due to acetylcholine hydrolysis. The
quantity of alkali, expended for titration, is accepted as a measure of enzyme activity.
Method. Three tubes are filled with a reagents according to the table (see below).
Reagents in tubes
N of tube
1
2
3
Blood serum (ml)
0,5
0,5
0,5
0,5 % solution of CaCl2,(drops)
--5
--0,5 % solution of phosphacol
----5
(drops)
Incubation at room temperature 5 min
2 % solution of acetylcholine
1,5
1,5
1,5
(ml)
Incubation for 10 min
Phenolphthaleine (drops)
2
2
2
Quantity of 0,1 М NaOH
expended for titration, ml
Explain the results and draw a conclusion..
Clinical and pharmaceutical significance. Cholinesterase catalyses hydrolysis of
acetylcholine, a known neuromediator with formation of choline and acetic acid. In
human blood there are two forms of cholinesterase. In blood plasma is present
nonspecific cholinesterase (EC 3.1.1.8), which cleaves not only acetylcholine, but also
other esters of choline. In red blood cells there is a specific, or true, cholinesterase,
which cleaves acetylcholine only (EC 3.1.1.7).
In normal conditions the activity of cholinesterase is about 44,4-94,4 ucat/l
(colorimetric method according to hydrolysis of acetylcholine chloride).
Physiologically active substances, which are inhibitors of cholinesterase, have an
important pharmacological and toxicological significance, as they cause a significant
increase in concentration of neuromediator in parts of CNS, as well as in a body in
general. Reversible inhibitors of acetylcholine esterase are employed in medicine for
enhancement of cholinergic activity, which is altered in some neurological diseases,
e.g. atonia of intestines or bladder. In these cases are used proserine, physostigmine,
galantamine.
Irreversible inhibitors of acetylcholinesterase are potent neurotoxins, which causes
a marked excitation of nerve system with convulsions, disorders of cardiovascular,
digestive and other systems of the organism. The most known irreversible inhibitors are
organic phosphates. They are employed for killing of harmful insects (dichlophos,
chlorophos, metaphos, etc.). Neuroparalytic toxins which are used as a warfare, are
zarine, soman, tabun and others.
14
High sensitivity of cholinesterase to these phosphororganic compounds permits to
use this enzyme as a marker for detection of these toxic substances in human body.
Examples of tests
1. In duodenum enzymes of pancreatic gland cleave different components of meal.
Which from listed below enzymes breaks α-glycosidic bonds of carbohydrates?
A. α-Amylase
B. Carboxypeptidase
C. Trypsin
D. Chymotrypsin
E. Urease
2. Note substance, which induce transformation of pepsinogen to pepsin
A. Hydrochloric acid
B. Gastrin
C. Enterokinase
D. Bile acids
E. Carboxypeptidase
3. Trypsinogen is produced in exocrine part of pancreatic gland and excreted to
duodenum, where it is activated by the next factor:
A. Enteropeptidase
B. Secretin
C. Bile acids
D. Cholecystokinine
E. Chymotrypsinogen
4. According to international convention as a unit of enzymatic activity is accepted 1
catal, which can be defined as follows:
A. Quantity of enzyme which transform 1 mole of substrate in 1 second
B. Quantity of enzyme which transform 1 umole of substrate in 1 minute
C. Quantity of enzyme which transform 1 umole of substrate in 1 second
D. Activity of 1 mg of pure enzyme
E. Number of substrate molecules transformed in 1 minute
5. In a patient the disorder of protein digestion in small intestines is observed. What
enzymes insufficiency can cause this disorder?
A. Pepsin
B. Galactosidase
C. Lipase
D. Trypsin and chymotrypsin
E. Alanyl aminotransferase
References.
15
1. Bhagavan N.V. Medical biochemistry. Jones and Bartlett Publishers International:
Boston-London. – 1992, 980 p.
2. Trudy McKee, James McKee. Biochemistry. Wm C Brown Publishers. 1966,
654 p.
3. Murrey R.K., Granner D.K., Mayes P.A., Rodwell V.W. Harper’s Biochemistry, 26th Ed. 2003, 927 p.
4. Champe P.C., Harvey R.A. Biochemistry.2-nd Ed. Lippincott’sIllustrated reviews.
1994, 443 p.
5. Biological chemistry. Methodical instructions for practical exercises. Part I Lviv2002, 102 p.
6. Mardashko O.O., Yasinenko N.Y., Biochemistry. Texts for lectures. Jdessa State
Medical University. 2003, 416 p.
Topic № 4. Regulation of enzymatic processess enzymopathias and mechanisms of
their development. Application of enzymes as pharmaceutical preparations
The aim of the lesson: To learn the main principles of regulation of metabolic
pathways, the consequences of alteration of enzymatic activity in the cell and
employment of enzymes in medicine and pharmacy.
Actualithy of the theme: Enzymes are biocatalysts with a changeable activity,
which is submitted to regulatory influences. Estimation of enzymatic activity is
routinely used in laboratory investigations with diagnostic purposes. Additionally
enzymes are employed as medicinals and drugs in practical medicine.
Specific objectives:
 To explain the expediency in employment of enzymes for correction of metabolic
disorders in pathological states.
 To explain changes in course of enzymatic processes and accumulation of distinct
metabolic intermediates in the inborn and acquired defects of metabolism –
enzymopathias.
 To analyze changes in activity of indicatory enzymes in blood in pathology of
selected organs and tissues.
1.
2.
3.
4.
5.
6.
7.
Theoretical questions:
Multi-enzyme complexes and systems, peculiarities of their structure and
functions. Polyfunctional enzymes and their advantages.
Immobilized enzymes and their employment in pharmacy.
Multiple forms of enzymes (isozymes). Isozymes in diagnostics.
Enzymodiagnostics, enzymopathias and enzymotherapy.
Changes in enzymatic activity of blood plasma and serum as diagnostic (marker)
indexes of pathological processes in distinct organs
Inborn (hereditary) and acquired metabolic defects, their clinical and laboratory
diagnostics.
Application of enzyme inhibitors as medicinals and drugs: acetylsalicyclic acid,
allopurinol, sulfonamides.
16
8. Enzymotherapy – application of enzymes as drugs and medical preparations in
the treatment of disorders of digestive system, in purulent and necrotic processes,
as fibrinolytic preparations, enzymatic preparations of organ origin.
Practical part
Experiment 1. Quantitative determination of amylase activity in urine
according to Wohlgemuth.
Principle. A minimal quantity of an enzyme, which cleaves a distinct quantity of
substrate, is determined. Arbitrary unit of amylase activity in urine corresponds to the
quantity of 0,1 % solution of starch, which can be cleaved by 1 ml of urine at 45 °0 in 15
min. time interval.
Method: Into 9 numerated tubes is added 1 ml of saline. To the first tube 1 ml of
urine is added, mixed and transfered 1 ml of the first tube content to the second tube.
With the same manner 1 ml of the mixture after mixing is transferred to the third tube
and so on. A series of dilution of urine is obtained as a geometric progression with a
coefficient 2.
Into each tube 1 ml of saline is added, thereafter 2 ml of 0,1 % of starch solution.
Content of tubes is substancially mixed and tubes are placed on a water bath at 45°C for
15 min.
After incubation tubes are taken out of the bath, cooled by tap water and to each
tube 2 drops of Lugol solution (iodine in potassium iodide solution) are added. A colour
reaction in tubes is registered. Results are registered as a table and the activity of
amylase is calculated.
Tube N
1
2
3
4
5
6
7
8
9
Urine dilution 1:2 1:4 1:8 1:16 1:52 1:64 1:128 1:256 1:512
Colour with
yel. yel. yel. yel. blue Blue blue blue blue
iodine
Starch
+
+
+
+
cleavage
Example. 1/16 ml of urine cleaved 2 ml of 0,1 % sol. of starch (yellow colour in
tubes NN 1-4). In the and subsequent tubes starch was not cleaved (blue colour).
Amylase activity:
X = 1×2/ 1/16 = 2×16 / 1 =32 (un.)
16 - urine dilution, 2 - quantity of starch added.
Explain the results and draw a conclusion.
Clinical and pharmaceutical significance.The determination of amylase activity in
biological fluids is widely used in clinical practice for diagnosis of pancreas diseases.
Normally amylase activity in blood serum and in urine consists around 16-64 units
(Wohlgemuth units). Increase of amylase activity to more than 256 units indicates to
acute inflamation of pancreatic gland. In renal insufficiency amylase is absent in urine.
Experiment 2. Quantitative determination of pancreatic proteinases activity.
Principle. The following method is based on the property of proteinases to
hydrolyze peptide bonds. The result is an increase in the quantity of free amino and
17
carboxyl groups. After blocking of amino groups with formaldehyde, carboxyl groups
acidify the medium and can be measured by Zorensen’s titration method with indicator,
phenolphthalein.
Method. Put into each of two tubes 5 ml of 6% casein solution and 2 ml phosphate
buffer, pH 7.6. Add into the first tube a suspension of 0.5 g of pancreatin in small
volume of water and mix well. Place both tubes (the first one is the test; the second the
control) in a water bath at 37 C. After 30 min of incubation, allow tubes to cool.
Transfer their contents into titration flasks. Into both flasks add 2-3 drops of
phenolphthalein and then titrate with 0.2 n NaOH solution until a faint pink colour
appears (pH - 8.3). At this pH the quantity of amino groups is equal to the quantity of
carboxyl. Then, add into both flasks 5 ml of formalin mixture. Formaldehyde binds
with amino groups causing the carboxyl groups to shift the pH to acidic side.
OH-CH2 \
NH3 – CH – COO + 2НСНО 
+
N – CH – COO- + H+
-

R
OH-CH2 /

R
Both probes are titrated with 0.02 n NaOH solution till faint pink colour (pH 8.3)
and additionally several drops of 0.02 n NaOH are added to attain a bright red colour
(pH 9.1).
Calculation: The volume of 0.02 n NaOH solution, expended for titration of the
test sample is determined as the difference between test and control probes. The
activity of proteinases is expressed in arbitrary units. 1 arbitrary unit corresponds to 1
ml of 0.02 n NaOH solution expended for titration of test sample
Explain the results and draw a conclusion.
Clinical and pharmaceutical significance. Proteinases and peptidases are
hydrolytic enzymes, decomposing proteins and peptides to amino acids. In living
organism these enzymes are found inside living cells. They are also excreted by
specialized exocrine glands into the digestive tract. The highest activity exhibit by
proteinases and peptidases are those of the pancreas - trypsin (E.C. 3.4.21.4),
chymotrypsin (E.C. 3.4.4.5), and carboxypeptidase (E.C. 3.4.2.1).
In blood plasma there exists a potent antiproteolytic system, which binds and
inactivates proteinases appearing in blood. This system includes xiantitrypsin,
aiantichymotrypsin, antiplasmin, and aimacroglobulin. Proteolytic activity can be
detected in blood in some disease states, when the quantity of proteinases exceeds the
binding capacity of antiproteolytic system. This situation can take place during acute
pancreatitis, gastric ulcers, heavy combustions, and acute hepatitis.
There are a group of specialized proteinases, which perform limited proteolysis
and generate biologically active peptides and proteins. They include blood coagulation
enzymes (such as the Hageman factor, Stewart-Prower factor, thrombin, plasmin),
kallikrein-kinine system, and renin. The last generates angiotensin I peptide and
transforms it to angiotensin II to increase blood pressure.
Limited proteolysis is a process of a great importance for regulation of vital
processes in the cell.
18
Examples of tests:
1. Researchers isolated 5 isoenzymic forms of lactate dehydrogenase from the human
blood serum and studied their properties. What property indicates that the isoenzymic
forms were isolated from the same enzyme?
A. Catalyzation of the same reaction
B. The same molecular weight
C. The same physicochemical properties
D. Tissue localization
E. The same electrophoretic mobility
2. Marked increase of activity of МВ-forms of CPK (creatinephosphokinase) and LDH1 were revealed on the examination of the patient's blood. What is the most likely
pathology?
A. Miocardial infarction
B. Hepatitis
C. Rheumatism
D. Pancreatitis
E. Cholecystitis
3. 12 hours after an accute attack of retrosternal pain a patient presented a jump of
aspartate aminotransferase activity in blood serum. What pathology is this deviation
typical for?
A. Myocardium infarction
B. Viral hepatitis
C. Collagenosis
D. Diabetes mellitus
E. Diabetes insipidus
4. Desulfiram is widely used in medical practice to prevent alcocholism. It inhibits
aldehyde dehydrogenase. Increased level of what metabolite causes aversion to
alcochol?
A. Acetaldehyde
B. Ethanol
C. Malonyl aldehyde
D. Propionic aldehyde
E. Methanol
5. In a treatement of some infection diseases sulfanilamide drugs are used, which inhibit
the growth of bacteria. What is the mechanism of their action?
A. They are structural analogs of p-aminobenzoic acid, needed for biosynthesis of
folic acid
B. Allosteric inhibition of bacterial enzymes
C. They are involved in oxidative-reductive reactions
D. Inhibit the absorption of folic acid
19
E. Irreversibly inhibit biosynthesis of folic acid, needed for normal bacterial
survival
Individual independent work of students
1. Employment of isoenzymes in enzymodiagnostics
2. Application of enzymes and inhibitors of enzymes as pharmaceutical preparations
1.
2.
3.
4.
5.
References:
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee.. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Topic 5. Role of cofacors and coenzymatic vitamins in catalytic activity of
enzymes. Fat-soluble vitamins
The aim of the lesson: To learn the structure, principles of classification and
funtion of coenzyme vitamins, vitaminoids, antivitamins. To master the methods of
qualitative and quantitative determination of water soluble and fat soluble vitamins.
Actuality of the theme: Water soluble vitamins are participants of metabolism as
coenzymes and activators of many enzymatic reactions.
Restriction in vitamin supply of the body or disorders of their metabolism which is
caused by alteration of their obsorption, transformation into coenzyme forms,
substantially decrease the intensity of energetic and plastic metabolism. This is
accompanied with functional disorders of brain, heart, liver and other organs,
suppression of immune response to infection, loss of ability to adapt effectively to
unfavorable enviremental conditions.
Specific objectives:
 To interpret the role of water soluble and fat soluble vitamins and their precursors
as nutritional components in metabolic and physiological processes, their role as
pharmaceuticals.
 To explain application of antivitamins as enzyme inhibitors in contagious diseases
and in disorders of homeostasis.
 To evaluate the role of water soluble and fat soluble vitamins in metabolism,
development of hypo- and hyper- vitaminoses, their prevention and treatment.
 To explain the role of vitaminoids in metabolic processes.
 To explain the role of biologically active supplements as nutritional components and
their effect in the organism.
 To explain the application of antivitamins as inhibitors of enzymes in contagious
diseasesand in disorders of homeostasis.
 To explain the role of metals in mechanisms of enzymatic catalysis.
20
 To classify distinct groups of coenzymes according their chemical nature and type of
the reaction which they catalyse.
Theoretical questions:
1. Vitamins as essential nutritional components. History of vitamins discovery and
development of vitaminology.
2. Causes of exo- and endogenous hypo- and avitaminoses.
3. Vitamin B1 and B2, their structure, biological function, sources of supplement, daily
requirement. Symptoms of hypovitaminosis.
4. Structure and properties of vitamin H and pantothenic acid. Their participance in
metabolism, sources of supplement, daily requirement. Metabolic significance of
CoA.
5. Antianemic vitamins (B12, folic acid), their structure, biological function, sources of
supplement, daily requirement. Symptoms of hypovitaminosis.
6. Vitamins B6 and PP, their structure, biological function, nutritional sources, daily
requirement. Symptoms of hypovitaminosis.
7. Vitamin C and P, their structure, biological function, nutritional sources, daily
requirement. Functional interrelations between vitamin C and P, manifestations of
insufficiency in human organism.
8. Vitamins of D group, their structure, biological function, nutritional sources, daily
requirement. Symptoms of hypo- and hyper-vitaminosis, avitaminosis.
9. Vitamin A, its structure, biological function, nutritional sources, daily requirement.
Symptoms of hypo- and hyper- vitaminosis.
10.Vitamins E, F, their structure, biological role, nutritional sources, mechanism of
action, daily requirement. Symptoms of insufficiency, application in medicine.
11.Antihemorrhagic vitamins (K2, K3) and their water soluble forms, structure,
biological function, nutritional sources, mechanism of action, daily requirement,
symptoms of insufficiency, application in medicine.
12.Provitamins, antivitamins, mechanism of action and employment in practical
medicine.
13.Vitaminoids, their structure and biological activity.
14.Modern vitamin drugs, theirm application in treatment and prevention of diseases.
Biologically active supplements.
Practical part
Experiment 1. Detection of vitamin E with ferric chloride.
Principle of the method. Alcohol solution of α-tocoferol is oxidized by ferric
chloride to tocoferylquinone, which possess a red color.
Performance. Into dry tube are placed 4-5 droplets of 0,1% alcohol solution of
tocoferol, thereafter is added 0,5 ml of 1% solution of ferric chloride. The content is
intensively mixed and heated on a flame up to the change of color.
Explain the result, draw a conclusion.
Clinical and pharmaceutical significance. Due to the modern ideas the main
function of tocopherols consists is to serve as antioxidants in relation to unsaturated
lipids. By the meaning hydrogen atom in its molecule α-tocopherol co-operates with
21
the peroxide radicals of lipids, redusing them into hydroperoxides and thus breaking the
chain reaction of peroxidation.
Most displays of tocopherol insufficiency depend from stopping of carried out by
the vitamin inhibiting action on autooxidation of the unsaturated fat acids which are
included in composition of cellular and subcellular membranes: hemolytic anemia in
prematurely born childs, atrophia of testicles and sterility; dissolving of embryo on the
early stages of pregnancy; muscular dystrophy which is accompanied with loss of
cellular nitrous components and muscle proteins. Direct reason of muscular dystrophy
is freeing of lysosomal hydrolases as a result of lysosom membrane defect.
Recommended dietary allowance for the grown man per day 20 – 30 mg,
concentration in the blood whey 3500 – 8000 nmole/l. With the membrane pathology,
presumably, are linked the areas of necrosis, which are observed at vitamin E
avitaminosis in a liver, brain tissue, especially in cerebellum.
Most enriched with the vitamin E vegetable butters are: sunflower, corn, cotton
and olive. Especially high maintenance of it is in butter, got from the wheat embryos,
oat, green a pea. Synthetic preparation of -tocopherol acetate in vegetable butter are
produced for an internal reception and for intramuscular injections. It is used as an
antioxidant at muscular dystrophy, violation of reproductive function in women and
men, to hemolytic anemia in new-born, in complex therapy of heart and vessels
diseases, eye and hepatic diseases etc.
Experiment 2. Reaction of vicasol with cysteine.
Principle of the method. Vicasol solution in presence of cysteine in alkaline
medium develops a lemon-yellow color.
Performance. In a tube are placed 5-10 droplets of 0,05% alcohol solution of
vicasol and then are added 5-10 droplets of 0,025% cysteine and 2,5 ml of 20% solution
of NaOH. The appearance of bright lemon-yellow color is observed.
Explain the result, draw a conclusion.
Clinical and pharmaceutical significance.The main active form of vitamin K is
menachinon MC-4, that appears from naphtochinons of vegetable and bacterial origin in
tissues.
The most studied function of vitamin K is his connection with the process of
blood coagullation. He is nessesary for a synthesis in the liver of protein factors of
coagulation: prothrombin (factor II), proconvertin (factor VII), Christmas factor (IX),
Stuart-Prower factor (X).
Vitamin K stimulates inserting of additional carboxyl groups in glutamate
residues in protrombin precursors. The synthesis of “complete” molecule of
protrombinu is thus completed, in other words - its posttranslational modification.
Joining of additional СОО - groups is nessesary for optimal binding of Са 2+, that
activates converting of protrombin into trombin.
Its role in this process consist in transport of НСО3--ions, that are inserted in position glutamine acid, or in activating of hydrogen -carbon atom of glutamine acid, or
in activating of one of enzymes in the reaction of carboxylation.
Modern data allows to consider that vitamin K, like other fat-soluble vitamins,
influences on the state of cell membranes and subcellular structures, being the
22
component part of lipoproteins of these membranes. Recommended dietary allowance
for the adult man of 1 – 2 mg per day, concentration in the whey of blood 400 – 600
nmol/l.
The lack of this vitamin more frequent develops as endogenous, caused by
violation in its biosynthesis in an intestine (sterilization of intestine by sulfonyl amide
preparations or antibiotics), or by violation of absorbtion (insufficient products of bile
or impassability of bile ducts, liver diseases). To insufficiency can lead also using of
preparations with properties of antivitamins K (for example, anticoagulants of mediocre
action). Basic signs of insufficiency are bleeding at small damages, hemorragia in newborn (before appearance of microflora in the intestine).
Naftochinons enters organism of man, mainly, with the meal of vegetable origin:
spinach, fruits, and also synthesized by the bacteria of thin bowel. Content of vitamin K
in food considerably exceeds minimum daily necessities and that is why, insufficiency
of this vitamin at a normal feed and physiology conditions of absorbtion of lipids, is
rare phenomenon.
In medical practice are used preparations of vitamin K and his synthetic watersoluble analogue – vikasol. It is prescribed at the pathological states which are
accompanied with hypotrombonemia and bleeding.
Experiment 3. Quantitative determination of ascorbic acid in urine.
Principle. Ascorbic acid is readily reduced to form a specific dye - 2,6dichlorophenol-indophenol with a change in color from blue to pink. Urine is titrated
with a solution of the stain and the quantity of ascorbic acid is determined taking into
account, that 1 ml of dye solution corresponds to 0.1 mg of ascorbic acid.
Method. Pour into two flasks 5 ml of freshly obtained urine, add 5 drops of
concentrated acetic acid. Titrate one flask with a solution of dichlorophenolindophenol
to a blue color. The quantity of ascorbic acid is calculated in a portion of urine and in
daily excreted urine.
Explain the results and draw a conclusion.
Clinical and pharmaceutical significance. In many diseases of the digestive
system absorption of vitamins through the intestinal wall to the blood is impaired and
decomposition of vitamins in the digestive tract is intensified. This is observed in
patients with ulcers, gastritis, enteritis, cholecystitis, etc.
The level of ascorbic acid in blood, urine and in foods is measured in order to
assess whether the organism receives sufficient amounts of the vitamin. Normal blood
level of vitamin C of an adult is 39.7 – 113.6 μmol/L.
Ascorbic acid and the products of its decomposition are excreted with urine. A
healthy person excretes daily 20 – 30 mg. or 113.55 – 170.33 μmol of vitamin C.
Intensified decomposition of ascorbic acid occurs in patients with hypoacidic gastritis,
ulcers, enteritis. Excretion of vitamin C below the normal level indicates vitamin C
hypovitaminosis – insufficient supply of vitamin C to the organism.
Vitamin C hypovitaminosis causes scurvy – a disease, characterized by bluish
lips and nails, gingival bleeding, dry and pale skin, subcutaneous hemorrhage, loosening
and falling teeth, joint pains and slow healing of injuries.
23
Examples of tests
1. Hydroxylation of endogenous substrates and xenobiotics requires a donor of protons.
Which of the following vitamins can play this role?
A. Vitamin C
B. Vitamin P
C. Vitamin B6
D. Vitamin E
E. Vitamin A
2. According to clinical indications a patient was administered pyridoxal phosphate.
What processes is this medication intended to correct?
A. Transamination and decarboxylation of aminoacids
B. Oxidative decarboxylation of ketonic acids
C. Desamination of purine nucleotide
D. Synthesis of purine and pyrimidine bases
E. Protein synthesis
3. There is observed inhibited fibrillation in the patients with bile ducts obstruction,
bleeding due to low level of absorbtion of some vitamin. What vitamin is in deficit?
A. К
B. А
C. D
D. Е
E. Carotene
4. While examining the child the doctor revealed symmetric cheeks roughness, diarrhea,
disfunction of the nervous system. Lack of what food components caused it?
A. Nicotinic acid, tryptophane
B. Lysine, ascorbic acid
C. Threonine, pantothenic acid
D. Methionine, lipoic acid
E. Phenylalanine, pangamic acid
5. A patient has pellagra. Interrogation revealed that he had lived mostly on maize for a
long time and eaten little meat. This disease had been caused by the deficit of the
following substance in the maize:
A. Tryptophan
B. Tyrosine
C. Proline
D. Alanine
E. Histidine
Induvudual independent student’s work
1. Role of vitamins in the mechanism of action of complex enzymes
2. Biologically active dietary supplements. Their significance in pathological states
prevention and in well balanced nutrition.
24
1.
2.
3.
4.
5.
6.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2003,
Part II, P. 3-23.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.- 416p.
Sense module 2.
General principles of turnover of substances and energy
Topic № 6. General principles of turnover of substances and energy.
Functioning of citric acid cycle
Aim of the lesson: To learn the sequence of reactions in tricarboxylic acids (TCA)
cycle and biological significance of TCA cycle as the final stage of catabolic pathway
in the cell. To make an aquaintance with methods of TCA cycle investigation in
mitochondria and to examine the effect of malonic acid upon this process.
Actuality of the theme: The peculiarities of TCA cycle functioning have an
important significance in elucidation of its role for providement of the cell with energy
as well as for understanding of its amphibolic significance. The analysis of TCA cycle
function is necessary for estimation of its role in turnover of matter and energy in the
cell.
Specific aims:
 To comment biochemical principles of metabolic pathways: catabolic, anabolic,
amphibolic pathways;
 To explain biochemical mechanisms of regulation of catabolic and anabolic
metabolic reactions;
 To comment biochemical principles of TCA cycle functioning and its anaplerotic
reactions and their amphibolic sense;
 To explain biochemical regulatory mechanisms in TCA cycle and its principal
position in turnover of material and energy.
1.
2.
3.
Theoretical questions:
Conception of turnover of material and energy (metabolism).
Characterization of catabolic, anabolic and amphibolic reactions and their
significance. Exergonic and endergonic biochemical reactions.
The role of ATP and other macroergic phosphate containing compounds in their
coupling.
25
4.
Catabolic transformation of biomolecules: proteins, carbohydrates, lipids, its
characterization.
5.
Tricarboxylic acid (TCA) cycle (cellular location of TCA cycle enzymes,
sequence of TCA cycle reactions, characterization of enzymes and coenzymes
participating TCA cycle, reactions of substrate phosphorylation in TCA cycle, the
effect of allosteric modulators upon TCA cycle reactions, energetic effect of TCA
cycle).
Practical part
Experiment 1. Investigation of TCA cycle functioning in mitochondria and the
effect of malonate upon this process.
Principle. The transformations of acetyl-CoA in presence of mitochondrial
enzymes is accompanied with production of CO2 . As a source of acetyl-CoA, which is
further incorporated into CTA cycle, is used pyruvate. The last under the action of
multimeric pyruvate dehydrogenase complex is submitted to oxidative decarboxylation
and acetyl-CoA and CO2 are produced. If TCA cycle is inhibited with malonate bubbles
of gaze does not occur. Malonate is a classic competitive inhibitor of succinate
dehydrogenase – enzyme of TCA cycle. It binds with active center of this enzyme and
hinder binding of true substrate – succinic acid (or succinate).
For binding of released CO2 into incubation medium is added Ca(OH)2. At the end
of incubation a bound CO2 is detected due to a production of gaze bubbles after addition
of sulphuric acid solution into incubation medium.
Method. Three tubes – a control one, experimental N1 and N 2 – are filled with
reagents as indicated in the table.
Content of tubes
Control
2,0
0,5
Tubes
Exp. N 1
2,0
0,5
Phosphate buffer, pH 7,4, ml
Sodium pyruvate solution, ml
Malonic acid, ml
Saline, ml
0,5
0,5
Ca(OH)2 solution, ml
0,5
0,5
Suspension of mitochondria
0,5
Boiled suspension of mitochondria
0,5
Incubation in thermostate 15 min at 37 oC
0,1 M solution of sulphuric acid
1,0
1,0
Result: production of CO2 bubbles
Exp. N 2
2,0
0,5
0,5
0,5
0,5
1,0
All tubes are placed in a thermostate for 15 min at 37oC. Thereafter into each
tube is added 1,0 ml of 0,1 M solution of sulphuric acid and appearance of CO2 bubbles
is observed and registered.
Explain the results and draw a conclusion.
26
Experiment 2 Investigation of TCA cycle activity in mitochondria according to
the rate of reductive equivalents production and the influence of malonate upon this
process.
Principle. During the oxidation of acetyl-S-CoA in TCA cycle NAD and FAD are
reduced by hydrogen taken from the substrates. Reduced NAD and FAD in turn reduce
methylene blue and make it colorless. Time of decoloration of reaction medium,
containing methylene blue, corresponds to the intensity of reactions in TCA cycle of
mitochondria.
Performance. Three tubes (control, experiment 1 and experiment 2) are prepared
as indicated in a table.
Reagents added
Control
Phosphate buffer, pH 7,4, ml
2,0
Solution of sodium pyruvate, ml
0,5
Solution of malonate, ml
Saline
0,5
Suspension of mitochondria, ml
Suspension of boiled mitochondria, ml
0,5
Solution of methylene blue, ml
0,5
o
Incubation at 37 C in thermostate
Results: time of medium decoloration
Explain the results and draw a conclusion.
Tubes
Exp.N1
2,0
0,5
0,5
0,5
0,5
Exp.N2
2,0
0,5
0,5
0,5
0,5
Clinical and pharmaceutical significance. Many substances, including
medicinals and drugs, may influence the bioenergetics of the cell by changing the
effectiveness of oxidative phosphorylation and ATP production. They can be devided
into activators and inhibitors of energetic metabolism. Activators are represented by
acid participants of TCA cycle (citric, succinic, malic acids) as well as by other
compounds (glucose, amino acids, etc.). They are used in medical practice.
Citric acid as sodium salt is additionally used as anticoagulant, as well as a
component of some other drugs.
.
Examples of tests
1. A patient was admitted into hospital with a diagnosis diabetes mellitus type I. In
metabolic changes the decrease of oxaloacetate synthesis rate is detected What
metabolic passway is damaged as a result?
A. Tricarboxylic acid cycle
B. Glycolysis
C. Cholesterol biosynthesis
D. Glycogen mobilization
E. Urea synthesis
27
2. Substrate phosphorylation is a process of phosphate residue transfer from macroergic
donor substance to ADP or some other nucleoside diphosphate. What enzyme of
tricarboxylic acid cycle participates in reaction of substrate phosphorylation.
A. Succinyl thiokinase
B. Citrate synthase
C. Succinate dehydrogenase
D. Fumarase
E. Alpha-ketoglutarate dehydrogenase complex
3. Which of the following compounds would you expect to liberate the highest free
energy on hydrolysis?
A. Phosphoenolpyruvate
B. ATP
C. ADP
D. AMP
E. Phosphocreatine
4. The number of molecules of ATP produced by the total oxidation of acetyl CoA in
TCA cycle is:
A. 12
B. 6
C. 8
D. 10
E. 15
5. Enzymes of tricaboxylic acid cycle are located:
A. In the mitochondrial matrix
B. On the outer surface of the outer mitochondrial membrane
C. On the inner surface of the outer mitochondrial membrane
D. In the inner mitochondrial membrane
E. In the intermembrane space
Topics for individual educational work of students
1. Anaplerotic and amphibolic role of tricarboxylic acid cycle
2. Role of the most important metabolites ( pyruvate, α-ketoglutarate, acetyl-CoA,
succinyl-CoA) in the integration of metabolism.
1.
2.
3.
4.
References:
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee.. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
28
5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Topic № 7. Biological oxidation, oxidative phosphorylation and ATP
synthesis. Inhibitors and uncouplers of tissue respiration and oxidative
phosphorylation in respiratory chain of mitochondria
The aim of the lesson: to learn general principles of enzymatic respiratory chain
organization in mitochondria, distinct oxido-reductases, their functional significance in
tissue respiration. To learn the mechanisms of tissue respiration and oxidative
phosphorylation. To master the methods of investigation of the next oxido-reductases:
phenol oxidase, aldehyde dehydrogenase and peroxidase.
Actualithy of the theme: oxido-reductases catalyze reactions connected with
transfer of electrons and protons and are in the background of macroergic compounds
production. Investigation of their activity is necessary for detailed understanding of
the mechanisms of tissue respiration and its changes in different functional status of the
body as well as for the the correction with different medicinals and drugs.
Specific aims:
 To explain processes of biological oxidation of different substrates in the cell and
reservation of released energy in a form of macroergic bonds of ATP.
 To analyze reactions of biological oxidation and their role in providement of
fundamental biochemical processes in tissues.
 To explain the structural organization of electron transport chain and its
macromolecular complexes.
 To interpret role of biological oxidation, tissue respiration and oxidative
phosphorylation in generation of ATP in aerobic conditions.
 To explain the main principles of chemiosmotic theory of oxidative
phosphorylation.
 To analyse the action of inhibitors and uncouplers of oxidative phosphorylation of
natural and artificial origin and their physiologicasl significance.
1.
2.
3.
4.
7.
8.
Theoretical questions:
Modern concepts about structure and functions of the mitochondria. Molecular
organization of electron transport chain.
The significance of redox potentials in transport of electrons and protons
Electrochemical gradient of protons (Н+), which is developed during the
functioning of electron transport chain and its role in a coupling of electron transport
in mitochondria and ATP synthesis.
Inhibitors of electron transport in a respiratory chain of mitochondria.
Points of introduction of reducing equivalents (electrons and protons) into
respiratory chain of mitochondria.
Chemiosmotic thery of oxidative phosphorylation – molecular mechanism of ATP
production in course of biological oxidation.Molecular structure and principles of
functioning of ATP-synthetase.
29
9. Endo- and exogenous uncouplers of electron transport and oxidative phosphorylation
in a respiratory chain of mitochondria.
10.The respiratory control and its regulation in the cell. Alterations of ATP synthesis in
cases of action of medicinals and drugs.
Practical part
Experiment 1. Study of phenoloxidase activity.
Principle. Method is based on the phenomenon of pyrocatechol oxidation by
oxygen in the presence of phenoloxidase.
Method. The source of enzyme is fresh potato juice. 1 - 2 g of potato are minced in
a mortar, 25 ml of distilled water are added, mixed and press the juice into tube. (Iron
instruments must not be used as ions of iron induce the darkening of juice).
Into 4 ennumerated tubes 0,5 ml of potato juice are added. To the first tube add 0,5
ml of pyrocatechol solution, to the second – 1 - 2 drops of inhibitor Na2S and 0,5 ml of
pyrocatechol. The juice in the third tube is boiled and then O,5 ml of pyrocatechol is
added. To the fourth tube O,5 ml of water are added. Tubes are placed on a water bath at
37o C for 30 min. The content is periodically shaken for better aeration. In the first tube
brown colour appears due to oxidation of pyrocatechol. The color of other tubes does
not change due to the presence of inhibitor (2 tube), denaturation of enzyme (3 tube) and
absence of substrate (4 tube).
Explain the results and draw a conclusion.
Experiment 2. A study of aldehydedehydrogenase activity.
Principle. Method is based on the decoloration of methylen blue stain during its
reduction by aldehyde in presence of an enzyme aldehyde dehydrogenase
Method. Into three ennumerated tubes the following solutions are added: 1 - 1 ml
of fresh non boiled milk , 1-2 drops of neutralized formaldehyde, 2 drops of methylene
blue solution. 2- 1 ml of fresh milk and 2 drops of methylene blue. 3 - 1 ml of boiled
milk, 1-2 drops of formaldehyde and 2 drops of methylene blue. Tubes are placed for 30
min on a water bath at 37 C.
Result. Content of the first tube is decolorized due to the enzyme activity. Colour
in the rest tubes does not change , as in the 2 tube substrate is absent, in the 3 - enzyme
is heat denaturated.
Explain the results and draw a conclusion.
Experiment 3. A study of peroxidase activity.
Principle. Method is based on the oxidation of benzidine by hydrogen peroxide in
the presence of peroxidase. Blue product is formed during the oxidation of benzidine.
Method. Into 3 ennumerated tubes the following reagents are added: (1) - 3-4
drops of benzidine solution in acetic acid, 3-4 drops of 3 % solution of H2O2 , 3-4
drops of horseradish extract. (2) - 3-4 drops of benzidine solution, 3 - 4 drops of H2O2
solution, 3 - 4 drops of a boiled horseradish extract. (3)-3-4 drops of benzidine solution,
peroxidase inhibitor, 3 - 4 drops of horse radish peroxidase and 3-4 drops of H2O2
solution. In the first tube a blue colour is developed due to oxidation of benzidine by
H2O2 under catalysis of peroxidase. In the 2 and 3 tubes colour is not developed.
30
Explain the results and draw a conclusion.
Examples of tests
1. Mitochondria – subcellular organelles, located in cytoplasm of all cells exluding red
blood cells, bacteria, blue-green algae. What is principal function of mitochondria?
A. Oxidative phosphorylation
B. DNA replication
C. Hydrolytic reactions
D. Protein biosynthesis
E. Secretory function
2. Enzymes of respiratory chain perform oxidation of substrates and transfer of
reductive equivalents to oxygen with production of water molecules. Where they are
located?
A. On inner mitochondrial membrane.
B. On cytoplasmic membrane
C. In cytoplasm
D. In nucleus
E. On outer mitochondrial membrane
3. Cyanides are cellular poisons, inhibiting electron transport on terminal segment of
respiratory chain in mitochondrias. What is the mechanism of their toxic effect?
A. Formation of a complex with Fe+3 form of cytochrome oxidase
B. Block up of electron transport on a level of NAD H –coenzyme Q-reductase.
C. Block up of electron transport from cytochrome bc1.
D. Inhibition of ATP-synthase function
E. Uncoupling of respiration and oxidative phosphorylation in mitochondria
4. Patient P. is working in chemical industry, connected with cyanic acid production. He
complains in attacks of suffocation and dizziness. What is the mechanism of cyanides
effect upon tissue respiration?
A. Interaction with heme of cytochrome a3
B. Attacment to nitrogen atom in pyrimidine cycle of NAD
C. Block the activity of cytochrome c
D. Block bonding with an oxygen
E. Block the transfer of electrons from cytochrome bc1
5. During the necropsy of a 20-year-old girl a pathologist concluded that the death of
the patient had resulted from poisoning by cyanides. The activity of what enzyme is
mostly inhibited by cyanides?
A. Cytochrome oxydase
B. Malate dehydrogenase
C. Heme synthase
D. Aspartate aminotransferase
E. Lactate dehydrogenase
31
Individual independent students work
1. Composition, localisation and function of multienzyme complexes in aerobic
oxidation of substrates
2. Structure, conditions of activity and regulation of ATP synthase in the inner
mitochondrial membrane
1.
2.
3.
4.
5.
References:
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee.. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Sense module № 3
Metabolism of carbohydrates and its regulation
Topic № 8 Anaerobic and aerobic oxidation of glucose, alternative pathways of
carbohydrate metabolism
The aim of the lesson: To learn fundamental principles of intracellular oxidation
of glucose in anaerobic and aerobic conditions and pathways of its regulation. To
interpret the role of coenzymes and enzymes in glycolytic pathway. To explain
reactions of oxidative phosphorylation and ATP synthesis.
Actuality of the theme: Carbohydrate metabolism plays an important role in
providing an organism with energy. In process of glycolysis, as well as in alcohol
fermentation, initially are produced phosphate esters of hexoses and trioses, which are
further oxidized with production of ATP. During phosphorylation of carbohydrate
metabolites the concentration of inorganic phosphate in the medium is diminished and
this permits to follow the process of phosphorylation, resp. glycolysis.
Specific aims:
 To interpret biochemical pathways of intracellular oxidation of glucose in anaerobic
conditions
 To analyze peculiarities of glycolytic reactions, which occur with involvement of
ATP
 To analyze peculiarities of substrate phosphorylation and production of ATP in this
way
 To interpret role of coenzymes and enzymes in glycolytic reactions
 To analyze regulatory mechanisms of glucose oxidation in anaerobic and anaerobic
conditions
 To explaine the functioning of shuttle mechanisms of oxidation of glicolitic NADH2
32
Theoretical questions:
1. Classification, structuren and biological significance of certain classes of
carbohydrates for the human organism.
2. Digestion of carbohydrates in the digestive tube.
3. Anaerobic and aerobic oxidation of glucose: stages, biological role, location in the
cell, energetic effect.
4. Shuttle mechanisms of transfer of glicolitic NAD-H2 electrons from cytosole to
mitochondria.
5. Mechanisms of regulation of the rate of reactions in anaerobic and aerobic glycolysis.
Practical part
Experiment 1. Quantitative determination of lactic acid in blood serum after
Buchner method.
Principle. By heating with concentrated sulfuric acid lactic acid is converted to
acetic aldehyde, which, when followed by a reaction with hydroquinone, forms a redbrown substance.
Method. Pour into 2 clean tubes 6 ml of water. To the first add 1 ml of standard
solution of lactate and to the second tube add l ml of blood serum. For protein
precipitation add to each tube l ml of metaphosphoric acid and after several minutes
filter the solutions. Add to each filtrate l ml of copper sulfate (10% sol.) and 0.5g of
calcium hydroxide. Mix the probes with a glass rod and filter after 5 min. One ml of
each filtrate are added to 2 tubes, then add 0.1 ml of copper sulfate and 4 ml of
concentrated sulfuric acid to each tube and place into a boiling water bath for 1.5 min.
After cooling add 0.1 ml solution of alcohol solution of hydriquinone and incubate the
tubes in boiling water bath for 15 min. Cool the tubes and measure optical density at
blue light filter.
Calculations. Lactic acid concentration is calculated according to a formula:
Cstand·Eprobe
C lac=
Estand
Where C is concentration and E is optical density. Explain ths results and draw a
conclusion.
Clinical and pharmaceutical significance. Venous blood of the healthy person
contains 0.5 - 2.2 mmol/1 of lactic acid. An increase of lactic acid content can be
associated with strenous muscular effort during short time term intervals when there is a
deficiency of oxygen. In this case the oxidative decarboxylation of pyruvate to acetylCoA does not occur and the production of the lactate takes place. Lactate can be utilized
during a restoration period with a sufficient supply of oxygen. The increase production
of lactic acid is also observed curing epilepsy, tetanies, convulsions and hypoxia, and it
is associated with cardiac and pulmonary failure, malignant tumors, liver diseases and
other pathologies.
33
Experiment 2. Quantitative determination of pyruvic acid in urine by
colorimetric method.
Principle. Pyruvic acid and 2,4-dinitrophenyl hydrazine form in alkaline medium
in alkaline medium form 2,4-dinitrophenylhydrazone pyruvate which has brown-red
color. The intensity is proportional to tne pyruvic acid content and is evaluated
colorimetricaly.
Materials and reagents. Sample of urine, standard solution of pyruvic acid (625
mg in 100 ml of water), 0,1% solution of 2,4-dinitrophenylhydrazine in 2 n
hydrochloric acid, 12% solution of sodium hydroxide, distilled water, tubes, pipettes,
colorimeter.
Method. To one tube is added 0,1 ml of tested urine, to another tube – 0,1 ml of
standard pyruvate solution. To each tube is added 0,9 ml of distilled water. Thereafter
is added 0,5 ml of 2,4-dinitrophenylhydrazine and tubes are leaved for 20 min in a dark
place. Then to each tube is added 1 ml of 12% solution of sodium hydroxide and after
10 min the intensity of color is measured
In colorimeter with a blue light filter.
Calculation: .
Pyruvic acid concentration is calculated using the formula
Cexp = Cstand x Aexper. x V
Astand x a
where:
Cstand. – concentration of standard pyruvate
Cexper. – concentration of pyruvate in a sample of urine
Aexper.- optical density of tested urine
Astand. – optical density of standard pyruvate
V – daily volume of excreted urine
a – 0,1 ml of urine, taken for analysis.
Compare the obtained result with normal value. Draw the conclusion.
Examples of tests
1. Buffer capacity of blood was decreased in the worker due to exhausting muscular
work. Entry of what acid substance to the blood can this state be explained?
A. Lactate
B. Pyruvate
C. 1,3-bisphosphoglycerate
D. alpha-ketoglutarate
E. 3-phosphoglycerate
2. A 7-year-old girl manifests obvious signs of anemia. Laboratory tests showed the
deficiency of pyruvate kinase activity in erythrocytes. The disorder of what biochemical
process is a major factor in the development of anemia?
A. Anaerobic glycolysis
B. Deamination of amino acids
C. Tissue respiration
34
D. Oxidative phosphrylation
E. Breaking up of peroxides
3. The high speed sprint causes a feeling of pain in skeletal muscles of untrained people
that occurs due to lactate accumulation. The activation of what biochemical process is it
resulting from?
A. Glycolysis
B. Gluconeogenesis
C. Pentose phosphate pathway
D. Lipogenesis
E. Glycogenesis
4. After a sprint an untrained person develops muscle hypoxia. This leads to the
accumulation of the following metabolite in muscles:
A. Lactate
B. Ketone bodies
C. Acetyl CoA
D. Glucose 6-phosphate
E. Oxaloacetate
5. When blood circulation in the damaged tissue is restored, then lactate accumulation
comes to a stop and glucose consumption decelerates. These metabolic changes are
caused by activation of the following process:
A. Aerobic glycolysis
B. Anaerobic glycolysis
C. Lipolysis
D. Gluconeogenesis
E. Glycogen biosynthesis
1.
2.
3.
4.
5.
6.
Individual independent students work
Humoral regulation of carbohydrate metabolism
References:
Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London, 1992.- 980 p.
Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.
Biochemistry / Trudy McKee, James R. McKee, 1999.- 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews, 1994.- 443 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416 p.
35
Topic № 9. Catabolism and biosynthesis of glycogen.Regulation of glycogen
metabolism. Biosynthesis of glucose – gluconeogenesis.
Objective. To learn reactions of synthesis and breakdown of glycogen;
mechanisms of humoral regulation of glycogen metabolism in liver and muscles. To
interpret reactions of gluconeogenesis, their peculiarities and principles of their
regulation.
Actuality of the theme: Glycogen is a principal form of carbohydrate reserve in
the body. It is deposited as granular particles mainly in liver and muscle cells. The
synthesis (glycogenesis) and decomposition (glycogenolysis) of glycogen are under
strict regulation and control depending from nutrition, silent or active state of the body,
etc. The principal regulatory role play the key enzymes of glycogen metabolism –
glycogen phosphorylase and glycogen synthetase. Their activity is regulated by
reversible phosphorylation and partially by mechanism of allosteric regulation.
Specific aims:
 To explain characteristic features of glycogen breakdown and biosynthesis.
 To analyze mechanisms of humoral regulation of glycogen metabolism in liver
and muscles.
 To explain hereditary disorders of glycogen metabolism.
 To analyze specific features of gluconeogenesis reactions and substrates of this
process.
 To explain and interpret regulatory mechanisms of gluconeogenesis.
Theoretical questions:
1.
Structure and biological significance of polysaccharides. Features and functions
of the homo-and heteropolysaccharides in humans.
2. Glycogenesis and glycogenolysis: localization, enzymatic reactions, biological
significance.
3. Role of adrenalin, glucagone and insuline in hormonal regulation of glycogen
metabolism in liver and muscles
4. Hereditary disorders in enzymes of glycogen synthesis and breakdown.
Glycogenoses, aglycogenoses, their characterization and causes.
5. General understanding of the metabolism glycosaminoglycans. Genetic disorders of
their metabolism.
6. Gluconeogenesis. Relations between glycolysis and gluconeogenesis (Cori cycle).
Glucose-lactate and glucose-alanine cycles.
Practical part
Experiment 1. Reaction on polysaccharides.
Principle. Iodine in presence of starch produce complex compounds with blue
color. Blue coloration is explained due to iodine adsorption of starch and formation of
complex compounds with iodine:
Reagents: 1% solution of starch, Lugol solution (iodine dissolved in potassium
iodide), tubes, pipettes.
36
Method. Into the tube is introduced 0,5 ml of starch solution and 1-2 drops of
Lugol solution are added. The appearance of blue color is observed. Changes in color
during heating and cooling are observed. Note the results, draw the conclusions.
Make a conclusion.
Experiment 2. Detection of glycogen in the liver.
Principle. Glycogen in presence of iodine produces complexes with a red-violet
color (starch gives clear blue color).
Reagents and materials. Fresh or frozen liver tissue, Lugol solution (iodine
dissolved in potassium iodide), 1% solution of acetic acid, porcelain mortar, water bath,
paper filters, pipettes.
Method. 0,5 g of liver tissue is minced with scissors, mixed with 4 ml of boiling
distilled water , transferred to the tube and boiled 2-3 min in order to inactivate
enzymes, then transferred to the mortar and grinded to uniform mass. This homogenate
is transferred to the tube, mortar washed with water and combined with the homogenate
and boiled on a water bath for 20 min. It is recommended to add 5-10 droplets of acetic
acid solution.
Precipitated proteins are eliminated by filtration through paper filter. To the filtrate
are added 2-3 droplets of Lugol solution. In presence of glycogen a red-violet color
develops.
Clinical and pharmaceutical significance. Glycogen is a polysaccharide, which
serves as a main reserve of carbohydrates in the body. It is stored mainly in liver and
muscles. Normal blood level – 16,2–38,7 mg/l/. Increased concentration of glycogen in
blood is observed in some infection diseases, which are accompanied with leukocytosis,
diabetes mellitus, malignancies.
An important clinical significance has cytochemical detection of glycogen in blood
cells, bone marrow cells and liver cells.
1. Glycogen – polysaccharide that has the ability to congest in the liver and
muscles. The processes of synthesis and decomposition of glycogen in the cells is
controlled by the inclusion of the mechanisms of phosphorylation of such key enzymes
of glycogen exchange:
А. Glycogen synthase and glycogen phosphorylase
В. Glycogen phosphorylase and lipase
С. Glycogen synthase and protein kinase
D. Phosphoprotein kinase and protein kinase
Е. Adenylate cyclase and lipase
2. In a patient a lowering in ability to physical load was revealed, while in skeletal
muscles the glycogen content was increased. The decrease in activity of what enzyme
may cause this condition?
A. Phosphofructokinase
B. Glucose 6-phosphate dehydrogenase
C. Glycogen phosphorylase
D. Glycogen synthase
E. Glucose 6-phosphatase
37
3. During biochemical investigation of blood in a patient was detected hypoglycemia in
fasting condition. Investigation of liver bioptates revealed the failure of glycogen
synthesis. What enzyme deficiency may cause such status?
A. Glycogen synthase
B. Phosphorylase
C. Aldolase
D. Fructose bis-phosphatase
E. Pyruvate carboxylase
4. Phosphorolysis of carbohydrates plays a key role in mobilization of polysaccharides.
Under the action of phosphorylase from glycogen is produced the next substance:
A. Glucose -1-phosphate
B. Glucose 1,6-bis-phosphate
C. Glucose 6-phosphate
D. Glucose
E. Fructose 6-phosphate
5. In an infant with point mutations in genes the absence of glucose 6-phosphatase,
hypoglycemia and hepatomegaly were revealed. What disease is characterized by these
symptoms?
A. Edison disease
B. Gierke disease
C. Parkinson disease
D. Cori disease
E. Mac Ardle disease
Individual independent students work
1. Hereditary disorders of synthesis and breakdown of glycogen and glycoconjugates.
1.
2.
3.
4.
5.
References:
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London, 1992. – 980 p.
Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.
Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003. – 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews, 1994. – 443 p.
Topic № 10. Mechanisms of metabolic and humoral regulation of
carbohydrate metabolism. Disorders of carbohydrate metabolism.
Objectives: To understand the sequence of enzymatic reactions of pentose
phosphate pathway. To interpret the role of hormones in regulation and maintenance of
38
constant blood glucose level. To learn the peculiarities of changes in metabolism of
carbohydrates, lipids and proteins during diabetes mellitus.
Actuality of the theme: The concentration of glucose in blood depends from the
equilibrium between its secretion into blood and utilization by tissues. The regulatory
system involves hormones insulin, glucagon, epinephrine, glucocorticoids, as well as
interaction between distinct organs, i.e. liver, muscles, brain, etc. Determination of
blood glucose level in clinical laboratory investigations is of great importance in
diagnostics of diabetes mellitus and many other diseases and disorders. The system of
regulatory mechanisms including hormones insulin, glucagon, adrenaline,
glucocorticoids, as well as metabolic interaction between liver, muscle, brain, etc.
Specific aims:

To interpret biochemical patterns of alternative pathways of monosaccharide’s
metabolism: pentose phosphate pathway, conversion of fructose and galactose.

Explain the molecular basis of hereditary disorders of fructose and galactose
metabolism.

To analyze the principal sources and metabolic pathways of utilization of blood
glucose

To explain the role of hormones in maintenance of constant glucose level in blood

To interpret terms, hyper-, hypoglycemia, glycosuria as pathological conditions
of glucose metabolism.
Theoretical questions:
1. Pentoso phosphate pathway of glucose oxidation; enzymatic reactions and
biological significance.
2. Metabolic pathways of fructose and galactose metabolism; enzymatic disorders of
their metabolism.
3. Hormonal regulation of carbohydrate metabolism (insulin, its structure, mechanism
of action, role in carbohydrate metabolism; adrenalin and glucagon, mechanism of their
regulatory effects on carbohydrate metabolism; Glucocorticoids, their effect on
carbohydrate metabolism).
4. Characterization of hypo- and hyperglycemia, glucosuria.
5. Disorders of carbohydrates, lipids and proteins metabolism during diabetes mellitus
6. Insulin dependent and noninsulin dependent forms of diabetes mellitus.
7. Pharmaceuticals for the treatment of diabetes mellitus.
Practical part
Experiment 1. Quantitative determination of glucose concentration in blood by
orto-toluidine method.
Principle. Heating of glucose with ortotoluidine in acetic acid gives compound of
blue-green color, the intensity of color is
proportional to glucose concentration.
39
Reagents and materials. Blood sample, 3% solution of trichloroacetic acid
(TCA), orto-toluidine reagent, glucose standard (4 mM/L, i.e. 720 mg of glucose
dissolved in 1 l of water), distilled water, tubes, pipettes, micropipette 0,1 ml,
centrifuge, centrifuge tubes, electrocolorimeter, water bath.
Procedure. Into each of two centrifuge tubes 0,9 ml of trichloroacetic acid are
added. Into test tube 0,1 ml of blood specimen is introduced, into control tube – 0,1 ml
standard glucose solution. Tubes are centrifuged at 3000 rpm for 10 min. From each
tube 0,5 ml of supernatant is transferred to the clean tubes and 4,5 ml of orto-toluidine
reagent is added. Tubes are placed on a boiling water bath for 8 min. Thereafter tubes
are cooled and the intensity of color is measured in a colorimeter at wavelength 630 nm
(red filter) in 1 cm pathway cuvette.
Calculation. The concentration of glucose is calculated using the formula:
Ctest = Cstand × Atest/Astand
where: Ctest – concentration of glucose in blood , mmoles/L;
Cstand – concentration of glucose in standard solution
Atest – optical density of test probe
Atest – optical density of standard glucose probe.
Compare the obtained result with normal value, draw the conclusion.
Experiment 2. Construction of the sugar curve of a healthy person and patient
with diabetes.
In the study 0.1 ml of blood is taken and a glucose solution (at the rate of 1 g
glucose in 1 kg of body weight) is drinking. Then, every half hour the blood is re-taken
for 3 hours and the level of sugar in it is determined by the Hahedorn-Jensen’s method.
After glucose intake the increasing in the concentration of glucose in blood sugar,
which reaches a maximum of 60-th minute is observed. After 120 minutes blood sugar
can be even lower from baseline. After 180 minutes blood sugar in a healthy person is
usually normal. Based on the obtained data students have to build a sugar curve by
plotting on the vertical axis the amount of sugar in mmol / l and the horizontal - the time
in minutes.
Investigation of sugar load on the blood sugar levels is particularly important for
the diagnosis of latent forms of diabetes, pancreatic differentiation and renal glycosuria
as well as the influence of insulin on carbohydrate metabolism.
Here are two types of sugar curves:
40
Number 1 - a healthy person and number 2 – with diabetes mellitus
The content of glucose, mmol / l
11,5
9,2
№2
6,9
4,6
№1
2,3
30
60
90
120
150
180
min
Qualitative and quantitative determination of sugar in the blood and urine is of
great clinical and diagnostic value. Pathological hyperglycemia is often associated with
diseases of the endocrine system. They are observed in diabetes mellitus, tumors of
adrenal and pituitary glands, severe liver function disorders, thyroid hyperfunction,
organic lesions of the nervous system. Substantial clinical and diagnostic value has a
diagnostic test for ketone bodies in blood and urine.
Hypoglycemia occurs during adenoma of the pancreas as a result of increased
production of insulin by β – cells, insufficiency of the thyroid gland, adrenal glands,
pituitary. In addition, hypoglycemia can be caused by starvation, heavy physical labor,
after overdose of insulin treatment, kidney disease, accompanied by reduced renal
threshold for glucose.
Clinical and pharmaceutical significance. Pathological hyperglycemia is often
associated with diseases of the endocrine system. They are observed in diabetes
mellitus, tumors of adrenal and pituitary glands, severe liver function disorders, thyroid
hyperfunction, organic lesions of the nervous system. Substantial clinical and diagnostic
value has a diagnostic test for ketone bodies in blood and urine.
Examples of tests:
1. Increase in blood glucose concentration under the action of glucagone is caused by
activation of the following enzyme:
A. Hexokinase
B. Glucokinase
C. Aldolase
D. Glycogen phosphorylase
E. Glycogen synthase
2. During the investigation in some person was revealed blood glucose concentration on
a level 4,5 mMoles/l. It may be the next interpretation of obtained result:
A. A person is healthy
41
B.
C.
D.
E.
A person has diabetes mellitus
A person possess increased tolerance toward glucose
A person has diabetes insipidus
A person has steroid diabetes
3. A patient was admitted to a hospital in comatose state. The accompanying mates
explained that the patient loss his consciousness during the training on the last stage of
marathon distance. What coma type can be recognized?
A.
B.
C.
D.
E.
Hyperglycemic
Hypoglycemic
Acidotic
Hypothyroid
Hepatic
4. A woman 46 years old complains on the feeling of dryness in mouth, thirst, frequent
urination. In biochemical investigation of blood hyperglycemia was detected. In urine
are present glucose, ketone bodies. In a patient can be suggested:
A.
B.
C.
D.
E.
Alimentary hyperglycemia
Diabetes mellitus
Acute pancreatitis
Diabetes insipidus
Cardiac ischemia
5. A patient addressed to physician with complaints on permanent thirst. In laboratory
investigation it was revealed hyperglycemia, polyuria and increased content of 17ketosteroids in urine. What disease is the most probable?
A.
B.
C.
D.
E.
1.
2.
3.
4.
5.
Steroid diabetes
Insulin dependent diabetes mellitus
Addison disease
Glycogenosis of the 1 type
Myxoedema
References:
Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London, 1992. – 980 p.
Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.
Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003. – 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews, 1994. – 443 p.
Sense module 4: Lipid metabolism and its regulation
42
Topic № 11. Catabolism and biosynthesis of triacylglycerols and phospholipids.
Intracellular ipolysis and molecular mechanisms of its regulation
Objective. To learn the processes of biosynthesis of phospholipids and triacylglycerols
and the main pathways of intracellular metabolism of lipids. To perform the methods of
determination of phospholipids and activity of lipase and interpret the obtained results.
Actuality of the theme: The knowledge of principal pathways of intracellular
metabolism of lipids in normal conditions and in pathology is necessary to medical
students in further studies of general pathology, pharmacology and related clinical
disciplines for correct interpretation of results of laboratory investigations and
recognition of metabolic disorders in distinct cases.
Specific aims:
 To interpret biochemical function of simple and complex lipids in organism: their
involvement in formation of structure and function of biological membranes, reserve
and energetic significance, the role as precursors in biosynthesis of biologically
active compounds of lipid nature.
 To explain the principal pathways of intracellular lipid metabolism.
 To explain enzymatic reactions of catabolism and biosynthesis of triacylglycerols.
 To interpret enzymatic reactions of synthesis of phospholipids and sphingolipids.
 To analyze the main pathways of lipid metabolism in human body in normal
conditions and in pathology.
 To explain hormonal regulation of lipid metabolism.
1.
2.
3.
4.
5.
6.
7.
Theoretical questions:
Biological functions of simple and complex lipids in human body (reserve, energetic,
thermoregulatory, production of biologically active substances)
Involvement of lipids in formation of structure and function of biological
membranes. Fluid mosaic model of biomembranes. Liposomes. Application of
liposomes in medical practice.
Circulatory transport and deposition of lipids in adipose tissue. Endothelial
lipoproteinase.
Catabolism of triacylglycerols: characterization of intracellular lipolysis, its
biological significance; enzymatic reactions; neurohumoral regulation of lipolysis:
role of epinephrine, norepinephrine, glucagone, insulin; energetic balance of
triacylglycerol oxidation.
Biosynthesis of triacylglycerols and phospholipids, the significance of phosphatidic
acid as a precursor.
Metabolism of sphingolipids. Genetic anomalies of sphingolipid metabolism –
sphingolipidoses. Lysosomal diseases.
Humoral regulation of lipolysis involving adrenaline, noradrenalin, glucagon and
insulin
Practical part
Experiment 1. Quantitative determination of phospholipids in blood serum.
43
Principle: phospholipids are precipitated with trichloroacetic acid together with
plasma proteins. After mineralization of sediment the quantity of phosphorus is
determined colorimetrically and content of phospholipids is calculated.
Method. Into the centrifuge tube 0,2 ml of blood serum and 2 ml of water are
added. 3 ml of 10 % solution of trichloroacetic acid is added and after 2–3 min the
mixture is centrifuged 5 min at 3000 rpm. Supernatant is carefully discarded. To
sediment, which contains lipoproteins, 1 ml of 56 % HClO4 is added and heated on a
boiling water bath for 30 min. The final solution must be colorless.
Thereafter to the tube are added 5 ml of water, 1 ml of ammonium molybdate and 1
ml of 1% solution of ascorbic acid. Simultaneously a standard solution of phosphorus is
prepared for comparison by mixing 1 ml of standard phosphorus solution (0,05 mg/ml),
5 ml of water, 1 ml of ammonium molybdate and 1 ml of ascorbic acid solution. After
15-20 min the extinction of probes is measured in a colorimeter.
Calculation: conducted according to formula:
Total phospholipids in serum = (Eexp × 0,05 / Est × 0,2) x 25 mg/ml or g/l
where Eexp – extinction of a sample probe,
Est – extinction of phosphorus standard
0,05 – concentration . of phosphorus in standard (mg/ml)
0,2 – volume of analyzed serum
25 – coefficient for calculation of total phospholipids content.
Clinical and pharmaceutical significance. The determination of phospholipid
content in blood has an important diagnostic significance. The concentration of total
phospholipids in blood serum of healthy adult is 1,5 – 3,6 g/l. Increasing of
phospholipids level in blood serum (hyperphospholipidemia) is observed in heavy form
of diabetes mellitus, nephrosis, obturative jaundice. Decreasing of phospholipids level
(hypophospholipidemia) is observed in atherosclerosis, anemia, fever, alimentary
distrophias, liver diseases.
Experiment 2. The influence of bile on the lipase activity.
Principle. The lipase activity is estimated according to the production of fatty acids
during hydrolysis of fat. The quantity of fatty acids is determined by titration with alkali
in the presence of phenolphthalein. Bile activates lipase thus accelerating the cleavage of
lipids.
Method. Into two tubes or flasks 10 ml of milk and 0.5 ml pancreatine solution are
added. Into the first tube 1 ml of water is added, into the second one - 1 ml of bile. After
the mixing 1 ml of the mixture from each tubes is taken off and transferred to the other
tubes, into which 1-2 drops of 0.5% phenolphthalein solution are added. The mixture is
titrated with 0.05 N NaOH up to the pink color, which doesn’t disappear during 30
seconds. The volume of alkali is registered. The first two tubes with digestive mixture
are placed at 38°C. Every 15 min 1 ml of the mixture from each tube is taken off and
transferred to the clean tubes and the titration is conducted. 4-5 subsequent
determinations are conducted. The results are registered as ml of alkali solution,
expended for titration. Obtained results are noted in the table.
44
Time, min
0
15
30
45
60
Quantity of NaOH expended for
titration, ml
Lipase
activity in
presence of
bile
Lipase
activity in
absence of
bile
L
L
Explain the results, draw a conclusion.
Clinical and pharmaceutical significance. Digestion of lipids occurs
predominantly in intestines under the action of active pancreatic lipase, which acts only
on emulsified lipids, e.g. milk fat. In gastric juice the activity of lipase is negligible.
Lipids must be emulsified, which is achieved due to the action of bile acids. As bile
emulsifies lipids and activates lipase, hydrolysis of lipids proceeds more quickly. As
lipase is the main enzyme in lipid digestion, changes in its activity may indicate on
disorders in some processes of lipid metabolism. Deficiency of pancreatic lipase causes
pancreatogenic steatorrhea, which is observed in chronic pancreatitis, hereditary or
acquired deficiency of pancreatic gland, in mucoviscidosis. The amount of bile
pigments in feces in such conditions is accompanying with the lowering of non
esterified fatty acids content and significant increase in unhydrolized lipids. The
alteration in emulsification, digestion and absorption of lipids and fat soluble vitamins
as well may take place in diseases of liver, gall bladder or biliary ducts (cholelythiasis).
Examples of tests:
1. Arachidonic acid as essential nutrient is needed for normal growth and development
of animal and man. It is precursor of biologically active substances. Indicate what
compounds are synthesized from arachidonic acid
A. Choline
B. Noradrenalin
C. Ethanolamine
D. Triiodothyronine
E. Prostaglandin E1
2. After consumption of fat meal in a patient appears nausea and heartburn, steatorrhea
also develops. A cause of such symptoms may be:
A. Amylase insufficiency
B. Hypersecretion of lipase
C. Disorder in phospholipase synthesis
D. Disorder in trypsin synthesis
45
E. Bile acids insufficiency
3. A victim of a snake’s beat almost dyed due to very intensive hemolysis. Blood
analysis detected unusually high content of lysolecitine. The toxic effect of snake’s
venom was caused by the presence of:
A. Phospholipase A1
B. Phospholipase A2
C. Phospholipase C
D. Phospholipase D
E. Neuraminidase
4. The form in which most dietary lipids are packaged and exported from the intestinal
mucosa cells is as follows:
A. Free fatty acids
B. Mixed micelles
C. Free triacylglycerol
D. 2-monoacylglycerol
E. Chylomicrons
5. Which of the following substances is involved in the synthesis of both glycerolcontaining phospholipids and triacylglycerol?
A. Acetoacetyl CoA
B. Choline
C. Phosphatidic acid
D. CDP-Ethanolamine
E. 3-Hydroxyburyrate
References:
1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London, 1992. – 980 p.
2. Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.
3. Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.
4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003. – 927 p.
5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews, 1994. – 443 p.
Topic № 12. -Oxidation of fatty acids and their biosynthesis. Investigation of
fatty acids ketone bodies metabolism.
Objective. To learn reactions of biosynthesis and oxidation of fatty acids. To know
metabolic pathways of ketone bodies formation in normal conditions and in pathology
states, to determine their concentrations in urine.
Actuality of the theme: Oxidation of lipids, as well as ketone bodies metabolism
is an important constituent of energetic metabolism in sense of providing tissues and
46
cells with ATP. Determination of ketone bodies concentration in blood and in urine has
an important significance in diagnostics of several pathological processes.
Specific aims:
 To interpret biochemical patterns of β-oxidation of long chain fatty acids.
 To interpret biosynthesis of long chain fatty acids and regulation of biosynthetic
process on the level of acetyl-CoA-carboxylase and fatty acid synthetase.
 To analyze the metabolism of ketone bodies.
 To explain the mechanism of excessive accumulation of ketone bodies in diabetes
mellitus and in starvation.
Theoretical questions:
1.
β-Oxidation of long chain fatty acids: localization of the process of β-oxidation of
fatty acids; activation of fatty acids, the role of carnitine in transport of fatty acids into
mitochondria; the sequence of enzymatic reactions in β-oxidation of fatty acids;
energetic balance of β-oxidation of fatty acids.
2.
Mechanism of glycerol oxidation; reactions and bioenergetics of this process.
3.
Biosynthesis of long chain fatty acids: localization of biosynthesis of long chain
fatty acids; metabolic sources for biosynthesis of fatty acids; stages in synthesis of
saturated fatty acids; characteristic of the synthetase of long chain fatty acids, the
significance of acyl transporting protein and biotin; sources of NADP H2 for
biosynthesis of long chain fatty acids; the sequence of enzymatic reactions in
biosynthesis of long chain fatty acids regulation of biosynthetic process on level of
acetyl-CoA-carboxylase and fatty acid synthetase; elongation of carbon chain of fatty
acids; synthesis of unsaturated fatty acids.
4.
Metabolism of ketone bodies: enzymatic reactions of ketone bodies biosynthesis
(ketogenesis); reactions of ketone bodies utilization (ketolysis), energetic effect;
metabolism of ketone bodies in pathology. Mechanism of excessive accumulation of
ketone bodies in diabetes mellitus and in starvation; the notions of ketoacidosis,
ketonemia, ketonuria.
Practical part
Experiment 1. Qualitative reaction on acetone and acetoacetic acid (Lange
probe).
Principle. It is based on a property of acetone to produce violet color compound
after an overlaying of concentrated ammonia solution over the mixture of test sample,
containing sodium nitroprussid and acetic acid.
CH3 – CO – CH3 + Na2 [Fe (CN)5 NO] + 2 NaOH →
Na4 [Fe (CN)5 NO = CHCOCH3] + 2 H2O
Acetoacetic acid can also form cherry-red colored complex compound of iron
chloride (III).
47
Na4 [Fe (CN)5 NO =CHCOCH3] + CH3COOH →
Na3 [Fe (CN)5 NOCH3COCH3] + CH3COONa
The rate of color ring formation between two layers of liquids depends from
acetone concentration. It is assumed, that appearance of the ring after 3-4 min
corresponds to the acetone concentration 0.0085 g/l (8,5 mg/ l).
Reagents and materials. Urine of patient with diabetes mellitus and urine of
healthy person, 10% sodium nitroprussid solution, concentrated acetic acid, 10%
solution of NaOH solution, ammonium sulfate, concentrated ammonia solution, 10%
solution of iron chloride (III), pipettes, test tubes, funnel, filter paper
Method. Into two tubes 0.5 ml of tested urine is added: to the first tube - from
healthy person (control), to the second one – from patient with diabetes mellitus (test
sample). To both tubes 2 drops of concentrated acetic acid, 5 drops of fresh 10% sodium
nitroprussid solution are added; thereafter the content of the tubes is mixed. An equal
volume of concentrated ammonia is overlaid carefully omitting the mixing of two
liquids. It may be observed an appearance of red-violet or violet colored layer in a form
of ring at the border of two layers in the second tube with a test sample, containing
ketone bodies.
Explain the results, draw a conclusion.
Acetoacetic acid can also produce a complex compound of cherry red color after
interaction with ferric chloride. This is used in Gerhardt probe for detection of
acetoacetic acid in urine.
Method. Into the tube 2 ml of tested urine are added and by droplets a solution of
FeCl3 is introduced up to complete precipitation of iron phosphates. The precipitate is
eliminated by filtration and several additional droplets of FeCl 3 solution are added. In
presence of acetoacetic acid a cherry red color appears.
Explain the results.
Experiment 2. Quantitative determination of ketone bodies in urine.
Principle. (See previous experiment).
Method. 8 tubes are taken for the experiment. Into each tube, except the first one, 1
ml of distilled water is added. Into the first and the second tubes 1 ml of urine of the
patient with diabetes mellitus are added. Thus, in the first tube urine is undiluted; in the
second one the urine is two times diluted. After the mixing 1 ml of the mixture from the
second tube is taken off and transferred to the third tube, after mixing 1 ml from the
third tube – into the following one and so on. Into each tube 8 drops of 50% ammonium
sulfate solution are added for increasing the density of the solutions, thereafter 8 drops
of concentrated acetic acid and sodium nitroprussid are added. The content of the tubes
is mixed and into each tube 1 ml of concentrated solution of ammonia is overlaid
carefully beginning from the last tube (be careful with concentrated ammonia!). An
appearance of red or violet ring in the tubes is registered. The last tube in which a
colored ring appears between the 3-d and the 4-th min is registered and the dilution of
urine sample is noted.
Calculation:
X = 0.85 × A × 15, were
X – content of acetone in urine (mg/day);
48
0,85 – empirical coefficient, corresponding to 0,85 mg of acetone in 100 ml
A – the dilution of the urine sample;
15 – coefficient for calculation on the daily volume of urine (1500 ml).
Explain the results.
Clinical and pharmaceutical significance. Ketone bodies include the following
substances – acetoacetic acid, -hydroxybutyric acid and acetone. Biosynthesis of
ketone bodies (ketogenesis) takes place in liver from intermediates of fatty acid
oxidation, namely, from acetyl-CoA.
Acetoacetic acid, produced in liver, is transported to body tissues (brain, muscles,
kidneys, heart etc.), where it serves as an energetic material. In healthy persons the
content of ketone bodies in blood is in ranges of 13-185 moles/l (1,5-20 mg/l). With
urine is excreted 20-40 mg of ketone bodies daily, which are preferentially acetoacetic
and -hydroxybutyric acids. Acetone appears in pathological conditions.
The increase of ketone bodies concentration in blood (ketonemia) and in urine
(ketonuria) is observed in diabetes mellitus, deficiency of sugar in nutrition,
overproduction of hormones, antagonistic to insulin (corticosteroids, thyroxine,
hormones of adenohypophysis). The decrease of ketone bodies content has no clinical
value. In early childhood prolong disorders of digestion (toxicosis, dysenteria) may lead
to ketonemia due to the permanent starvation and exaggeration.
Examples of tests
1. Lipids are obvious energetic material for the body. What is the main pathway of fatty
acids metabolism in mitochondria?
A. Reduction
B. Decarboxylation
C. β-Oxidation
D. α -Oxidation
E. -Oxidation
2. In diabetes mellitus and starvation there is an increase of ketone bodies content in
blood, which are utilized as energetic material by tissues. Note the substance which is
used in ketone bodies synthesis.
A. Citrate
B. Acetyl-CoA
C. Succinyl-CoA
D. α –Ketoglutarate
E. Malate
3. During the prolong starvation in blood of the person an increase in ketone bodies
content occurs. It is caused by the next factors:
A. Decrease of free fatty acid level in blood plasma
B. Mobilization of high density lipoproteins
C. Production of acetyl-CoA.
D. Enhance of fatty acids oxidation in liver
E. Decrease of triacylglycerols in adipose tissue
49
4. In a patient suffering from diabetes mellitus in blood was detected acetone. Note the
process of its production in the body.
A. In course of -oxidation of fatty acids
B. In course of β-oxidation of fatty acids
C. By condensation of two molecules of acetyl-CoA
D. In course of -oxidation of fatty acids
E. In tricarboxylic acid cycle.
1.
2.
3.
4.
5.
References:
Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London, 1992. – 980 p.
Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.
Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003. – 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews, 1994. – 443 p.
Topic № 13. Biosynthesis and biotransformation of cholesterol/ Regulation
of lipid metabolism and its disorders.
Objective. To learn general pathways of cholesterol metabolism and principal
disorders of lipid metabolism.
Actuality of the theme: Disorders in cholesterol biotransformation processes
cause a variety of diseases, such as atherosclerosis, obesity et al. In this connection the
investigation of lipid metabolism indexes is obvious for diagnostics and treatment of
different diseases.
Specific aims:
 To interpret stages of cholesterol biosynthesis
 To explain regulation of cholesterol production in human body
 To analyze pathways of cholesterol biotransformation: esterification, synthesis of
bile acids, steroid hormones, vitamin D3 and excretion of cholesterol from the
body.
 To explain biochemical bases of genetic disorders of lipids, lipoproteins and
cholesterol metabolism. Atherosclerosis, diabetes mellitus, obesity, steatorrhea.
Theoretical questions:
1. Biosynthesis of cholesterol in human body: localization of the process and its
significance; stages of cholesterol biosynthesis; enzymatic reactions of biosynthesis of
mevalonic acid; regulation of cholesterol synthesis.
2. Pathways of cholesterol biotransformation (esterification, production of bile
acids and steroid hormones, synthesis of vitamin D3, excretion from the body).
3. Atherosclerosis, mechanism of its development, role of genetic factors,
hypercholesterolemia, WHO classification of hyperlipidemias.
50
4. Disorders of lipid metabolism in diabetes mellitus.
5. Pathological processes which leads to the development of obesity
6. Lipoproteins of blood plasma, structure and physiological significance.
Practical part
Experiment 1. Qualitative reaction on bile acids (Petenkoffer reaction)
Principle. The reaction is based on formation of colored products after
condensation of bile acids with hydroxymethylfurfurol. The last is produced under the
action of sulfuric acid upon fructose, which in turn is formed from sucrose due to its
hydrolysis with sulfuric acid.
Reagents and materials. Bile, 20% sucrose solution, concentrated H2SO4, tubes.
Method. 5-6 droplets of bile are introduced into tube and then 10-15 droplets of
fresh 20% sucrose solution are added. This mixture is overlaid carefully onto 1 ml of
conc. sulfuric acid in another tube without mixing two fluids. In the borderline between
two fluids the precipitation of bile acids is observed and appearance of violet color ring.
Clinical and pharmaceutical significance. Bile acids are produced in liver, about
10-15 g daily. Bile acids include cholic, deoxycholic, chenodeoxycholic, lithocholic et
al., which are excreted in bile in free state or conjugated with glycine or taurine. Bile
acids emulsify lipids, activate lipase, are involved in absorption of fatty acids, form
choleinic complexes, stabilize cholesterol. Deficiency of bile acids in intestines may be
caused with liver diseases (occlusive jaundice, hepatitis, cirrhosis), disorders in
gallbladder or bile ducts (cholelithiasis, tumors of bile ducts).
Experiment 2. Determination of low-density lipoprotein (LDL).
Principle. The method is based on precipitation of LDL in the presence of СаCl2
and heparin, causing turbidity of the solution, which is proportional to the concentration
of LDL.
Reagents and materials. Blood serum, 0.025 M calcium chloride solution,
pipettes, micropipettes.
Method. In the test tube are added 2 ml of 0.025 M calcium chloride, 0,2 ml of
blood serum. The content is mixed thoroughly and the optical density is measured (E1)
at a wavelength of 630 – 690 nm (red light filters) against distilled water. The solution
is poured from into a tube, filled up with 0.04 ml 1% solution of heparin and carefully
mixed. In 4 min. the optical density of solution (E2) is determined under similar
conditions.
The difference (E1 – E2) indicates the optical density, due to the presence of LDL.
Calculations should be performed according to the formula:
Х = (Е1 – Е2) × 100
The content of LDL is expressed in units of optical density, multiplied on the
dilution (100). Normal values of LDL is 35 – 55 cu
Explain the result. Make a conclusion.
Clinical and pharmaceutical significance. LDL are synthesized in the liver and
represent about 5% of blood plasma proteins. In the lipid part of LDL predominate
sterols: cholesterol and its esters – 45% phospholipids – 25%, triglycerides – 10%. High
content of cholesterol and small sizes of molecules facilitate their infiltration into aorta.
51
This leads to atherosclerosis. In this regard, this fraction of lipoprotein is called
atherogenic lipoproteins.
In normal conditions the content of LDL and HDL is 3.6 and 2.6 g /l, respectively.
The ratio between LDL and HDL in the organism is essential. For example increasing
of concentration of LDL is observed in atherosclerosis, jaundice, hepatitis, diabetes,
obesity etc.
Examples of tests:
1. A man 67 years old suffers from brain vessels atherosclerosis. After investigation
hyperlipidemia was detected. What class of lipoproteins in blood plasma will be
increased most of all in biochemical investigation?
A. HDL
B. Non esterified fatty acids in complex with albumin
C. Chilomicrons
D. LDL
E. VLDL
2. A child 5 years old suffers from transient abdominal pains. On a skin – xanthoma
plaques are found. After investigation a hepatosplenomegala is detected. Blood serum is
turbid in fasting conditions. Cholesterol content – 4,3 mmoles/l, total lipids – 18 g/l. For
precisement of diagnosis electrophoresis of blood lipoproteins is administered. What
classes of lipoproteins are expected to be increased?
A. HDL
B. IDL
C. Chylomicrons
D. LDL
E. VLDL
3. In a patient suffering from diabetes mellitus an increase in concentration of VLDL
and triacylglycerols was detected. Cholesterol and HDL content in normal values. What
type of lipid metabolism disorder can be classified such changes of indicated data?
A. Hyperlipoproteinemia type II
B. Hyperlipoproteinemia type Y
C. Hypelipoproteinemia type III
D. Hyperlipoproteinemia type IY
E. Hyperlipoproteinemia type II b
4. Bile acids are derivatives of:
A. Heme
B. Cholesterol
C. Sphingomyeline
D. Phosphatidyl choline
E. Long chain fatty acids
52
5. In course of investigation of the patient with symptoms of atherosclerosis there was
detected substantial decrease of HDL, increase of LDL, cholesyterol concentration 11
mM/L. Decrease of activity of what enzyme is the most possible cause of changes
observed?
A. Blood lipoprotein lipase
B. Tissue lipases
C. Pancreatic phospholipases
D. Cholesterol esterase
E. Lecitin cholesterol acyl transferase
References:
1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. – 1992, 980 p.
2. Biochemistry / Trudy McKee, James R. McKee. – 1996, 654 p.
3. Biochemistry / Trudy McKee, James R. McKee. – 1999, 288 p.
4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. – 2003, 927 p.
5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. – 1994, 443 p.
53