Download LABORATORY MANUAL ON BIOLOGICAL CHEMISTRY

DANYLO HALYTSKYI
LVIV NATIONAL MEDICAL UNIVERSITY
DEPARTMENT OF BIOLOGICAL CHEMISTRY
LABORATORY MANUAL ON BIOLOGICAL
CHEMISTRY
for students of pharmaceutical faculty
PART І
LVIV – 2016
1
Methodical instructions prepared by:
Prof. Sklayrov A.Ya., M.D., Ph.D.
Fomenko I.S., Ph.D.
Klymyshin D.O, PhD.
Nasaduk Ch.M., M.D., PhD
Editor: prof. Sklayrov A.Ya., M.D., Ph.D.
Reviewed by: prof. Pinyazhko O.R., M.D., PhD
2
PREFACE
Biological chemistry is a fundamental medical discipline. A competence in
biochemical processes, which take place on different levels of organization – cellular,
organ, tissues and in a whole body – is necessary to medical students for
understanding of metabolic processes and turnover of substances in tissues, energy
production, anabolic and catabolic reactions, transfer of genetic information,
processes providing elementary physiological functions as well for interpretation of
biochemical data and their diagnostic and prognostic value.
In each practical lesson students would be learning topics, which are
determined by a program, will perform investigations of different substances and
compounds, and interpret their significance. In each sense modul the themes for
independent studies are proposed
A considerable attention is paid to clinical aspects of biological chemistry –
inborn and acquired disorders of metabolism, enzymopathias, to employment of
enzymes as medicinals, as well as to a pathochemical processes, which take place in
development and in course of diabetes mellitus, atherosclerosis, rheumatism,
myocardial infarction, diseases of digestive system, etc.
In order to improve the learning of educational material students are
recommended to use modern textbooks and instruction materials, prepared by
leading ukrainian specialists as well by coworkers of the Department of Biochemistry
of Lviv national medical university.
The final aims in learning of the subject “Biological and bioorganic chemistry“
are determined with a thesis, that student in his future professional activity ought to
be able:
 To analyze a correspondence of bioorganic compounds structure to
physiological function, which fulfill these compounds in the body.
 To interpret peculiarities of physiological status of organism and development
of pathological processes on basis of laboratory investigations data.
 To analyze reaction ability of carbohydrates, lipids and amino acids which
provides their functional properties and metabolic transformations in the body.
 To interpret peculiarities of structure and transformations in tissues of
bioorganic compounds as a background of their pharmacological activity as
medicinals and drugs.
 To interpret biochemical mechanisms of pathogenesis of different disorders in
human body and principles of their correction.
 To explain the main mechanisms of biochemical effect and directed action of
different classes of pharmacological agents.
 To explain biochemical and molecular basis of physiological functioning of
cells, organs and systems of human body.
 To analyze enzymatic reactions which occur in cell membranes and organelles
for explanation an integration of metabolic processes
 To classify the results of biochemical investigation and changes of biochemical
and enzymatic data, which are used in diagnostics of the most spread human
diseases
 To interpret the significance of biochemical processes and their regulation in
providement of normal function of organs, systems and whole body
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LABORATORY SAFETY
Avoid loose or synthetic clothing for lab work; remove loose jewelry; secure
hair and clothing away from flames, equipment, and chemical contamination.
A laboratory is a workplace. The list of things not permitted in chemistry
labs is long – begin with eating, drinking, cooking, applying makeup, smoking and
anything else that might increase the chance of ingesting lab chemicals. Careful
workers do not touch hands to their faces while working in lab. Professional and
serious behavior is expected at all times; rowdy or boisterous play or pranks of any
kind will be deemed cause for expulsion from lab.
Housekeeping. Store backpacks and other extra materials away from work areas
and off floors to protect them and to keep walkways clear. Keep work areas clear;
store extra glassware and materials as soon as you finish with them, keeping only
essential materials on the workbench.
Use cotton towels to dry wet hands & clean surfaces. Use paper toweling for
absorbing hazardous materials. Dispose of dirty waste paper in trash receptacles.
 Clean your work area:
 Check to be sure all reagents and waste containers are securely closed;
 Clean lab surfaces with sponges;
 Paper with absorbed hazardous chemicals should be placed in solid hazardous
waste containers, not in the general trash.
 Broken glass contaminated with hazardous or smelly materials can be rinsed
with appropriate solvent before placing shards in the broken glass container.
Handle hazardous materials with correct techniques.
Never touch hazardous chemicals with bare hands; use tools such as tongs and
scoops.
Never remove chemicals from the laboratories. Do not attach samples to lab
reports or notebook pages. In addition to causing disposal problems, taking samples
from lab creates the potential for an accidental exposure.
Anhydrous materials (such as NaOH, CaCl2, MgSO4 or NaSO4) may absorb
water from the air and MUST be kept tightly closed between uses. Left in the air,
NaOH or KOH pellets will absorb moisture and produce a puddle of concentrated
corrosive liquid on the work bench – a serious skin exposure hazard.
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Thematic plan of practical lessons in Semester 1
N
Topics of lessons
1. Introduction to biochemistry. Methods of biochemical investigation.
Amino acid composition, structure, physico-chemical properties,
classification and functions of simple and conjugated proteins
2. Enzymes: structure, physico-chemical properties, classification and
mechanism of action of enzymes. Methods of detection of enzymes in
biological material.
3 Kinetics of enzymatic reactions. Regulation of enzymatic activity,
determination of enzymatic activity.
4. Regulation of enzymatic processess enzymopathias and mechanisms of
their development. Application of enzymes as pharmaceutical
preparations.
5. Role of cofacors and coenzymatic vitamins in catalytic activity of
enzymes.
6. General principles of turnover of substances and energy.
Functioning of citric acid cycle.
7 Biological oxidation, oxidative phosphorylation and ATP synthesis.
Inhibitors and uncouplers of tissue respiration and oxidative
phosphorylation in respiratory chain of mi9tochondria
8 Glycolisis – anaerobic oxidation of glucose, alternative pathways of
carbohydrate metabolism.
9 Aerobic oxidation of glucose. Alternative pathways of carbohydrate
metabolism.
10 Catabolism and biosynthesis of glycogen. Regulation of glycogen
metabolism Biosynthesis of glucose - gluconeogenesis,
11 Mechanisms of metabolic and humoral regulation of carbohydrate
metabolism. Disorders of carbohydrate metabolism.
12 Catabolism and biosynthesis of triacylglycerols and phospholipids.
Intracellular ipolysis and molecular mechanisms of its regulation.
13 -Oxidation of fatty acids and their biosynthesis. Investigation of fatty
acids ketone bodies metabolism.
14 Biosynthesis and biotransformation of cholesterol/ Regulation of lipid
metabolism and its disorders.
Hours
2
2
2
2
2
2
2
2
2
2
2
2
2
2
5
Thematic plan of lectures in Semester 1
N
1
2
3
4
5
6
7
8
9
10
Theme of the lecture
Hours
Introduction to biochemistry. Methods of biochemical investigations.
2
Enzymes: structure, properties and classification. Mechanism of action
and regulation of enzymatic activity. Kinetics of enzymatic reactions.
2
Role of cofactors in catalytic activity of enzymes.
Regulation of enzymatic processes and analysis of enzymopathias
2
appearance. Medical enzymology
General principles of turnover of biomolecules and energy.
2
Tricarboxylic acid cycle.
Molecular principles of bioenergetics. Biological oxidation. Oxidative
2
phosphorylation and its regulation.
Carbohydrates: structure, classification, functional significance.
Metabolism of monosaccharides, anaerobic and aerobic oxidation of
2
glucose. Gluconeogenesis.
Metabolism of polysaccharides. Gluconeogenesis. Alternative pathways
2
of carbohydrate metabolism.
Regulation of carbohydrate metabolism and its disorders.
2
Lipids: structure, classification and functional significance. Transport
2
forms of lipids in blood. Metabolism of simple lipids.
Metabolism of complex lipids and its regulation. Correction of disorders
2
of lipid metabolism by pharmaceutical preparations.
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Topic №1. Introduction to biochemistry. Methods of biochemical investigation.
Amino acid composition, structure, physico-chemical properties, classification
and functions 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.
Actuality of the theme: Biochemistry is a science which investigates chemical
composition of living organisms, chemical structure of constituents, their properties,
localization, the pathways of their appearance and transformations, as well as
chemical processes, which take place in living cell and provide turnover of matter
and energy in the cell.
Biochemistry is on the way of solution of important problems and questions of
natural history and medicine, e.g. problem of protein synthesis, life span
prolongation, etc.
Modern physical, chemical and mathematical methods are used in biochemical
investigations. Biochemical data are used in medical diagnostics, treatment and
prevention of diseases
Specific aims:

To know principal stages and regularities in origin and development of
biochemistry as fundamental medical and biological science and educational
discipline.

To recognize priciples of methods of investigation of functional status in human
body in health and disease.

To interprete data of biochemical investigations and evaluate the status of
selected metabolic pathways

To determine optical density of colored solutions at distinct light wavelength
using a photocolorimeter, to interpret obtained results properly.
1.
2.
3.
4.
5.
Theoretical questions:
The objectives and assignments of biochemistry and its principal trends and parts.
A short history of biochemistry and its main periods.
Methods of biochemichemical analysis:
 Optical methods in biochemical investigations.
 Electrophoresis.
 Chromatography.
 Radioisotopic methods.
 Enzyme immunoassays (ELISA).
General characterization of amino acids. Classification of amino acids (due to
their structural, electrochemical, biological properties).
Biologically active peptides, their significance and employment in medicine
(glutathione, hormones of hypophysis and hypothalamus, insulin etc.).
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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. Classification of proteins. Characterization of simple proteins.
8. Conjugated proteins, their characteriristics.
Essay of the history of biochemistry unit
Unit of Biological Chemistry was founded in 1894 at the Medical department of
Lviv University by Prof. V. Niemilovych (1863-1904) who had arrived from Vienna
in 1891 upon invitation to work as an associate professor in Pharmacognosy and as a
lecturer in chemistry.
From 1904 to 1919, the unit was headed by Prof. Bondzinski (1862-1929). He
was the founder and the first president of the Academy of the Medical Sciences of
Poland. Research of the unit's faculty under Prof. S. Bondzinski was the so-called
oxyproteins - the products of protein turnover (metabolism) excreted in small
amounts within urine.
From 1919 to 1922, the unit was supervised by Prof. W.Morachewsky, who also
became rector of the Academy of Veterinary medicine in Lviv. W. Morachewsky
was certified to teach medical chemistry at the University of Lviv in 1918 and he
began his work as Professor at Lviv Academy of Veterinary medicine in 1921.
From 1922 until 1941 the unit was headed by the world-well known scientist
Jakub Oscar Parnas. His most significant work at the Lviv schoole was connected
with enzymatic products, their formation in association with muscular activity and
alcohol fermentation. Some of his investigations have become classic works in
biochemistry. They concern the rapid autolytic increase of ammonium content in
extravasate blood and "postmortem" or traumatic ammoniogenesis in muscular tissue.
Significantly important was his discovery of the higher phosphorylated derivatives of
adenylic acid found in muscular fibers and erythrocytes, namely ATP and ADP. As
alternatives to adenylic acid they are not subject to enzymatic deamination in cells.
J.Parnas and his schoole studied biological and chemical properties of muscular
adenylic acid and its derivatives and introduced analytical methods for its quantitative
determination. J. Parnas achieved worldwide recognition in his research of anaerobic
degradation of carbohydrates. His research is called in biochemical literature as the
Embden-Meyerhof-Parnas pathway or "EMP scheme of glycolysis ". J. Parnas was
the first who introduced isotopic methods into biochemical investigations.
From 1944 to 1973, the unit was headed by Prof. B.A. Sobchuk who began his
scientific activity under J. Parnas conduction. B. A. Sobchuk investigated
carbohydrate metabolism in the muscular tissue and yeast. He was awarded the
degree of Doctor of Medicine (1937) for his research on the significance of pyruvate
in glycogenolysis. For his thesis, "Synthesis of xanthopterin and its derivatives and
their effect on experimental tumors in animals", B.A .Sobchuk was awarded the
degree of Doctor of Biological Sciences in 1959. A year later he attained
professorship of biochemistry.
The co-workers of department studied the
peculiarities of carbohydrate metabolism in tumor cells (the Krebtry's effect), as well
as the synthesis and specific effect of xanthopterin and its derivatives iodopterin and
porphyropterin on tumors. Another significant direction of the unit's work was the
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investigation of iodine metabolism, function of the thyroid gland and toxicology of
carbon monoxide.
From 1974 to 1995, the unit was headed by prof. M.P. Shlemkevych. In close
collaboration with the unit of oncology, the department of biochemistry studied the
mechanisms of sensitivity and resistancy of tumor cells of stomac cancer to
chemotherapeutic agents, namely 5-fluorouracyl.
From 1995 to 1998, the head of biochemistry unit was prof. M.F. Tymochko.
The main goal of his scientific research was the investigation of the fundamentals of
adaptive-compensatory processes under various experimental conditions with
emphasize on changes in oxygen-dependent reactions. He studied metabolic
backgrounds of oxygen homeostasis in different functional conditions and dealt with
solving of a wide range of important problems in medical practice. He also
investigated the effect of harmful environmental factors on the functions of digestive
system, the role of energy exchange in the pathogenesis of chronic diseases of the
liver and cardiovascular system. He determined the degree of risk in surgery in
abdominal cavity, endocrine and cardiovascular surgery and endogenous intoxication
in oncological diseases.
Since 1998, the Biochemistry unit has been headed by O. Ja. Sklyarov, who
started his scientific and educational activities at the unit of normal physiology. In
1993, for his thesis, "Mechanisms of combined action of mediators and hormones on
the secretory function of gastric glands", he obtained the scientific degree doctor of
medical sciences. In 1997 he was nominated as honorary associate professor by the
fund of Soros and was noted by the fund "Revival". Prof. O.Ja.Sklyarov is concerned
with the study of the mechanisms of regulation of gastric secretion under the
influence of the combined action of neurohormonal substances. Alongside his
research, prof. O.Ja.Sklyarov pays significant attention to educational work.
Currently, the unit's researchers are investigating the systemic influence of
malignant tumors and endogenous intoxication in patients and in experimental
animals. They are also studying the effects of stress on cytoprotective and
ulcerogenic mechanisms of gastric mucosa, the ion-transport and metabolic processes
in it and the action of exogenous factors on endoecological conditions.
Practical part
Determination of optical density of colored solutions according to their
concentration
The photoelectrocolorimetric method of analysis serves for determination of
substances concentrations in coloured solutions, biological liquids or tissue extracts.
It may be also used for determination of concentrations of colourless substances if
they can be transformed into coloured state with the specific reagents. The method is
one of the most widespread in biochemistry and clinical medicine.
Experiment 1. Measurement of optical density (A) of solutions which contain
different quantities of phosphorus.
Principle. Phosphates in presence of sulfuric acid form phosphomolybdate
complexes with molybdate, which are then reduced to a compound called molybdene
blue. The amount of phosphates is then measured colorimetrically according to a
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color intensity of molybdene blue complex.
Method. Pipette the solutions into five labelled test tubes according to the table:
Tube number
№
1
2
3
4
5
Reagents
Standard phosphate solution (10 mg/ml),
ml
Distilled water, ml
Molybden reagent, ml
Phosphorus concentration, mg%
Results of optical density measurement
1
-
2
0.5
3
1.0
4
2.0
5
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 color develops. After 5 min measure the optical density on a
photoelectrocolorimeter with a red light filter. Then draw a plot (calibration curve)
according to obtained results.
Experiment 2. Protein precipitation with sulfosalicylic acid.
Principle. Proteins dissolve in water with formation of homogenous solutions.
There are two main factors of the stabilization of proteins in solutions. The first, the
existence of electric charges, and, the second, the formation of hydrate envelopes,
which are due to the attachment of water molecular dipoles round charged
hydrophilic residues of amino acids. The electric charge of protein molecules
depends from the ionization of the functional groups of side chains of amino acids.
Thus, the solubility of proteins is due to their amino acid composition, the unique
structural organization of the protein molecule and the properties of the solvent.
Neutral salts increase solubility of proteins because of the interaction of their ions
with the polar groups in proteins. There are reversible and irreversible (denaturation)
methods for the precipitation of proteins. The reversible precipitation occurs with
high concentration of neutral salts (salting out), alcohol, and acetone (the last two
conducted at below zero temperatures). The mechanism of precipitation consists in
removing the hydrate envelopes and masking the charges of the protein molecule.
During the denaturation process the alteration of spatial structure of protein molecule
occurs. This is accompanied by decrease or complete loss of solubility, changes in
chemical properties, and loss of biological activities of a protein. The biological
activity of proteins such as hormone effect, enzymatic activity, virulence of
microorganisms is lost after denaturation (called inactivation). Cations of heavy
metals, organic acids (trichloroacetic, sulfosalicylic, tannic acids), concentrated
mineral acids, heating, ultraviolet and ionizing radiation exhibit a denaturing effect
on proteins. Though denaturation is an irreversible process, it is not connected with
destruction of the primary structure of a protein.
Method. Add 1.0 ml of protein solution into the tube, and 3-5 drops of 20%
sulfosalicylic acid and observe the appearance of a precipitate.
Clinical and diagnostic significance. Proteins are important cell constituents.
They play an important role in metabolic processes. Denaturation is usually met in
natural conditions and in experimental research. Denatured proteins are readily
10
digested by proteolitic enzymes. Gastric hydrochloric acid causes the denaturation of
proteins of food. As a result they are more readily hydrolyzed by digestive enzymes.
Milk proteins, such as casein, bound heavy metals and are used in treatment of
poisoning with heavy metals. In clinical laboratories the proteins of blood and serum
are denatured by precipitation with organic acids like trichloroacetic acid and
sulfosalicylic acid.
Methods of quantitative determination of proteins
Protein quantity in the specimen can be estimated by physical and chemical
methods. Physical methods include refractometry, spectrophotometry, etc.
Chemical methods employ color reactions of proteins and the intensity of color
is measured by colorimetry (e.g. biuret test, Lowry method). Chemical methods
include also determination of nitrogen by Quieldahl method.
3
Standard tube
Serum A
4
5
1-
Serum C
2
Serum B
1
Control tube
Experiment 3. Biuret method of protein quantitation.
Principle. Proteins in alkaline medium interact with copper sulfate forming a
compound with a violet colour. Optical density of this compound solution is directly
proportional to the concentration of protein.
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).
Method. Take five clean dry tubes and do the following procedures:
1. Add to the first tube (control) 0.1 ml of saline.
2. Add to the second tube 0.1 ml of standard protein solution.
3. Add to the third, fourth and fifth tubes 0.1 ml of tested blood serum A, B and C
respectively.
4. Add 5 ml of biuret reagent to all tubes, mix and incubate for 10-15 min.
5. Measure the extinction (optical density) on a photocolorimeter using a green
light filter against control solution.
2-
- 0.1 ml of standard solution
3-
- 0.1 ml of blood serum A
4-
- 0.1 ml of blood serum B
5-
- 0.1 ml of blood serum C
+ 5 ml of biuret
reagent
- 0.1 ml of saline
Incubation for 10-15 min (room temperature)
Measurement of the optical density
using a green light filter
C
RESULTS:
BA
BB
BC
Calculation. The concentration of protein in the test solution is calculated
according to the formula:
X= (AB)/C, where
X – protein concentration in test solution, (g/l),
A – protein concentration in standard solution (50 g/l),
B – extinction of tested protein solution,
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C – extinction of standard protein solution.
Clinical and diagnostic 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 decrease 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, in 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 cirrhosis as well as in
diseases accompanied with dehydration, that is during diarrhea, vomiting, and
diabetes insipidus. As a result of heavy mechanic 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.
Examples of Krock-1 tests
1. Choose from listed below methods ONE, which is used for fractionation of
protein mixtures and isolation of individual proteins (enzyme, hormone, toxin
etc):
A. Affinity chromatography
D. Proteolysis
B. Precipitation with nitric acid
E. Radioimmunoassay
C. Boiling of extracts
2. Determination of C-reactive protein (CRP) in blood plasma is conducted with
the use of antisera, containing specific antibodies against CRP. What type of
analytical method is used in this case?
A. Immunoprecipitation
D. Chromatography
B. Spectrophotomenry
E. Polarography
C. Electrophoresis
3. Proteins are biopolymers of principal significance in cell building, they are
composed from amino acids as monomers, which are connected into chain by the
next main type of chemical bond:
A. Peptide bond
C. Ionic bond
B. Phosphodiester bond
D. Hydrogen bond
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E. Glycosidic bond
4. Protein preparations from human blood plasma are frequently used in clinical
medicine for treatment of many diseases. Fractionation of blood plasma and
preparation of distinct protein fractions is achieved by the next method:
A. Fractional precipitation with
C. Precipitation with salts of heavy
ammonium sulphate
metals
B. Fractional precipitation with ethanol
D. Electrophoresis in agarose gel
by Cohn YI method
E. Ultracentrifugation
Individual independent students work
1. History of biochemistry and its main periods. The significance of biochemistry in
the development of medical sciences and practical health care.
2. The fundamental discoveries in a branch of structural and functional significances
in proteins and nucleic acids.
References:
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry.
2nd edition” – 2008. – 488 p.
8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M.. – 2012. – 308 p.
Topic № 2. Enzymes: structure, physico-chemical properties, classification and
mechanism of action of enzymes. Methods of detection of enzymes in biological
material.
Objective: Life in all its diverse manifestations is an extremely complex system of
chemical reactions which are catalyzed by enzymes. The last play vital role in nearly
all life processes. They are involved in living organisms in a vast multitude of
interrelated chemical reactions such as synthesis, degradation and interconversion of a
large number of chemical compounds. An understanding of their implications provides
a deep insight into the sense and innermost enigmas of the fascinating phenomenon that
we call life.
Specific aims:
 To interpret biochemical principles of structure and functioning of different
13
classes of enzymes.
 On the basis of physical and chemical properties of enzymes as proteins to
explain the dependence of enzymatic activity from pH of medium, temperature
and other factors.
 To analyze values of the activity of enzymes in blood plasma in dependence from
their localization in the cell, tissue or organ.
 To analyze methods of determination of enzymatic activity for an optimal
application in clinical biochemistry.
1.
2.
3.
4.
5.
6.
7.
8.
Theoretical questions:
Enzymes: definition, properties of enzymes as biological catalysts, difference
between enzymes and inorganic catalysts.
Nomenclature and classification of enzymes.
Physico-chemical properties of enzymes.
Simple and conjugated enzymes. Role of non-protein part of conjugated enzymes.
Structure of enzymes: active centres and allosteric sites.
Levels of structural organization of enzymes. Multi-enzyme complexes, their
advantages.
Specificity of enzymes.
The localization of enzymes in cells and organs.
Practical part
Salivary amylase
Amylase – is an enzyme that catalyses the hydrolysis of starch into sugars.
Amylase is present in the saliva of humans and some other mammals, where it begins
the chemical process of carbohydrates digestion. Salivary gland makes amylase to
hydrolyse α-1,4-glycosidic bonds of starch into disaccharides (maltose) which are
converted by other enzymes to glucose to supply the body with energy. As diastase,
amylase was the first enzyme to be discovered and isolated (by A. Payen in 1833).
Experiment 1. Investigation of the thermolability of salivary amylase.
Principle. Salivary amylase is a protein and consequently is thermo-sensitive.
Boiling inactivates enzymes due to denaturation, low temperature (near 0 oC)
substantially lowers the rate of reaction. The hydrolysis of starch is monitored by
probes with iodine and the appearance of the of maltose, which is the final product of
starch hydrolysis by salivary amylase. Starch with iodine gives a blue coloured
complex. Dextrins (intermediate products of starch hydrolysis) 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’s reaction.
Method.
I. Dilution of saliva. To obtain diluted saliva (the source of amylase), rinse one’s
mouth with distilled water during 1-2 min and collect that portion of fluid
(saliva + water) into separate tube and use it for the analysis.
14
II.
Preparation of experimental mixtures and incubation. Take 3 clean tubes
and do the following procedures:
1. Add 2 ml of diluted saliva to the first tube and boil it for two min on a flame.
2. Add 2 ml of diluted (nonboiled) saliva to the second and the third tubes.
3. Add 5 ml of starch solution into each tube.
4. Place the first and the second tubes into the termostate at 37 oC for 10 min.
5. Place the third tube in a cold water (near 4-6 oC) for 10 min.
6. Divide the content of each tube into two equal portions.
III. Color reactions for the detection of starch and maltose.
1. Add 3 drops of iodine to the first portion of each test solution. Register changes in
color.
2. Use the second portion of the probes for the performance of the Trommer’s
reaction.
Preparation of mixtures and incubation
2
1
- 2 ml of boiled diluted saliva
3
- 2 ml of diluted saliva
- 5 ml of starch solution
Incubation for 10 min
(T=4-60C)
Incubation for 10 min (T=370C)
Color reactions for the detection of starch and maltose
1a
Trommer’s reaction
(+CuSO4+NaOH)
1b
+ I2
2a
2b
Trommer’s reaction
(+CuSO4+NaOH)
+ I2
3a
Trommer’s reaction
(+CuSO4+NaOH)
3b
- 2.5 ml of mixture after
incubation
+ I2
Results:
–
+
+
–
–
+
Trommer’s reaction. Add 5 ml of 5% NaOH and several drops of CuSO4 solution,
mix the content of these tubes and heat it 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. The investigation of the influence of pH of medium on amylase
activity.
Principle. Each enzyme has an optimum pH at which the velocity is maximum.
Below and above this pH, the enzyme activity is much lower and at extreme pH, the
enzyme becomes totally inactive. Most of the enzymes of higher organisms show
optimum activity around neutral pH (6-8).
Method.
I. Dilute saliva as described above (Experiment 1)
15
II. Preparation of experimental mixtures and incubation. Take 3 clean tubes and
do the following procedures:
1. Add 2 ml of starch solution to each of the three tubes.
2. Add to the first tube 2 ml of phosphate buffer, pH 5.0.
3. Add to the second 2 ml of phosphate buffer, pH 7.0.
4. Add to the third 2 ml of phosphate buffer, pH 9.0.
5. Add into each tube 1 ml of diluted saliva.
6. Incubate tubes at 37 oC for 10 minutes.
7. Divide the content of each tube into two equal portions.
Stage 1 Preparation of mixtures and incubation
2
1
- 2 ml of of starch solution
3
- 2 ml of phosphate buffer
pH 9
pH 7
pH 5
- 1 ml of diluted saliva
Incubation for 10 min (T=370C)
Stage 2. Color reactions for the detection of starch and maltose
1a
Trommer’s reaction
(+CuSO4+NaOH)
1b
+ I2
2a
2b
Trommer’s reaction
(+CuSO4+NaOH)
+ I2
3a
Trommer’s reaction
(+CuSO4+NaOH)
3b
- 2.5 ml of mixture after
incubation
+ I2
Results:
–
+
+
–
–
+
III. Color reactions for the detection of starch and maltose.
1. Add 3 drops of iodine to the first portion of each test solution. Register changes in
color.
2. Use the second portion of the probes for the performance of the Trommer’s
reaction (see Experiment 1).
Explain the results and draw a conclusion.
Experiment 3. Investigation of substrate specificity of salivary amylase and
yeast sucrase.
Principle. Enzymes exhibit selectivity to substrates, which is called substrate
specificity. In many cases this property is the essential characteristic that distinctly
differs enzymes from inorganic catalysts. The high specificity of enzymes depends
from the conformational complementarity between the molecules of enzyme and
substrate due to the unique structure of active centre of the enzyme.
16
Method:
I. Specificity of amylase:
1. Add 1 ml of two fold diluted saliva in each of two tubes.
2. Add 2 ml of 1% solution of starch into the first tube.
3. Add 2 ml of 1% solution of sucrose into the second tube.
4. Incubate both tubes at 37oC in thermostat for 15 min.
5. Conduct the Trommer’s reaction (see Experiment 1) with the content of both
tubes.
II. Specificty of yeasts sucrase:
1. Add 1 ml of sucrase from yeasts solution in each of two tubes.
2. Add 2 ml of 1% solution of sucrose to the first tube
3. Add 2 ml of 1% starch solution to the second tube.
4. Incubate both tubes at 37oC in thermostat for 15 min.
5. Conduct the Trommer’s reaction (see Experiment 1) with the content of both
tubes.
Part 3. Note the results of the experiment in the table.
Tube №
Enzyme
Substrate
Result of Trommer
reaction
1
Amylase
Starch
2
Amylase
Sucrose
3
Sucrase
Sucrose
4
Sucrase
Starch
Explain the results and draw a conclusion.
Clinical and diagnostic significance. Amylase (E.C. 3.2.1.1) hydrolyzes starch
and dextrins. It is produced predominantly in salivary and pancreatic glands, although
amylase activity can be detected in tissues of liver, kidneys, intestines and lungs.
During normal conditions amylase activity in blood serum corresponds to 0.42-0.96 g
of starch, hydrolyzed by 1 m1 of enzyme in 1 minute.
An increase of amylase activity in serum is observed in pancreatitis, peritonitis,
mesenteric vessels thrombosis, rupture of oviduct in case of extrauterine pregnancy
and after the injection of some drugs, e.g. morphine, caffeine, ACTH and cortisol.
The increase of the activity of amylase in saliva is observed in renal insufficiency,
stomatitis, neuralgia. A decrease in amylase activity is noted in some cases of
psychoses, accompanied by depression or excitation and in gastric secretion disorders
(anaciditas).
Examples of Krock-1 tests
1. After the addition of an extract of pancreatic gland to the tube with starch
solution a blue coloration of the sample with iodine have disappeared, which
indicates on starch hydrolysis. What pancreatic enzyme is involved in this
reaction?
A. Amylase
D. Lipase
B. Trypsin
E. Aldolase
C. Chymotrypsin
17
2. In human saliva there is an enzyme able to hydrolyze the α[1→4] glucosidic bonds
in the molecule of starch. Name this enzyme:
A. α-Amylase
D. β-Galactosidase
B. Phosphatase
E. Lysozyme
C. Fructofuranosidase
3. In a patient was detected disorder in digestion of protein in stomach and small
intestines. What group of enzymes may cause this disorder?
А. Proteinases
D. Lyases
В. Amylase
Е. Aminotransferases
С. Lipase
4. The first position in classification of enzymes is occupied by:
A. Oxidoreductases
D. Hydrolases
B. Transferases
E. Ligases
C. Isomerases
Individual independent students work
1. Multi-enzyme complexes and their advantages.
2. The employment of enzymes in biochemical investigations.
1.
2.
3.
4.
5.
6.
7.
8.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry.
2nd edition” – 2008. – 488 p.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M.. – 2012. – 308 p.
Topic № 3. Kinetics of enzymatic reactions. Regulation of enzymatic activity,
determination of enzymatic activity.
Objective: To show the catalytic role of enzymes on the basis of pancreatic
proteinases and amylase activity measurements. To estimate clinical and diagnostic
significance of determination of amylase activity in urine.
Actuality of the theme: Enzymes are biocatalysts with changeable activity,
submitted to regulatory influences. The knowledge of kinetics of enzymatic reactions
is important for the understanding of metabolic processes in cells, tissues and organs
of human body.
18




Specific aims:
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 interprete changes in activity of enzymes in biological fluids under conditions
of different pathological processes.
To explain the employment of inhibitors and activators of enzymes in cases of
metabolic disorders or distinct pathology
To analyse the mechanism of enzyme action using an example of chymotrypsin
and acetylcholinesterase.
Theoretical questions:
1. Enzyme kinetics. Factors affecting enzymatic activity:
 concentration of enzyme
 concentration of substrate;
 effect of temperature;
 effect of pH
2. Michaelis-Menten constant and equation;
3. Lineweaver-Burk double-reciprocal plot;
4. Mechanism of enzyme action:
 substrate-enzyme complex formation and dissociation;
 lock and key model or Fisher’s template theory;
 induced fit theory or Koshland’s model;
 substrate strain theory.
6. Units of enzymatic activity.
7. Methods of enzymatic activity assays.
Practical part
Methods of enzymatic activity assays
Methods for the determination of enzymatic activity can be divided into: direct,
indirect and coupled assays. The direct method is based on measurement of the
substrate or product concentration as a function of time. For example, the enzyme
cytochrome c oxidase catalyzes the oxidation of the heme-containing protein
cytochrome c. In its reduced (ferrous iron) form, cytochrome c displays a strong
absorption band at 550 nm, which is significantly diminished in intensity when the
heme iron is oxidized (ferric form) by the oxidase. One can thus measure the change
in light absorption at 550 nm for a solution of ferrous cytochrome c as a function of
time after addition of cytochrome c oxidase; the diminution of absorption at 550 nm
that is observed is a direct measure of the loss of substrate (ferrous cytochrome c)
concentration.
In some cases the substrate and product of an enzymatic reaction do not provide
a distinct signal for convenient measurement of their concentrations. Often, however,
product generation can be coupled to another, nonenzymatic, reaction that does
produce a convenient signal; such a strategy is referred to as an indirect assay.
Dihydroorotate dehydrogenase (DHODase) provides an example of the use of an
indirect assays. This enzyme catalyzes the conversion of dihydroorotate to orotic acid
in the presence of the exogenous cofactor ubiquinone. During enzyme turnover,
19
electrons generated by the conversion of dihydroorotate to orotic acid are transferred
by the enzyme to a ubiquinone cofactor to form ubiquinol. It is difficult to measure
this reaction directly, but the reduction of ubiquinone can be coupled to other
nonenzymatic redox reactions.
A third way of following the course of an enzyme-catalyzed reaction is referred
to as the coupled assays method. Here the enzymatic reaction of interest is paired
with a second enzymatic reaction, which can be conveniently measured. In a typical
coupled assay, the product of the enzyme reaction of interest is the substrate for the
enzyme reaction to which it is coupled forconvenient measurement. An example of
this strategy is the measurement of activity for hexokinase, the enzyme that catalyzes
the formation of glucose 6-phosphate and ADP from glucose and ATP. None of these
products or substrates provide a particularly convenient means of measuring
enzymatic activity. However, the product glucose 6-phosphate is the substrate for the
enzyme glucose 6-phosphate dehydrogenase, which, in the presence of NADP+,
converts this molecule to 6-phosphogluconolactone. In the course of the second
enzymatic reaction, NADP+ is reduced to NADPH, and this cofactor reduction can be
monitored easily by light absorption at 340 nm.
Experiment 1. Quantitative determination of amylase activity in urine
according to Wohlgemuth.
Principle. Wohlgemuth’s method is based on the ability of amylase to
breakdown (hydrolyze) the starch. A minimal quantity of an enzyme, which is able to
hydrolyze 1 ml of 0.1% starch solution at 45 °0 during 15 min, is considered as an
enzymatic unit of amylase activity
Method:
Take 9 clean tubes and do the following procedures:
1. Add 1 ml of saline into each of 9 enumerated tubes.
2. Add 1 ml of urine to the first tube, mix it and transfer 1 ml of the mixture from the
first tube to the second tube. With the same manner, transfer 1 ml of the mixture
after mixing from the second to the third tube and so on. Then remove 1 ml of
mixture from tube 9.
3. Add 1 more ml of saline into each tube.
4. Add 2 ml of 0.1 % of starch solution.
5. Mix substantially the content of tubes and place tubes are into incubator (45°C for
15 min).
6. Cool tubes by tap water.
7. Add 2 drops of Lugol solution (iodine in potassium iodide solution) to each tube.
8. Registered a colour of solution in each tube. Results write in a table and calculate
the activity of amylase.
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
iodine
Starch cleavage
20
1
2
3
4
5
6
7
8
9
- 1 ml of saline
- 1 ml of urine
- 1 ml of mixture from the
previous tube
Add 1 ml of saline into each tube and add 2 ml of 0.1 % of starch solution
1
2
3
4
5
6
7
8
9
Blue
Blue
- 2 ml of 0.1 % of starch
solution
Incubation for 15 min (T=450C)
+ I2
Results:
Yelow
Yelow
Yelow
Yelow
Blue
Blue
Blue
Example. 1/16 ml of urine cleaved 2 ml of 0.1 % starch solution (yellow color in
tubes NN 1-4). In the end subsequent tubes starch was not cleaved (blue colour).
Amylase activity:
X = 1×2/ 1/16 = 2×16 / 1 = 32 (un.), where
16 – urine dilution, 2 – quantity of starch added.
Explain the results and draw a conclusion.
Clinical and diagnostic 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). An increase of amylase activity in urine to more than 256 units
indicates is called amylasuria. It may be a sign of: acute inflammation of the
pancreatic gland (acute pancreatitis), alcohol consumption, cancer of the pancreas,
ovaries, or lungs, cholecystitis, Gallbladder disease, intestinal obstruction, pancreatic
duct obstruction, pelvic inflammatory disease. Decreased amylase levels may be due
to: damage to the pancreas, kidney disease, pancreatic cancer, toxemia of pregnancy
Experiment 2. Quantitative determination of pancreatic proteinases activity.
Principle. The following method is based on the ability of proteinases (proteases,
peptidases) to hydrolyze peptide bonds of proteins. Thus free amino acids become
released from proteins. After amino groups of amino acids can be blocked by
formaldehyde, then carboxyl groups can be measured by Zorensen’s method of
titration with NaOH in presence of phenolphthalein.
Method.
21
1. Put into each of two flasks 5 ml of 6% casein solution and 2 ml phosphate buffer,
pH 7.6.
2. Add into the first flask a suspension of 0.5 g of pancreatin in small volume of
water and mix well.
3. Place both flasks (the first one is the test; the second the control) in a thermostat at
37 C for 30 min.
4. Add into both flasks 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.
5. 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.
6. Titrated both flasks with 0.02 n NaOH solution till faint pink colour (pH 8.3) and
add additionally several drops of 0.02 n NaOH to attain a bright red colour (pH
9.1).
Test
Control
- 5 ml of 6% casein solution
- 2 ml phosphate buffer, pH 7.6
- 0.5 g of pancreatin
Incubation for 30 min, 37oC
Test
Control
- 2-3 drops of phenolphthalein
Titration by 0.2N NaOH
to appearance of faint pink colour
Test
pH - 8.3
Control
- 5 ml of formalin mixture
Titration by 0.2N NaOH
to appearance of pink colour
+ several drops of 0.02N NaOH to attain a bright red colour
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 diagnostic significance. Proteinases and peptidases are hydrolytic
enzymes, decomposing proteins and peptides to amino acids. In living organism these
22
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 α-1 antitrypsin, α-1
antichymotrypsin, antiplasmin, and α-2 macroglobulin. Proteolytic activity can be
detected in blood in some diseases, when the quantity of proteinases exceeds the
binding capacity of antiproteolytic system. This situation can take place during acute
pancreatitis, gastric ulcers, heavy combustions.
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
which is transformed to angiotensin II and increases blood pressure.
Limited proteolysis is a process of a great importance for regulation of vital
processes in the cell.
Examples of Krock-1 tests
1. Michaelis-Menten constants of two enzymes are 1,3x10-5 M/l and 2,3x10-3 M/l
subsequently. Indicate true statement about the affinity of these enzymes to
substrate.
A. The second enzyme has higher
to substrate
affinity to substrate
D. For decision an information on
B. Enzymes possess equal affinity to
concentration of enzyme is needed
substrate
E. Data are incomplete and it is
C. The first enzyme has higher affinity
impossible to draw a conclusion
2. In an enzyme assay the substrate concentration was taken much higher than
Km. In this conditions the rate of the reaction will be as follows:
A. Shows zero-order kinetics
D. Is
independent
of
enzyme
B. Approaches 50 % value of Vmax
concentration
C. Is
proportional
to
substrate
E. Is independent of temperature
concentration
3. Activity of many enzymes depends from the presence of free thiol groups in
active center. What amino acid residue provides presence of these groups in
enzyme molecule?
A. Cysteine
D. Methionine
B. Lysine
E. Serine
C. Tryptophan
4. Michaelis-Menten constant (Km) reflects the next property of enzyme:
A. Affinity to substrate
D. Affinity to a product of reaction
B. Sensitivity to pH of medium
E. Sensitivity to competitive inhibitors
C. Thermolability
23
1.
2.
3.
4.
5.
6.
7.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M. – 2012. – 308 p.
Topic № 4. Regulation of enzymatic processes enzymopathias and mechanisms of
their development. Application of enzymes as pharmaceutical preparations
Objective: 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.
Actuality of the theme: Enzymes are biocatalysts with changeable activity,
submitted to regulatory influences. Estimation of enzymatic activity is routinely used in
laboratory investigations with diagnostic purposes. Enzymes are also employed as
medicines and drugs in practical medicine.
Specific aims:
 To analyze pathways and mechanisms of regulation of enzymatic reactions as a
background of metabolism in health and disease.
 To explain the application of activators and inhibitors of enzymes as medicines and
pharmaceuticals for correction of metabolic disorders in pathology.
 To explain changes in metabolic pathways and accumulation of distinct metabolic
intermediates in the inborn (hereditary) and acquired disorders of metabolism –
enzymopathias.
 To analyze changes in activity of indicatory enzymes in blood plasma in pathology of
distinct organs and tissues.
Theoretical questions:
1. Enzyme inhibition (reversible, irreversible, competitive, non-competitive).
2. Regulation of enzyme activity in the living system:
 allosteric regulation;
 feedback regulation;
 covalent modification of enzymes;
 activation of latent enzymes by limited proteolysis;
 cyclic nucleotides in regulation of enzymatic processes.
3.
Control of enzymes synthesis (constitutive and adaptive enzymes).
4. Application of enzymes:
24
 enzymes as therapeutic agents;
 enzymes as analytic agents;
 immobilized enzymes.
5. Diagnostical importance of enzymes (plasma specific and non-plasma specific
enzymes.
6. Changes in enzymatic activity of blood plasma and serum as diagnostic indexes
(markers) of pathological processes in distinct organs – myocardial infarction, acute
pancreatitis, liver disease, pathology of muscle tissue.
7. Isoenzymes, their role in enzymodiagnostics.
8. Inborn (hereditary) and acquired metabolic defects, their clinical and laboratory
diagnostics.
9. Application of enzyme inhibitors as medicinal and drugs – acetylsalicylic acid,
allopurinol, sulfonamides.
Practical part
Experiment 1. Study 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 metabolic intermediates. 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.
I. Dilution of saliva. Dilute saliva two fold.
II. Preparation of experimental mixtures and incubation. Take 3 clean dry tubes
and do the following procedures:
1. Add 1 ml of distilled water into the first tube.
2. Add 0,8 ml of distilled water and 0,2 ml of 1 % NaCl into the second tube.
3. Add 0,8 ml of distilled water and 0,2 ml of 1% solution of CuSO4 into the third tube.
4. Add 1 ml of diluted saliva into each tube
5. Add 2 ml of 1% starch solution into each tube. Mix them well and place tubes to
thermostat at 37C during 15 min.
III. Color reaction. Add 0,1% solution of iodine in 0,2% sodium iodide). Changes in
color development are observed and registered in a table (see below).
№ of tube
Tube content
Water (ml)
NaCl, 1 %-solution (ml)
CuSO4, 5 %-solution (ml)
Saliva 2 fold diluted (ml)
Starch, 1 %-solution (ml)
1
2
3
1
1
2
0,8
0,2
1
2
0,8
0,2
1
2
25
Final color after addition of
iodine
Explain the results and draw a conclusion.
Clinical and diagnostic 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. The influence of phosphacol and calcium ions on activity of
cholinesterase.
Principle. Organic phosphate compounds are irreversible inhibitors of cholinesterase
and acetyl-cholinesterase due to irreversible binding with an active site of enzyme and
suppress its activity. Preparations of phosphorganics are strong poisons 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 acetylcholine receptors of postsynaptic membrane and hydrolytic cleavage
by cholinesterase. Besides, calcium ions are strong acticvators of cholinesterase.
Method of quantitative determination of cholinesterase is based on the titration with
alkali the acetic acid, which is liberated after acetylcholine hydrolysis. The quantity of
alkaline solution, expended for titration, is a measure of enzyme activity.
Method. Take three clean dry tubes and add reagents according to the table:
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 (ml)
1,5
1,5
1,5
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 diagnostic 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).
26
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 some parts of CNS, as well as in body in
general. Reversible inhibitors of acetylcholine esterase are employed in medicine for
enhancement of cholinergic impulsation, which is alterede in some neurological diseases,
e.g. atonia of intestines or bladder. In these cases are used preparations proserine,
physostigmine, galantamine.
Irreversible inhibitors of acetylcholinesterase are potent neurotoxins, which causes a
marked excitation of nerve system manifested as convultions, disorders of cardiovascular,
digestive and other systems of the organism. The most wide spreded irreversible inhibitors
are organic phosphates. They are employed for killing of harmfull insects (dichlophos,
chlorophos, metaphos, etc.). Neuroparalytic toxins which are used as warfare, are zarine,
soman, tabun and others.
High sensitivity of cholinesterase to this phosphorganic compounds permits to use
this enzyme as a marker for detection of traces of phosphorganic compounds in human
body.
Examples of Krock-1 tests
1. Marked increase of activity of МВ-forms of CPK (creatinephosphokinase) and
LDH-1 were revealed on the examination of the patient's blood. What is the most
likely pathology?
A. Miocardial infarction
D. Pancreatitis
B. Hepatitis
E. Cholecystitis
C. Rheumatism
2. 12 hours after an accute attack of retrosternal pain a patient presented an increase
of aspartate aminotransferase activity in blood serum. What pathology is this
deviation typical for?
A. Myocardium infarction
D. Diabetes mellitus
B. Viral hepatitis
E. Diabetes insipidus
C. Collagenosis
3. 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
D. Propionic aldehyde
B. Ethanol
E. Methanol
C. Malonyl aldehyde
4. In course of tuberculosis treatement a patient was administered isoniazide – a
structural analogue of nicotinamide and pyridoxine. What type of inhibition by
mechanism of action exhibits isoniazide?
A. Competitive
D. Allosteric
B. Irreversible
E. Noncompetitive
C. Uncompetitive
27
Individual independent students work
1. Regulators of enzymatic activity and their employment in clinical practice.
2. Enzymodiagnostics. The use of enzymes in enzymodiagnostics.
References:
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
7. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M.. – 2012. – 308 p.
Topic № 5. The role of cofactors, vitamins and their coenzyme forms in enzyme
catalysis.
Objectives: To learn the structure, principles of classification and funtion of
coenzymatic vitamins. To learn the methods of qualitative and quantitative determination
of vitamins.
Actuality of the theme: Water soluble vitamin take part in metabolism as coenzymes
and activators for many enzymatic reactions.
Deficiency in vitamin supply of the body or disorders of their metabolism which is
caused by alteration of their absorption or 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 accommodate effectively to unfavorable
environmental conditions.
Specific aims:
 To interprete the role of vitamins and their biologically active derivatives in mechanism
of catalysis by enzymes of different classes.
 To explain the application of antivitamins as inhibitors of enzymes in contagious
diseases and in disorders of homeostasis.
 To explain the role of metals in mechanisms of enzymatic catalysis.
 To classify distinct groups of coenzymes according to their chemical nature and type of
the reaction, which they catalyze.
Theoretical questions:
1. Cofactors, coenzymes and prosthetic groups of enzymes.
2. Role of metal ions in function of enzymes
3. Classification of coenzymes due to their chemical nature and type of catalytic reaction.
28
4. Coenzymes as transporters of hydrogen atoms and electrons (examples of distinct
reactions):
 NАD+, NАDP+ coenzymes – derivatives of vitamin РР;
 FАD, FМN coenzymes – derivatives of vitamin В2 – riboflavin;
 Role of vitamin C in oxidative-reductive reactions
 metalloporphyrins
5. Coenzymes as transporters of chemical groups (examples):
 pyridoxal phosphate;
 НS-CоА – coenzyme of acylation;
 lipoic acid;
 THF – derivatives of folic acid
6. Coenzymes of isomerisation, synthesis and cleavage of C-C bonds (examples) :
 thiamine pyrophosphate – coenzyme form of vitamin В1;
 biocytin – coenzyme form of vitamin Н – biotin;
 methylcobalamin and deoxyadenosylcobalamin – coenzyme forms of vitamin В12
Practical part
In complex enzymes a protein component (apoenzyme) can be found out with the
biuretic reaction. An unprotein component, containing a derivative of any vitamin, can be
opened with the appropriate qualitative reactions for each vitamin.
Experiment 1. A method for the detecton of nicotinic acid.
Principle. Heating of a mixture of nicotinic acid and copper acetate gives a blue
sediment of copper nicotinate.
Method. Dissolve 10 mg of nicotinic acid in 10-20 drops of 10% acetic acid. Heat
the tube to boiling and then add an equal volume of 5% solution of copper acetate. The
liquid turns blue and after standing blue sediment appears.
Explain the results and draw a conclusion.
Experiment 2. Probe for pyridoxine with FeCl3..
Principle. In the presence of pyridoxine FeCl3 solution turns red.
Method. Add to 5 droplets of pyridoxine solution one drop of 5% solution of FeCl 3.
The liquid in tube becomes red due to formation of complex, a compound of iron of the
phenolate type.
Explain the results and draw a conclusion.
Experiment 3. Reduction of K3Fe(CN)6 by ascorbic acid.
Principle. Ascorbic acid reduces K3Fe(CN)6 to K4Fe(CN)6. The latter reacts with
FeCl3 to produce a bright blue pigment – Berliner blue.
Method.
1. Add into two tubes one drop of 5% solution of K3Fe(CN)6 and one drop 1% solution of
FeCl3.
2. Add to the first tube 5-10 drops of an extract from canine rose fruits. Color changes to
blue (sometimes a blue sediment of Berliner blue pigment appears).
3. Add into the second 5-10 drops of distilled water. No changes occur in the second tube.
Explain the results and draw a conclusion.
29
Examples of Krock-1 tests
1. In case of enterobiasis acrihine – the structural analogue of vitamin B2 - is
administered. The synthesis disorder of which enzymes does this medicine cause in
microorganisms?
A.FAD-dependent dehydrogenases
D. NAD-dependet dehydrogenases
B. Cytochromeoxidases
E. Aminotransferases
C. Peptidases
2. Hydroxylation of endogenous substrates and xenobiotics requires a donor of
protons. Which of the following vitamins can play this role?
A. Vitamin C
D. Vitamin E
B. Vitamin P
E. Vitamin A
C. Vitamin B6
3. A newborn child has convulsions that have been observed after prescription of
vitamin B6. This most probable cause of this effect is that vitamin B6 is a component
of the following enzyme:
A. Glutamate decarboxylase
D. Aminolevulinate synthase
B. Pyruvate dehydrostase
E. Glycogen phosphorylase
C. Netoglubarate dehydromine
1.
2.
3.
4.
5.
6.
7.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M. – 2012. – 308 p.
Topic № 6. Metabolic pathways and bioenergetics. Tricarboxylic acid cycle and
its regulation.
Objectives: 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 evaluation of its role for providement of the cell with
energy as well as for understanding of its amphibolic significance. The analysis of
30
TCA cycle function is necessary for estimation of its role in turnover of matter and
energy in the cell.
Specific aims:
 To interprete biochemical principles of metabolic pathways: catabolic, anabolic,
amphibolic pathways.
 To explain biochemical mechanisms of regulation of catabolic and anabolic
reactions.
 To interprete 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 matter and energy.
1.
2.
3.
4.
5.
6.
7.
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, role of ATP and other
macroergic phosphate containing compounds in their coupling.
Intracellular location of metabolic pathways, compartmentalization of metabolic
reactions in the cell. Methods of investigation of metabolism.
Catabolic transformation of biomolecules: proteins, carbohydrates, lipids, its
characterization.
The most important metabolites of amphibolic pathways in turnover of proteins,
carbohydrates, lipids, their significance for integration of metabolism in the cell.
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.
Anaplerotic and amphibolic reactions of TCA cycle.
Practical part
Experiment 1. Isolation of mitochondria from liver by method of differential
centrifugation (a demonstration).
Principle. Liver cells contain a great amount of specific subcellular organelles –
mitochondria, in which reactions of tricarboxylic acid (TCA) cycle take place as well
as processes of tissue respiration and oxidative phosphorylation.
Enzymes of TCA cycle are localized in mitochondrial matrix, while components
of respiratory chain – on inner mitochondrial membrane.
Mitochondria are isolated from the tissue after its breakdown (homogenization)
in special devices, called homogenizer. There exists several types of homogenizers,
the most frequently is used glass homogenizer with a teflon pestle. Tissue is
homogenized in 0.25 M sucrose solution. Thereafter homogenate is submitted to
31
centrifugation at different speeds (revolutions per minute - rpm). At low speeds are
sedimented nuclei and cell debris, which are discarded, mitochondria are sedimented
at higher speeds (about 7-10 thousands rpm).
Method. Liver of experimental animal (albino rat) is washed from the blood,
minced with a scissors and homogenized in 10 fold volume of cold solution N1 (0,25
M sucrose in 0,01 M solution of EDTA, pH 7,6). All subsequent procedures are
conducted at 1-20 C. Homogenate is centrifuged at 700 rpm during 6 min in order to
eliminate nuclei and large cell debris. Supernatant is collected, transferred to another
tube and submitted to centrifugation 10 min at 7000 rpm. The sediment of
mitochondria is washed with solution N1 by suspending it in original volume of the
solution and recentrifugation 10 min at 7000 prm. The obtained sediment is
suspended in a small volume of solution N2 (0,25 M sucrose in 0,02 M tris buffer, pH
7,5), protein concentration is determined and obtained suspension is used for
investigation of TCA cycle reactions, tissue respiration, oxidative phosphorylation
etc.
Experiment 2. 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. Fill three tubes – a control one, experimental 1 and 2 with reagents as
indicated in the table:
Content of tubes
Control
Tube 1
2,0
0,5
Tubes
Exp. 1
Tube 2
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 a thermostate for 15 min at 37oC
0,1 M solution of sulphuric acid
1,0
1,0
Result: production of CO2 bubbles
Exp. 2
Tube 3
2,0
0,5
0,5
0,5
0,5
1,0
32
Place tubes in a thermostat for 15 min at 37 oC. Thereafter add 1,0 ml of 0,1 M
solution of sulphuric acid into each tube and observe the appearance of CO2 bubbles.
Explain the results and draw a conclusion.
Experiment 3. 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.
Method. Fill three tubes – a control one, experimental 1 and 2 with reagents as
indicated in the table:
Reagents added
Tubes
Control
Exp.N1
Exp.N2
Tube 1
Tube 1
Tube 1
Phosphate buffer, pH 7,4, ml
2,0
2,0
2,0
Solution of sodium pyruvate, ml
0,5
0,5
0,5
Solution of malonate, ml
0,5
Saline
0,5
0,5
Suspension of mitochondria, ml
0,5
0,5
Suspension of boiled mitochondria, ml
0,5
Solution of methylene blue, ml
0,5
0,5
0,5
o
Incubation at 37 C in a thermostate
Results: time of medium decoloration
.
Explain the results and draw a conclusion.
Clinical and diagnostic 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 Krock-1 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
D. Glycogen mobilization
B. Glycolysis
E. Urea synthesis
C. Cholesterol biosynthesis
33
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
D. Fumarase
B. Citrate synthase
E. Alpha-ketoglutarate dehydrogenase
C. Succinate dehydrogenase
complex
3. In a patient are manifested symptoms of intoxication with arsenic compounds.
What metabolic process is damaged taking into account that arsen containing
substances inactivate lipoic acid?
A. Oxidative decarboxylation of αD. Coupling of oxidation and
ketoglutarate
phopsphorylation
B. Fatty acids biosynthesis
E. Microsomal oxidation
C. Neutralization of superoxide anions
4. Mitochondria are subcellular organelles and are present in a cytoplasm of
every cell exept mature red blood cells, bacteria, blue-green algae. What
method is used principally for their isolation?
A. Differential centrifugation
D. Spectrophotometry
B. Chromatography
E. Gel-filtration
C. Electrophoresis
Individual independent students work
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.
5.
6.
7.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M. – 2012. – 308 p.
34
Topic № 7. Biological oxidation and oxidative phopshorylation. Mechanisms of
ATP synthesis.
Objective: to learn general principles of enzymatic respiratory chain
organization in mitochondria, distinct oxidoreductases and their functional
significance in tissue respiration. To master the methods of investigation of the next
oxido-reductases: phenol oxidase, aldehyde dehydrogenase and peroxidase.
Actuality of the theme: oxidoreductases 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.
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.
 Malate-aspartate and glycerophosphate shuttle systems of transmembrane
transfer of reduced NADH2 into mitochondria and their significance
 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.
Theoretical questions:
1. Biological oxidation of substrates in cells.
2. Reactions of biological oxidation and their functional significance.
3. Pyridine dependent dehydrogenases, structure of NAD and NADP, their role in
reactions of oxidation and reduction.
4. Flavine dependent dehydrogenases. Structure of FAD and FMN, their role in
reactions of oxidation and reduction.
5. Cytochromes and their role in tissue respiration. Structure of their prosthetic
group.
6. Molecular organization of electron transport chain of mitochondria.
7. Supramolecular complexes of respiratory chain in inner membrane of
mitochondria.
8. Oxidative phosphorylation. Sites of oxidative phosphorylation. P/O ratio.
9. The scheme of chemiosmotic mechanism of coupling of electron transport in
respiratory chain with ATP synthesis. Molecular structure and principles of
functioning of ATP-synthetase.
10.Inhibitors of electron transport in a respiratory chain of mitochondria.
11.Uncouplers of electron transport and oxidative phosphorylation in a respiratory
chain of mitochondria.
Practical part
Oxidoreductases
35
Oxidoreductases are enzymes that catalyze the transfer of electrons from one
molecule, the reductant, also called the electron donor, to another the oxidant, also
called the electron acceptor. Phenol oxidase, aldehyde dehydrogenase, peroxidase
and many others belong to oxidoreductases.
Polyphenol oxidase is an enzyme that catalyses the hydroxylation of
monophenols to o-diphenols. They can also further catalyse the oxidation of odiphenols to produce o-quinones. It is the rapid polymerisation of o-quinones to
produce black, brown or red pigments (polyphenols) that is the cause of fruit
browning.
O
OH
+ Ѕ О2
Phenol oxidase
OH
Hydroquinon (o-diphenol)
+ H2O
O
Quinon
Aldehyde dehydrogenases are a group of enzymes that catalyse the oxidation
(dehydrogenation) of aldehydes. To date, nineteen ALDH genes have been identified
within the human genome. These genes participate in a wide variety of biological
processes including the detoxification of exogenously and endogenously generated
aldehydes.
Peroxidases are a large family of enzymes that typically catalyze a reaction of
the form:
ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH
For many of these enzymes the optimal substrate is hydrogen peroxide, but
others are more active with organic hydroperoxides such as lipid peroxides
Experiment 1. Study of phenol oxidase activity.
Principle. Method is based on the phenomenon of pyrocatechol oxidation by
oxygen in the presence of phenol oxidase.
Method.
I. The source of enzyme is fresh potato juice. Mince in a mortar 1 - 2
g of potato add 25 ml of distilled water, mix it and press the juice
into tube. (Iron instruments must not be used because ions of iron
induce the darkening of juice).
II. Add 0.5 ml of potato juice into 4 tubes. Add 0.5 ml of pyrocatechol solution to the
first tube; 1-2 drops of Na2S (an inhibitor of phenol oxidase) and 0,5 ml of
pyrocatechol – to the second; boiled juice and 0.5 ml of pyrocatechol - to the third
and 0.5 ml of water to fourth tube. Place tubes into the thermostat at 37o C for 30
min. Periodically shake the tube’s content for better aeration.
36
2
1
3
- 0.5 ml of potato juice
4
- 0.5 ml of boiled potato juice
- 0.5 ml of pyrocatechol solution
- 1-2 drops of Na2S
- 0.5 ml of water
Incubation for 30 min (T=370C)
Result:
1. brown 2. colorless 3. colorless 4. colorless
III. Result. In the first tube brown colour appears due to oxidation of pyrocatechol.
The colours of other tubes don’t 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 decolouration of methylene blue during its
reduction by aldehyde in presence of an enzyme aldehyde dehydrogenase (ADH).
О
ОН
ADH
+ Н2О
Н С
НС ОН
Н
ОН
Formaldehyde
НС
Hydrated form of formaldehyde
ОН
ADH
ОН + Мb
ОН Methylene blue
Н
С
О
ОН
Formiatic acid
Method. Add the following solutions into three ennumerated tubes:
(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.
Place tubes to the termostate for 30 min to at 37 °C.
1
2
3
- 1 ml of fresh non boiled milk
- 1 ml of boiled milk
- 1-2 drops of neutralized formaldehyde
- 2 drops of methylene blue solution
Incubation for 30 min (T=370C)
Result:
1. colorless
2. blue
3. blue
37
Result. Content of the first tube is decolorized due to enzyme activity. Color in
the rest of the tubes does not change, as in the second tube – substrate is absent, in the
third – enzyme is heat denaturated.
Explain the results and draw a conclusion.
Clinical and diagnostical importance. Aldehyde dehydrogenase plays a crucial
role in maintaining low blood levels of acetaldehyde during alcohol oxidation. In this
pathway, the intermediate structures can be toxic, and health problems arise when
those intermediates cannot be cleared. When high levels of acetaldehyde occur in the
blood, facial flushing, light headedness, palpitations, nausea, and general “hangover”
symptoms occur.
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. Add the following reagents into 3 tubes:
(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.
Place
Result. In the first tube a blue color is developed due to oxidation of benzidine
by H2O2 under catalysis of peroxidase. In the 2 and 3 tubes color is not developed.
1
2
3
- 3-4 drops of horseradish extract
- 3 - 4 drops of a boiled horseradish extract
- 3-4 drops of 3 % solution of H2O2
- 3-4 drops of benzidine solution in acetic acid
Incubation for 30 min (T=370C)
1. blue
- 3-4 drops of peroxidase inhibitor
Result:
2. colorless 3. colorless
Explain the results and draw a conclusion.
Clinical and diagnostical importance. Horseradish peroxidase can be
conjugated to a labeled molecule. It produces a coloured, fluorimetric, or luminescent
derivative of the labeled molecule when incubated with a proper substrate, allowing it
to be detected and quantified. Horseradish peroxidase is often used in conjugates
(molecules that have been joined genetically or chemically) to determine the presence
of a molecular target. For example, an antibody conjugated to horseradish peroxidase
may be used to detect a small amount of a specific protein in a western blot. Here, the
antibody provides the specificity to locate the protein of interest, and the horseradish
peroxidase enzyme, in the presence of a substrate, produces a detectable signal.
Horseradish peroxidase is also commonly used in techniques such as ELISA and
38
Immunohistochemistry due to its monomeric nature and the ease with which it
produces coloured products. Peroxidase, a heme-containing oxidoreductase, is a
commercially important enzyme which catalyses the reductive cleavage of hydrogen
peroxide by an electron donor.
Examples of Krock-1 tests
1. 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.
D. On outer mitochondrial membrane
B. On cytoplasmic membrane
E. In nucleus
C. In cytoplasm
2. Cytochromes are components of respiratory chain in mitochondrias, which
transfer electrons from ubiquinon to molecular oxygen. What part of
cytochrome molecule take part in oxydative-reductive reactions?
A. Iron atom
D. Proteinous part
B. Vinyl residue
E. Methen bridge
C. Pyrrole cycle
3. The next process occurs in suspension of mitochondria with ruptured inner
membrane and provided with malate and oxygen:
A. Transport of electrons along
D. Increase of pH in mitochondrial
enzymes of respiratory chain
matrix
B. Phosphorylation of ADP
E. Oxydative phosphorylation will
C. Decrease of pH in the external
take place
medium
4. Redox potential (EO volts) of ubiquinone, OX/RED system is:
A. +0.04
D. +0.29
B. +0.08
E. + 0.35
C. +0.10
5. The correct sequence of cytochrome carriers in respiratory chain is:
A. Cyt b→cyt c1→cyt c→cyt aa3
D. Cyt b→cyt aa3→cyt c→ cyt c
B. Cyt aa3→ cyt b→cyt c→cyt c1
E. Cyt aa3→ cyt c1→cyt c→ cyt b
C. Cyt b→cyt c→cyt c1→cyt aa3
Individual independent students work
1. Development of knowledge on biological oxidation, works of A. Bakh, V.
Palladin, O.Warburg in this field.
2. Modern data about the organization and functioning of respiratory chain as a single
polyenzyme complex.
References:
39
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
2.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
3.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
4.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa.
The Odessa State Medical University, 2003.-416p.
6.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
7.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in
Biochemistry. 2nd edition” – 2008. – 488 p.
8.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M.. – 2012. – 308 p.
Topic № 8. Glycolisis – anaerobic 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: The occurrence of uninterrupted glycolysis is very
essential in skeletal muscle during strenuous exercise where oxygen supply is very
limited. Glycolysis in the erythrocytes leads to lactate production, since mitochondria
- the centres for aerobic oxidation - are absent. Brain, retina, skin, renal medulla and
gastrointestinal tract derive most of their energy from glycolysis. Concentration of
lactate in blood increases after hard muscle exercises and in some diseases. Under
anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6
ATP are synthesized-depending on the shuttle pathway that operates.
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 conditions
Theoretical questions:
1. Glucose as an important metabolite in carbohydrate metabolism: general scheme of
sources and turnover of glucose in the organism
2. Anaerobic oxidation of glucose:
 the sequence of reactions in glycolysis;
 enzymatic reactions of anaerobic and aerobic glycolysis;
40
 characterization of glycolytic reactions, which occur with utilization of energy;
 characterization of enzymatic reactions of substrate phosphorylation in
glycolysis;
 mechanism of glycolytic oxido-reduction and reactions, which provide this
process.
3. The contribution of works of Embden, Meyerhof and Parnas in detection of
sequence of enzymatic glycolysis reactions.
4. The role of lactate dehydrogenase (LDH) in glycolysis, mechanism of reaction and
its peculiarities. Isoenzymes of LDH and their clinical diagnostic significance.
6. Energetic effect of anaerobic oxidation of glucose.
7. Alcohol fermentation, common and different reactions in glycolysis and
fermentation.
Practical part
Experiment 1. The utilization of inorganic phosphate by yeast cells in fermentation
process.
A. The preparation of fermentation mixture.
1. Put 2 g of dry glucose into a test tube; afterwards add 5 ml of phosphate buffer
(pH 7.2);
2. Incubate the solution at 37° C.
3. Add 4 ml of the yeast suspension and mix the solution.
B. The investigation of phosphate binding rate in probes. From the fermentation
mixture take 1 ml probes and allow 0, 30, 60 and 90 min for incubation.
a) Precipitation of yeast cells. Add to 1 ml of mixture 3 ml of 0.1 n acetic acid. In a
weak acidic medium yeast cells form a sediment, which is eliminated by filtration.
b) Dilution of phosphates.
1. Add to 1 ml of the filtrate 19 ml of water.
2. From this solution take 1 ml and mix with 4 ml of water; repeat (a) and (b) 3
times after every 30 min.
c) Determination of phosphate concentration by colorimetry.
1. To the 5ml of the above obtained solution add 4.5 ml of molybdate reagent and
subsequently 0.5 ml of ascorbic acid (0.5%).
2. Mix the content of tubes and after 2 min (exactly)
3. Measure the optical density (A) in a colorimeter at red light. The concentration
of phosphate is determined according to calibration curve, which is obtained as
indicated below. Explain the results and draw a conclusion.
The calibration curve for phosphate: In 5 tubes are mixed the following reagents
(see the table):
№
Reagents
1
2
3
4
1.
2.
3.
4.
Standard solution of
phosphate
Distilled water
Molybdate reagent
Ascorbic acid solution
0
0.5
1.0
2.0
5
4.5
0.
4.5
4.5
0.5
4.0
4.5
0.5
3.0
4.5
0.5
41
5.
Phosphate concentration
0
50
100,0 200,0
(mg%)
6.
Optical density
0.01 0.06
0.11
0.21
According to optical density values a calibration curve is constructed, where the
concentration of phosphate is on the abscissa and corresponding optical density
values is on ordinate axis.
Using a calibration curve the concentration of phosphate in the examined probes
is determined.
From data obtained a graph (the dependence of inorganic phosphate utilization
in the course of fermentation) is constructed.
Calibration curve (example)
Graph of concentration of inorganic
phosphate from the incubation time
(example)
Experiment 2. Quantitative determination of lactic acid in blood serum after
Buchner method.
The occurrence of uninterrupted glycolysis is very essential in skeletal muscle
during strenuous exercise where oxygen supply is very limited. Glycolysis in the
erythrocytes leads to
lactate
production,
since mitochondria-the
centres for aerobic
oxidation-are absent.
Brain, retina, skin,
renal medulla and
gastrointestinatl
ract
derive most of their energy from glycolysis.
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.
Preparation of a filtrate.
1. Pour into 2 clean tubes 6 ml of water.
2. Add to the first tube 1 ml of standard solution of lactate (test tube) and to the
42
second one add l ml of blood serum (control tube).
3. For protein precipitation add to each tube l ml of metaphosphoric acid and
after several minutes filter the solutions.
4. Add to each filtrate l ml of copper sulfate (10%) and 0.5g of calcium
hydroxide. Mix the probes with a glass rod and filter after 5 min.
Method.
1. Add 1 ml of each filtrate into 2 clean, dry tubes,
2. Add 0.1 ml of copper sulfate, mix the solutions
3. Add 4 ml of concentrated sulfuric acid to each tube and place them into a
boiling water bath for 1.5 min.
4. After cooling add 0.1 ml of alcohol solution of hydroquinone and incubate
the tubes in boiling water bath for 15 min.
5. Cool the tubes and measure optical density at blue light filter.
Calculations. Lactic acid concentration is
calculated according to a formula:
Where, C is concentration and A is optical density. Explain the results and draw a
conclusion.
Clinical diagnostic significance. Venous blood of healthy person contains 0.5 2.2 mmol/1 of lactic acid. An increase of lactic acid content is associated with
strenuous muscular effort during short time term intervals when there is a deficiency
of oxygen. In this case the oxidative decarboxylation of pyruvate to acetyl-CoA 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 during epilepsy, tetanies, convulsions and hypoxia, and it
43
is associated with cardiac and pulmonary failure, malignant tumors, liver diseases and
other pathologies.
Examples of Krock-1 tests
1. An untrained person who has not been practicing physical exercises for a long
time complains of a muscle pain as a result of intensive manual work. What is
the probable reason of the pain syndrome?
A. Accumulation of lactate in muscles
D. Accumulation of creatinine in
B. Decreasing of lipids level in
muscles
muscles
E. Increase of ATP level in muscles
C. Increased disintegration of muscle
proteins
2. 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
D. Lipogenesis
B. Gluconeogenesis
E. Glycogenesis
C. Pentose phosphate pathway
3. 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
D. Oxidative phosphrylation
B. Deamination of amino acids
E. Breaking
up
of
peroxides
C. Tissue respiration
4. During consumption of cakes or sweets in mixed saliva a transient increase in
lactate level takes place. Activation of what biochemical process causes this
effect?
A. Anaerobic glycolysis
D. Gluconeogenesis
B. Tissue respiration
E. Microsomal oxidation
C. Aerobic glycilysis
5. In yeast cells occurs a process which is similar to glycolysis- an alcohol
fermentation. In course of this process through several stages from pyruvate is
produced:
A. Lactate
D. Glyceraldehyde
B. Ethanol
E. Pyruvate
C. Acetaldehyde
Individual independent students work
1. Disorders of carbohydrate metabolism and its pharmacological correction.
2. Principles of regulation of glucose metabolism. Characterization of regulatory
enzymes in glycolysis.
44
References:
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792
p.
2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa.
The Odessa State Medical University, 2003.-416p.
6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in
Biochemistry. 2nd edition” – 2008. – 488 p.
8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M.. – 2012. – 308 p.
Topic № 9. Glucose oxidation under aerobic conditions and alternative
metabolic pathways of monosaccharides metabolism.
Objective: To learn the role of pyruvate dehydrogenase multienzyme complex
in aerobic metabolism of glucose. To interpret metabolic pathways of fructose and
galactose in human body. To learn the sequence of enzymatic reactions in Pentose
Phosphate Pathway (PPP).
Actuality of the theme: Metabolism of carbohydrates includes all complex
process of carbohydrates transformation starting from digestion, absorption, transport
and utilization in cells up to formation of end products – CO2 and H2O. In aerobic
conditions pyruvate, as a product of glycolysis, releases CO2 and is transformed to
acetyl-CoA, which is further oxidized in tricarboxylic acid cycle (Crebs cycle) to CO2
and H2O. The rate of reactions in TCA cycle depends from requirements of the cell in
ATP. Regulatory reactions in TCA cycle are synthesis of citrate and oxidative
decarboxylation of alpha-oxoglutarate, which are regulated by amount of ADP,
succinyl-CoA and NADH2.




Specific aims:
To interpret mechanisms of monosaccharides transformation to final metabolic
products and energetic effect in aerobic conditions
To analyze structural and functional peculiarities of pyruvate dehydrogenase
complex
To explain the sequence of reactions in PPP and significance of this process
To analyze metabolic pathways of fructose and galactose transformations in
human body.
45
1.
2.
3.
4.
5.
6.
Theoretical questions:
Stages of aerobic oxidation of glucose.
Oxidative decarboxylation of pyruvic acid.
 structure of multienzyme pyruvate dehydrogenase complex.
 peculiarities of function of pyruvate dehydrogenase complex.
 mechanism of oxidative decarboxylation of pyruvate.
 role of vitamins and coenzymes in transformation of pyruvate to acetyl-CoA.
Energetic effect of aerobic oxidation of glucose.
Pentose phosphate pathway (PPP) of glucose utilization:
 scheme of reactions in oxidative and nonoxidative stages of PPP;
 enzymes and coenzymes of PPP reactions;
 biological significance of PPP;
 disorders of PPP in red blood cells;
 enzymopathias of glucose-6-phosphate dehydrogenase.
Enzymatic reactions of fructose turnover in human body. Hereditary
enzymopathias of fructose metabolism.
Enzymatic reactions of galactose metabolismr in human body. Hereditary
enzymopathias of galactose metabolism.
Practical part
Experiment 1. Quantitative determination of pyruvic acid in urine by
colorimetric method.
Pyruvate is converted to acetyl CoA by oxidative decarboxylafion. This is an
irreversible reaction, catalysed by a multienzyme complex, known as pyruvate
dehydrogenase complex(PDH), which is found only in the mitochondria. High
activities of PDH are found in cardiac muscle and kidney. The enzyme PDH requires
five cofactors (coenzymes), namely-TPP, lipoamide, FAD, coenzyme A and NAD+
(lipoamide contains lipoic acid linked to amino group of lysine).
Principle. Pyruvic acid and 2.4-dinitrophenyl hydrazine form in alkaline
medium a 2.4-dinitrophenylhydrazone pyruvate (brown-red color compound). The
intensity of the color is proportional to the pyruvic acid concentration and is
evaluated calorimetrically.
46
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 (Cexp) is calculated according to the formula:
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 (1500 ml)
a – 0.1 ml of urine, taken for analysis.
47
Compare the obtained result with normal value. Draw the conclusion.
Examples of Krock-1 tests
1. A newborn child with the signs of cataract, growth and mental retardation,
who manifested vomiting and diarrhea, was brought to an emergency clinic. A
presumptive diagnosis of galactosemia was made. The deficiency of what enzyme
occurs in case of this disease?
A. Galactose-1-phosphate uridyl
C. UDP-galactose-4-epimerase.
transferase.
D.Hexokinase.
B. Glucokinase.
E. Glucose-6-phosphate dehydrogenase
2. A cataract and fatty degeneration of the liver develop in the conditions of high
galactose and low glucose level in blood. What disease do these symptoms testify
to?
A. Galactosemia.
D. Steroid diabetes.
B. Diabetes mellitus.
E. Fructosemia.
C. Lactosemia.
3. In a patient are manifested symptoms of intoxication with arsenic compounds.
What metabolic process is damaged taking into account that arsenic containing
substances inactivate lipoic acid?
A. Oxidative decarboxylation of
D. Coupling of oxidation and
pyruvate
phopsphorylation
B. Fatty acids biosynthesis
E. Microsomal oxidation
C. Neutralization of superoxide anions
4. A 2-year-old boy has the increase of liver and spleen sizes detected and eye
cataract present. The total sugar level in blood is increased, but glucose
tolerance is within the normal range. The inherited disturbance of the
metabolism of what substance is the cause of the indicated state?
A. Galactose.
D. Maltose.
B. Fructose.
E. Saccharose.
C. Glucose.
Individual independent students work
1. Pathobiochemistry of disorders in pentosophosphate pathway.
2. Pathobiochemistry of enzymopathias in connection with genetic defects in
synthesis of enzymes of fructose and galactose metabolism.
References:
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
48
4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry.
2nd edition” – 2008. – 488 p.
8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M. – 2012. – 308 p.
Topic № 10. Catabolism and biosynthesis of glycogen. Regulation of glycogen
metabolism Biosynthesis of glucose – gluconeogenesis
Objectives: To learn reactions of synthesis and breakdown of glycogen and
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 the storage form of glucose in animals, as
is starch in plants. It is stored mostly in liver (6-8 %) and muscle (1-2 %). Due to
more muscle mass, the quantity of glycogen in muscle (250 g) is about three times
higher than that in the liver (75 g). Glycogen is stored as granules in the cytosol,
where most of the enzymes of glycogen synthesis and breakdown are present.
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.
1.
2.
3.
4.
5.
6.
7.
8.
Theoretical questions:
Mechanism and peculiarities of enzymatic reactions of glycogenesis.
Glycogenolysis, common and different reactions with glycolysis.
Cascade mechanisms of ATP-dependent regulation of glycogen phosphorylase
and glycogen synthase activities.
Peculiarities of hormonal regulation of glycogen metabolism in liver and muscles.
Hereditary disorders in enzymes of glycogen synthesis and breakdown.
Glycogenoses, aglycogenoses.
Metabolic pathways and substrates of gluconeogenesis, mechanisms of regulation,
compartmentalization of enzymes, biological significance of the process.
Relations between glycolysis and gluconeogenesis (Cori cycle). Irreversible
reactions of glycolysis and their shunt pathways. Glucose-lactate and glucosealanine cycles.
Regulation of gluconeogenesis in human organism.
49
Practical part
Experiment 1. Iodine Test for Starch.
Principle. Starch is a polysaccharide consisting of glucose units joined together
by glycosidic bonds. The chains formed during the condensation reaction are either
linear or highly branched molecules.
Linear - both straight and helical - molecules of starch are referred to as
Amylose.
Whereas branched molecules of starch are called Amylopectin.
The Iodine Test for Starch is used to determine the presence of starch in
biological materials. The chains formed during the condensation reaction are either
linear or highly branched molecules. Iodine on its own (small non-polar molecule) is
insoluble in water.Therefore Potassium triiodide solution - Iodine dissolved in
potassium iodide solution - is used as a reagent in the test. To be more specific,
potassium iodide dissociates, and then the Iodide ion reacts reversibly with the Iodine
to yield the the triiodide ion. A further reaction between a triiodide ion and an iodine
molecule yields the pentaiodide ion.
(C6H10O5)n  I2
starch
heating
(C6H10O5)n I2
complex of starch with Iodine
Since molecular iodine is always present in solution, the bench iodine solution
appears brown; the iodide and triiodide pentaiodide ions are colourless.
The triiodide and pentaiodide ions formed are linear and slip inside the helix of
the amylose (form of starch).
Reagents: 1% solution of starch, Lugol solution (iodine dissolved in potassium
iodide), tubes.
Method.
1. Add 1ml of the starch solution to a clean, dry test tube.
2. Add about 5 drops of Lugol solution to the test tube.
50
3. Note any colour changes. Write a conclusion.
Experiment 2. Detection of glycogen in the liver.
Principle. A positive test for glycogen is a brown-blue color. A negative test is
the brown-yellow color of the test reagent. Glycogen, as well as starch, forms a
colored compound with iodine (starch forms blue, glycogen - a red-brown
compound). It is thought that starch and glycogen form helical coils. Iodine atoms
can then fit into the helices to form a starch-iodine or glycogen-iodine complex.
Starch in the form of amylose and amylopectin has less branches than glycogen. This
means that the helices of starch are longer than glycogen, therefore binding more
iodine atoms. The result is that the color produced by a starch-iodine complex is more
intense than that obtained with a glycogen-iodine complex.
Reagents. Fresh or frozen liver tissue, Lugol solution (iodine dissolved in
potassium iodide), 1% solution of acetic acid, porcelain mortar, water bath, paper
filters.
Generation of filtrate
1. Put 0.5 g of liver tissue into a tube, add 4 ml of distilled water and boil it (2-3
min in order to inactivate enzymes).
2. Transfer liver into the mortar and grind it.
3. Transfer the obtained homogenate into the tube, add 1 ml of distilled water
and boil the solution on a water bath (20 min). Add 5-10 droplets of acetic acid
solution for protein precipitation.
4. Eliminate precipitated proteins by filtration using paper filters.
Method.
1. Take a clean, dry tube. Put 1 ml of filtrate.
2. Add 2-3 droplets of Lugol solution.
3. Note any colour changes. (In presence of glycogen a red-violet color is
observed). Compare the color with a color obtained in the previous
experiment. Explain the results.
Clinical diagnostic 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. The prime function of liver glycogen is to
maintain the blood glucose levels, particularly between meals. Liver glycogen stores
increase in a well-fed state which are depleted during fasting. Muscle glycogen serves
as a fuel reserve for the supply of ATP during muscle contraction.
The metabolic defects concerned with the glycogen synthesis and degradation
are collectively referred to as glycogen storage diseases. These disorders are due to
defects in the enzymes which may be either generalized (affecting all tissues) or
tissue-specific. The inherited disorders are characterized by deposition of normal or
abnormal type of glycogen in one or more tissues. Increase in blood glycogen
concentration is observed in some infection diseases, which are accompanied with
leukocytosis, diabetes mellitus, and malignancies.
Examples of Krock-1 tests
1. In a weak apathic infant an enlarged liver was detected, which in investigation
of biopcia pieces showed an excess of glycogen. Blood glucose concentration is
under the normal value. What may be the cause of this disease?
51
A. Lowered activity of glycogen
phosphorylase in a liver
B. Lowered activity of glycogen
synthase
C. Lowered activity of glucose 6-
phosphate isomerase
D. Lowered activity of glucokinase
E. Deficiency of gene responsible for
synthesis of glucose 1-phosphate
uridyl transferase
2. 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
D. Fructose bis-phosphatase
B. Phosphorylase
E. Pyruvate carboxylase
C. Aldolase
3. In an infant with point mutations in genes the absence of glucose 6phosphatase, hypoglycemia and hepatomegalia were revealed. What disease is
characterized by these symptoms?
A. Gierke disease
D. Cori disease
B. Adison disease
E. Mac Ardle disease
C. Parkinson disease
4. 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. Glycogen phosphorylase
D. Glycogen synthase
B. Phosphofructokinase
E. Glucose 6-phosphatase
C. Glucose 6-phosphate
dehydrogenase
Individual independent students work
1. Principles of regulation of glycogen biosynthesis and breakdown.
2. Hereditary disorders of synthesis and breakdown of glycogen
glycoconjugates.
1.
2.
3.
4.
5.
6.
7.
and
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed.
New York: Wiley-Liss, 2002.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry.
2nd edition” – 2008. – 488 p.
52
8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O.,
Nasadyuk C.M. – 2012. – 308 p.
Topic № 11. Mechanisms of metabolic and humoral regulation of carbohydrate
metabolism. Disorders of carbohydrate metabolism
Objectives: To interpret the role of hormones in regulation and maintenance of
constant blood glucose level. To learn the peculiarities of changes in metabolism of
carbohydrates, lipids and proteins in diabetes mellitus.
Actuality of the theme: Diabetes mellitus is a clinical condition characterized
by increased blood glucose level (hyperglycemia) due to insufficient or inefficient
insulin. In other words, insulin is either not produced in sufficient quantity or
inefficient in its action on the target tissues. As a consequence, the blood glucose
level is elevated which spills over into urine in diabetes mellitus. Determination of
blood glucose level in clinical laboratory investigations is of great importance in
diagnostics of diabetes mellitus and many other diseases and disorders.
Specific aims:
 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 explain disorders in metabolism of carbohydrates in diabetes mellitus.
Theoretical questions:
1. Biochemical processes which provides the constant blood glucose level. Role of
different pathways of carbohydrate metabolism in regulation of blood glucose level.
2. Hormonal regulation of carbohydrate metabolism:
 Insulin, its structure, mechanism of action, role in carbohydrate metabolism.
 Adrenalin and glucagone, mechanism of their regulatory effects on carbohydrate
metabolism.
 Glucocorticoids, their effect on carbohydrate metabolism.
3. Characterization of hypo- and hyperglycemia, glucosuria.
4. Insulin dependent and noninsulin dependent forms of diabetes mellitus.
5. Characterization of metabolic disorders in diabetes mellitus
6. Biochemical tests for evaluation of conditions of patients with diabetes mellitus.
Glucose tolerance test and its alteration in diabetes mellitus. Biochemical criteria of
diabetes mellitus.
Practical part
Experiment 1. Estimation of blood glucose by o-toluidine method.
Principle. In this method of glucose determination, a primary aromatic amine, otoluidine, reacts in hot glacial acetic acid with the terminal aldehyde group of glucose
to produce a blue-green color. The absorbance of this product is measured
photometrically and glucose concentration can be calculated. The absorbance in 600 700 nm region is directly proportional to the glucose concentration.
53
Reagents and materials. Blood sample, 3% solution of trichloroacetic acid
(TCA), o-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.
Preparation of supernatant. Add 0.9 ml of trichloroacetic acid into two
centrifuge tubes. Into test tube add 0.1 ml of blood specimen, into control tube – 0.1
ml of standard glucose solution. Centrifuge the tubes at 3000 rpm for 10 min.
Separate test and control supernatants for further investigations.
Method.
1. Take two clean, dry tubes. Into the first tube add 0.5 ml of control supernatant,
into second – 0.5 ml of tested supernatant.
2. Add 4.5 ml of o-toluidine reagent to each tube.
3. Place the tubes on a boiling water bath for 8 min.
4. Cool tubes and measure optical density (A) in a colorimeter at wavelength 630
nm (red filter).
Calculation. The concentration of glucose is calculated using the formula:
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.
Clinical diagnostic significance. The fasting blood glucose level in normal
individuals is 3.3-5.5 mmol/l and it is very efficiently maintained at this level. When
the blood glucose concentration falls, the symptoms of hypoglycemia appear. The
manifestations include headache, anxiety, confusion, sweating, slurred speech,
seizures and coma, and, if not corrected, death. All these symptoms are directly and
54
indirectly related to the deprivation of glucose supply to the central nervous system
(particularly the brain) due to a fall in blood glucose level.
Elevation of blood glucose concentration is the hallmark of uncontrolled
diabetes. Hyperglycemia is primarily due to reduced glucose uptake by tissues and its
increased production via gluconeogenesis and glycogenolysis. When the blood
glucose level goes beyond the renal threshold, glucose is excreted into urine
(glycosuria).
Examples of Krock-1 tests
1. A 46-year-old woman complains of dryness in the oral cavity, thirst, frequent
urination, general weakness. Biochemical research of the patient's blood
showed hyperglycemia and hyperketonemia. Sugar and ketone bodies are
revealed in the urine. Diffuse changes in myocardium are marked on the
electrocardiogram. Make an assumptive diagnosis of the illness.
A. Diabetes insipidus.
D. Diabetes mellitus.
E. Ischemic cardiomyopathy.
B. Alimentary hyperglycemia.
C. Acute pancreatitis.
2. A patient was admitted to a hospital in comatous 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. Hypoglycemic
D. Hypothyroid
B. Hyperglycemic
E. Hepatic
C. Hypovolemic
3. A patient addressed to physician with complaints on permanent thirst. In
laboratory investigation it was revealed hyperglycemia, polyuria and increased
content of 17-ketosteroids in urine. What disease is the most probable?
A. Steroid diabetes
D. Glycogenosis of the 1 type
B. Insulin dependent diabetes mellitus
E. Myxoedema
C. Addison disease
55
4. A 38-year-old man is receiving treatment for schizophrenia in hospital. Fhe
initial levels of glucose, ketone bodies and urea in the blood are within the
normal range. Shock therapy put into practice by regular insulin injections
resulted in the development of the comatose state which improved the clinical
status of the patient. What is the most probable cause of insulin coma?
A. Hypoglycemia.
D. Ketonemia.
B. Dehydratation of tissues.
E. Hyperglycemia.
C. Metabolic acidosis.
1.
2.
3.
4.
5.
6.
7.
8.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd
edition” – 2008. – 488 p.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M. – 2012. – 308 p.
Topic № 12. 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 learn the
methods of determination of phospholipids concentration and activity of lipase and to
interpret the obtained results.
Actuality of the theme: The knowledge of main pathways of intracellular
metabolism of lipids under normal conditions and in pathology are necessary for 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.
56
 To analyze the main pathways of lipid metabolism in human body in normal conditions
and in pathology.
 To explain hormonal regulation of lipid metabolism.
Theoretical questions:
1. Classification of lipids. Biological functions of simple and complex lipids in human
body (reserve, energetic, thermoregulatory, production of biologically active
substances).
2. Structure and role of fatty acids (saturated, unsaturated and polyunsaturated).
3. Triacylglycerols (TAG) their structure and properties.
4. Phospholipids: structure of main glycerophospholipids (phosphatidic acid, lecithin,
cephalin, phosphatidylinosit, phosphatidylserine, plasmalogen) and sphingolipids
(sphlngomyeliln). Their biological role.
5. Structure and occurrence of main steroids.
6. Amphipathic lipids Involvement of lipids in formation of structure and function of
biological membranes. Liposomes. Application of liposomes in medical practice.
7. Digestion of lipids.
8. 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.
9. Biosynthesis of triacylglycerols and phospholipids, the significance of phosphatidic
acid as a precursor.
10.Metabolism of sphingolipids. Genetic anomalies of sphingolipid metabolism –
sphingolipidoses. Lysosomal diseases.
Practical part
Experiment 1. Quantitative determination of phospholipids in blood serum.
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.
I. Precipitation of phospholipids with trichloroacetic acid (Attention!!!! This part of
experiment was previously done by technitions):
1. Take a clean dry centrifuge tubes.
2. Add 0.2 ml of blood serum, 2 ml of distilled water and 3 ml of 10 % solution of
trichloroacetic acid.
3. Mix the content of the tube and after 2-3 min. centrifuge it during 5 min at 3000 rpm.
4. Carefully remove the supernatant. Sediment contains lipoproteins. Add to the sediment
1 ml of 56 % HClO4 put the tube into a bath with a boiling water for 30 min. The final
mineralizate must be colorless.
II. Color reaction:
1. Take 2 clean dry tubes.
57
2. To the 1st (experimental) tube add 5 ml of mineralizate, 1 ml of ammonium molybdate
and 1 ml of 1% solution of
Exp
St
ascorbic acid.
3. To the 2nd tube (standard) add
5 ml of standard phosphorus
1 ml of 1% solution of
solution (0,05 g/l) 1 ml of
ascorbic acid
ammonium molybdate and 1
1 ml of ammonium molybdate
ml of 1% solution of ascorbic
5 ml of mineralizate
5 ml of standard
acid.
4. Mix the content of both tubes
and incubate 15-20 min at room temperature.
III. Measurement of optical desity:
After 5 min measure the optical density on a photoelectrocolorimeter with a red light
filter.
IV. Calculation: Use the following formula:
Aexp  0,05
[Total phospholipids in serum] = --------------  25 g/l
Аst  0,2
where Aexp – optical density of the experimental probe;
Ast – optical density of the standard probe;
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 diagnostic 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. The increase of phospholipids level in blood
serum (hyperphospholipidemia) is observed in heavy form of diabetes mellitus, nephrosis,
obturative jaundice. Decrease of phospholipid level (hypophospholipidemia) may be
observed in atherosclerosis, anemias, feaver, alimentary distrophias, liver diseases.
Experiment 2. The influence of bile on the lipase activity.
Principle. The lipase
activity is estimated according
2
1
to the production of fatty acids
1 ml of bile
during hydrolysis of fat. The
0.25 g of pancreatine
quantity of fatty acids is
determined by titration with
10 ml of milk
alkali in the presence of
Mix well
phenolphthalein. Bile activates
1 ml from
lipase thus accelerating the
tube 1
cleavage of lipids.
Method.
I.
Preparation of mixtures:
1 ml of H2O
1 ml from
tube 2
1-2 drops of
0.5% phenolphthalein
Titrate with 0.05 N NaOH to appearance the pink color, which
doesn’t disappear during 30 seconds.
58
1. Take 2 clean dry tubes.
2. Add 10 ml of milk and 0.25 g of pancreatine into both tubes.
3. Add 1 ml of bile into 1st tube and 1 ml of water into 2nd. Mix them well.
II. Titration:
1. Take 1 ml of the mixture from tube 1 (with bile) and transfer it to the flask 1.
2. Take 1 ml of the mixture from tube 2 (without bile) and transfer it to the flask 2.
3. Add 1-2 drops of 0.5% phenolphthalein solution into both flasks.
4. Titrate mixtures of both flasks with 0.05 N NaOH up to the pink color, which doesn’t
disappear during 30 seconds. Register the volume (in ml) of used alkali.
Time, min
0 min
15 min
30 min
45 min
Quantity of NaOH expended for titration, ml
Lipase activity in
presence of bile
Lipase activity in
absence of bile
60 min
Lipase without bile
Lipase without bile
III. Incubation. The first two tubes with digestive mixture (see I) place to the termostate
at 38C. Every 15 min take 1 ml of the mixture from each tube into flasks and repeat the
titration (see II). Conduct 4-5 subsequent determinations. The results are registered as ml
of alkali solution, expended for titration. Note the obtained result into the table bellow.
Explain the results, draw a conclusion.
Clinical and diagnostic 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 emulsify 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 emulcification, 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 Krock-1 tests
1. In patients suffering from diabetes mellitus an increase in a content of non
esterified fatty acids (NEFA) in blood is observed. It may be caused by:
A. Increase in activity of triacylglycerol
C. Activation
of
synthesis
of
lipase
apolipoproteins A1 , A2, A3
B. Stimulation of ketone bodies utilization
59
D. Decrease
in
activity
phosphatidylcholine-cholesterolacyltransferase in blood plasma
of
E. Accumulation
palmitoyl-CoA
in
cytosol
of
2. The essence of lipolysis, that is the mobilization of fatty acids from neutral fats
depots, is an enzymatic process of hydrolysis of triacylglycerols to fatty acids and
glycerol. Fatty acids that release during this process enter blood circulation and are
transported as the components of:
A. Serum albumins
D. LDL
B. Globulins
E. Chylomicrons
C. HDL
3. Which one of the following statements about the absorption of lipids from the
intestine is correct?
D. Fatty acids that contain ten carbons
A. Dietary triacylglycerol is partially
or less are absorbed and enter the
hydrolyzed and absorbed as free fatty acids
circulation primarily via the lymphatic
and monoacyl glycerol
system
B .Release of fatty acids from triacylglycerol
E. Formation of chylomicrons does not
in the intestine is inhibited by bile salts
require protein synthesis in the
C. Dietary triacylglycerol must be
intestinal mucosa.
completely hydrotyzed to tree fatty acids and
glycerol before absorption
4. After consumption of lipids in the body than begins their digestion and absorption
in intestines. What products of lipid hydrolysis are absorbed in the intestine?
A. Monoacylglycerol, fatty acids
D. Monosacharides
B. Amino acids
E. Lipoproteins
C. Polypeptides
5. After the consumption of animal food rich in fats, a patient feels discomfort, and
droplets of fats are found during laboratory investigation of his feces. Bile acids are
revealed in the urine. The cause of such state is the deficiency of ___ in the digestive
tract.
A. Bile acids.
D. Triacylglycerols.
B. Fatty acids.
E. Phospholipids.
C. Chylomicrons.
Individual independent students work
1. Metabolism of sphingolipids under normal conditions and in pathology. Abnormalies of
sphingolipid metabolism – sphingolipidoses.
References:
1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
60
4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd
edition” – 2008. – 488 p.
8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M.. – 2012. – 308 p.
Topic № 13. β –Oxidation and biosynthesis of fatty acids. Studies on metabolism of
fatty acids and ketone bodies.
The aim of the lesson: To learn reactions of biosynthesis and oxidation of fatty acids.
To know metabolic pathways of ketone bodies under normal conditions and in pathology
and to determine their amount in urine.
Actuality of the theme: Oxidation of lipids, respectively fatty acids, as well as
ketone bodies metabolism are important constituents of energetic metabolism in sense of
providing tissues and cells with ATP. Determination of ketone bodies concentration in
blood and in urine has important significance in diagnostics of several pathological
processes.
Specific aims:
 To study reactions 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 carnitin 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, 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 NADPH2 for biosynthesis of long chain fatty acids;
 the sequence of enzymatic reactions in biosynthesis of long chain fatty acids
61
 regulation of biosynthetic process on level of acetyl-CoA-carboxylase and fatty
acid synthetase;
 elongation of carbon chain of 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.
Practical part
Experiment 1. Qualitative reaction on acetone and acetoacetic acid (Lange
reaction).
Principle. It is based on a property of acetone and acetoacetic acid to produce
compounds with sodium nitroprussid of red color in the basic pH of medium.
CH3 – CO – CH3 + Na2 [Fe (CN)5 NO] + 2 NaOH →
→ Na4 [Fe (CN)5 NO = CHCOCH3] + 2 H2O.
Under the action of acetic acid it turns to a violet product:
Na4 [Fe (CN)5 NO =CHCOCH3] +CH3COOH →
Na3 [Fe (CN)5 NOCH3COCH3] + CH3COONa.
compound after an overlay of conc. ammonia solution over the mixture of test sample,
containing sodium nitroprussid and acetic acid. 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).
Method.
1. Take two clean dry tubes.
2. Add 0.5 ml of urine of a healthy person to the first tube (blank).
3. Add 0.5 ml of urine of a patient with diabetes mellitus to the second one (test sample).
4. Add 0.5 ml of 10 % NaOH and 5-7 drops of fresh 10% sodium nitroprussid solution
into both tubes. Color in the 2nd tube turns red.
5. Add 5-7 drops of acetic acid into both tubes. Color in the 2nd tube turns violet.
Explain the results, draw a conclusion.
Experiment 2. Quantitative determination of ketone bodies in urine.
Principle. (see the previous experiment).
Method.
1. Take 5 clean dry tubes.
2. Add 1 ml of distilled water into each tube (except the first one).
3. Add 1 ml of urine of the patient with diabetes mellitus into the first and the second
tubes. Thus, in the first tube urine is undiluted, in the second one the urine is two
fold diluted.
4. Mix the content of the second tube thereafter transfer 1 ml of this mixture to the third
tube and mix it. Now the third tube conatains 4 fold diluted urine.
62
5. Remove 1 ml from the third tube - into the forth and mix it - 8 fold dilution.
6. Remove 1 ml from the fourth tube - into the fith one and mix it - 16 fold dilution.
7. Add 8 drops of 50% ammonium sulfate solution into each tube for increasing the
density of the solutions
8. Add 8 drops of concentrated acetic acid into each tube.
9. Add 8 drops of sodium nitroprussid each tube. Mix well the content of the tubes
10. Overlay carefully 1 ml of concentrated solution of ammonia into each tube starting
from the last tube (be careful with concentrated ammonia!).
11. Register an appearance of red or violet ring in the tubes. Notice the last tube in
which a colored ring appears between the 3-d and the 4-th min.
Calculation:
X = 0.85  A  15, were
X – concentration of acetone in urine (mg/day);
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).
Draw the conclusions.
Clinical and diagnostic significance. Ketone bodies include the following
substances – acetoacetic acid, -hydroxybutyric acid, 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 person 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 under 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 Krock-1 tests
1. In a patient suffering from diabetes mellitus in blood was detected acetone. Note
the process of its production in the body.
A. By condensation of two molecules of
D. In course of γ-oxidation of fatty
acetyl-CoA
acids
B. In course of α-oxidation of fatty acids
E. In tricarboxylic acid cycle.
C. In course of β-oxidation of fatty acids
2. Aerobic oxidation of substrates is typical of a cardiac muscle. Which of the
following is the major oxidation substrate of a cardiac muscle?
A. Fatty acids.
D. Glucose.
B. Triacylglycerols.
E. Amino acids.
C. Glycerol.
63
3. Carnitine is recommended to a sportsman as a preparation that increases physical
activity and improves achievements. What biochemical process is mostly activated
under the action of carnitine?
A. Transport of fatty acids into
C. Lipids synthesis.
mitochondria.
D. Tissue respiration.
B. Ketone bodies synthesis.
E. Steroid hormones synthesis.
4. A 1-year-old child was brought to a clinic with signs of muscle weakness. Through
the inspection, the deficiency of carnitine in the muscles was determined. The
biochemical mechanism of the development of this pathology consists in the disorder
of the process of:
A. Transport of fatty acids into
C. Substrate level of phosphorylation.
mitochondria.
D. Utilization of lactate.
2+
B. Regulation of the level of Ca in
E. Synthesis of actin and myosin.
mitochondria.
5. Lipids are obvious energetic material for the body. What is the main pathway of
fatty acids metabolism in mitochondria?
A. β-Oxidation
D. Reduction
B. Decarboxylation
E. γ-Oxidation
C. α -Oxidation
1.
2.
3.
4.
5.
6.
7.
8.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd
edition” – 2008. – 488 p.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M.. – 2012. – 308 p.
Topic № 14. Biosynthesis and biotransformation of cholesterol. Pathology of lipid
metabolism: steatorrhea, atherosclerosis, obesity. Transport forms of lipids:
lipoproteins of blood plasma.
Objective: To learn the pathways of cholesterol metabolism and principal disorders
of lipid metabolism. To interpret free radical reactions, mechanisms of lipid peroxidation
and its significance in biological processes in normal conditions and in pathology.
64
Actuality of the theme: Disorders in cholesterol biotransformation processes cause
several 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.
Production of free radicals and products of peroxide oxidation of lipids are normal
metabolic processes. In normal conditions the level of products of peroxide oxidation of
lipids is regulated by antioxidative enzymatic system. Disorders in relations between
activity of pro- and anti- oxidative systems is manifested in form of different pathological
features. This makes necessary the investigation an evaluation of oxidative stress indexes
in pathology.
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, excretion of cholesterol from the body.
 To interpret pathology of lipid metabolism: atherosclerosis, diabetes mellitus,
obesity, steatorrhea.
 To explain processes of lipid peroxidation under normal conditions and in
pathology.
 To interpret regulation of free radical reactions in human body.
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.
3. Pathways of cholesterol biotransformation (esterification, production of bile acids
and steroid hormones, synthesis of vitamin D3, excretion from the body).
4. Atherosclerosis, mechanism of its development, role of genetic factors,
hypercholesterolemia. Hypercolesterolemia in diabetes mellitus, myxoedema,
obstructive jaundice, nephritic syndrome. Control of hypercholesterolemia
5. Lipoproteins: structure, classification, characteristics of apolipoproteins.
6. Metabolism of lipoproteins – a general view.
7. Disorders of plasma lipoproteins (classification of hyperlipoproteinemias,
characteristics of hypolipoproteinemias.
8. Fatty liver (steatosis), lipotropic factors.
9. Pathological processes which leads to the development of obesity.
10. Lipid peroxidation and mechanisms of antioxidant enzymatic system action:
 Lipid peroxidation under normal conditions and in pathology.
 Regulation of free radical reactions in human body.
 Characterization of prooxidants and antioxidants, their significance in peroxide
oxidation of lipids.
Practical part
65
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.
Method.
1. Take a clean dry tube.
2. Add 10-15 droplets of bile.
3. Add ~5 droplets of a fresh 20% sucrose solution and mix it.
4. Carefully overlay 1 ml of conc. sulfuric acid without mixing of two fluids.
5. Observe the precipitation of bile acids in the borderline between two fluids (appearance
of violet color ring).
6. Mix it well and observe the appearance of red-violet color.
Clinical and diagnostic significance. The bile acids and their salts are detergents
that emulsify fats in the gut during digestion. They are synthesized from cholesterol in the
liver by a series of reactions. The 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). In coprologic investigations decrease or complete
absence of bile pigments in feces are observed, as well as high content of soaps, espetially
calcium soaps.
Experiment 2. Investigation of antioxidative enzymatic system. Quantitative
determination of catalase activity.
Principle. Catalase belongs to a class of oxido-reductases and catalyzes the
transformation of hydrogen peroxide to water and oxygen:
2H2O2  2 H2O + O2
Biological significance of this enzyme consists in the defense of the body from
harmful effect of hydrogen peroxide, which forms in the course of intracellular oxidation
of different substances. According to chemical structure, catalase is a chromoprotein,
containing heme as a prosthetic group. Molecular weight of catalase is 225 – 240 kDa. It is
widely distributed and extremely active enzyme.
The determination of catalase is based on the estimation of hydrogen peroxide, which
is decomposed by the enzyme in a distinct period of time. Hydrogen peroxide is titrated
with permanganate, the reaction occurs according to the equation:
5H2O2 + 2 KMnO4 + 3 H2SO4 → K2SO4 + 2 MnSO4 + 8 Н2О + 5О2
Method. Take two flasks and do the following procedures:
1. Add 1 ml of diluted 1:1000 blood into both flasks then add 7 ml of water.
2. Add to the first flask (the test) 2 ml of 1% solution of hydrogen peroxide
3. and 5 ml of 10 % sulfuric acid to the other flask (the control). Leave both flasks at
room temperature for 30 min. Then, add to the test flask 5 ml of 10 % sulfuric acid, and
to the control flask 2 ml of 1% solution of H2O2. Titrate the content of flasks by 0.1 N
KMnO4 to appearance of pink colour.
66
Test
- 1 ml of diluted 1:1000 blood
Control
- 7 ml of water
- 2 ml of 1% solution of H2O2
- 5 ml of 10 % sulfuric acid
Incubation for 30 min (room temperature)
Test
Control
A - the volume (ml) of KMnO4 used for
titration of the control probe,
B - the volume (ml) of KMnO4 used for
titration of the test probe.
Titration by 0.1 N KMnO4
to appearance of pink colour
Calculate catalase activity according to formula:
Cf = (A – B)  1.7, where
A - the volume of KMnO4 solution, used for titration ofthe control probe,
B - the volume of KMnO4 solution used for titration of the test probe.
Explain the results and draw a conclusion.
Clinical and diagnostic significance. Catalase (E.C. 1.11.1.6. ) is one of the most
active and the most wide spread enzymes. It accelerates the breakdown of hydrogen
peroxide and its derivatives. Normal value of catalase activity in blood is in ranges of 10 –
15 units/L. The estimation of catalase activity data on red blood cells count in blood is of
low diagnostic value as catalase activity is tightly connected with red blood cells. In
clinical laboratory the catalase index determination is used, which is equal to catalase
activity in units over number of: red blood cells (in ml/mm3). In normal conditions this
index is in 2 – 3  10 – 6 ranges.
Examples of Krock-1 tests
1. A patient suffers from arterial hypertension due to atherosclerotic injury of blood
vessels. The consumption of what dietary lipid needs to be limited?
D. Monooleateglycerol.
A. Cholesterol.
E. Phosphatidylserine.
B. Oleic acid.
C. Lecithine.
2. Fats of phospholipids is disordered due to fat infiltration of the liver. Indicate
which of the presented substances can enhance the process of methylation during
phospholipids synthesis?
A. Methionine
D. Glycerin
B . Ascorbic acid
E. Citrate
C. Glucose
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3. In a patient after investigation it was detected an increased content of low density
lipoproteins in blood serum. What disease can be expected in this patient?
A. Atherosclerosis
D. Acute pancreatitis
B. Pneumonia
E. Kidney disease
C. Gastritis
4. A child 5 years old suffers from transient abdominal pains. 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. Chylomicrons
D. LDL
B. HDL
E. VLDL
C. IDL
5. In cases of complete or partial restriction of lipotropic factors in humans develops
a fat degeneration of liver. What substances can be considered as lipotropic?
A. Choline
D. Cholesterol
B. Pyridoxine
E. Triacylglycerols
C. Fatty acids
Individual independent students work
1. Lipid peroxidation, its role under normal conditions and in pathology.
1.
2.
3.
4.
5.
6.
7.
8.
References:
Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes,
Victor W. Rodwell, 2003.- 927 p.
Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.
Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and
Company. – 2005. – 1010 p.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.-416p.
Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New
York: Wiley-Liss, 2002.
Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd
edition” – 2008. – 488 p.
MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk
C.M.. – 2012. – 308 p.
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