THE STATE MEDICAL EDUCATIONAL INSTITUTION

1 

Ministry of Public Health of Ukraine 

Central methodical cabinet 

for higher medical education 

State Medical Educational Institution 

“Ukrainian Medical Stomatological Academy” 

Biological and bioorganic chemistry 

study guide 

Poltava - 2011

2 

Authors: MD, Professor L.M. Tarasenko, MD, Professor K.S. Neporada, Ph D, 

Assistant Proffesor V.C. Grigorenko, MD, Assistant Proffesor L.G. Netyuhaylo, Ph D 

M.V. Bilets, O.E. Omelchenko, N.N. Slobodyanik. 

Translation: A.M. Manko, M.V. Bilets 

Study guide on biological and bioorganic chemistry (Modules ІІ-V) for self-preparatory 

work of second-year students of the dentistry faculty.– Poltava, 2011. –94 p., English. 

Study guide for self-preparatory work of second-year students on biological and 

bioorganic chemistry (Modules ІI-III) of higher medical institutions of the ІV accreditation 

level are prepared according to the program “ Biological and bioorganic chemistry ”. The 

program is composed by the stuff of the leading department of bioorganic, biological and 

pharmacological chemistry of National medical university named after O.O. Bogomoltsa. 

The study material is divided into 3 modules. It is consistent with the teaching standarts, 

the principles of Bologna process and improves students performance. The manual is 

made for 2-hour classes, it contains sequence of studying actions, guidelines to studying 

actions, list of practical skills and kinds of individual and self-preparatory work, and also 

the list of the compulsory and additional literature. 

Reviewers: 

Head of department of medical and bioorganic chemistry of KharkivN 

national Medical University, PhD Professor, G.O. Sirova 

Head of department of chemistry and teaching methods of chemistry of Poltava National 

Pedagogical university named after Korolenko and bioorganic chemistry 

PD, Professor N.I. Shiyan

3 

FOR STUDENT’S ATTENTION! 

The subject “ BIOLOGICAL AND BIOORGANIC CHEMISTRY ” 

is divided into 3 modules: 

Module 1. Biologically important classes of bioorganic compounds. 

Biopolymers and their structural components. 

Module 2. General principles of metabolism. Metabolism of 

carbohydrates, lipids, amino acids and its regulation. 

Module 3. Molecular biology. Biochemistry of intercellular 

communications. Biochemistry of tissues and physiological functions. 

Structure of the 

subject 

Studying plan on the subject “ Biological and bioorganic chemistry ” 

for students of dentistry faculty 

ECTS credits 9,0 

Module 1 75hrs/2,5 

ECTS 

credits 


ECTS 

credits 


ECTS 

credits 

Summary control 90hrs/3,0 

for four modules ECTS 

credits 

Weekly amount 

Number of hours, i.e. 

Auditorium 

Total Lectures 

Practical 

classes 

SPW 

Year of 

study 

270 20 130 120 1-2 

10 40 25 1 

10 40 55 2 

- 50 40 2 

The note: 1 ECTS credit – 30 hours 

Auditorium work - 55 %, SRW – 45% 

- 

6,75hrs / 0,23 ECTS credits 

Types of control 

Current and summary 

(standardized) 


(standardized) 


(standardized)

4 

MODULE 2 

GENERAL PRINCIPLES OF METABOLISM. METABOLISM OF 

CARBOHYDRATES, LIPIDS, AMINO ACIDS 

AND ITS REGULATION. 

Theme 1. Subject and tasks of biochemistry. The purpose and 

methods of carrying out of biochemical researches, their clinicaldiagnostic 

value. Research of a structure and physical and chemical 

properties of proteins - enzymes. 

Actuality of the theme. 

Biochemistry – is the fundamental science and a subject matter which studies a 

chemical composition of alive organisms and chemical transformations of biomolecules. 

Only the knowledge of molecular mechanisms of functioning of an organism provides deep 

understanding of mechanisms of pathogenesis, biochemical diagnostics, preventive 

maintenance and treatment of the major diseases of mankind. Knowledge of biochemistry 

are necessary for studying physiology, microbiology, pharmacology, the general pathology 

and clinical disciplines. The formation of the highly skilled doctor is impossible without the 

strong base of theoretical disciplines. 

Enzymes are biological catalysts of reactions of a metabolism. Protein nature of 

enzymes causes their high lability depending on many factors. So, depending on a body 

temperature of the person, and also on рН of the internal environment of an organism, the 

activity of enzymes can change that results in the development of pathological processes. 

Purpose and initial level of knowledge. 

General purpose. 

Studying of a chemical composition of alive organisms and chemical reactions of 

biomolecules which are a part of these organisms. To study physical and chemical 

properties of proteins - enzymes. 

Concrete purposes: 

1. To analyze stages and principles of becoming of biochemistry as medical-biologic 

science and subject matter. 

2. To explain principles and bases of methods of biochemical researches of a 

functional condition of an organism of the person in norm and at a pathology. 

3. To analyze mechanisms of regulation of the basic metabolic processes; 

4. To treat biochemical principles of a structure and functioning of different classes of 

enzymes; 

5. To analyze physical and chemical properties of proteins - enzymes. 

Initial level of knowledge - abilities: 

Initial level of knowledge - abilities. To know a chemical composition of alive 

organisms and structure of bioorganic compounds. To know the structure of proteins, 

classification and structure of protein-genetic amino acids. 

Reference card for the separate study

5 

of educational literature for the lesson preparation. 

Table of contents and sequence of Guidelines to the educational actions 

actions 

The control of an initial level of 

knowledge. 

1. Definition of biochemistry as 

biological science. Tasks of 

biochemistry. 

1.1. Biochemistry as a science. 

1.2. Tasks of biochemistry. 

1.3. Value of biochemistry for diagnostics and 

treatments of the basic diseases of the person. 

2. Sections of biochemistry. 2.1. Static biochemistry. 

2.2. Dynamic biochemistry. 

2.3. Functional biochemistry. 

2.4. Clinical biochemistry as section of 

biochemistry. 

3. Practical studying physical and 

chemical properties of proteins - 

enzymes. 

4. Enzymes as biological catalysts 

of reactions of a metabolism. 

5. Structure of enzymic proteins. 

Simple and complex enzymes. 

3.1. To prove the protein nature of enzymes by 

a biouret reaction, Fol’s reaction. To explain 

principles of the method. 

3.2. To explain thermolability of enzymes by 

the example of determination of amylase 

activity of a saliva which is preliminary heated 

or cooled and preliminary is not processed. 

3.3. To study the dependence of amylase 

activity of a saliva on рН environment. 

4.1. Physical and chemical properties of 

proteins - enzymes. 

4.2. Electrochemical properties, solubility. 

4.3. Thermodynamic stability of protein 

molecules of enzymes; denaturation. 

5.1. Apoenzyme, coenzyme (prosthetic group). 

Oligomer proteins - enzymes, multienzymes 

complexes. 

5.2. Membrane-assotiated enzymes. 

Individual self-preparatory work of students. 

To prepare the report from the history of the development of biochemistry: 

а) Directions of the development of biochemistry; 

b) Basic discoveries in the branch of structure and a functional role of proteins. 

Rules of work in biochemical laboratory. 

1. To begin laboratory researches after instructing by the teacher, strictly adhering to a 

methodology (algorithm) of researches. 

2. To be attentive, accurate and exact at carrying out of laboratory researches. 

3. To carry out the work with dangerous reagents under the hood. 

4. To adhere to instructions to the work on devices and the equipment. 

The operating procedure on FEC: 

1. To switch on the colorimetre for 15 minutes prior to the beginning of measurements. 

During warming up cavetti branch should be open. 

2. To expose a color optical filter necessary for measurement. 

3. To set the minimal sensitivity of a colorimetre.

4. Before measurement to check up the setting of the pointer of a colorimetre on "0" at 

closed cavetti branch. 

5. To place a cavetti with a solution at a light beam . 

6. To close a cover of a cavetti branch. 

7. To write down parameters from the scale of colorimetre. 

8. To switch off the device from a network. 

The operating procedure with the using of a water bath: 

1. To place a bath on a support or a table. 

2. The water level in a bath should be not more than 2/3 volumes of utensils. 

3. To dip into a bath a support for test tubes. 

4. On a complex plug of a bath to set the switching of capacity to position “ 700 W.А. ”, 

that corresponds to the accelerated heating. To insert the plug into an electric network. 

5. After boiling turn the switch to position “ 350 W.А. ”, that corresponds to the operating 

conditions. 

6. During the work to ensure the necessary water level in a bath. 

1. Studying of thermolability of enzymes. 

To dissolve the saliva in 5 times (1 ml of a saliva + 4 ml of water). To boil 2 ml of the 

dissolved saliva for 5 minutes. To pour on 10 drops of 1 % solution of starch in 3 test 

tubes. To add 10 drops of water in the first test tube (control), over the second – 10 drops 

of the cooled boiled saliva, in a third – 10 drops of the dissolved saliva. 

To place all test tubes in a water bath or thermostate at temperature 38 ºС for 10 

minutes. After that to execute qualitative reactions to starch and products of its hydrolysis 

(glucose and maltose). 

Reaction to starch: To add to 5 drops of a researched solution 1 drop of Lugol’s 

solution (KI 3 ). At the presence of starch there is a dark blue colouring. 

Feling’s reaction: To add to 5 drops of a researched solution 3 drops of a Feling’s 

solution (CuSO 4 + NaOH) to warm up. At the presence of glucose or maltose the yellow 

deposit of cuprous hydrate (Cu(OH) 2 ) or a red of cuprous oxide (CuO 2 ) falls out. 

2. Studying of dependence of enzymes activity on рН environment. 

To dissolve saliva in 10 times (1 ml of a saliva + 9 ml of water). To pour in 3 test tubes 

on 1 ml of buffer solutions with рН 3; 7,4; 14. To measure in each test tube on 1 ml of the 

dissolved saliva, on 1 ml of a solution of 1 % starch, to mix and incubate for 10 minutes at 

temperature 38º C. After this under the control of a display paper to neutralize the contents 

of a test tube with рН 3 - by a solution of alkali, and the contents of a test tube with рН 14 

– by a solution of 0,1n HCl. 

To reveal the result of experience with the help of Lugol’s solution, adding it on 1-2 

drops. In each test tube, to stir, to note colouring and to make a conclusions. 

Theme 2. Determination of the enzymes activity. Research of ferment 

processes by the type of reactions of the basic classes of enzymes. 

Research of the mechanism of enzymes action and the kinetics of 

ferment catalysis. 


Quantitative determination of the enzymes activity in a biological material is used for 

diagnostics of different diseases. So at a sharp pancreatitis the amylase activity in blood 

and urine rises. 

6

The complex structure and the functional organization of enzymes in part is a key to 

understanding of characteristic properties of enzymes – high specificity and velocity of 

catalysis. Classical works of Michaelis and Menten who have developed regulations about 

enzyme - substratum complexes have played the great role in development of 

representations about the mechanism of enzymes action . 

7 



To analyze the change of the amylase activity in biological liquids. Studying of the 

international classification of enzymes by the type of reactions. 

Studying of mechanisms of enzymes action and the kinetics of ferment catalysis. To 

know methods of determination of enzymes activity, and also to be able to interpret these 

methods. 


1. To determine quantitatively the urine amylase activity by Wolgelmut, to treat the 

received results. 

2. To treat the international classification and the nomenclature of enzymes by the type 

of reactions. 

3.To analyze mechanisms of enzymes action. 

4. To explain methods of determination of enzymes activity. 

5. To explain the kinetics of ferment catalysis. 

Initial level of knowledge - abilities: to know gradual hydrolysis of a molecule of starch 

to maltose through a stage of dextrins. To know types of chemical reactions. To know a 

biological role and structure of enzymes, physical and chemical properties of proteins. 

Reference card for the separate study 



actions 

1. Practical studying of 

1.1. Determination of the saliva amylase 

determination of the saliva activity. 

amylase. 

2. Methods of determination of the 

enzyme’s activity. 

2.1. At concrete examples explain principles of 

methods of determination of the enzyme’s 

activity. 

а) By quantity of a product that is formed under 

action of enzyme for a time unit; 

b) By quantity of the spent substratum for a 

time unit; 

c) spectrophotometric methods of 

determination of the enzymes activity and 

visualization of results of ferment reactions. 

2.2. Units of measurements of activity and 

quantity of enzymes: 

а) International units; 

b) Katal; 

c) Specific activity of enzyme.

3. The international classification 

and the nomenclature of enzymes 

by type of reactions. 

3.1. To explain the principle of the international 

classification and the nomenclature of 

enzymes. 

3.2. To name six classes of enzymes and to 

explain types of catalytic reactions by them. 

4. Mechanisms of enzymes action. 4.1. Thermodynamic principles of ferment 

catalysis. 

4.2.The active centers of enzymes. Differences 

in structure of the active centers in simple and 

complex enzymes. 

4.3. The ferment transformation of substrata at 

catalytic action of enzyme by the example of 

chymotrypsin and acetylcholinesterase action. 

Sequence of stages of catalytic process. 

5. The kinetics of ferment reactions. 5.1. Dependence of speed of reactions on 

concentration of enzyme, a substratum, рН and 

temperatures. 

5.2. Constant of Michaelis-Menten, their 

semantic value. 


To prepare the abstract report on the theme: ” The role of a pancreas in the origin of 

hyperamylasemia and hyperamylasuria ; « The mechanism of enzymes action ». 

Algorithm of laboratory work. 

Quantitative determination of the amylase activity. 

Principle of the amylase activity determination (Wolgelmut method): 

The method is based on amylase ability of hydrolizing the starch. Find such cultivation 

of a saliva, that splits 2 mg of starch (in 2 ml of 0,1 % solution of starch) for 30 minutes at 

t=38 ºС. Then calculate the quantity of starch that is splited by 1 ml of not dissolved saliva. 

In norm the saliva amylase activity is – 160-320 cond. units 

Course of work. 

In 10 test tubes pour on 1 ml of water. In the 1-st test tube add 1 ml of the saliva 

dissolved in 10 times, mix. To collect in a pipette 1 ml of solution and transfer it in the 2-nd 

test tube, from it – in the same way in 3-rd and so on up to 10-th test tube. To pour out 1 

ml of a mix from the 10-th test tube. In all test tubes add 1 ml of water and 2 ml of starch, 

mix and leave in thermostate for 30 minutes at t=38 ºC. In 30 minutes take out test tube 

and add 1 drop of Lugol’s solution, mix. The liquid in test tubes is colored in yellowish, pink 

and violet colors. Mark last test tube with yellow color where hydrolysis of starch has 

passed completely and carry out the calculation. 

Calculation: 

Mark a test tube where hydrolysis of starch has passed completely. For example, 

yellow color has appeared in 4-th test tube where the saliva is dissolved in 160 times. 

Accordingly, 1/160 ml of a saliva splits 2 ml of 0,1 % solution of starch, and 1 ml of not 

dissolved saliva will split X ml of 0,1% solution of starch: 

1/160 ml - 2 ml 

1ml - X ml 

2 1160 

X = = 320 ml of 0,1 % solution of starch. 

1 

Thus, the saliva amylase activity equals 320 c.u. 

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9 

Theme 3. Research of regulation of enzyme processes. 

Actuality of the theme. Course of biochemical reactions in possible only under 

condition of presence of catalytically active forms of enzymes. There are some ways of 

regulation of enzymes activity: change of catalytic activity of enzyme, change of the 

amount of enzyme. Due to activation or braking of activity of enzymes the velocity of 

biochemical reactions that are in the processes of ability to live increases or decreases. 

Infringement of the regulation of enzymes activity results in the development of 

pathological processes in an organism of the person. 


General purpose. To study regulation of enzyme processes. 


1. To analyze mechanisms of the regulation of the basic metabolic processes. 

2. To analyze ways and mechanisms of regulation of the ferment processes as basis of 

metabolism in an organism in norm and at pathologies. 

Initial level of knowledge - abilities: to know properties of enzymes, their structure, the 

active centers. 



Table of contents and 

Guidelines to the educational actions 

sequence of actions 

1. Practical studying of specificity 

breaking and activation of 

enzymes. 

1.1. In a writing-book of reports of experiences to 

write out algorithm of laboratory work. 

2. Specificity of action of enzymes. 2.1. Explain the biological importance of specificity 

of enzymes action, by what it is caused? What 

kinds of specificity of enzymes are known to you? 

Give examples. 

3. Regulation of enzyme 

processes. 

3.1. Regulation of enzymic activity by change of 

enzyme catalytic activity: 

а) allosteric regulation of enzymes activity; 

b) covalent updating of enzymes; 

c) activation of enzymes by limited proteolysis; 

d) action of regulatory proteins – effectors (of 

calmodulin, proteinases, proteinase inhibitors, cyclic 

nucleotides). 

3.2. Regulation of activity of enzymes by change of 

amount of enzyme. 

4. Inhibitors, activators of enzymes. 4.1. Convertible and irreversible inhibition of 

enzymes. 

4.2. Physiologically active connections and 

xenobiotics as convertible (competitive and not 

competitive) and irreversible inhibitors of enzymes. 

5. Isoenzymes – plural molecular 

forms of proteins, result of 

expression of different genes. 

5.1. On an example of isoenzymes of lactatedehydrogenase 

(LDH), creatine phosphokinase 

explain 

value of their determination in clinic. 

5.2. Proenzymes, their biological role and value.


To prepare the abstract message on the theme: “ Proteinases. Proteinase inhibitors ”. 


1. Specificity of enzymes. 

To dissolve saliva in 5 times (1 ml of a saliva + 4 ml of water). To pour in 2 test tubes 

on 5 drops of the dissolved saliva. To add 10 drops of 1 % solution of starch in the first 

test tube, in the second – 10 drops of 1 % solution of sucrose. To place both test tubes for 

10 minutes in thermostate at temperature 38 0 С, to execute the Feling’s reaction to 

carbohydrates. 

Feling reaction: To add 3 drops of Feling’s solution (CuSO 4 + NaOH) to 5 drops of 

a researched mix, to warm up. At the presence of glucose or maltose the yellow deposit of 

cuprous hydrate (Cu(OH) 2 ) or a red of cuprous oxide (CuO 2 ) falls out. 

2. Influence of activators and inhibitors on the activity of saliva amylase. 

To pour on 1 ml of the saliva dissolved in 10 times (1 ml of a saliva + 9 ml of water) in 

3 test tubes. To add 1 drop of 1 % solution of sodium chloride in the first test tube, in the 

second – 1 drop of 1 % solution of sulphate of copper, in the third – 1 drop of waters, then 

in each test tube to add 5 drops of 1 % solution of starch, to mix and leave at room 

temperature for 4-5 minutes. To carry out reaction to starch with the Lugol’s reagent(KI 3 ). 

Reaction to starch: To add 1 drop of Lugol’s solution to 5 drops of a researched 

mix to add. At the presence of starch there is a dark blue colouring. 

Theme 4. Medical enzymology. 


The reduction of concentration or full absence of any enzyme in alive system is the 

result of genetic inability to its biosynthesis and is shown by a specific pathology. Now it is 

known about 150 of such enzymopathies (in the exchange of simple and complex proteins, 

in the exchange of carbohydrates, lipids and the nitrogenous bases). 

The change of activity of enzymes can serve in tissues as a criterion of biochemical 

diagnostics, and studying of dynamics of these changes specifies the efficiency of 

treatment. 

Pure enzymes and their mixes are widely used as medical products in therapy, 

surgeries, ophthalmology and other areas of medicine. 



To explain changes of a course of ferment processes and accumulation of 

intermediate products of a metabolism at the congenital (hereditary) and got defects of a 

metabolism – enzymopathies. 


1. To analyze the changes of activity of display enzymes of the blood plasma at a 

pathology of the certain organs and tissues. 

2. To explain the application of ferment preparations and inhibitors of enzymes as 

pharmacological preparations at the certain pathological states. 

Initial level of knowledge - abilities: 

1. To know a structure of glutarate, pyruvate and α-amino acids. 

2. To know a principle of colouremetry and the operating procedure on FEC. 

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Table of contents and sequence of actions Guidelines to the educational actions 

1. Practical studying of determination of 

АsАТ and АlАТ activity in whey of blood. 

1.1. In a writing-book of reports to write 

down 

algorithm of laboratory work. 

2. Medical enzymodiagnostics. 2.1. Modern aspects of 

enzymodiagnostics: 

cellular, secretory and excretory enzymes. 

2.2. Isoenzymes in enzymodiagnostics, 

tissue 

specificity of distribution of enzymes. 

2.3. Change of activity of enzymes of 

plasma 

and whey of blood as diagnostic 

parameters 

of development of pathological processes 

in 

an organism. 

2.4. Application of enzymodiagnostics in 

cardiology, hepathology, nephrology, 

urology, oncology, pulmonology, 

orthopedy, 

and so forth (examples). 

3. Enzymopathology. 3.1. Infringement of the course of ferment 

processes: hereditary and got 

enzymopathies. 

3.2. Congenital defects of a metabolism 

and 

their clinical-laboratory research. 

4. Enzymotherapy in medical practice. 4.1. Use of enzymes as medical products. 

5. Application of inhibitors of enzymes in 

medicine. 


To prepare the abstract message on a theme: 

1. Enzymopathology. 

2. Enzymodiagnostics of a myocardium, a liver diseases. 

3. Enzymotherapy. 

5.1. Inhibitors of enzymes as medical 

drugs. 


Quantitative determination of АsАТ and АlАТ activity in whey of blood. 

Principle of method: the optical density of a solution of hydrazone ketoglutarate and 

pyruvate is determined, that are formed at interaction of alanine (or aspartate) with 

ketoglutarate. Determination is carry out in 1 sm of ditch at length of a wave λ – 500-530 

nanometers. At determination of AsAT – pyruvate is formed by decarboxylation of 

oxaloacetate. 

Reagents: 

1. Reference 2 mM solution of a pyruvate. 

2. 1 mM solution of 2,4-dinitrophenilhydrazine in 1 M НCl. 

3. Solution of NаОН (16 p. in 1000 ml of H 2 O).

12 

4. Substratum of АsАТ: а) 0,1 mM solution of aspartate; 

b) 0,2 mM solution of ketoglutarate; 

c) phosphatic buffer рН-7,4. 

5. Substratum of АlАТ: а) 0,2 mM solution of alanine; 

b) 0,2 mM solution of ketoglutarate; 

c) phosphatic buffer рН-7,4. 

Determination of АsАТ activity. 

Reagents, sequence of 

Skilled test 

The control 

action 

Reagent 4 0,25 ml 0,25 ml 

Physiological solution --- 0,05 ml 

Whey of blood 0,05 ml --- 

Incubation up to 60 minutes. 

At t = 23 о С + + 

Reagent 2 0,25 ml 0,25 ml 

We mix and leave for 20 

minutes at t = 23 о С + + 

Solution of alkali 2,5 ml 2,5 ml 

We mix and in 10 minutes determine the optical density of skilled test against the 

control in a ditch 1sм at the length of a wave λ – 500-530 nanometers. 

Determination of АlАТ activity is carried out the same, but instead of a reagent 4, 

take a reagent 5. 

For calculation of activity we make up a calibrating schedule with solutions 1 and 2. 

In whey of blood of the healthy person the activities of: 

АsАТ = 0,1 – 0,45 mM / g / L 

АlАТ = 0,1 – 0,68 mM /g / L 

Theme 5. Research of a role of cofactors and coenzyme vitamins in 

catalytic activity of enzymes. 


Enzymes as catalysts take part in all without exception biochemical processes that lie 

in the basis of the ability to live of an alive matter. Coenzymes are part of the majority of 

enzymes and practically determine the activity of the last ones. 

Coenzyme (active) forms of water-soluble vitamins take part in regulation of the vital 

biological processes, and also find wide application in clinical practice for correction of 

infringements of processes of a metabolism. 


General purpose: to be able to treat biochemical principles of functioning of vitamins 

(and their coenzyme forms) as components of the person’s food and regulators of ferment 

reactions and exchange processes. To be able to investigate precisely the interrelation of 

pathogenesis of frustration at different hypovitaminoses with functioning of the certain 

parts of a metabolism. 

Concrete purposes: to have precise concept about biological functions of watersoluble 

coenzyme vitamins B 2 , РР, B 6 . To be able to write formulas of the most important

coenzymes, and to understand principles and the purpose of the determination of their 

predecessors in biological liquids. 

Initial level of knowledge - abilities. To interpret the mechanisms of participation of 

vitamins in construction of coenzymes, that catalyse biochemical reactions in organism (on 

an example of NAD + ). 




actions 

1. Practical studying of qualitative 

reactions to vitamins B 2 , РР, B 6 . 

2. Non-protein part of complex 

enzymes. 

1.1. In a writing-book of reports of practical 

occupations to write out algorithm of laboratory 

work. 

2.1. Definition of concept "coenzymes". Watersoluble 

vitamins as predecessors in 

biosynthesis of coenzymes. 

2.2. Definition of concept "cofactors", their 

influence on structure and catalytic activity of 

enzymes. 

3. Coenzymes. 3.1. Structure and properties of coenzymes. 

3.2. Classification of coenzymes by the 

chemical nature. 

3.3. Classification of coenzymes by the type of 

a reaction, that is catalysed: 

а) coenzymes that are the carriers of hydrogen 

protons and electrons; 

b) coenzymes that are the carriers of chemical 

groups; 

c) coenzymes of synthesis, isomerization and 

splittings of carbon - carbon bonds. 

4. The characteristics and 

4.1. Participation of coenzyme forms of 

properties of coenzyme forms of vitamins B 2 and РР in oxidation-reduction 

vitamins B 2 , РР, B 6 . 

reactions. 

4.2. Influence of coenzyme derivative of 

vitamins B 6 on separate parts of a metabolism. 


1. To make up a scheme on a theme “ Coenzyme vitamins ” in an electronic variant. 

2. To write a structural formulas of coenzyme vitamins and explain the mechanism of 

formation 

their biologically active (coenzyme) forms. 


1) Qualitative reaction to vitamin B 2 . 

Principle of method: reaction to vitamin B 2 is based on its ability to restore easily, thus 

solution of B 2 of a yellow color, gets a pink color at the begining, and then becomes 

colourless, because restored form of B 2 is formed. 

Course of work: To add 5 drops of HCl and an iron shaving to 10 drops of vitamin B 2 . 

Observe the release of bubbles of hydrogen. The solution is gradually painted in a pink 

color, and then becomes colourless. 


Principle of method: vitamin B 6 at interaction with a solution of chloric iron forms a 

complex salt such as phenolate of iron of a red color. 

13

Course of work: To add 5 drops of 1 % solution of chloric iron to 5 drops of vitamin B 6 . 

The complex of a red color is formed. 

Theme 6. Research of a role of cofactors and coenzyme vitamins in 

catalytic activity of enzymes. 


Vitamins take part in regulation of life-important processes, in particular, they are 

necessary for functioning a citric acid cycle, bioenergetic processes and other. Vitamins 

find wide application in clinical practice for correction of different infringements of a 

metabolism. 

The principle of qualitative reactions to vitamins underlies many methods of 

determination of the last ones in biological liquids and food stuffs. 



To be able to apply knowledge on biochemistry to an explanation of pathogenetic 

frustrations at vitamin insufficiency, and also their preventive maintenance and 

substantiations of therapy of diseases, in symptomatic complex of which absolute or 

relative insufficiency of vitamins takes place. 

Concrete purposes: to treat a role of vitamins and their biologically active derivatives 

in mechanisms of catalyse at participation of the basic classes of enzymes. To analyze 

ways and mechanisms of regulation of enzyme processes, as the bases of a metabolism 

in an organism in norm and at pathologies. 

Initial level of knowledge - abilities. To know the structure of bioorganic compounds 

which are a part of some vitamins. 




actions 

1. Practical studying of qualitative 

reactions to vitamins. 

2. Theoretical studying of 

coenzyme vitamins. 

1.1. In a writing-book of reports of practical 

occupations to write out algorithm of laboratory 

work. 

2.1. In a writing-book of self-preparation to write 

down formulas of vitamins, their biological role 

and the mechanism of action. 

2.2. To define concepts: "vitamins", 

"antivitamins", "provitamins" (conditions of their 

transformation into vitamins). Explain clinical 

application of vitamins and antivitamins on 

concrete examples. 

2.3. Coenzyme of acetylization (Coenzyme-А) – 

a derivative of pantothenic acid. Biological 

properties of vitamin B 3 , the mechanism of 

action. 

2.4. Coenzymes – derivatives of a folic acid. 

Vitamin Вс (folic acid): biological properties, the 

mechanism of action. 

2.5. Lipoic acid: coenzyme in reactions of 

14

15 

oxidative decarboxylation of ketoacids and 

aerobic oxidation of glucose. 

2.6. Coenzyme thiamine pyrophosphate. Vitamin 

B 1 (thiamine): a structure, biological properties, 

the mechanism of action. 

2.7. Coenzyme carboxybiotin. Vitamin Н (biotin): 

biological properties, the mechanism of action. 

2.8. Coenzymes – derivatives of vitamin B 12. 

Vitamin B 12 (cobalamin): biological properties, 

the mechanism of action. 


Preparation of the review of the scientific literature on the chosen themes: 

1. Role of vitamin C in a metabolism of a connective tissue. 

2. Medical application of vitamin C. 

3. Medical application of vitamin B 12 . 

4. Vitamins and metabolism. 



Principle of method: in alkaline environment thiamin is oxidated in tiochrom by 

ferricianid of potassium. Tiochrom is able to fluoresce by blue color at ultra-violet 

irradiation of a solution on fluoroscope. 

Course of work: To add 5 drops of 5 % solution of K 3 Fe(CN) 6 to 0,5 ml of vitamins, 5 

drops of 30% solution of NaOH and 15drops of butanol (mix carefully!). Observe blue 

fluorescence on fluoroscope. 

2) Quantitative determination of vitamin C in urine. 

Principle of method: the method is based on the ability of vitamin C to reduce 2,6- 

dichlorfenol-indofenol which in the sour environment has red color, and at reduction 

becomes colourless. 

Course of determination: in retort measure 10 ml of urine and 10 ml of H 2 O, add 20 drops 

of 10 % HCl and titrate by 0,001n solution of 2,6- dichlorfenol-indofenol (DNF) up to pink 

color. 

Calculation: 

0,088 А В 

X = 

, where X – the contents of vitamin C in mg/day 

Б 

0,088 - amount of vitamin C, mg (empirical size) 

A – the result of titration by 0,001 n solution of DNF, ml 

B – the volume of urine, taken for titration (10 ml) 

C - daily diureasis (1500-2000 ml) 

Norm: 20 - 30 mg/day. 

113,55-170,33 µmol/day. 

Theme 7. Fundamental principles of metabolism 

Research of functioning of a tricarboxylic acid cycle. 

and energy. 


The tricarboxylic acid cycle is the general way of catabolism of organic substances and 

the important stage of biological oxidation of organic substances. Infringement of

functioning of a tricarboxylic acid cycle results, first of all, in hypoenergetic conditions and 

increase of alternative ways of using of acetyl - CоА, in particular of ketogenesis. 



To study biochemical principles of functioning of a tricarboxylic acid cycle and its 

regulation. 


1. To treat biochemical principles of course of a metabolism. 

2. To treat biochemical principles of functioning of a tricarboxylic acid cycle, its anaplerotic 

reactions and amphibolic role. 

3. To explain biochemical mechanisms of regulation of processes of anabolism and 

catabolism. To explain biochemical mechanisms of regulation of a tricarboxylic acid 

cycle and its key role in a metabolism and in exchange of energy. 

Initial level of knowledge - abilities: to know a structure of organic (bi-and tricarboxylic) 

acids. 




actions 

1. Practical studying of method of 

quantitative determination of a citric 

acid. 

2. Metabolic ways. To define 

concepts catabolic, anabolic and 

amphibolic ways of a metabolism. 

3. Three general stages of 

biomolecules catabolism. 

4. General characteristic of a 

tricarboxylic acid cycle. 

1.1. To write down in a writing-book of reports 

– experiments the algorithm of laboratory work. 

2.1. Catabolism as a set of processes of 

splitting of biomolecules (glucose, amino acids, 

fatty acids up to end-products). 

2.2. Anabolism as a synthesis of the 

biomolecules necessary for construction of 

cellular structures. 

3.1. Stage 1 – disintegration of complex 

macromolecules up to simple components. 

3.2. Stage 2 – endocellular catabolism of 

carbohydrates, lipids and amino acids. 

3.3. Acetyl - CоА – the general end-product of 

the second stage of an endocellular 

metabolism of carbohydrates, lipids and amino 

acids. 

3.4. Stage 3 – oxidation of acetyl - CоА up to 

end-products – CO 2 and H 2 O. 

3.5. General characteristics of TCA and system 

of electron transport in mitochondrion 

membranes (tissue breathing) and interfaces 

with oxidative phosphorylation. 

4.1. Biochemical value of the tricarboxylic acid 

cycle. 

4.2. The scheme of functioning, sequence of 

reactions. 

4.3. Ferment reactions of tricarboxylic acid 

cycle: the characteristics of enzymes. 

4.4. Features of functioning of α-ketoglutarate 

16

17 

5. Regulation of a tricarboxylic acid 

cycle. 

dehydrogenase multienzyme complex. 

4.5.Reactions of substratum phosphorylation in 

the tricarboxylic acid cycle. 

4.6.Total balance of АТP molecules which are 

formed at functioning of a cycle. 

4.7.Anaplerotic and amphibolic reactions of 

tricarboxylic acid cycle. 

5.1. Explain biochemical mechanisms of 

regulation of tricarboxylic acid cycle and its key 

role in a metabolism and in exchange of energy. 


Quantitative determination of a citric acid. 

Principle of a method: the method is based on reaction of interaction of a citric acid 

with salts of copper and iron at their simultaneous presence. Products which are formed in 

reaction, have a yellow-green color; intensity of painting is proportional to amount of a 

citric acid. 

Course of determination: In a test tube measure 1 ml of researched whey, add 7,5 ml 

of water, and then 1ml of 10 % solution of copper sulfate and 0,5 ml of 15 % solution of 

iron - ammoniacal alums in 5 % nitric acid. Parallelly put the standard: the same reagents 

and in the same sequence, but instead of whey we take 1 ml of a citric acid solution of 

known concentration. The contents of test tubes (separately) mix and in 5 minutes 

determine the optical density of solutions on FEC at a blue optical filter in 5 mm ditchs. 

On the basis of got values of optical density of the standard and whey we make a 

proportion and we calculate the contents of a citric acid in whey. 

In norm the concentration of a citric acid in blood is 88 - 156 µmol/l (1,7 – 3,0 mg %). 

Theme 8. Bioenergetic processes: biological oxidation, oxidative 

phosphorylation. Chemiosmotic theory of oxidative phosphorylation. 

Inhibitors and uncouplers of oxidative phosphorylation. 


Biological oxidation is a final stage of disintegration of carbohydrates, lipids and 

proteins in alive organisms. It is realized by multienzyme complexes of internal 

membranes of mitochondria, accompanied by absorption of oxygen and allocation of CO 2 , 

water and energy that is partially accumulated in bonds of АТP that is synthesized. 

Hypoxia, both some natural and synthetic compounds break biological oxidation or 

oxidative phosphorylation, that results in an energy crisis and irreversible changes in an 

organism. 

Mitochondrial system of interface of oxidative processes with generation high-energy 

intermediate АТP, name the oxidative phosphorylation. 

Oxidative phosphorylation allows to organism to absorb a significant share of 

potentially free energy of oxidation of substrata. Chemiosmotic theory allows to make a 

substantiation of the mechanism of oxidative phosphorylation. Oxidative phosphorylation is 

very important process, infringement of its course is incompatible with a life.



To know the bases of bio-energetics of tissues, mechanisms of 

development of the lack of energy and irreversible changes in an organism at 

hypoenergetic states. To study the chemiosmotic theory of oxidative phosphorylation 

and conditions of its effective course. 


1. To treat a role of biological oxidation, tissue breathing and oxidative phosphorylation 

in generation of АТP at aerobic states. 

2. To analyze infringement of synthesis of АТP at conditions of action on an organism 

of the person of pathogenetic factors of a chemical, physical and biological origin. 

Initial level of knowledge - abilities: to know features of oxidation-reduction reactions, to 

be able to explain a biological role of vitamins РР and B 2 and enzymes of the first class in 

these reactions. 




actions 


determination of cytochromoxidase 

activity of mitochondria. 

1.1. To what class and a subclass of enzymes 

these enzymes belong? 

1.2. To write down in a writing-book of reports 

– experiments the algorithm of laboratory work 

and explain a principle of a method of 

determination of cytochromoxidase activity of 

mitochondria. 

2. Reactions of biological oxidation. 2.1. Types of reactions (dehydrogenasation, 

oxidesation, oxigenasation) their biological 

value. 

3. The molecular organization of a 

mitochondrial chain of biological 

oxidation. 

3.1. Components of a respiratory chain as 

oxidation-reduction pairs of cofactors. 

3.2. Molecular complexes of internal 

membranes of mitochondria. 

4. Oxidative phosphorylation. 4.1. Clearing energy in a respiratory chain and 

sites of formation of АТP. 

4.2. Coeficient of oxidative phosphorylation, 

points of interface. 

5. The chemiosmotic theory of 

oxidative phosphorylation – the 

molecular mechanism of generation 

of АТP during biological oxidation. 

6. Inhibitors and uncouplers of 

tissue breathing. 

5.1. Electrochemical gradient of protons (ΔμН 

+ ). Physical and chemical components of an 

electrochemical gradient of protons. 

6.1. Inhibitors of electron’s transport (rotinon, 

amital, cyanids, CO). 

6.2. Uncouplers of oxidative phosphorylation 

(2,4-dinitrophenol, hormones of a thyroid gland, 

free fatty acids). 

6.3. Infringement of synthesis of АТP in 

conditions of action on an organism of the 

person of pathogenic factors of a chemical, 

18

19 

physical and biological origin. 


Determination of cytochromoxidase activity – as a component of a respiratory 

chain of mitochondria. 

Principle of method. Cytochromes – are the complex proteins-hemoproteins, concern 

to a class of enzymes – oxido-reductases, are presented in all animal and vegetative cells. 

Due to that atoms of iron in cytochromes easily change the valency, cytochromes are the 

components of a respiratory chain of mitochondria and carriers of electrons from restored 

ubiquinone to oxygen. In cytochrome system to transfer electrons to oxygen can only 

cytochrome a-a 3 – the cytochromoxidase the structure of which includes copper. 

The method of determination of cytochromoxidase activity is based on ability of 

dimethil-paraphenilen-diamine-chloride (DPPhD) to be the donor of electrons for 

cytochrome c. DPPhD nonenzymatic restores cytochrome c, and itself being oxidized, 

transformed into red pigment, the quantitative formation of which is proportional to 

cytochromoxidase activity of mitochondria. 

Course of work. Fill two test tubes: control and skilled with the reagents under the 

table: 

The contents of test tubes 

Test tube 

Control Skilled 

The phosphate buffer, рН = 7,4 1,0 ml 1,0 ml 

Solution of cytochrome c 2 drops 2 drops 

Suspension of mitochondria 0,5 ml 0,5 ml 

Solution of DPPhD 0,5 ml 0,5 ml 

Ethyl alcohol (ethanol) 1,0 ml --- 

Physiological solution --- 1,0 ml 

Incubation in the thermostate for 5 minutes at 37 о С 

Results: occurrence of a red colouring 

Addition of ethanol inactivates the cytochromoxidase and the ferment transformation of 

cytochrome c. Place test tubes in thermostate for 5 minutes at 37 о С and observe the 

occurrence of a red colouring. 

To draw conclusions by results of the carried out experiment. 

Theme 9. Research of glycolysis – anaerobic oxidation of glucose. 

Gluconeogenesis. 

Actuality of theme. 

Release of the sun energy accumulated in chemical bonds of glucose, begins at 

glycolysis in reaction of substratum oxidative phosphorylation. In this process the part of 

energy of glucose is transformed to energy of macroergic phosphatic bonds which are 

used for resynthesis of АТP, that is the form of energy accessible to use by all tissues at 

performance of different kinds of work (synthesis, mechanical, transport of substances 

through membranes).

Anaerobic conditions temporatily arise at hard work of muscles and then the basic 

process that provides muscles by energy becomes glycolysis – anaerobic oxidation of 

glucose. In norm an end-product of glycolysis – is a lactic acid, - it is quickly resynthesised 

in a liver in glucose and glycogen and partially is oxidized to the water and carbonic gas. 

Many diseases are accompanied by tissue’s hypoxia (cardiovascular diseases, 

respiratory diseases, etc.), that results in accumulation in tissues of a lactic acid, to 

development of acidosis, and, accordingly, to frustration of a metabolism and functions of 

organs. Determination of a lactic acid in blood is the test for an estimation of intensity of 

glycolysis. Hyperlactatemia – a marker of the block of anaerobic ways of glucose 

oxidation. 



To be able to use knowledge about anaerobic exchange of carbohydrates for an 

explanation of a condition of an organism in norm and at a pathology that are 

accompanied by hypoxia. 


1. To treat biochemical laws of an endocellular metabolism of carbohydrates: anaerobic 

glycolysis. 

2. To be able to write the chemism of reactions of glycolysis. 

Initial level of knowledge - abilities: to be able to write a structure of carbohydrates 

(bioorganic chemistry): glucose, fructose. To be able to write a structure of alcohols, 

aldehydes, carbonic acids. 




actions 

1. Practical studying method of 1.1. Determination of lactic acid by method of 

determination of a lactic acid. Uffelman. 

2. Anaerobic glycolysis. Biological 2.1. General characteristics of anaerobic 

role, chemism, the mechanism of oxidation of glucose. 

regulation. 

2.2. Sequence of reactions and enzymes of 

glycolysis. 

2.3. Glycolytic oxidoreduction: substratum 

phosphorylation and shuttle mechanisms of 

glycolytic oxidation of NADP + . Pasteur's effect. 

2.4. Regulation of glycolysis. 

2.5. Alcoholic and other kinds of fermentation. 

2.6. Ways of recycling of a lactic acid. 

2.7. The reasons and consequences of 

3. Theoretical studying of 

gluconeogenesis process. 

hyperlactatemia. 

3.1. What process refers to 

"gluconeogenesis"? In what tissues does it 

actively pass? 

3.2. Physiological value of gluconeogenesis. 

3.3. Metabolic way of gluconeogenesis; 

irreversible reactions of glycolysis, reactions 

and enzymes that allow to evade them. 

3.4. Compartmentalisation of transformation of 

20

21 

pyruvate in phosphoenolpyruvate. 

3.5. Substrata of gluconeogenesis. Lactate and 

alanine as substrata of gluconeogenesis. 

3.6. Glucose-lactate cycle (cycle of Cori). 

3.7. Glucose-alanine cycle. 

3.8. Metabolic and hormonal regulation of 

gluconeogenesis. Regulatory enzymes. Illness 

of Cushing (steroid diabetes). 


1. To write the fermentative reactions of transformation of intermediates in glycolysis. 

2. To make up the scheme of glycolysis. 

3. To create the scheme of regulation of glucose exchange . 


Determination of the end-product of anaerobic glycolysis – lactic acid 

by the method of Uffelman. 

Principle of reaction. At interaction of complex compound of iron phenolate of violet 

color with lactic acid lactate of iron of yellow-green color is formed . 

Course of work: place in a test tube 1 ml of bloods whey, add 5 drops of 1 % solution 

of FeCl 3 and 1,0 ml of phenol solution. We observe occurrence of yellow-green color. 

Theme 10. Research of aerobic oxidation of glucose. 


Aerobic oxidation of glucose plays the important role during ability to live as the main 

process of energy-forming (especially for a nervous tissue, a cardiac muscle). At a 

pathology that is accompanied by hypoxia of tissues (an insult, the heart attack of a 

myocardium, obliterating diseases of vessels, diseases of respiratory organs) an aerobic 

exchange of carbohydrates is broken, it results in frustration of other kinds of a metabolism 

and infringement of functions of organs and tissues. 



To be able to use knowledge about aerobic oxidation of glucose for an explanation of 

energy processes of an organism in norm and their infringement in anaerobic conditions. 

To be able to use in clinic of determination of pyruvic acid for an estimation of an energy 

exchange of the person. 


1. To treat biochemical laws of an endocellular metabolism of carbohydrates: aerobic 

oxidation of glucose. 

2. To analyze changes of the level of pyruvate in clinic for an estimation of a power 

exchange of the person and maintenance with vitamin B 1 . 

Initial level of knowledge - abilities: to be able to write structures of glucose, ketoacids. 

To know a structure of choloenzymes and a role of coenzymes. To know the energy 

exchange.

22 

Reference card for the separate stydy 



actions 


1.1. Determination of pyruvic acid in urine. 

determination of pyruvic acid in 

biological liquids. 

2. Studying of aerobic oxidation of 2.1. Stages of aerobic oxidation of glucose. 

glucose. 

2.2. Oxidative decarboxylation of pyruvate. 

2.2.1. Multienzymatic pyruvate dehydrogenase 

complex – features of functioning at 

participation of three enzymes and five 

coenzymes. The total equation of process. 

2.3. The comparative characteristic of bioenergetics 

aerobic and anaerobic oxidation of 

glucose. 

2.4. Pasteur's effect – switching of anaerobic at 

aerobic oxidation of glucose, feature of 

regulation. 

2.5. Shuttle mechanisms of oxidation of 

glycolytic NADH. Malate-aspartate shuttle of 

transport of regenerative equivalents of 

glycolytic NADH in mitochondrion at aerobic 

3. Clinical value of pyruvic acid 

determination in bioobjects. 

conditions. 

3.1. The reasons and consequences of 

hyperpyruvatemia. 


To prepare the report on a theme: ” Participation of water-soluble vitamins in a 

carbohydrate exchange and their clinical application ”. 


Quantitative determination of pyruvic acid in urine. 

Principle of method: pyruvic acid (PVA) at interaction with 2,4-dinitrophenilhydrazine 

(2,4-DPhG) in the alkaline environment forms the painted complex of yellow-orange color. 

Intensity of painting is directly proportional to the amount of PVA. 


(To use dry utensils). In the first test tube pour 0,5 ml of urine; in the second – 0,5 ml 

of the standard. Then in both test tubes add 1 ml of a solution 2,4-DPhG; and in 5 minutes 

– add 4 ml of concentrated solution of KOH. 

Mix and in 5 minutes determine the optical density (extinction) of solutions at FEC, in 

a ditch on 5 mm with a dark blue optical filter. On the basis of the values of extinction the 

standard and experiment calculate the contents of PVA in daily volume of urine. 

In norm for a day with urine it is allocated: 

10-25 mg (114 – 284 µmol) PVA. 

Increase of excretion is observed at the lack of vitamin B 1 and hypoxia of different 

origin.

Theme 11. Alternative ways of an exchange of monosaccharides. 

Metabolism of fructose and galactose. 


Pentose phosphate and glucuronate ways of an exchange of glucose, an exchange of 

fructose and galactose belong to alternative ways of an exchange of monosaccharides . 

The pentose-phosphate pathway of an exchange of glucose – the source of pentoses 

for synthesis of nucleotides, nucleic acids, coenzymes, except for it is a source of restored 

NADPН which are used at synthesis of fatty acids, cholesterol, steroid hormones and other 

compounds. 

Fructose and galactose are included in a way of an exchange of glucose. At 

infringement of transformation of fructose and galactose fructosemia and galactosemia are 

developed. 


General purpose: to study alternative ways of an exchange of monosaccharides. 

Concrete purposes: to treat biochemical laws of ways of an exchange of 

monosaccharides, pentose-phosphate pathway of oxidation of glucose, a way of uronic 

acids. 

Initial level of knowledge - abilities: to be able to write a structure of monosaccharides, 

hexuronate acids. 




actions 

1. Practical studying of method of 1.1. Quantitative determination of pentoses in whey in 

determination of pentoses in blood. 

2. Pentose-phosphate pathway 

(PPhP) of glucose exchange. 

23 

blood. 

2.1. Biological value and features of functioning of 

pentose-phosphate pathway for different tissues. 

2.2. Sequence of ferment reactions of PPhP: 

а) oxidative stage; 

b) stage of isomeric transformations. 

2.3. Infringement of pentose-phosphate pathway of an 

exchange of glucose in erythrocytes: enzymopathy of 

glucose-6-phosphate-dehydrogenase. 

3. Metabolism of fructose. 3.1. Metabolic way and ferment reactions of 

transformation of fructose in the person organism. 

3.2. Hereditary enzymopathies, connected to genetic 

defects of enzymes synthesis of fructose metabolism – 

intolerance of fructose (fructosemia). 

4. Metabolism of galactose. 4.1. Metabolic way and ferment reactions of 

transformation of galactose in the person organism. 

4.2. Hereditary enzymopathies, connected to genetic 

defects of enzymes synthesis of a galactose 

metabolism – galactosemia. 

Individual self-preparatory work of students: 

1. To make up schemes of metabolic ways of an exchange of carbohydrates. 

2. To estimate a condition of a carbohydrate exchange on biochemical parameters at 

pathological processes. 

3. Clinical value of galactose test.


1. Quantitative determination of pentoses in blood. 

Principle of method: the method is based on color reaction between pentose (ribose or 

deoxiribose) and orcine in the sour environment at presence Fe 3 + . At their interaction the 

complex of green color is formed. Intensity of painting depends on quantity of pentoses. 

Course of work: 

To add in a test tube to 0,5 ml of researched solutions 0,5 ml of 1 % orcine (prepared 

on 0,1 % solution of FeCl 3 in concentrated HCl). Simultaneously to put the standard: to 

take 0,5 ml of pentose solution known concentration and to add 0,5 ml of orcine reagent. 

To place test tubes in a boiling water bath for 40 minutes. Then to extend, add on 5 ml 

of water; to mix the contents of test tubes and to determine optical density of solutions on 

FEC in a ditch on 5 mm at a red optical filter (λ = 665 nanometers). 

On the bases of values of optical density of the standard and a researched solution to 

make a proportion and to calculate the contents of pentoses. 

In norm concentration of pentoses: in blood – it is less 133 µmol/l (2mg %), 

in urine – on the average about 250 mg / day. 

24 

Theme 12. Research of catabolism and biosynthesis of glycogen. 

Regulation of glycogen exchange. 


Knowledge by a doctor of biochemical mechanisms of synthesis and disintegration of 

glycogen as processes of regulating the concentration of glucose in blood, has the great 

importance for an estimation of a carbohydrate exchange condition, understanding of 

pathogenesis of separate hereditary and not hereditary diseases and their diagnostics. 


General purpose: To learn and write schemes of biochemical processes of synthesis and 

disintegration of glycogen, their regulation and importance in pathogenesis of glycogen 

storage diseases type І, type ІІ and aglycogenoses. 


1. To treat functional features and biological value of synthesis and disintegration of 

glycogen in tissues. 

2. To explain molecular-biological bases hereditary of enzymopathies connected with an 

exchange of glycogen. 

Initial level of knowledge - abilities: to be able to write structure of monomers and to 

determine types of bonds in molecules of the most widespread of homo-and 

heteropolysaccharides. 



Table of contents and sequence of Guidelines to educational actions 

actions 

1. Practical studying of revealing of 1.1. Revealing of glycogen in a liver. 

glycogen in a liver. 

2. Synthesis of glycogen and its 

infringements. 

2.1. Structure of glycogen and its biological 

role. 

2.2. Explain mechanisms of biosynthesis of

25 

3. Catabolism of glycogen. 

Regulation of an exchange of 

glycogen. 

glycogen (reactions, enzymes). 

2.3. Genetic infringements of enzyme systems 

of glycogen synthesis, the characteristic of the 

most widespread aglycogenoses. 

3.1. Explain biochemical mechanisms of 

disintegration of glycogen in a liver and 

muscles: reactions, enzymes, a biological role. 

What are the differences of glycogen 

disintegration in a liver and muscles? 

3.2. Hormonal regulation of an exchange of 

glycogen: the cascade mechanism of 

regulation of activity c-АМP-depending 

glycogen phosphorylase and glycogen 

synthetase. 

3.3. Genetic infringements of process of 

disintegration of glycogen (glycogenoses). 


Creation of the scheme in an electronic variant: “ Regulation of processes of synthesis 

and disintegration of glycogen ”. 


Quantitative determination of glucose in blood on an empty stomach. 

Principle of method: glucose at interaction with ortotoluidine forms the product of bluegreen 

color. Intensity of painting is directly proportional to quantity of glucose. 

In norm the concentration of glucose in blood is: 

3,3-5,5 mmol/L. 


In a dry test tube bring 2,0 ml of ortotoluidine reagent (measure by a centrifusion 

measured test tube) and add to it by micropipette 0,2 ml filtrate of blood (filtrate receive by 

mixing blood of 0,2 ml about 1,8 ml of a solution of 3 % three-chloracetic acid with the 

following filtering). 

In other dry test tube bring 2,0 ml of ortotoluidine reagent and 0,2 ml of a standard 

solution of glucose (4,5 mmol/L). 

Place test tubes for 8 minutes in a boiling water bath; then cool the contents of test 

tubes photometrate on FEC in 5 mm a ditch at a red optical filter (length of a wave = 620 

nanometers). 

On the basis of got values of extinction calculate the concentration of glucose in blood. 

Example of calculation: 

extinction of filtrate - 0,28 

extinction of standard - 0,25 

concentration of glucose in a filtrate - X 

concentration of glucose in the standard – 4,5 mmol/L 

4,5 mmol/L - 0,25 

x - 0,28 

x = 

4,5 

0,28 

= 5,0 mmol/L 

0,25

26 

Theme 13. Research of mechanisms of metabolic and hormonal 

regulation of carbohydrates exchange. Diabetes mellitus. 


Regulation of an carbohydrates exchange, at all stages of their synthesis and 

disintegration, is provided by neuro-humoral way. Insulin, modifying different ways of 

carbohydrates metabolism, reduces a level of glucose in blood. Somatotropic, 

аdrenocorticotropic, thyrotropic hormones, thyroxine, adrenaline, glucocorticoids and 

glucagon cause hyperglycemia. 

At the same time, the carbohydrate exchange is adjusted by different factors that 

influence on the enzymes activity by concentration: substrata, metabolites, coenzymes, 

presence or absence of oxygen. 



To know integration of all stages of an exchange of carbohydrates and the control of 

the contents of glucose over blood that is achieved by close interaction of neuro-geneous 

and humoral factors, the main among them hormones which carry out hypo-and 

hyperglycemic influence. 

The most widespread disease which basis is absolute or relative insulin insufficiency is 

the diabetes mellitus. Each future doctor should acquire well mechanisms of hormonal 

regulation of an exchange of carbohydrates as the given knowledge explain biochemical 

and clinical diagnostics, and also approaches to treatment of pathological changes of 

carbohydrates exchange . 


1. To analyze changes of glucose level in blood, the mechanism of its hormonal 

regulation (insulin, glucagon, adrenaline), pathological displays of infringements of 

glucose exchange, diabetes, starvation. 

Initial level of knowledge - abilities. 

To be able to write formulas of mono- and disaccharides (bioorganic chemistry). To know 

endocrine glands which take part in regulation of an exchange of carbohydrates (biology, 

histology). 




actions 

1. Practical studying of the glucose- 

1.1. Principle of the glucose-tolerant test. 

tolerant test. 

2. Hormones - regulators of an 

exchange of glucose, effects and 

mechanisms of influence on a level 

of glucose. 

3. Glucosemia: normal condition 

and its infringement. 

2.1. Mechanisms of hypoglycemic effect of 

insulin. 

2.2. The mechanism of influence on a 

carbohydrate exchange of glucagon, adrenaline, 

glucocorticoids. 

3.1. Normoglycemia: mechanisms of its support. 

3.2. Hypoglycemia: the reasons of occurrence 

and a consequence for an organism. 

3.3. Hyperglycemia – the reasons and

27 

4. Diabetes mellitus: insulindependent 

(type I) and insulinindependent 

(type ІІ). 

complications. 

4.1. Clinical-biochemical characteristic. 

4.2. Diagnostic criteria of a diabetes mellitus: 

а) glucose-tolerant test; 

b) double sugar loading. 

c) glycosilative НbA С1 . 


To prepare the abstract report: “ Metabolic changes and complications at diabetes 

mellitus ”. 


Oral glucose-tolerant test. 

Principle of method: in the morning on an empty stomach determine concentration of 

glucose in the blood taken from a finger. After that give to the patient give to drink a 

solution of glucose at the rate of 1 g glucose on 1 kg of weight of a body. And then, during 

3 hours, in each hour take blood and determine in it the concentration of glucose by 

ortotoluidine method (see the previous lesson). 

On the found sizes build a sugar curve: across postpone time in hours, and on a 

vertical – concentration of glucose in blood. 

In norm in blood of the healthy person: 

1) The maximal increase of a level of glucose is observed in 1 hour after sugar 

loading; but does not exceed “ kidney’s threshold” 

2) by the second o'clock the level of glucose in blood is reduced below initial level. 

3) by the third o'clock concentration of glucose in blood comes back to norm. 

C, mmol/L 12,0 

10,0 

8,0 

6,0 

2 

4,0 1 

2,0 

1 2 3 hour 

1 - sugar curve in norm; 

2 - sugar curve at the latent diabetes mellitus.

Theme 14. Research of catabolism and biosynthesis of 

triacylglycerols and phospolipids. An establishment of molecular 

mechanisms of regulation of lipolysis. 


Lipids in organism of the person carry out energetic, depository, regulatory and 

structural functions, take part in construction and functioning of biological membranes of 

cells. Knowledge of pathobiochemistry of lipid exchange is necessary for the 

understanding of pathogenesis of the most widespread diseases of the person (an 

atherosclerosis, obesity, steatohepatitis, etc.) 



To be able to use knowledge of mobilization of fat from fatty depots, oxidation of lipids 

in tissues in clinic with the purpose of an explanation of pathogenesis of some diseases 

(separate endocrine diseases, obesity, hereditary illnesses). 


1. To treat biochemical functions simple and complex lipids in an organism: participation 

in construction and functioning of biological membranes of cells, spare energetic 

functions, use as predecessors in biosynthesis of biologically active compounds of 

lipid nature. 

2. To treat biochemical laws of an endocellular lipid metabolism: catabolism and 

biosynthesis of triacylglycerols, phospholipids, hormonal regulation of lipolysis. 


1. To know classification of lipids. 

2. To write formulas of glycerol, fatty acids, triacylglycerols, phospholipids. 




actions 

1. Practical value of determination 1.1. Determination of the contents of the 

of the general lipids in whey of general lipids in whey of blood after Councel 

blood. 

method. 

2. Ways of metabolism of simple 

lipids (triacylglycerols). 

Mechanisms of regulation. 

2.1. Biological functions of the main classes of 

lipids: energetic, structural, regulatory. 

2.2. Catabolism of triacylglycerols in adipocytes 

of adipose tissue: sequence of reaction, 

mechanisms of regulation of triglyceridlipase 

activity. 

2.3. Neuro-humoural regulation of lipolysis with 

participation of adrenaline, noradrenaline, 

glucagon and insulin. 

2.4. Oxidation of glycerol: enzyme reactions, 

bio-energetics. 

2.5. Biosynthesis of triacylglycerols. 

2.6. Adipocytes of adipose tissue and their role 

in lipid exchange and bioenergetic processes in 

organism. 

28

29 

2.7. Pathochemistry of obesity. 

3. Exchange of compound lipids. 3.1. Biological role of compound lipids. 

3.2. Biosynthesis of phospotedylcholine. 

3.3. What lipotropic factors (irreplaceable 

components of meal) are necessary for 

synthesis of phosphotedylcholine? 

3.4. Infringement of an exchange of compound 

lipids – steatosis of liver. 


1. To prepare the abstract on the theme "Obesity". 

2. To prepare the abstract report: „ Biochemical mechanisms of steatohepatitis 

development ”. 

3. Make up the scheme and to write chemical reactions of triacylglycerol’s catabolism. 

4. Make up the scheme and to write chemical reactions of glycerol oxidation. 

5. Make up the scheme and to write chemical reactions of triacylglycerols 

biosynthesis. 

6. Make up the scheme and to write chemical reactions of phosphoglycerids 

biosynthesis. 

7. Make up the scheme and to write chemical reactions of lipid exchange. 

Algorithm of laboratory work 

Quantitative determination of lipids by Councel method. 

Principle of method: water-soluble lipid complexes of blood at interaction with the 

water-sated phenol collapse with formation of free lipids. There of whey grows turbid; 

intensity of turbidity depends on lipid quantity in whey. 


To 1,6 ml of Councel reagent (the water-sated phenol) add 0,2 ml of researched 

whey, mix, in 30 min, colorimetrate on FEC in a ditch of 3 mm with a colorless optical filter. 

(At very high turbidity mix is diluted by Councel reagent in 2 times and colorimetrate. 

Multiply result on 2). 

Calculation: multiply value of extinction on 100. 

Norm is 35 - 45 units of optical density. 

Norm of concentration of the general lipids in blood whey is 4 – 8 g/L. 

Take blood for the analysis of all parameters of an lipids exchange in 12 hours after 

meal (on empty stomach). 

Clinical value of the analysis: expressed hyperlipidemia is observed at patients with a 

diabetes mellitus, lipid nephrosis, by a sharp or chronic nephritis, an atherosclerosis and 

some other diseases. It is frequently observed at hypovitaminoses, IHD, pancreatitis, 

abusing alcohol. 

Theme 15. β-oxidation and biosynthesis of fatty acids. Ketone 

bodies. 


Free fatty acids in plasma of blood are supplied after hydrolysis of triglycerids of 

adipose tissue and are the main energy substrata for cells. Determination of fatty acids in 

whey of blood has a diagnostic value. Hyperlipoacidemia it is observed at a diabetes 

mellitus, at hypersecretion of adrenaline and glucocorticoids, at nephroses, starvation. 

Hypolipacidemia is marked at hypothyroidism, the increased secretion of insulin.

Ketone bodies: acetoacetic acid, β-hydroxipropionic acid are synthesized in a liver and 

carry out energetic function. In norm they are formed in small amounts and are oxidized in 

tissues up to carbon oxide and water. Urine of the healthy person gives negative reaction 

on ketone bodies. At diabetes mellitus, Basedow's illness, starvation there is an amplified 

disintegration of fats (and proteins) that’s why ketone bodies are collected in blood, 

causing displacement of blood pH in the sour side (acidosis). It results in development of 

organism intoxication (ketosis). 


General purpose: to study the process of β-oxidations of fatty acids and ways of use of 

acetyl-CоА in organism, and also biosynthesis of ketone bodies and consequence of 

ketosis. 


1. To treat biochemical laws of fatty acids metabolism. 

2. To explain biochemical bases of occurrence and development of infringements of 

lipids exchange. 

Initial level of knowledge - abilities: to be able to write a structure of fatty acids, ketone 

bodies. 




actions 

1. Practical value of determination 1.1. Determination of ketone bodies in urine. 

of ketone bodies in urine. 

2. Oxidation of fatty acids. 2.1. Oxidation of fatty acids (β-oxidation) 

а) Activation of fatty acids; 

b) Role of carnitine in transport of fatty acids in 

mitochondria; 

c) Sequence of ferment reactions. 

2.2. Energetics of β-oxidation of fatty acids. 

3. Biosynthesis of fatty acids. 3.1. Biosynthesis of the supreme fatty acids, metabolic 

sources. 

3.2. Biosynthesis of the saturated fatty acids 

(palmitate). 

3.3. Synthesis of malonyl-CoА. 

3.4. Features of fatty acids synthase structure, acetyltransport 

protein. Sources of NADPH for biosynthesis 

of fatty acids. 

3.5.Regulation of biosynthesis of fatty acids. 

3.6.Elongation of the saturated fatty acids. 

4. Exchange of ketone bodies. 4.1. Ketone bodies. Reactions of biosynthesis and 

recycling of ketone bodies, their physiological value. 

4.2. Metabolism of ketone bodies in conditions of a 

pathology. Mechanisms of superfluous rising of the 

contents of ketone bodies at a diabetes mellitus and 

starvation. 

4.3. Consequences of ketosis. 

Individual self-prepratory work of students. 

30

To create schemes: 

1. Transport and deposition of lipids. 

2. To prepare the abstract report „ Reasons and consequences of ketosis ”, „ Acetonemia 

syndrome of children ” . 


1. Revealing of acetone (ketone bodies) in urine (reactions with sodium nitropruside 

and chloride of iron). Revealing of ketone bodies in urine by express - method. Value of 

revealing of ketone bodies in blood and urine for medicine. Determination of ketone bodies 

in urine. 

Principle of method: acetone and acetoacetic acid at interaction with sodium 

nitropruside (Na 2 [Fe (CN) 6NO]) in the alkaline environment form products of reaction of 

orange color. Add an acetic ice acid (100 %)these products get cherry color. 


In centrifugal test tube pour 0,5 ml of researched urine, then 0,5 ml of 10 % of a 

solution of alkali (NaOH) and 0,5 ml of sodium nitropruside solution. At hashing the orange 

coloring develops. Addition of an ice acetic acid of 0,5 ml causes cherry painting. 

In blood concentration of ketone bodies is equaled 0,034-0,43 mmol/L (1-2 mg per %). 

In norm in urine ketone bodies are not found out. They appear at a sugar and steroid 

diabetes, at starvation, at lack of carbohydrates of meal. 

31 

Theme 16. Biosynthesis and biotransformation of cholesterol. 

Research of infringements of lipid exchange: steatorrhea, 

atherosclerosis, obesity, hyperlipoproteinemias. 


The most widespread diseases of mankind – cardiovascular illnesses and their heavy 

complications – ischemic illness of heart (IHD), ischemic illness of a brain and the bottom 

extremities are connected with infringement of cholesterol exchange and development of 

an atherosclerosis. Obesity – the epidemic of 21-st century. Its pathogenetic basis is made 

with infringement of triacylglycerols exchange. 

The diabetes mellitus is accompanied by development of micro- and 

macroangiopathies, last caused by frustration of an exchange of cholesterol. 

Knowledge of lipid metabolism is necessary for mastering of ethyology, pathogenesis, 

risk factors of an atherosclerosis and its metabolic and pharmacological correction. 


General purpose: studying of a metabolism and biotransformation of cholesterol is 

necessary for the deep analysis of different forms of a pathology of lipid exchange which 

make a molecular basis of the most widespread diseases of mankind. 


1. To treat biochemical laws of regulation of cholesterol biosynthesis and its 

biotransformation: etherification, formations of bilious acids, steroid hormones, 

vitamin D 3 . 

2. To analyze changes in the system of circulatory transport of lipids at a pathology of 

an lipid exchange(atherosclerosis, obesity, diabetes mellitus). 

3. To explain biochemical bases of development of a pathology of lipid exchange: 

genetic and got (atherosclerosis, adiposity, diabetes mellitus).

32 

Initial level of knowledge - abilities: to know a structure and functions of lipids, 

hormones of glands of internal secretion. 




actions 


1.1. Determination of contents of cholesterol in 

determination of cholesterol in whey of blood. 

whey of blood by Ilc. 

2. Biosynthesis of cholesterol. 2.1. Biological role of cholesterol. 

2.2. Circulatory transport of cholesterol. Norm of 

the contents of cholesterol in whey of blood. 

Transport of cholesterol, change in system of 

lipoproteins at a pathology, their functional 

value. 

2.3. The scheme of reactions of cholesterol 

synthesis. Key reaction of biosynthesis. 

3. Ways of biotransformation of 

cholesterol. 

2.4. Regulation of cholesterol synthesis. 

3.1. The mechanism of cholesterol etherefication 

. 

3.2. Biosynthesis of bile acids from cholesterol. 

3.3. Biosynthesis of steroid hormones from 

cholesterol. 

3.4. Formation of vitamin D 3 from cholesterol. 

4. Pathology of lipid exchange. 4.1. Mechanisms of development of an 

atherosclerosis. 

4.2. Mechanisms of obesity development. 

4.3. Infringement of lipid exchange at a diabetes 

mellitus (macroangiopathies, ketosis), 

mechanisms of their development. 

4.4. Steatorrhea, the mechanism of its 

development. 

4.5. The clinical-biochemical characteristic of 

primary and secondary lipoproteinemias by 

WHO classification. 


1. Creation of the scheme in an electronic variant on the theme “ Biosynthesis of 

cholesterol and its regulation ”, “ Transport of lipids ”. 

2. Estimation of infringements of lipid exchange at pathological conditions by biochemical 

parameters . 


Quantitative determination of cholesterol in blood by Ilc. 

Principle of method: Cholesterol at interaction with acetic anhydride at the presence of 

the concentrated sulfuric and acetic acids forms products of reaction of blue-green color. 

Intensity of painting is directly proportional to quantity of cholesterol. 

Course of work.

Test tubes and micropipettes should be dry. In two centrifugal measuring test tubes 

pour 2 ml of Ilc reagent (carefully, the concentrated acids!!!); then in one of them measure 

0,1 ml of a standard solution of cholesterol, and in the second - 0,1 ml of researched whey. 

Cautiously shake the contents of test tubes cautiously shake for hashing and leave for 20 

minutes. Then photometrate solutions on FEC at a red optical filter (λ = 630-690 

nanometers). On the basis of got values of optical density (exstinction) of the standard and 

whey make a proportion and calculate the concentration of cholesterol in researched 

whey. 

In norm concentration of cholesterol (general) in whey of the adult person is 3,1 – 5,2 

mmol/L (120-250 mg of %). Ethero-bonded cholesterol of whey is 2/3 of general 

cholesterol. 

33 

Theme 17. Research of transformations of amino acids 

(transamination, deamination, decarboxylation). 


In the process of decarboxylation of amino acids in tissues active compounds – 

biogenic amines (GABA, dopamine, histamine, serotonin, noradrenaline, adrenaline) and 

amines – endogenic toxins (putrescine, kadaverine) are formed at rotting tissues in 

intestines. 

In the process of deamination of amino acids ketoacids are formed which can be used 

in the process of transamination for synthesis of essential amino acids. 

Alanine aminotransferase (АlАТ) is hepatospesific enzyme, aspartate 

aminotransferase (АsАТ) is cardiosresific enzyme. These enzymes are used for 

enzymodiagnostics of diseases of a liver and a myocardium. 


General purpose: 

1. To acquire a principle of a method and an estimation of clinical value of 

determination of AlAT and AsАТ. 

2. To reproduce in experiment the process of reamination with the using of glutamic 

and pyruvatic acids. 


1. To treat biochemical laws of an endocellular metabolism of amino acids: processes of 

transamination, deamination, decarboxylation to explain biological action of formed 

biogenic amines: serotonine, histamine, scale – amino-propionic acid. 

2. To write the equation of reaction of reamination of glutamic and pyruvatic acids. 

Initial level of knowledge - abilities: to be able to write formulas of 20 amino acids. 

To apply a method of a chromatography to revealing amino acids. 




actions 


1.1. Determination of aminotransferase activity 

determination of aminotransferase in whey of blood. 

activity in whey of blood. 

2. Poole of free amino acids in an 

organism. 

2.1. Ways of obtaining of free amino acids in 

tissues.

34 

3. The general ways of 

transformation of amino acids. 

2.2. Ways of using of free amino acids in 

tissues. 

3.1. Transamination of amino acids: reactions 

and their biochemical value. 

3.2. To write the equation of reamination 

reactions of glutamic acid and pyruvate. 

3.3. The mechanism of aminotransferases 

action. 

3.4. Direct and indirect of deamination of free 

L-amino acids in tissues. 

3.5. Decarboxylation of L-amino acids in the 

organism of the person. Physiological value of 

the formed products. 

3.6. Oxidation of biogenic amines. 


1. To make up schemes and to write biochemical reactions of transformation of amino 

acids in metabolic processes of deamination, transamination and decarboxylation. 

2. To analyze and treat molecular mechanisms of regulation of amino acids exchange 

and separate metabolic ways. 

3. To prepare the abstract message: ” Clinical value of aminotransferases determination 

”. 


1. To reproduce in experiment process of transamination, using glutamic and pyruvatic 

acids. 

Principle of method: is based on the fact that in process of ferment transfer of an 

amino group with glutamic acid to ketoacid (PVA) alanine is formed. Conclusion about 

reamination is based on the occurence of alanine on a chromatogramme. 

Course of work: in two test tubes measure on 0,5 ml of a glutamic acid solution, 0,5 ml 

of PVA solution, 1 ml of a potassium carbonate solution and 0,25 ml of a 

monobromacetate solution. 

In 1 test tube (experiment) add 0,5 ml of resh muscular mess, in the second test tube - 

muscular mess which is preliminary boiled during 1-2 minutes. Put both test tubes in 

thermostate at t = 37 о С for 15 minutes. Then add in each 0,25 ml of an acetic acid and boil 

2-3 minutes before full sedimentation of proteins. Filter contents of test tubes. 

Chromatographate filtrates. With this purpose put a drop of filtrates from test tubes on 

a line of start of two lines of filtering paper (experiment and control), each time drying with 

air. Strips of paper put in test tubes at the bottom of which there is phenol sated with 

water. Place test tubes in a support in thermostate for 1 hour at 35-40 о С. Take out strips of 

a paper after that, mark a front line (finish) of solvent, dry for 10-15 minutes at 100 о С, and 

then use a solution of ninhydrin. Dry again. 

For each certain stain calculate factor Rf under the formula: Rf = 1/h 

where, 1-is the distance from start to the center of a stain, 

h- is the distance from start to finish (distance which the solvent passed). 

Conclude the received data glue the chromatogramme to the report.

35 

Rf amino acids (for phenol): 

Aspartic acid – 0,07 Arginine – 0,41 

Glutamic acid – 0,16 Tyrosine – 0,52 

Cysteine – 0,19 Alanine – 0,55 

Glycine – 0,30 Phenylalanine – 0,78 

Methionine – 0,39 Leucine – 0,79 

2. Determination of aminotransferase activity. 

Principle of method. Aminotransferases – enzymes that contain 

phosphopyridoxamines as coenzymes, catalyse reverse transfer of amino groups with α- 

amino acids to α-ketoacids. Determination of concentration of α-ketoacids which are 

formed at transamination underlies dinitrophenil hydrasine method of determination of 

transaminases activity. 

Course of work. Determination of aspartate aminotransferase (АsАТ). Bring 0,5 ml 

of a substratum-buffer mix in a test tube for determination of АsАТ, place in thermostate at 

t =37 о С for 5 minutes, add 0,1 ml of whey and incubate at t =37 о С for 60 minutes. Then 

add 0,5 ml of a dinitrophenil hydrasine solution and leave for 20 minutes at a room 

temperature. Add 5 ml of 0,4 M of NaOH solution, mix and leave for 10 minutes. 

Measure optical density of tests that are investigated at λ = 560 nanometers. 

Determination of alanine aminotransferaseactivity (АlАТ). Bring in a test tube 0,5 

ml of a substratum-buffer mix for determination of АlАТ, place in thermostate at t =37 о С for 

5 minutes, add 0,1 ml of whey and incubate at t =37 о С for 30 minutes. Then add 0,5 ml of 

a dinitrophenyl hydrasine solution and leave for 20 minutes at a room temperature. Add 5 

ml of 0,4 M of NaOH solution, mix and leave for 10 minutes. Measure optical density of 

tests that are investigated. Process control tests like the experimental ones, but add whey 

after incubation of tests. 

Carry out the calculation of activity of enzymes by a calibrating schedule. At the 

creation of the calibrating schedule on an axis of ordinates mark values of optical density, 

and on an axis of absciss mark the contents of PVA. 

Physiological levels: for АsАТ – 0,1-0,5, for АlАТ – 0,1-0,7 µmol of PVA on 1 ml of 

whey for 1 hour of incubation at t =37 0 С. 

Theme 18. Research of processes of ammonia detoxication and 

biosynthesis of urea. 


Understanding of biochemical processes which underlie neutralization of ammonia, 

and also correct treatment of change of concentration of urea in blood and urine, are 

extremely necessary for the practising doctor for correct determination of the diagnosis, an 

estimation of a condition of sick and dynamic supervision over efficiency of the appointed 

treatment. Measurement of the contents of urea is the important parameter of a final stage 

of protein exchange. In norm on the average 25-30g of urea is allocated with urine in a 

day. At hepatic insufficiency synthesis of urea is weakened, therefore its concentration in 

blood decreases. 

At kidney or cardiovascular insufficiency allocation of urea with urine decreases, and 

the level in blood raises, therefore the uremia - a poisoning of an organism with products 

of a nitrogenous exchange develops. The quantity of urea in blood increases also at

amplification of protein’s catabolism (radiation sickness, malignant formations, 

intoxications, fever, etc.). The analysis is obligatory at inspection and estimations of a 

condition of the sick with kidney’s diseases and a liver. 



To be able to analyze changes of concentration of urea in blood and to interpret the 

received results. To master the unified method of quantitative determination of urea in 

blood. To study stages of biosynthesis of urea. 


1. To treat metabolic laws of formation and neutralization of ammonia, circular transport 

of ammonia, biosynthesis of urea. 

2. To analyze changes in systems of transport and neutralization of ammonia at genetic 

anomalies of enzymes of a metabolism of ammonia. 

Initial level of knowledge - skills: to know chemical structure of urea and substances 

which take part during neutralization of ammonia. 




actions 


1.1. Determination of the contents of urea in 

determination of the contents of whey of blood. 

urea in whey of blood. 

2. Formation of ammonia and 

processes of its urgent 

neutralization in an organism. 

2.1. Ways of ammonia formation. 

2.2. To explain molecular mechanisms of toxic 

influence of ammonia on an organism. 

2.3. Circulatory transport of ammonia. 

2.4. Explain 4 molecular mechanisms of urgent 

neutralization of ammonia. What ways of use 

by an organism of the formed nitrogenous 

products exist? 

3. Biosynthesis of urea. 3.1. What processes deliver free ammonia at 

the first stage of synthesis of urea? Write the 

reactions of glutamine and carbomailphosphate 

synthesis. 

3.2. To give a general characteristic of process 

of biosynthesis of urea, chemism of reactions, 

the name of enzymes. 

4. Clinical value of research of urea 

in blood and urine. 

4.1. Primary and secondary hyperamoniemias 

(the reasons and consequences). 

4.2. Explain changes of protein exchange at 

uremia, hepatic and kidney insufficiency, 

radiation sickness. 

4.3. Name the digital values of the contents in 

norm in blood of ammonia, urea, daily their 

allocation with urine. At disease of what organs 

and systems is it necessary for the doctor to 

appoint the analysis for the contents of urea in 

blood and urine? 

36


1. To estimate biochemical parameters of infringement of processes of ammonia 

neutralization at the congenital and got defects of a metabolism. 

2. To prepare schemes in electronic variant: 

a) оrnithine cycle of ammonia neutralization; 

b) Interrelation of process of urea formation with deamination, transamination of amino 

acids and an energetic exchange. 


Quantitative determination of the contents of urea in blood with diacetylmonooxime. 

Principle of method: urea forms with diacetylmonooxime the complex of rose-red color. 

Intensity of painting is proportional to the concentration of urea in blood. 


1 test tube – experiment 2 test tube – standard 3 test tube – control 

0,1 ml of experiment 

2 ml of working solution 

0,1 ml of the standard 


0,1 ml of water 


To boil all test tubes on a water bath for 10 minutes, after cooling to colorimetrate contrary 

to the control at λ =540 nanometers (a green optical filter), a ditch of 5 mm. 

Calculation: 

Еex. 

Urea =---------* 16,65 = mmol/L, where Еex. – extinction of skilled test, 

Est. 

Est. – extinction of standard test (in 1 ml is 16,65 mmol/L of urea) 

Concentration of urea in whey of blood is 3,3-8,3 mmol/L 

Excretion with urine is 20-30 g/day 

37 

Theme 19. Analysis of amino acids using in the biosynthetic 

processes (biosynthesis of glutathione, creatine and porphyrins) . 


Creatinine is an end-product of an exchange of tissue’s creatine phosphate. Increased 

excretion of creatinine in urine is observed at patients with a fever sharp infections, at a 

diabetes mellitus. Creatinine – nitrogenous slag, but due to nitrogen excretion function of 

kidneys clears blood At a pathology of kidneys that is accompanied by infringement of 

nitrogen excretion function, and also at sharp heart insufficiency, creatinine collects in 

blood, and its allocation with urine is reduced. Therefore quantitative determination of 

creatinine in blood and urine is one of obligatory analyses at diseases of kidneys. Creatine 

phosphate is applied in clinic for the treatment of patients with cardiovascular insufficiency. 

As a result of an exchange in an organism of hemoglobin, mioglobin, cytochromes and 

other hemoproteids that contain tetrapyrole’s prostetic groups, there is a formation of 

pigments – porphyrins. There are hereditary infringements of porphyrins synthesis – 

porphyrias. Depending on a place of display of a specific fermental defect, erythropoietic 

and hepatic porphyrias are distinguished. The basic clinical displays of porphyrias are 

increase of light sensitivity, neurologic infringements.

38 

The purpose and initial level of knowledge. 


To acquire a method of quantitative determination of creatinine in urine, to be able to 

estimate results of the analysis. To know color reaction on creatine with pycrine acid, to be 

able to calculate the result of the analysis. 

To study the process of porphyrins synthesis. To be able to use knowledge of 

hereditary infringements of an porphyrins exchange. 


1. To explain biochemical mechanisms of formation of creatine and creatinine. 

2. To analyze clinical value of infringements of an creatine and creatinine exchange. 

3. To treat a role of glutathione in an exchange of organic peroxides. 

4. To explain biochemical principles of regulation of an porphyrins exchange, occurrence 

and developments of hereditary infringements of porphyrins synthesis – porphyrias. 

Initial level of knowledge – abilities: to be able to write a structure of amino acids – 

predecessors of creatine and glutathione synthesis; to be able to write a structure of 

predecessors of pyrrole’s rings of porphyrins (glycine, succinyl-CоА). To know a structure 

of porphyrins. 




actions 

1. Practical value of creatinine 1.1. Determination of creatinine in urine. 

determination in urine. 

2. Exchange of creatine. 2.1. Biological role of creatinphosphate. 

2.2. Biosynthesis of creatine. 

2.2.1. Precursors of creatine biosynthesis. 

2.2.2. Features of the second stage of creatine 

biosynthesis – transmetilation of glycociamine 

(guadenine acetate). Sources of CH 3 - groups. 

2.3. Reaction of creatine phosphorylation. 

3. Clinical-biochemical value of 

infringements of an creatine 

exchange. 

3.1. Clinical value of determination of the 

creatine and creatinine contents in blood and 

urine. 

3.2. Clinical value of creatine phosphokinase 

determination. Isoforms of creatine 

phosphokinase. 

4. Glutathione. 4.1. Precursors of glutathione biosynthesis. 

4.2. Role of glutathione in an exchange of 

organic peroxides. 

5. Metabolism of porphyrins. 5.1. Porphyrins: structure, biological role. 

5.2. Reactions of protoporphyrin IX 

biosynthesis; formation of heme. 

5.3. Regulation of porphyrins synthesis. 

5.4. Hereditary infringements of an porphyrins 

exchange (enzymopathies): erythropoetic 

porphiria, hepatic porphyrias, neurologic 

infringements, photodermatites.


To prepare the report on the theme: 

1. Modern biochemical methods of research of function of kidneys. 

2. Biological role of glutathione system in antioxidant protection. 

3. To make the scheme of heme synthesis and to specify on it enzymes which 

infringement of synthesis results in development of porphyrias. 

4. To make the comparative scheme of erythropoetic and hepatic porphyrias (a kind of 

porphyria, defect of enzyme, plasma of blood, urine, excrement, a dominating 

syndrome). 


Quantitative determination of creatinine in urine. 

Principle of method: creatinine at interaction with pycrine acid in the alkaline 

environment forms the painted compounds which intensity of painting is proportional to 

creatinine concentration in urine. 

Ecscretion of creatinine : ♂ - 1,0-2,0 g/day 

♀ - 0,8-1,8 g/day 

Course of work: 

In a flask in volume of 100 ml pour 1 ml of urine, 1 ml NaOH, 1,5 ml of pycrine acid 

solution, mix and leave for 10 minutes. Then make volume up to 100 ml by dist. water. 

Parallely control: 1 ml of H 2 O, 1 ml of NaOH, 1,5 ml of pycrine acid, mix and make the 

volume up to 100 ml dist. water. 

Photometrate contrary to control on FEC with a green optical filter in a ditch on 5 mm. 

With the help of calibrating schedule determine the quantity of creatinine in 1 ml of urine 

and calculate its quantity in urine for day (1500-2000 ml). 

Calibrating schedule of quantitative determination of creatinine in 1 ml of urine. 

Е 

39 

0,25 

0,20 

0,15 

0,10 

0,51,01,52,0 C, mg/ml

40 

FINAL MODULAR CONTROL. 

Module 2. GENERAL PRINCIPLES OF METABOLISM. METABOLISM OF 

CARBOHYDRATES, Lipids, AMINO ACIDS 

AND ITS REGULATION. 

Purpose. 1. To apply knowledge of the general principles of a metabolism during the 

subsequent study and professional work for the analysis of pathogenesis and 

clinical diagnostics of diseases, and also the control of medicamentous therapy 

and a substantiation of metabolic therapy of diseases. 

2. To use theoretical and practical skills on biochemistry and pathobiochemistry 

at 

studying of clinical disciplines and in clinical biochemistry. 

The list of questions for the final modular control. 

І. Theoretical preparation. 

1. Biochemical components of a cell, their biochemical functions. Classes of 

biomolecules. Hierarchy of biomolecules, their origin. 

2. Enzymes: definition; properties of enzymes as biological catalysts. 

3. Classification and the nomenclature of enzymes, the characteristic of separate classes 

of enzymes. 

4. Structure and mechanisms of action of enzymes. Active and allosteric (regulatory) 

centers. 

5. Cofactors and coenzymes. Structure and properties of coenzymes; vitamins as 

predecessors in biosynthesis of coenzymes. 

6. Coenzemes: types of reactions, that catalyse separate classes of coenzymes. 

7. Isoenzymes, features of a structure and functioning, importance in diagnostics of 

diseases. 

8. Mechanisms of action and kinetics of ferment reactions: dependence of speed of 

reaction on the concentration of a substratum, рН and temperature. 

9. Activators and inhibitors of enzymes: examples and mechanisms of action. 

10. Types of inhibition of enzymes: convertible (competitive, non-competitive) and 

irreversible inhibition. 

11. Regulation of ferment processes. Ways and mechanisms of regulation: allosteric 

enzymes; covalent modification of enzymes. 

12. Cyclic nucleotides (cАМP, cGМP) as regulators of ferment reactions and biological 

functions of a cell. 

13. Enzymopathies – are congenital (hereditary) defects of a metabolism of carbohydrates, 

amino acids, porphyrins, purines. 

14. Enzymodiagnostics of pathological processes and diseases. 

15. Enzymotherapy – application of enzymes, their activators and inhibitors in medicine. 

16. Principles and methods of revealing of enzymes in bioobjects. Units of measurements 

of activity and amount of enzymes. 

17. Metabolism - the general principles of course of catabolic and of anabolic processes. 

18. The general stages of endocellular catabolism of biomolecules: proteins, 

carbohydrates, lipids. 

19. Tricarboxylic acid cycle. Localization, sequence of ferment reactions, importance in a 

metabolism.

20. Energetic balance of a tricarboxylic acid cycle. Physiologic importance of reactions 

TCA. 

21. Substratum phosphorylation of ATP. 

22. Reactions of biological oxidation; types of reactions (dehydrogenasational, 

oxidasational, oxigenisational) and their biological importance. Tissue breathing. 

23. Enzymes of biological oxidation in mitochondria: pyridin-, flavin-dependent 

dehydrogenasis, cytochromes. 

24. Sequence of components of a respiratory chain of mitochondria. Molecular complexes 

of internal membranes of mitochondria. 

25. Oxidative phosphorylation: points of interface of electron’s transport and of 

phosphorylation, coeficient of oxidative phosphorylation. 

26. Chemiosmotic theiry of oxidative phosphorylation, АТP-synthetase of mitochondria. 

27. Inhibitors of electron’s transport and uncouplers of oxidative phosphorylation. 

28. Microsomal oxidation: cytochrome P-450; the molecular organization of a chain of 

electron’s carrying. 

29. Aerobic and аnaerobic oxidation of glucose, general characteristic of processes. 

30. Аnaerobic oxidation of glucose. Sequence of reactions and enzymes of glycolysis. 

31. Aerobic oxidation of glucose. Stages of transformation of glucose up to CO 2 and H 2 O. 

32. Oxidative decarboxylatoin of puryvate. Enzymes, coenzymes and sequence of 

reactions in multienzyme complex. 

33. Glycolytic oxidoreduction: substrate phosphorylation and shuttle mechanisms of 

oxidation of glycolytic NADH. 

34. The comparative characteristic of bio-energetics of aerobic and anaerobic oxidations of 

glucose, Pasteur's effect. 

35. Phosphorylytic way of glycogen splitting in a liver and muscles. Regulation of glycogen 

phosphorylase activity. 

36. Biosynthesis of glycogen: enzyme reactions, physiologic value. Regulation of glycogen 

synthase activity. 

37. Mechanisms of reciprocal regulation of glycogenolysis and glycogenesis due to 

cascade cАМP-dependent phosphorylation of proteins-enzymes. 

38. Role of adrenaline, glucagon and insulin in hormonal regulation of an glycogen 

exchange in muscles and liver. 

39. Genetic infringements of glycogen metabolism (glycogenosis, aglycogenosis). 

40. Gluconeogenesis: substrata, enzymes and physiologic value of process. 

41. Glucose-lactate (cycle of Cori) and glucose-alanine cycles. 

42. Glucose of blood (glucosemia): normoglycemia, hypo- and hyperglycemias, glucosuria. 

Diabetes mellitus – pathology of glucose exchange. 

43. Hormonal regulation of concentration and exchange of glucose of blood. 

44. Pentose-phosphate pathway of glucose oxidation: the scheme of process and 

biological value. 

45. Metabolic ways of fructose and galactose transformations; hereditary enzymopathies of 

their exchange. 

46. Catabolism of triacylglycerols in adipocytes of an adipose tissue: sequence of 

reactions, mechanisms of regulation of triglycerydlipase activity. 

47. Neuro-humoral regulation of lipolysis at participation of adrenaline (epinephrine), 

noradrenaline (norepinephrine), glucagon and insulin. 

48. Reactions of fatty acids oxidation (β-oxidation); role of carnitine in transport of fatty 

acids in mitochondria. 

49. Energetic value of β-oxidations of fatty acids in cells. 

50. Oxidation of glycerol: enzyme reactions, bio-energetics. 

41

51. Ketone bodies. Reactions of biosynthesis and recycling of ketone bodies, physiological 

value. 

52. Infringement of ketone bodies exchange in conditions of a pathology (diabetes mellitus, 

starvation). 

53. Biosynthesis of the supreme fatty acids: reactions of biosynthesis of the saturated fatty 

acids (palmitate) and regulation of process. 

54. Biosynthesis of triacylglycerols and phophoglycerids. 

55. Metabolism of sphingolipids. Genetic anomalies of an sphingolipids – sphingolipidoses. 

56. Biosynthesis of cholesterol: the scheme of reactions, regulation of cholesterol 

synthesis. 

57. Ways of biotransformation of cholesterol: etherification; formations of bile acids, steroid 

hormones, vitamin D 3 . 

58. Circulatory transport and lipids deposition in the adipose tissue. Lipoproteinlipase of 

endotelium. 

59. Lipoproteins of blood plasma: lipid and protein (аpoprotein) structure. 

Hyperlipoproteinemias. 

60. Pathologies of lipid exchange: atherosclerosis, adiposity, diabetes mellitus. 

61. Poole of free amino acids in an organism: ways of receipt and use of free amino acids 

in tissues. 

62. Transamination of amino acids: reactions and their biochemical value, mechanisms of 

aminotransferases action. 

63. Direct and indirect deamination of free L-amino acids in tissues. 

64. Decarboxylation of L-amino acids in the person organism. Physiological value of the 

formed products. Oxidation of biogenic amines. 

65. Ways of formation and neutralization of ammonia in organism. 

66. Biosynthesis of urea: sequence of enzyme reactions of biosynthesis, genetic anomalies 

of enzymes of urea cycle. 

67. The general ways of carbon skeletons metabolism of amino acids in the person 

organism. Glucogenic and ketogenic amino acids. 

68. Biosynthesis and biological role of creatine and creatine phosphate. 

69. Glutathione: structure, biosynthesis and biological functions of glutathione. 

70. The specialized ways of cyclic amino acids metabolism – phenylalanine and tyrosine. 

71. Hereditary enzymopathies of cyclic amino acids exchange – phenylalanine and 

tyrosine. 

72. Exchange of cyclic amino acid of tryptophan and its hereditary enzymopathies. 

73. Metabolism of porphyrins: structure of heme; the scheme of reactions of 

protoporphyrins IX and heme biosynthesis. 

74. Hereditary infringements of porphyrins biosynthesis, types of porphyrias. 

42

43 

ІІ. Practical preparation. 

1. Preparation of a material (biological liquids, cells, subcellular organels) to carrying out 

off biochemical researches. 

2. Making up of schedules of dependence of speed of ferment reaction on the 

concentration of a substratum, changes of рН of environment and temperature. 

3. To explain the mechanism of transformation of a substratum at catalytic action of 

enzymes. 

4. Writing structural formulas of coenzyme vitamins and to explain the mechanism of 

formation of their biologically active (complex) forms. 

5. To explain the mechanism of course of ferment reactions at participation of 

coenzymes. 

6. Reproduction of consecutive stages of the general principles of catabolism of proteins, 

carbohydrates and lipids. 

7. Writing the sequence of reactions of transformation of intermediates in a tricarboxylic 

acid cycle. 

8. To draw the scheme and to explain a structure and the mechanism of action of a chain 

of electron’s transport. 

9. To explain on the basis of chemiosmotic theory the mechanism of interface, oxidation 

and phosphorylation, synthesis of АТP in a respiratory chain. 

10. Revealing of glucose in solutions by Trommer’s and Feling’s reactions. To write the 

equation. 

Determination of glucose in blood by glucose-oxidative (Gorodetsky's) method. To 

write the equation of reaction which underlies this method. What is a normal 

concentration of glucose in blood of the person? 

11. Determination of glucose in blood by Hagendorn-Iensen method. Explain the 

principle. 

12. Revealing of fructose by Selivanov's reaction. Principle of method. 

Revealing of glycogen in a liver. How can glycogen be used in a liver? Where is 

glycogen collected in organism of the person? 

13. Determination of an end-product of anaerobic glycolysis – lactic acid by Uffelman 

method. Principle of method. 

14.Revealing of acetone (ketone bodies) in urine (reactions with nitroprusside sodium 

and chloride of iron). Revealing of ketone bodies in urine by express-method. 

Principles of methods. Value of revealing of ketone bodies in blood and urine for 

medicine. 

15. Revealing of acetone by iodine-phorme reaction. To write this reaction. 

16. Determination of the contents of pyruvatic acid in biological liquids by colorimetric 

method. Explain the principle. How is the calibrating curve built? 

17. Determination of sialic acids by Gess's reagent. What clinical value has the 

determination of sialic acids concentration in blood? 

18. Revealing of cholesterol by Salkovskiy method. Principle of method. 

19. Revealing of cholesterol by Liberman - Burhard method. Principle of method. What 

is normal concentration of cholesterol in blood of the person? 

20. Studying of kinetics of pancreas lipase actions. What compounds in an organism 

activate lipase? Illustrate the answer with results of practical work. 

21. Reproduce in experiment the process of reamination with using of glutamic and 

pyruvatic acids. Use of chromatography method for an estimation of results. Write the 

equation of corresponding reactions. 

22. Determination of alanine aminotransferase and aspartate aminitransferase activity. 

Principle of method. Value of determination of these enzymes for medicine.

23. Determination of urea in urine by color reaction with diacetylmonooxide. To write 

reactions of formation of urea in an organism. 

24. Determination of ammonia in urine. Principle of method. 

25. Determination of creatinine in urine by Yaffa’s color reaction. Under what conditions 

is the increase or reduction of creatinine amount in urine is observed? 

26. Revealing of bilious pigments in urine by Gmelin’s reaction. Explain process of 

formation of bilious pigments in an organism. 

27. Revealing of urobiline in urine by Bogomolov’s reaction. Principle of method. When 

is urobiline present among bilious pigments in urine? 

28. Qualitative reaction on phenylpyruvate acid (Feling’s test). Principle of method. At 

what disease does phenylpyruvatic acid appears in urine? 

44

45 

MODULE 3 

MOLECULAR BIOLOGY. BIOCHEMISTRY OF INTERCELLULAR COMMUNICATIONS. 

BIOCHEMISTRY OF TISSUES AND PHYSIOLOGICAL FUNCTIONS 

Theme 1. Research of biosynthesis and catabolism of purine and pyrimidine 

nucleotides. Determination of end products of their metabolism. 


Purine and pyrimidine mononucleotides are structural components of DNA and RNA. 

Their biosynthesis provides formation of information molecules of nucleic acids needed for 

realization of genetic information. 

Degradation of nucleic acids in tissues is accompanied by formation of end products. The 

characteristic end product of metabolism of purine nucleotides is uric acid, its excessive 

formation promotes development of gout. 

Gout can be caused by insufficient kidney nitrogen-excretion function, heart diseases, 

hereditary infringements of purine nucleotides metabolism, and also excessive use of 

products, rich in nucleoproteids. 



To know biochemical dynamics (changes) of nucleotide transformation, basis of their 

pathochemistry and biochemical diagnostics. 

Specific purposes: 

1. To analyze sequence of reactions of biosynthesis and catabolism of pyrimidine 

nucleotides. 

2. To analyze sequence of reactions of biosynthesis and catabolism of purine 

nucleotides, infringement of synthesis of uric acid and biochemical basis of gout 

development. 


1. To be able to carry out titration (general chemistry). 

2. To be able to write structure of nitrogenous bases and mononucleotides. 

Reference card for self-preparation study 

of educational literature for the lesson. 


actions 

1. Practical studying of quantitative 1.1. Write the algorithm of the laboratory work in 

determination of uric acid. the practicum notebook. 

2. Biosynthesis of purine 

2.1. The scheme of reactions of IMP synthesis 

nucleotides. 

and sequence of AMP, GMP, ATP, GTP 

formation. 

2.2. Regulation of biosynthesis of purine 

nucleotides by the principle of negative feedback 

inhibition. 

3. Biosynthesis of pyrimidine 3.1. Initial substrates, reactions and regulation. 

nucleotides. 

4. Biosynthesis of deoxyribonucleotides. 

4.1. Formation of thymidine nucleotides. 

4.2. Inhibitors of dTMP biosynthesis. dTMP 

derivatives as antitumor agents (structural 

analogues of dTMP, derivatives of pterine).

d 

0,75 

c 

46 

5. Catabolism of purine nucleotides. 5.1. Hereditary infringements of uric acid 

metabolism. 

5.2. Clinical and biochemical characteristics of 

hyperuricemia, gout, Lesch-Nyhan syndrome. 

Student’s individual self-preparatory work: 

Prepare a report on the theme: « Gout, possible causes and clinical symptoms». 


Quantitative determination of uric acid in blood plasma. 

Principle of the method. Uric acid reduces tungsten phosphate reagent to a bluecolored 

compound, which quantitatively reacts with K 3 [Fe(CN) 6 ] with discolouration in an 

alkaline medium. Compare results of the experiment and standard samples. 

Course of determination. Use two flasks for experiment (E) and standard (S) tests. 

1) To the flask (S) add 2 ml of a standard solution of uric acid (0,5 mg/ml, there is 1 

mg of uric acid in the flask), to the flask (E) add 2 ml of urine. To both flasks add 1 ml of 

NaOH solution and 1 ml of tungsten phosphate reagent. Mix, the solutions become dark 

blue. 

2) Titrate the contents of the flasks until discolouration with a solution of K 3 [Fe(CN) 6 ], 

register the volume of the solution used for titration of experiment and standard samples. 

3) Calculate with the proportion. We assume, that S ml of K 3 [Fe (CN) 6] solution was 

used for titration of the standard sample (S), and E ml of the solution was used for titration 

of the experiment sample (E). Assume that daily excretion of urine is 1,5L and calculate as 

following: 

E/S * 0,75= g/day 

Norm of excretion of uric acid with urine: 0,25 - 0,75 g/day 

Theme 2. Research of DNA replication and RNA transcription. 


The genetic information stored in chromosomal DNA can be realized through 

replication, or with the help of recombination, transposition and conversion. These 

processes are the basis of variability of organisms that bring about their ability to adapt, 

but they can cause development of pathological conditions. Alterations of DNA 

transcription are manifested at the level of protein biosynthesis and result in different 

metabolic infringements in the cell. Knowledge of mechanisms of replication and 

transcription allows to answer questions about normal physiology and pathophysiology of 

diseases at the molecular level. 



To learn biochemical mechanisms of DNA replication and RNA transcription. 

Specific purposes: 

3. Explain molecular biological laws of preservation and transfer of genetic information, 

role of the enzyme systems which provide the half-conservative mechanism of DNA 

replication in prokaryotes and eukaryots. 

4. Explain mechanisms of functioning of the enzyme system of RNA transcription.

47 

5. Analyze sequence of stages of DNA replication and RNA transcription. 

Initial level of knowledge - abilities: to know chemical structure of DNA and RNA. 

Reference card for self-preparation study 

of educational literature for the lesson. 


actions 

1. Practical study of DNA 

replication and RNA transcription. 

1.1. Explain the mechanism of formation of DNA 

double helix. 

1.2. Explain antitumor action of antibiotics. 

2. Replication of DNA. 2.1. Biological role of DNA replication. Essence 

of the DNA replication discovery by D.Watson 

and F.Crick (1953). 

2.2. Half-conservative mechanism of replication; 

the scheme of Meselson - Stahl experiment. 

2.3. General scheme of DNA biosynthesis. 

2.4. Enzymes of DNA replication. 

2.5. Molecular mechanisms of DNA: topological 

problems (Okazaki fragments). 

3. Transcription of RNA. 3.1. General scheme of transcription; coding and 

non-coding chains of DNA. 

3.2. RNA polymerases. 

3.3. Stages and enzymes of RNA synthesis. 

3.4. Signals of transcription: promotor, initial, 

termination sites of genome. 

3.5. Processing as posttranscriptional 

modification of RNA. 

3.6. Antibiotics – inhibitors of transcription. 

Individual self-preparatory work of the student. 

1. Prepare a report on themes: «Antibiotics – inhibitors of transcription», «Essence of 

Watson and Crick (1953) discovery ». 

2. Create a scheme of DNA replication. 

3. Create a scheme of RNA transcription. 

Theme 3. Biosynthesis of protein on ribosomes. Research of processes of 

initiation, elongation and termination in synthesis of polypeptide chain. 


Understanding of mechanisms of protein synthesis and the nature of hereditary 

diseases is based on knowledge of the genetic code which forms a set of codons. 

Biosynthesis of abnormal proteins underlies hereditary diseases caused by mutations – 

changes in nucleotide sequence of a gene. Antibacterial action of antibiotics is based on 

their ability to cooperate with proteins of prokaryote ribosomes and inhibit synthesis of 

bacterial proteins. Knowledge of molecular basis of protein biosynthesis is necessary to 

explain processes of regeneration, wound healing and ways of their correction. It is also 

important for analysis of pathological processes, that are based on infringement of 

anabolism and catabolism (protein deficiency, children hypotrophy, exhaustion, disfunction 

of endocrine glands, starvation, etc.).

THE STATE MEDICAL EDUCATIONAL INSTITUTION

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