MINISTRY OF HEALTHCARE OF THE RUSSIAN FEDERATION

Federal State Budget Educational Institution of Higher Education

Siberian State Medical University

LABORATORY MANUAL
FOR PRACTICAL BIOCHEMISTRY

Tutorial

Tomsk
Siberian State Medical University
2019
 УДК 577.1:615.01(075.8)
ББК Е072Я7 + Р282Я7
L11

Authors
Nosareva O.L., Stepovaya E.A., Fedorova T.S., Timin O.A., Shakhristova E.V.,
Spirina L.V., Serebrov V.Yu.

Laboratory Manual for Practical Biochemistry / Nosareva O.L., Stepo-

L11 vaya E.A., Fedorova T.S., Timin O.A., Shakhristova E.V., Spirina
L.V., Serebrov V.Yu. – Tomsk: Siberian State Medical University
2019. – 174 pp.

Biochemistry is a constantly changing and evolving academic discipline that re-

quires continuous study to keep one's knowledge current. The practical book is writ-
ten in accordance with the Federal State Educational Standard of Higher Professional
Education for students studying in the basic vocational and educational programs –
specialty programs: "General Medicine" and "Pediatrics".
There are laboratory works in each unit, which should be carried out by students
duringtheir practical lessons. The methods presented in the tutorial are used in clini-
cal diagnostic laboratories. They assist the physician to diagnose the disease and to
monitor the efficacy of treatment. Each unit is divided into several parts: questions
for self-study, test tasks and case studies for testing the knowledge gained.
The tutorial contributes to the formation of professional competencies, as well as
the identification of the patient's basic pathologies, clinical signs, disease syndromes,
nosologies.

УДК 577.1:615.01(075.8)
ББК Е072Я7 + Р282Я7
Reviewer:
Akbasheva О.Е. – Professor of the Department of Biochemistry, Molecular Bi-
ology and clinical laboratory diagnostics at Siberian State Medical University, Dr. of
Med. Sci., Associate Professor.

The tutorail has been approved and recommended for publication: the Central
Methodical Council of the Federal State Budget Educational Institution of Higher
Education Siberian State Medical University of Ministry of Healthcare of the Russian
Federation (Protocol No.94, April 19, 2018).

Siberian State Medical University, 2019

Nosareva O.L., Stepovaya E.A., Fedorova T.S.,
Timin O.A., Shakhristova E.V., Spirina L.V., Serebrov V.Yu, 2019

2
 UNIT 1
STRUCTURE, FEATURES AND FUNCTIONS of PROTEINs

THEME 1.1. AMINO ACID STRUCTURE AND CLASSIFICATION.

PROTEIN STRUCTURE

INTRODUCTION
In fact, proteins are known to be the the main members of human metabolism.
They regulate all biochemical processes, act as enzymes, hormones, receptors, anti-
bodies and are required for the structural integrity of cells. The plethora of protein
functions and structures is associated with amino acid variety. Amino acids are in-
volved in the formation of biogenic amines and nitrogenous bases of mononucleo-
tides, neurotransmitter, etc. Therefore, protein-based therapies are highly successful
in clinical practice.

THE AIM OF THE PRACTICAL IS TO:

Obtain simplified knowledge about amino acid structure.
Practically apply this knowledge by performing amino acid color and protein
precipitation reactions.

SELF-STUDY QUESTIONS
1. Amino acid classifications.
2. Amino acids differ according to:
• their biological role (essential and non-essential);
• their physical and chemical features (neutral, acidic, basic, hydrophilic, hydro-
phobic);
• their chemical structure (with aliphatic radicals, with additional functional
group, with aromatic and heterocyclic group, imino acids);
• their solubility in water (non-polar, polar positive charged, polar negative
charged).
3. The structure and formula of proteinogenic amino acids.
4. Physical and chemical properties of amino acids, their functional group role.
5. The iso-electic point of amino acids and proteins, its dependence on different
factors.
6. The influence of pH solution on amino acid charge.
7. The peptide bond, its formation. Peptide bond properties.
8. Influence of pH changes on protein charge and solubility.
9. Color qualitative amino acid reactions. Their principles and practical use.

3
 Practical
COLOR QUALITATIVE PROTEIN AND AMINO ACID REACTIONS
Reagents
1) 1% albumin solution 2) 0.5% ninhydrine, 3) 30% NaOH solution, 4) 10%
NaOH solution, 5) 5% Pb(CH3COO)2 solution, 6) 5% sodium nitroprusside solution,
7) concentrated solution of HNO3, 8) 5% CuSO4 solution
Material of the investigation
The object of investigation is 1% albumin solution with a complex set of amino
acids.

BIURET TEST (PEPTIDE BOND)

Biuret test is specific for proteins – to differentiate between proteins and amino
acids.
Principle:
The Biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in
proteins to form a violet complex known as the "Biuret complex". Two peptide bonds
are at least required for the formation of this complex, this is why amino acids give
negative results with Biuret test.
Procedure and observation:
Add 1.0 ml of 10% sodium hydroxide solution and 2-3 drops of 1% copper sul-
fate solution to 1.0 ml of protein solution in a test tube. Mix well; a violet color is ob-
tained.

NINHYDRIN TEST
Ninhydrin is specific for amino acids and proteins – to differentiate between car-
bohydrates and amino acid andproteins.
Principle
When heating ninhydrin reacts with α-amino acids (–NH2) in proteins giving a
purple colored complex, except for proline and hydroxy proline gives yellow col-
or(no –NH2).
Procedure and observation:
Add 2-3 drops of ninhydrin reagent to 1.0 ml amino acid solution in a test tube.
Put it into a boiling water bath and observe the formation of a purple color.

XANTHOPROTEIC ACID TEST

Aromatic amino acids, such as phenyl alanine, tyrosine and tryptophan, respond
to this test.
Principle
In the presence of concentrated nitric acid, the aromatic phenyl ring is nitrated to
give yellow colored nitro-derivatives. At alkaline pH, the color changes to orange due
to the ionization of the phenolic group.
Procedure & observation:
Add 2-3 drops of concentrated nitric acid to 1.0 ml amino acid solution in a test
tube. Put in a boiling water bath and observe the formation of a yellow color. When
4
the solution doesn’t become yellow, add 1-2 drops of concentrated nitric acid. At
presence of 30% sodium hydroxide the color in test tube could change to orange.

TESTS FOR SULPHUR CONTAINING AMINO ACIDS AND PROTEINS

Principle
Sulphur containing amino acids, such as cysteine and cystine upon basic hydrolysis,
yield sodium sulphide. This reaction occurs due to partial conversion of the organic
sulphur to inorganic sulphide.
• Lead sulphide test (Fole test). Sodium disulfide is detected by precipitating it
to lead sulphide, using lead acetate solution
• Nitroprusside’s test. Sodium disulfide reacts with nitroprussideand andgives a
red-purple colour called "Morner test".
Procedure & observation
Add 5 drops of 30% NaOH solution to 1.0 ml of protein solution containing Cyste-
ine/Cystine in a test tube and boil the test tube. Divide the receivedsolution in two
parts for carring out a) and b) reactions.
a) Lead sulphide test (Fole test)
after boiling add 1 drop of lead acetate solution to 5 drops of received protein
solution and heat it till brown or black residual matter appears.
b) Nitroprusside’s test
after boiling add 3 drops of a 5% solution of sodium nitroprusside to 5 drops of
received protein solution. Mix well and add few drops of ammonia solution, a deep
red-purple color appears; called also Morner test.

Practical use of color reactions

Color reactions can beuniversal (Biuret and ninhydrine reactions) and specific
(lead sulfide test, xanthoprotein reaction). They allow to estimate and identificate the
protein and amino acid content. For instance, ninhydrin iscommonly used as a foren-
sic chemical to detect "fingerprints", as amines left over from proteins sloughed off in
fingerprints react with ninhydrin giving a characteristic purple color. Principles of
these reactions are also used for quantitative determination of amino acids and pro-
teins in biological fluids.

Laboratory exercise:
Using the provided solutions of albumin perform the tests in the table below and
write down your observations. The color intensity is denoted as follows: "–" color ab-
sence; "+" weak color; "++"intensive color; "+++" very intensive color.
Tests
object of study
Biuret test Ninhydrin Xanthopro- Lead sulfide test Reaction with ni-
reaction tein reaction (Fole test) troprusside

Albumin solu-
tion

5
 In conclusion the possibility of peptide bond detection and presence of specific
amino acids using color tests shoud be noted.

THEME 1.2. STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF

PROTEINS

INTRODUCTION
The protein isolation as well as salting out, denaturation or precipitation are used
in modern medical practice. Theirmain roles are the disease diagnostic, experimental
investigation, production and purification of proteins as medicine.

THE AIM OF THE PRACTICAL IS TO:

Study the physical and chemical properties of proteins (molecular weight, shape,
ionization, hydration, solubility) and the basic types and structures of protein mole-
cules.
Studythe protein precipitation reaction of proteins by different reagents and their
use in clinical practice.

SELF-STUDY QUESTIONS
1. The structure of proteinogenic amino acids. The peptide bond formation, dif-
ference between peptides and proteins.
2. The organization of protein molecules (primary, secondary, tertiary, quater-
nary structures).
3. Bonds, involved in the formation of the protein structure. Functional groups of
amino acids responsible for the formation of these relations.
4. The quaternary structure of proteins. The complementarity of protomers. The
cooperative protomers conformation changes.
5. Properties of proteins: amphoteric ionization (charge), hydration, solubility.
What is the isoelectric point?
6. The molecular weight peptides and proteins. Methods of its determining (ul-
tracentrifugation, gel filtration).
7. Properties of protein solutions. Factors that stabilize the protein molecule in
the solution. The colloidal properties of proteins.
8. Denaturation of proteins. Factors that cause the proteins denaturation (physi-
cal, chemical, biological). Properties of denatured proteins.
9. Protein renaturation, its mechanism.
10. Protein removal methods. Precipitation reactions by acids, heavy metals, or-
ganic solvents in solution. The principle of protein precipitation reactions.
11. Protein purification methods from impurities in solutions (salting out, gel fil-
tration, dialysis). Their principles.

TOPICS FOR REPORTS

1. Chromatography of proteins, its types and the practical application.

6
 2. First aid and medical aid in case of poisoning by organic and inorganic acids,
salts of heavy metals. Biochemical basics.
3. The dialysis and plasmapheresis application in clinical practice: their princi-
ples, efficacy and application.

Practical 1
PRECIPITATION REACTIONS OF PROTEINS (SALTING OUT)
Salting out is the protein precipitation reaction due to the action of alkali and
neutral salts. This process is reversible. Native protein features are preserved.

Reagents
1) Saturated ammonium sulfate solution (NH4)2SO4, 2) ammonium sulfate crys-
tals (NH4)2SO4, 3) 10% NaOH solution, 4) 1% CuSO4 solution.
Material of investigation
Serum.
Principle
Protein molecules contain both hydrophilic and hydrophobic amino acids. In
aqueous medium, hydrophobic amino acids form protected areas while hydrophilic
amino acids form hydrogen bonds with surrounding water molecules (solvation lay-
er). When proteins are present in salt solutions (e.g. ammonium sulfate), some of the
water molecules in the solvation layer are attracted by salt ions. When salt concentra-
tion gradually increases, the number of water molecules in the solvation layer gradu-
ally decreases until protein molecules coagulate forming precipitates; this is known as
"salting out". As different proteins have different compositions of amino acids, dif-
ferent proteins precipitate at different concentrations of salt solution.

Procedure and observation

Add equal amount of saturated ammonium sulfate solutionto 2.0 ml of serum
Mix well and note the globulin is precipitated in the resulting half of saturated solu-
tion of ammonium sulfate.
Wait 5 minute and separate globulin by centrifugation or filtration and recover
the clear supernatant. The presence of proteins in the precipitate or on the filter is
proved by Biuret reaction (UNIT 1.1.)
Add Biuret reactive to 10 drops of supernatant or flowthrough and perform the
color qualitative test for protein presence. Add ammonium sulfate crystals gradually
to the remaining part of supernatant until full saturation occurs; another precipitate
(albumin) is obtained. Separate albumin by centrifugation.
Сlinical and diagnostic significance
Salting out is the method for the separation of serum albumin and globulin, the
albumin/globulin ratio (A/G) determination, which is previously used in clinical la-
boratory practice. The normal A/G ratio is 1.2-1.8. It could be changed at many
pathological situations. The A/G ratio can be decreased in response to a low albumin
or to elevated globulins. Total globulins may be increased in some chronic inflamma-
tory diseases (tuberculosis, syphilis), multiple myeloma, collagen disease, and rheu-
7
matoid arthritis. Decreased levels are seen in case of hepatic dysfunction, renal dis-
ease and various neoplasms.

Design of laboratory work

Note the principle, laboratory procedure, results of analysis and conclusion. The
possibilities of albumin and globulin separation due the use of this method have to be
précisedin it.

Practical 2
PROTEIN DENATURATION
Denaturation is a process when proteins lose thequaternary structure, tertiary
structure and secondary structure which is present in their native state and change
physical, chemical and biological properties. Application of some external stress or
compound such as a strong acid or base, a concentrated inorganic salt,
an organic solvent (chemical factors), radiation, utrasound or heat (physical fac-
tors) cause it.
Reagents
1) Acetone, 2) 10% trichloroacetic acid 3) concentrated HNO3, 4) 1% СuSO4,
5) concentrated H2SO4, 6) 5% lead acetate (Pb(CH3COO)2), 7) tannin, 8) 20% sul-
fosalicylic acid.
Material forthe investigation
1% Albumin solution.
Principle
Denaturation reduces the protein charge and hydration shells and leads to
thechange in dissolving features in water and stability.

Chemical denaturation.
Procedure and observation
Put 5 drops of albumin solution in 5 test tubes and add the reagents, which are
included in the following table. Signs "+" and "–" indicate the results of observation.
The intensity of denaturation indicates the number of "+" sign.
Reversibility and irreversibility of protein precipitation is proved by adding 20-
30 drops of water to the pellet.

8
 Probe Number
Reagents Mechanism and reaction features Result
number of drops
Denaturation by heavy metal salts
The metal ions bind with charged ami-
1 Coopersulfate 2
no groups, thereby change of the pro-
2 Lead acetate 2
tein spatial structure is observed
Denaturation by concentrated mineral acids
Concentrated acids cause the protein
3 Nitric acid 2
denaturation by changing its charge.
4 Sulfuric acid 2
Cationicgroups areneutralized.

The disappearance of the protein pre-

5 Nitric acid 10 cipitate by adding an excess of sulfuric
6 Sulfuric acid 10 acid group is the result of ionic re-
charging
Denaturation by organic acids
7 Trichloroacetic acid 2 Protein neutralizes the acid charge sys-
tem, destroy shydrogen bonds and
8 Sulfosalicylic acid 2 forms complexes with the protein
Denaturation by tannin
It is formed insoluble salt-like com-
9 Tannin 2 pound with basic amino groups of ami-
no acids
Denaturationby organic solvents
It destroys hydrophobic interactions in
10 Acetone 5
the protein molecule

Practical significance
Chemical denaturation reaction is used to precipitate the protein in biological
material with the following definition of low molecular weight substances in the fil-
trate. This test is performed to detect the presence of the protein in various body flu-
ids and its quantitative analysis.
In medical practice they are used in the treatment and prevention of poisoning of
heavy metal salts athome and at work. Their next significant functions arethe disposal
of waste in sanitary practices, disinfection of skin and mucous membrane.

Design of laboratory work

Note the principle, laboratory procedures, and results of analysis inthetable.
Make a conclusion about the most effective protein precipitation methods.

9
 THEME 1.3. PROTEIN CLASSIFICATION. PROTEIN STRUCTURE AND
FUNCTIONS IN HUMAN BODY. COMPLEX PROTEINS

INTRODUCTION
Peptides and proteins have multiple specific functions in the body. They are
structural, transport, hormonal, and enzymatic.
The change in the protein structure may underlie the development of pathologi-
cal processes like sickle cell anemia, or thalassemia. At the same time, many diseases
entail changes and ratios in protein level, particularly in blood proteins, which has a
diagnostic value.
Protein and the specific reactions of prosthetic group allow us to understand the
complex protein composition, as well as to use the data in research of the protein con-
tent carrying out the protein-specific reactions.

THE AIM OF THIS PRACTICAL SESSION IS TO:

Study the complex protein structure: phosphoprotein, nucleoproteins, glycopro-
teins, chromoproteins (hemo- and flavoproteins), metalloproteins, lipoproteins.
Learn how to allocate complex proteins of different objects and perform qualita-
tive reactions to complex protein components.

SELF-STUDY QUESTIONS
1. Structures of proteinogenic amino acids.
2. Protein classification according to their functional characteristics (safety,
structural, transport, contractile, hormonal, enzyme). Protein examples for each class.
3. The classes of proteins based on their structure: simple and complex, mono-
mers and polymers, globular and fibrillar. Protein examples of each class.
4. Characterization of simple proteins (albumin, globulins, histones, protamines).
Note the features of their structure and function.
5. Characteristics and features of the complex proteins structure classes:
• Nucleoproteins. Structure and properties of DNA and RNA. Differences be-
tween DNA and RNA. The structures of AMP, ADP, ATP, cAMP nucleotides. Types
of histones and their role in the formation of DNA and nucleosomes laying.
• Chromoproteins (hemoproteins, flavoproteins, retinal proteins). The chemical
structure concept, their examples and functions. Representation of the hemoglobin
molecule structure. The structure of heme.
• Glycoproteins. Structure, function in the body. Representation of the carbohy-
drate moiety structure. Proteoglycans, the structure, functions in the body. The chem-
ical structure of hyaluronic acid and chondroitin sulfates.
• Lipoproteins. The structure of the lipoprotein particles. The main transport
forms of plasma lipids – chylomicrons, very low density lipoproteins (VLDL), low
density lipoproteins (LDL), high density lipoproteins (HDL), their functions.
• Metalloproteins, representation of the structure, main examples. Metalloen-
zymes, definition, their examples.

10
 • Phosphoprotein. How does phosphate group join a protein? The main exam-
ples.
6. Analysis of the complex proteins chemical composition – glycoproteins and
phosphoproteins. The principle of methods.

TOPICS FOR REPORTS

1. The structural proteins: keratin, collagen, elastin. Features of the structure.
Functions.
2. Contractile muscle proteins: actin and myosin. Features of the structure. Func-
tion.

Practical
ISOLATION AND ANALYSIS OF PHOSPHOPROTEINS AND GLYCO-
PROTEINS CHEMICAL COMPOSITION
Qualitative test for non-protein components used for the detection of complex
proteins in various objects.
Reagents
1) 1% thymol solution in ethanol, 2) 10% solution NaOH, 3) molybdenum rea-
gent, 4) concentrated H2SO4, 5) 1% CuSO4 solution, 6) 10% CH3COOH solution,
7) 10% trichloroacetic acid.
Analysis of phosphoprotein chemical composition
Material of the investigation
Milk.
Procedure and observation
Take two test tubes and poure 1.0 ml of milk into them. Then add equal amoun-
tof distilled water and mix. Perform Molybdenum and Biuret tests according to the
following instructions.
Principle
Phosphoric acid presented in the precipitate interacts with ammonium molybdate in
Molybdenum nitric acid, forms painted in lemon-yellow color ammonium phosphomolybdate
test for phos- complex compound.
phoric acid Procedure
Take a half of the precipitate from the filter and put it into the test tube. Add 20 drops
of a molybdenum reagent in it and the ammonium phosphomolybdate precipitates.
Principle
A pink-violet or blue-violet color complex compound is formed in alkaline solution
at the protein present.
Biuret test
Procedure
Add 10 drops of 10% solution of NaOH and 1 drop of 1% solution of CuSO4 to the
precipitate on the filter.

Analysis of glycoprotein chemical composition

Material for the investigation
Saliva, collected after rinsing the mouth with water.

11
 Procedure and observation
Collect 2.0 ml of saliva in 2 test tubes; add 10% trichloroacetic acid drop by drop
till the precipitate appears.
Principle
Molisch's test The hydroxymethyl sulfuric acid is formed after pentoses dehydration. Its condensa-
(for the pres- tion with thymol hydroxymethyl furfural is associated with the development of red
ence color, and pink rings appear in vitro.
of carbohyd- Procedure
rates) Remove the liquid from the 1st test-tube and add 2-3 drops of thymol solution to the
clot. Mix gently and add the concentrated H2SO4.
Principle
A pink-violet or blue-violet color complex compound is formed in the alkaline solu-
tion with protein.
Biuret test Procedure
Add 10 drops of 10% solution of NaOH in the second test tube for the acid neutrali-
zation. Then put 10 drops of 10% solution of NaOH and 1 drop of 1% solution of
CuSO4.

Design of practical
Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about complex protein composition by component detection reac-
tions.

Object Complex proteins Component Coloring Conclusion

Protein
Saliva Glycoproteins
Carbohydrates
Protein
Milk Phosphoproteins
Phosphoric acid

TESTS
Choose one or more correct answers.

1. AN ESSENTIAL AMINO ACID IS

1) glycine
2) valine
3) tyrosine
4) serine

2. AT PH 7.4 POSITIVELY CHARGED AMINO ACIDS IS

1) proline
2) oxyproline
3) arginine
4) aspartate
12
3. SIMPLE PROTEIN IS
1) hemoglobin
2) somatotropin
3) albumin
4) ceruloplasmin

4. ALPHA HELIX IS STABILIZED BY

1) disulfide bonds
2) hydrophobic interactions
3) cooperative binding
4) peptide bonds
5) hydrogen bonds

5. THE TERTIARY STRUCTURE OF PROTEINS IS STABILIZED BY

1) amino acids radical electrostatic interactions
2) hydrogen bonds
3) Hydrophobic interactions
4) disulfide bondS
5) interaction with the prosthetic group

6. RECOGNIZING PROTEIN-LIGAND BINDING SITE IS

1) protein and non-protein cofactor binding site
2) "niche" of the peptide molecule
3) fragment of hydrophilic peptide
4) a region of the protein that is complementary to a specific molecule or
ion (ligand)

7. AT AN ELECTRIC FIELD AND AT PH OF 6.8 PEPTIDE ASP-LEU-GLU-

GLY WILL
1) move to the anode
2) stay put
3) move to the cathode

8. LIGAND BINDING SITE IS MADE OF

1) primary structure
2) tertiary structure
3) secondary structure
4) quaternary structure

9. THE SPECIFIC PROTEIN PROPERTY IS USED IN THE "ARTIFICIAL

KIDNEY"
1) the failure to penetrate through a semipermeable membrane (dialysis)
2) the ability to connect the polar molecules
13
 3) the formation of oncotic pressure
4) low rate of diffusion

10. COOPERATIVE INTERACTIONS IN PROTEIN ARE PERFORMED AT

THE LEVEL OF
1) primary structure
2) tertiary structure
3) secondary structure
4) quaternary structure

CASE STUDIES
1. Partial hydrolysis of insulin (B chain) is detected be recovering the tetrapep-
tide Glu-Ala-Glu-Leu.
Note the direction of the peptide in an electric field at a pH of 3.0 and 10.5.

2. The electrophoretic mobility of proteins is studied.

Note the direction of following proteins in the electric field: ovalbumin at pH 5.0
(isoelectric point pH 4.6); lactoglobulin at pH 5.0 and 7.0 (the isoelectric point pH
5.2); chymotrypsinogen at pH 5.0; 9.5 and 11.0 (isoelectric point pH 9.5).

3. Two peptides are obtained in partial hydrolysis and protein fractionation:

a) Gly-Ala-Val-Leu-Ile;
b) Thr-Asp-Lys-Tyr-Glu.
Indicate a compound that is more similar in properties to hydroxyl carbon.
Select a compound which is more soluble in nonaqueous fat-like medium.
Explain the features of each of these compounds in the procedure of Biuret, nin-
hydrin reactions, lead sulfide test and xanthoprotein reaction.
Indicate a compound capable to form the salt bridges.

14
 UNIT 2
VITAMIN STRUCTURE, THEIR CLASSIFICATION AND ROLE

THEME 2.1. FAT SOLUBLE VITAMINS

INTRODUCTION
Fat soluble vitamins are hydrophobic organic substances. They cannot be syn-
thesized and are essential food factors. The vitamin D is an exception, it could be
synthesized in the skin, but in insufficient quantities. The lack and insufficient intake
of vitamins in the body develops severe condition, leading to metabolic disorders.
The biological role of fat soluble vitamins is associated with the regulation of meta-
bolic processes. Additionally, these vitamins affect the synthesis of different structur-
al proteins and enzymes, especially in children.
Knowledge about vitamins, as well as practical skills in the qualitative determi-
nation of these substances in food are of great importance. They are an effective
method of the hypo-and avitaminosis prevention and used in the treatment of differ-
ent diseases (pathologies of skin, liver, muscle, and bone).

THE AIM OF THIS PRACTICAL SESSION IS TO:

Study chemical structure, features, classification and biological role of fat solu-
ble vitamins. Describe the clinical signs of avitaminosis.
Perform fat soluble quantitative reactions of vitamins with standardized solu-
tions.

SELF-STUDY QUESTIONS
1. General characteristics of vitamins, their role. Classification and nomenclature
of vitamins.
2. Characteristics of hypo-and avitaminosis, hypervitaminosis, their exogenous
and endogenous causes. Causes of hypovitaminosis in children.
3. Provitamins – beta carotene, ergosterol, 7-dehydrocholesterol. Conversion of
provitamins in the vitamin, beta carotene is an example. The concept of carotenoids
and their roles in the body.
4. The concept of antivitamin. Antivitamin as medicines. Dicumarol, mechanism
of its action.
5. Characteristics of the individual fat soluble vitamins under the plan:
• the structure of vitamins A, E, K, D2 and D3, F,
• the structure of the vitamin A and D active forms,
• food sources,
• the minimum daily requirement,
• biochemical functions, examples of reactions and / or processes that takes vit-
amin participation,
• clinical signs of hypo-, avitaminosis, hypervitaminosis.
15
 6. Biochemical manifestations of vitamin D deficiency, vitamin D-dependent
and vitamin D-resistant rickets. The role of liver and kidney diseases in the develop-
ment of clinical signs of hypovitaminosis.
7. Qualitative reaction for retinol, tocopherol, menadione, cholecalciferol. Oper-
ating principles of methods.
8. Make a table for the fat soluble vitamins.
(letter, chemical, physio-

hyper- and avitaminosis

Clinical signs of hypo-
The name of a vitamin

Chemical structure

Daily requirement

Biological role

Food sources

Medicines
logical)

TOPICS FOR REPORTS

1. History and discovery of vitamins. Works of Russian and foreign scientists.
2. Rickets – types, biochemical causes, prevention, treatment. Rickets-like condi-
tions.
3. Vitamin A, its active forms: retinal and retinoic acid. Participation in metabo-
lism and photochemistry of visual process?
4. Vitamin D, its active form. Participation in metabolism.

Practical 1
QUALITATIVE RECTIONS OF FAT SOLUBLE VITAMINS

RETINOL QUALIITATIVE REACTION

Principle
The method is based on the ability of concentrated sulfuric acid to take water
from retinol with the formation of colored products.
Reagents
1) Concentrated H2SO4, 2) butanol.
Material for investigation
VitaminА, 3.44% oil solution.
Procedure and observation
Add 2 drops of vitamin A solution and 5 drops of butanol in tube. Leave for 1
minute, shaking occasionally. Then add 5-7 drops of conc. H2SO4, and a blue color
turns into purple, then red-brown appears.

16
 CHOLECALCIFEROL QUALITATIVE REACTION
Principle
A red-violet color appears as a result of the interaction of vitamin D3 with hy-
droxymethylfurfural formed from sucrose due to the action of concentrated sulfuric
acid.
Reagents
1) Concentrated H2SO4, 2) butanol, 3) 20% sucrose solution.
Material for investigation
Vitamin D3, oil solution, 15 thousand IU/ml.
Procedure and observation
Take 3 drops of vitamin D3 and 5 drops of butanol, add 3 drops of sucrose solu-
tion and 5-7 drops of concentrated H2SO4. A red-violet color turns into black and
then converts into white.

TOCOFEROL QUALITATIVE REACTION

Principle
A compound of quinoid structure of red or yellowish-red color is formed as a re-
sult of the reaction of tocopherol with concentrated nitric acid.
Reagents
Concentrated HNO3.
Material for investigation
Vitamin Е, 30% oil solution.
Procedure and observation
Take 2 drops of vitamin E in a dry tube, and then add 10 drops of concentrated
HNO3. Shake the tube and observe the appearance of a red color. To accelerate the re-
action, the tube can be placed in a boiling water bath for 3 minutes.

VITAMIN K QUALITATIVE REACTION

Principle
Menadione (synthetic analogue of vitamin K1) in the presence of cysteine in a basic
medium turns to lemon yellow color.
Reagents
1) 0.025% cysteine solution, 2) 10% solution of sodium hydroxide.
Material for investigation
0.05% Menadione solution.
Procedure and observation
Add 5 drops of cysteine and 1 drop of 10% NaOH solution to 5 drops of mena-
dione. Yellow lemon color appears.

Clinical and diagnostic significance

Vitamin qualitative reaction allows us to establish the authenticity (confi-
dence) of vitamin drugs, as well as their use for the detection and quantification of
vitamins in food and medical plants.

17
 Design of laboratory work
Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about the ability of vitamin detection.

Vitamins Method and reagents Result

THEME 2.2. WATER SOLUBLE VITAMINS

INTRODUCTION
Water soluble vitamins are organic compounds of different chemical nature with
low molecular weight. They are the regulators of body's metabolism, which cannot be
synthesized in the body and belong to the essential food supplements. Being synthe-
sized in the liver vitamin PP is the exception. The insufficient intake of vitamins
leads to the development of severe metabolic disorder – hypo- and avitaminosis. The
biological role of water soluble vitamins is associated with the regulation of metabol-
ic processes in the body. Most of them are a part of coenzymes and prosthetic groups
of enzymes.
Knowledge about vitamins, acquiring the practical skills in the determination of
these substances is of great importance. They are used as an effective method of hy-
po-and avitaminosis prevention. Vitamins are therapeutic agents in a non-specific
treatment for a variety of diseases.

THE AIM OF THIS PRACTICAL SESSION IS TO:

Study the properties, chemical structure, classification, biological role of vitamins,
clinical sign of deficiency diseases (avitaminosis).
Master practical skills for conducting qualitative analysis of vitamins.

SELF-STUDY QUESTIONS
1. Characteristics of water soluble vitamins according to the following plan:
• Chemical structure of B1 (thiamine), B2 (riboflavin), B3 (PP, niacin, niacin am-
ide), B6 (pyridoxine), C (ascorbic acid), H (biotin). The structure of vitamin B5 (pan-
tothenic acid), B9 (folic acid), B12 (cobalamin),
• food sources,
• vitamin’s daily requirement,
• structure of coenzymes (TPP, FMN and FAD, NAD+ and NADP+, PLP),
• biochemical functions, examples of reactions and / or processes in which coen-
zyme takes part,
• the possible causes of hypo- and avitaminosis and their clinical signs.
2. The mechanism of antibacterial activity of sulfonamides.
3. Antivitamin – isoniazid, avidin, pteridines. The mechanism of their action.
Application of antivitamin as medicaments.

18
 4. Qualitative reactions of riboflavin, niacin, pyridoxine, cobalamin, ascorbic ac-
id. Operating principles of methods.

TOPICS FOR REPORT

1. Vitamin-like compounds, general characteristics, types, their role in metabo-
lism.
2. Vitamin P (bioflavonoids), its importance and the need for the children's body.
3. The mono and multivitamin preparations for nonspecific therapy. Advantages
and disadvantages of the use of these drugs in everyday life.
4. Hereditary metabolic disorders and functions of vitamin В12.

Practical
QUALITATIVE REACTIONS OF WATER SOLUBLE VITAMINS
Reagents
1) Concentrated НСl, 2) 1% solution of FеСl3, 3) 10 % thiourea solution, 4) 10%
CН3CООН solution, 5) 5% solution of Сu(СН3СОО)2, 6) metal zinc, 7) 0.01% meth-
ylene blue solution.
Equipment
Water bath.
Material for investigation
1% pyridoxine hydrochloride solution, dry nicotinic acid, 0.025% riboflavin so-
lution, 1% vitamin В12 solution, 1% ascorbic acid solution.

RIBOFLAVIN QUALITATIVE REACTION

Material for investigation
0.025% riboflavin solution, 1:5 dilutions.
Reducing reaction
Principle
The method is based on the reduction of riboflavin hydrogen released during the
addition of metallic zinc to concentrated HCl. The pink color product, riboflavin, ap-
pears which then transforms to colorless leucoform of the product.
Procedure and observation
Take 10 drops of riboflavin solution and add 5 drops of concentrated HCl and
the granule of metal zinc. The liquid gets pink and then becomes colorless.

NICOTINIC ACID QUALITATIVE REACTION

Principle
Heating vitamin PP solution with acetic acid copper is associated with blue pre-
cipitation sparingly soluble copper salt of nicotinic acid development.
Material for investigation
Nicotinamide powder.
Procedure and observation
Take 10.5 mg (pinch) of nicotinic acid, place it into tube with 10 drops of 10%
acetic acid solution, and dissolve the precipitate by heating. Then add equal amount
19
of acetic acid solution of copper (Cu(CH3COO)2) to the obtained solution. The liquid
becomes turbid.

PYRIDOXINE QUALITATUVEREACTION
Principle
Reaction of Vitamin В6 with FеСl3 is led to red color complex salt development.
Material for investigation
1% vitamin В6 solution.
Procedure and observation
Take 5 drops of 1% vitamin В6 solution and add equal quantity of 1% FеСl3 so-
lution. Then the red color appears.

COBALAMIN QUALITATIVE REACTION

Principle
Cobalt ions contained in a vitamin are interacted with thiourea. Then the ob-
tained solution is heated, and a green color of cobalt thiocyanate appears.
Material for investigation
1% vitamin В12 solution.
Procedure and observation
2-3 drops thiourea is applied on ashless filter, dried in hot air over the tiles. Then
add 1-2 drops of vitamin B12to the filter. The filter is dried again in hot air.
In the filter, on the edges the green color spots are appears, indicating the pres-
ence of cobalt.

ASCORBIC ACID QUALITATIVE REACTION

Material for investigation
1% ascorbic acid solution.
Principle
Ascorbic acid has the ability to restore the methylene blue. It is oxidized at the
same time to dehydroascorbic acid. Blue methylene during the restoring becomes
colorless.
Procedure and observation
Take 5 drops of 1% ascorbic acid solution to the first tube. Add 5 drops of distil-
lated water to the second tube. Put 1 drop of methylene blue to both tubes and place
them to the water bath at 40°С. The decoloration of liquid with vitamin is observed.
Design of laboratory work
Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about ability of vitamin detection.

Vitamins Methods and reagents Result

20
 TESTS

Choose one or more correct answers.

1. THE MAIN FUNCTION OF WATER-SOLUBLE VITAMINSIS
1) precursors of hormones
2) protection of biological membranes
3) precursors of coenzymes
4) carbohydrate precursors

2. THE COENZYME FORM OF VITAMIN В1 IS CALLED

1) pyridoxal phosphate
2) flavin mononucleotide
3) thiamindiphosphate
4) nicotinamide

3. THE COENZYME FORM OF VITAMIN В2 IS CALLED

1) pyridoxal phosphate
2) flavin mononucleotide
3) tetrahydrofolate
4) coenzyme A

4. THE PANTHOTHENIC ACID IS THE COMPONENT OF the COENZYME

1) coenzyme A
2) tetrahydrofolate acid
3) thiamin pyrophosphate
4) flavinmononucleotide

5. THE HYPOVITAMINOSIS B6 DURING THE LONG INTAKE OF ANTI-

BIOTICS AND SULFANILAMIDES IS ASSOCIATED WITH
1) suppression of intestinal microflora
2) binding of drugs with vitamin
3) the influence of drugs on the synthesis of coenzyme form
4) inhibition of pyridoxine-dependent enzymes

6. THE SCURVY IS A DISEASE CAUSED BY A DEFICIENCY OF VITA-

MIN C CHARACTERIZED BY
1) oxidation of SH-groups of enzymes
2) altered collagen synthesis
3) altered albumin synthesis
4) oxidation of the connective tissue cells lipid membrane

7. THERHODOPSIN PROSTHETIC GROUP, THE RECEPTOR PROTEIN OF

THE RETINA IS
1) riboflavin
2) calciferol
3) retinal
4) tocopherol
21
 8. THEVITAMIN F CONSISTS OF FATTY ACIDS
1) oleic
2) linoleic
3) linolenic
4) stearic acid
5) arachidonic

9. THEVITAMIN A DEFICEINCY IS CHARACTERIZED BY

1) hyperkeratosis
2) reduction in blood concentration of rhodopsin
3) bleeding
4) osteomalacia

10. THE MAIN ROLE OF VITAMIN K

1) is an antioxidant
2) increases platelet production
3) participates in the coagulation factors synthesis
4) participates in blood coagulation reactions

CASE STUDIES
1. Lactobacillus casei can grow in a simple culture medium containing vitamins
and riboflavin pyridoxine and 4 amino acids. If in the culture medium a complete set
of amino acids and riboflavin is added, pyridoxine amount needed for optimal growth
of bacteria will be reduced in 90%.
Explain the fact.

2. Sulfonamides were first used as antibacterial

medicines with wide spectrum. They have a similar
structure to para-aminobenzoic acid.
Justify the use of sulfonamides. Give further
guidance in the application of these drugs. Sulfanilamides structure

3. Patient will have a pre-operational treatment.

Consider the application of required vitamins.

22
 UNIT 3
ENZYMOLOGY

THEME 3.1. ENZYME STRUCTURE AND PROPERTIES.

APPLICATION IN MEDICINE

INTRODUCTION
Enzymes are protein molecules, which serve as biocatalysts in metabolic pro-
cesses. The study of their structure and functions are necessary for understanding the
biochemical features in variety of tissues and the ways of its possible regulation. The
pathogenesis of different diseases is found to be accompanied by the altered enzyme
function.

THE AIM OF PRACTICAL IS

to study the enzyme structure, properties and features;
to observe the influence of different factors on enzyme activity in vitro;
to obtain the practical skills for enzyme specificity investigation.

SELF-STUDY QUESTIONS
1. Simple and complex protein structure.
2. Coenzyme forms of vitamins B1 (TPP), B2 (FMN and FAD), PP (NAD+ and
NADP+), B6 (PLP).
3. The biological role of enzymes. The concept of an energy level and the activa-
tion energy of the reaction.
4. Stages of enzymatic catalysis.
5. Characteristics of the structural and functional enzymes organization accord-
ing to plan:
• simple enzymes,
• complex enzymes: term holoenzyme, apoenzyme, a cofactor, coenzyme, pros-
thetic group,
• active center (contact and catalytic sites),
• allosteric center.
6. Acid-base catalysis and covalent mechanisms.
7. The similarities and differences in the action of enzymes and inorganic cata-
lysts.
8. The general principles of quantifying enzyme activity. Units of enzyme activity.
9. A multi-complex structure, the principles of self-assembly, the role. Exam-
ples.
10. Isozyme, especially their structure on the example of creatine kinase and lac-
tate dehydrogenase.
11. The main properties of enzymes. The graphs of enzymatic reaction rate de-
pend on:
• temperature,
23
 • the pH of the environment,
• the substrate concentration,
• the enzyme concentration.
12. Specificity, types. Mechanisms of specificity (Fisher's theory and the theory
of Koshland).
13. Practical uses of enzymes in medicine: diagnostic and enzyme replacement
therapy. Examples.
14. Enzymopathies, primary and secondary forms. Examples. The role of the
lack of co-enzymes in enzymopathy development.
15. The study of enzyme action specificity on the example of amylase and ure-
ase. The principle of the method.
16. The dependence of the enzymatic reaction on temperature. Salivary amylase
as an example. The principle of the method.

TOPIC FOR REPORTS

1. The role of the essential microelements in the functioning and regulation of
enzyme activity.

Practical 1
DEPENDENCE OF ENZYME ACTIVITY ON TEMPERATURE
Investigation of influence of temperature on salivary amylase activity
Reagents
1) 1% starch solution, 2) Lughole’s solution.
Material of investigation
Saliva, diluted in 1:10 (source of α-amylase).
Principle
To investigate the dependence of enzymatic reaction activity on temperature in
the hydrolysis of starch using saliva amylase (diastase, 1.4-α-D-glucose hydrolase,
EC 3.2.1.1.). During the incubation of the substrate mixture (starch) and enzyme
(amylase of saliva) under different temperature conditions enzyme will hydrolyze dif-
ferent amounts of substrate.
Starch hydrolysis by amylase passes through the stage of dextrin formation and
then disaccharide maltose formation.

Starch + Н2О Maltose + Dextrins

The amount of split starch is evaluated by color reaction with iodine. Undigested
starch with iodine gives a blue color. The products of hydrolysis of starch (dextrin),
depending on the size of the molecules give iodine staining: ● amylodextrins – pur-
ple, ● erythrodextrins – red-brown, and maltose ● achrodextrins – no color reaction,
yellow color corresponds to the color of an aqueous solution of iodine.

24
 Procedure and observation.
Preparation of saliva (the student on duty performs the saliva for the whole
group).
Collect 1.0 ml of saliva into a measuring tube and dilute with distilled water to
10.0 ml, mix well (not shake!).
1. Add 10 drops of starch to the four tubes (1, 2, 3, 4). Bottle the starch solution
before use! Add10 drops of saliva diluted (solution of α-amylase) to the following
four tubes (5, 6, 7, 8). Divide the tubes into pairs – 1-5, 2-6, 3-7, 4-8.
2. To eliminate the enzymatic reaction after reaching the required temperature,
saliva and starch solutions should be initially heated separately:
Place 1st pair of tubes in an ice bath (0°C). Leave 2nd pair at room temperature
(20°C). Maintain the third pair at a temperature of 38-40°C. Place the 4th pair of
tubes in a boiling water bath (100°C) (water bath).
3. Wait for 3 minutes, and then combine the content of each pair of tubes, mix
and place immediately for 10 minutes in the same conditions.
4. Check the progress of the reaction. To do this, take 3 drops of mixture from
the third tube (38-40°C) and put it on the slide, then add 1 drop of Lughole’s reagent:
• the color of mixture is blue. It indicates a lower rate of starch hydrolysis. In
this case it is necessary to extend the incubation time.
• the appearance of red or yellow color indicates the completion of amylase
starch hydrolysis (you can proceed to step 5.).
5. After the hydrolysis of starch in the third sample add 2 drops of Lughole’s re-
agent in all tubes at the same time and compare the color in all tubes.
Design of laboratory work
Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about the optimal temperature of enzyme action.

N Temperature of Relative rate of enzy-

Color
tube incubation matic reaction
1 0°С
2 20°С
3 38°С
4 100°С

Practical 2
INVESTIGATION OF ENZYME SPECIFICITY
Reagents
1) 1% urea solution, 2) 1% thiourea solution, 3) 0.5% phenolphthalein alcohol
solution, 4) 1% starch solution, 5) 1% sucrose solution, 6) Felling reagents: Felling I
and Felling II.
Material for investigation
Urease solution made from the seeds of watermelon
Saliva, diluted in 1:10 (source of α-amylase).

25
 Detection of urease absolute specificity
Principle
The method is based on a comparison of the
possibility of similar substrates – urea and thiou-
rea to be hydrolyzed by urease (EC 3.5.1.5.).
The enzyme action is detected by a phenol-
phthalein color change in an alkaline medium,
which results in ammonia release by urea hydroly-
sis.

Urea + Н2О 2NH3 + CO2

Procedure
Urease preparation:
Grind clear 3-4 watermelon seed, corn in a mortar with 1 ml of distilled water.
Add 10.0 ml of water and filter the emulsion. Use it as urease.

Sample 1, Sample 2,
Drops Drops
1% urea solution 10 ––
1% thiourea solution –– 10
Urease 10 10
Phenolphthalein solution 1-2 1-2
Mix well. Leave for 3-5 minutes.
Observe the pink color appearance in one of the
tubes.

Design of laboratory work

Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about the reason of color absence in one of the tubes and speci-
ficity of an enzyme.

Substrate
Samples Color Presence of color
of reaction
Sample 1
Sample 2

Detection of salivary amylase specificity

Principle
The method is based on a comparative study of the enzyme amylase ability to
hydrolyze different substrates carbohydrate – starch polysaccharide and the disaccha-
ride sucrose (EC 3.2.1.1.)

26
 Substrate + Н2О Product (maltose, dextrins)

The enzyme action on the substrate is detected using qualitative response of car-
bohydrate to free aldehyde group (Trommer reaction).
Trommer reaction may be positive (red-orange color) only by splitting substrates
reducing sugars (maltose, glucose, etc.), which have a free aldehyde group and have
reducing properties. Reaction substrates (starches and sucrose) do not have free alde-
hyde group, so do not give a positive reaction to Trommer’s test.

Procedure and observation

Preparation of saliva (the student on duty performs the saliva for the whole
group).
Collect 1.0 ml of saliva into a measuring tube and dilute with distilled water to
10.0 ml, mix well (not shake!).

Sample 1, Sample 2,
Drops Drops
1% starch solution –– 10
1% sucrose solution 10 ––
Saliva solution 5 5
Mix well.
Incubate for 10 min at 37°С.
Felling I reagent 3 3
Felling II reagent 3 3
Mix well.
Incubate in a boiling water bath at a temperature of
100°C until the appearance of yellow-orange or reddish
color in one of the tubes.

Design of laboratory work

Note the principle, laboratory procedures, and results of analysis in the table.
Make a conclusion about the reason for color absence in one of the tubes and speci-
ficity of an enzyme.
Substrate of reac-
Samples Color Presence of color
tion
Sample 1
Sample 2

27
 THEME 3.2. ENZYME ACTIVITY REGULATION

INTRODUCTION
A plethora of drugs affects the enzymes activity in the body. Enzymes and af-
fecting their activity drugs can be used in medicine as therapeutic agents.
The rapidly expanding knowledge of enzyme activity regulation
is providing new targets for disease characterization, and early diagnosis.

THE AIM OF THE PRACTICAL IS

To know the features of enzymatic catalysis and to study the enzyme activity
regulation in the cell.
To introduce the methods of enzymes detection in tissues and biological fluids.
To determine the amylase activity in serum and urine.

SELF-STUDY QUESTIONS
1. Ways of enzymatic reactions regulation in the cell (in vivo):
• compartmentalization,
• change in the enzyme amount – the example: the effect of glucocorticoids on
gluconeogenesis,
• substrate availability change on an example of oxaloacetate and the citric acid
cycle,
• proenzymes and their limited proteolysis by the example of the enzymes in the
gastrointestinal tract,
• protein-protein interactions, for example, adenylate cyclase activation (join of
regulatory proteins) and the protein kinase A (dissociation of protein protomers).
Scheme of processes,
• allosteric regulation mechanisms of enzymes: a) changes in the scheme of en-
zyme activity when exposed to effector b) the role of allosteric regulation of metabo-
lism by the example of phosphofructokinase,
• covalent modification of the enzyme by the example of enzyme glycogen syn-
thase and glycogen phosphorylase. Mechanism of regulation.
2. Characteristics of enzyme inhibition. Competitive and non-competitive inhibi-
tion. Reversible and irreversible inhibition. Examples.
3. The use of enzymes inhibitors as drugs. Examples.
4. Determination of amylase activity in blood serum and urine. The principle of
the method. Clinical and diagnostic significance and reference values.

Practical
DETERMINATION OF AMYLASE ACTIVITY IN SERUM AND URINE
Principle
α-Amylase (diastase, 1.4-α-D-glycan hydrolase, EC 3.2.1.1.) catalyzes the hy-
drolysis of starch and glycogen α-1.4 glycosidic bonds to maltose and dextrin.

Starch + Н2О Maltose + Dextrin

28
 The amount of remaining starch proportional to the catalytic activity of the en-
zyme is determined by the color reaction with iodine.
Reagents
1) Substrate, 0.04% starch solution in distilled water, 2) working solution of io-
dine, 0.01 М.
Material for investigation
Serum, urine.
Procedure
Test 1, Test 2, Control,
ml ml ml
Starch solution 1.0 1.0 1.0
Incubate 5 minutes at 37°С
Serum 0.02 –– ––
Urine –– 0.02 ––
Incubate 5 minutes at 37°С
0,01 Мworking solution of iodine 1.0 1.0 1.0
Cold distilled water 8.0 8.0 8.0
Mix well. Measure the optical density of the test
and control solutions against the water at a 670 nm
wavelength (red filter).
Calculation:

Note: Еcontrol and Еtest – optical density of control and test samples, 240 – coeffi-
cient of calculation.

Normal values
Serum 16-30 g/l·h
Urine 28-160 g/l·h

Clinical and diagnostic significance

There are two amylase isozymes in human blood: pancreatic – P type (30%) and
salivary – S (70%), which are released into the bloodstream as a result of natural ag-
ing of salivary glands and pancreas cells. The enzyme has a relatively low molecular
weight (about 48.000 Da). It is filtered in the glomerulus and is contained in urine.
The ratio of isozymes in urine differed from the blood: P type – 70%, S type – 30%.
Serum and urine
The increased enzyme activity in the serum and urine occurs in the pancreatic
lesions. The enzyme activity in the blood reaches a maximum in 12-24 hours after the
disease onset and increases in 10 to 30 times in acute pancreatitis. The treatment of
lesions could lead to enzyme activity normalization during 2-6 days. The activity of
29
the enzyme is moderate in chronic pancreatitis. The increase in enzyme activity is de-
tected in lesions of salivary glands, cholecystitis, inflammation of biliary tract diseas-
es, during the pregnancy, renal failure, bowel obstruction, and diabetic ketoacidosis,
in some tumors as well as lung and ovarian cancers.
The reduced amylase activity is rarely detected in clinical practice. It usually
doesn’t have a diagnostic value. Sometimes it is seen in patients with liver disease
(cirrhosis), cancers, hypothyroidism, cachexia, with toxemia of pregnancy.

Design of laboratory work

Note the principle, laboratory procedures, and results of analysis, its clinical and
diagnostic significance. Make a conclusion about the pathologies associated with
changed amylase activity.

THEME 3.3. ENZYME CLASSIFICATION AND NOMENCLATURE

(SEMINAR)

INTRODUCTION
Enzymes are protein molecules which serve as biocatalysts in cells. General
principles of enzyme classification are found to be necessary for understanding the
role of biocatalysts in the metabolism.

THE AIM OF THE SEMINAR

To introduce the enzyme classification and to study specific reactions for each
enzyme class.

SELF-STUDY QUESTIONS
1. Role of enzymes and coenzymes in catalysis.
2. Coenzyme forms of vitamins (TPP, FMN and FAD, NAD+ and NADP+, PLP).
3. The principles of the modern classification and nomenclature of enzymes: ox-
idoreductases, transferases, hydrolases, lyases, isomerases, ligases (synthe-
tases) (Supplement 1).
4. Characteristics of each class of enzymes according to the plan:
• the name and number of the class,
• biochemical role,
• basic subclasses (subclasses 1-3),
• basic coenzymes,
• the rules of enzyme systematic names,
• examples of biochemical reactions (1-3 responses).

TOPIC FOR REPORTS

1. The application of enzymes and therapeutic enzyme inhibitors.
2. The application of enzymes in industry, in biochemical and immunological
investigations.

30
 TESTS
Chose the correct answer.

1. ENZYMES DIFFER FROM NON-PROTEIN CATALYSTS BY

1) the reduction the activation energy
2) the devoicing the consumption in the reaction
3) the absence of irreversible changes
4) the high specificity

2. A TRANSFER OF GROUPS WITHIN THE MOLECULE CATALYZES

1) isomerase
2) transferase
3) lyase
4) hydrolase

3. A REVERSIBLE REACTION OF SYNTHESIS AND BREAKDOWN

WITH FORMING A DOUBLE BONDBETWEEN GROUPSIS CATA-
LYZED BY
1) isomerase
2) ligase
3) lyase
4) transferase

4. A ALCOHOLDEHYDROGENASE IS A___________.
1) oxidoreductase
2) hydrolase
3) ligase
4) transferase

5. THE ENZYME FROM THE CLASS OF HYDROLASES IS

1) catalase
2) alcohol dehydrogenase
3) pepsin
4) hemoglobin

6. THE DECREASE IN ENZYME ACTIVITY AT 50°C ISPOSSIBLEDUE

1) the denaturation of apoenzyme
2) the denaturation of coenzyme
3) the breakdown of the holoenzyme
4) the hydrolysis

7. ENZYME SUBSTRATE SPECIFICITY EXISTS DUE TO

1) a set of determined functional groups in the active center
2) the compliance of the active chemical substrate center
3) the presence of coenzyme
4) the spatial conformity of the substrate active center
5) the complementarity of the substrate active center
31
 8. THE ACTION OF COMPETITIVE INHIBITORS CAN BE REMOVED BY
1) the increase the inhibitor concentration
2) the increase the substrate concentration
3) the reducing the enzyme concentration
4) the reaction conditions change: pH and temperature

9. THE COVALENT MODIFICATIONS DURING THE REGULATION OF

THE ENZYME ACTIVITY IS
1) the linkage of any chemical group
2) the intramolecular rearrangement of the enzyme structure
3) the addition or removal of a small portion of the substrate
4) the addition or removal of a small fragment of the enzyme

10. ENZYMES HAVE FOLLOWING PROPERTIES THAT DIFFER THEM

FROM THE INORGANIC CATALYSTS
1) enzymes accelerate the onset of the reaction
2) enzymes are adjustable
3) enzymes are consumed in the reaction process
4) enzymes do not catalyze the reaction impossible energetically

CASE STUDIES
1. If an allosteric enzyme aspartate carbamoyl-transferase (molecule consists of
12 protomers) is incubated for 60 minutes at 4°C, it loses its sensitivity to allosteric
inhibitor (CTP). But its enzymatic activity is preserved. All allosteric enzymes have
similar properties.
Identify possible mechanisms of an enzyme activity modification.

2. Explain the biochemical rules for enzymes storage and application in enzyme
replacement therapy:
• a dry drug dissolution must be performed by distilled water at room tempera-
ture,
• the drug must be stirred gently during the dissolving,
• the storage of the drug solution must be at a low temperature,
• a long-term storage of the drug is possible after its drying and sealing in evac-
uated vials.

3. Lipase is an enzyme of adipose tissue, provides neutral lipids metabolism. It

can be in two different forms: in the form of a simple protein and in a form of phos-
phoprotein.
Explain why the transition from one form to another is accompanied by a
change of its activity.
Indicate the condition when the lipase is active. It is known adrenaline induces a
cascade of reactions leading to phosphorylation of intracellular proteins during
physical activities.

32
 CHECKLIST FOR FINAL LESSON (UNITS 1, 2, 3)

1. Amino acids classification according to their biological role, chemical struc-

ture, physical and chemical properties, their solubility in water.
2. The structure of proteinogenic amino acids. Physical and chemical properties
of amino acids. The concept of an isoelectric point.
3. The peptide bond formation. The properties of the peptide bond.
4. The biological role of proteins. Classification of proteins according to the
function and structure. Physical and chemical properties of proteins and protein solu-
tions. Protein molecule stabilizing factors in the solution. The colloidal properties of
proteins.
5. pH changes and amino acids, proteins properties. Charge of amino acids and
proteins. Factors causing the precipitation of proteins. The properties of denatured
protein. Specific features of denaturation and renaturation.
6. Levels of protein structural organization. The types of bonds stabilizing the
protein structure. Amino acids forming these bonds.
7. Simple proteins (albumins, globulins, histones, protamines), their examples, a
role in the body.
8. Complex proteins: phosphoproteins, nucleoproteins, glycoproteins and prote-
oglycans, chromoproteins, metalloproteins, lipoproteins. Structure of nucleotides
AMP, ADP, ATP, cAMP. Scheme of heme, hyaluronic acid and chondroitin sulfates.
9. The principle of protein and amino acids color qualitative reactions. Possibil-
ity of their use in practice.
10. Removing proteins from the solution and purification of protein solutions
from impurities. Mechanisms of reactions. Their use in biochemistry and medicine.
11. Methods of protein precipitations, that could be used for the production of
proteins and enzymes in their native state.
12. Composition of random tetrapeptide with determined properties, the ability
to call them, the charge and solubility definition, the possible pH of their isoelectric
points.
13. Determination of phosphoprotein and glycoprotein composition components.
14. Note the properties of vitamins, their classes. Provitamins and antivitamins,
their examples. Common reasons of hypo-and avitaminosis development. Hypervit-
aminosis.
15. Characteristics of the fat-soluble vitamins A, D, E, K, F: physiological name,
chemical structure of vitamins A, D2, D3, E, K, F, active forms of vitamins A and D,
daily requirement, dietary sources. Biochemical functions and processes of vitamins.
Possible reasons and clinical features of hyper-, hypo- and avitaminosis. What are ca-
rotenoids? Note their role in the body.
16. Characteristics of water-soluble vitamins B1, B2, B3 (nicotinic acid), B5 (pan-
tothenic acid), B6, B9, B12, C, H: scientific and chemical names, chemical structure
(except vitamin B12, folic and pantothenic acid), the daily requirement, dietary
sources. Biochemical functions and reactions of vitamins. The structural formulas of

33
coenzymes (for B1, B2, B3, B6). Possible reasons and clinical features of hypo- and
avitaminosis development. The role of vitamins in child growth and development.
17. The mechanism of sulfonamides antibacterial activity.
18. Qualitative reaction of vitamins A, E, K, D3, B2, B3, B6, B12, C. Principles of
methods, the procedure of determination, practical significance.
19. Enzymes and their role in biochemical reactions. Comparison of inorganic
catalysts and enzymes.
20. Enzymes, structural and functional organization (the level of structure, sim-
ple and complex enzymes). Holoenzyme, apoenzyme, cofactor, coenzyme, prosthetic
group, active and allosteric centers. Apoenzyme and coenzyme role in catalysis. The
structure of cell multienzyme complexes.
21. Isozymes, structural features. Characteristics and examples of isozymes.
22. Classification of enzymes. The main sub-classes. The enzyme nomenclature.
What is the classification number? Examples of biochemical reactions of and en-
zymes.
23. Stages of enzymatic catalysis. Features of covalent and acid-base catalysis.
24. Quantitative determination of enzyme activity in biological objects. Units of
enzyme activity.
25. The basic properties of the enzymes, dependence of enzyme activity on vari-
ous factors. The enzyme specificity, its types. Specificity mechanisms (theory of
Fisher and Koshland).
26. The methods of metabolic activity regulation in the cell: compartmentaliza-
tion, change of enzyme concentration, substrate concentration change, the presence of
isozymes, mechanisms of enzyme allosteric regulation, covalent modification of en-
zymes, proenzymes and their limited proteolysis, protein-protein interactions.
27. The main types of enzyme inhibition: competitive and non-competitive, re-
versible and irreversible. Examples.
28. The use of enzymes in medicine. Enzyme and diagnostic. The use of enzyme
inhibitors as drugs. Examples.
29. The difference between primary and secondary forms of enzymopathy. Ex-
amples.
30. The effect of temperature on enzyme activity, salivary amylase as an exam-
ple. The principle of the method and the procedure.
31. The investigation of the enzyme action specificity on the example of salivary
amylase and urease. The principle of the method and the procedure.
32. The principle of the method and the procedure of amylase activity determina-
tion in serum and urine. Reference values, clinical and diagnostic significance.

34
 UNIT 4
BIOLOGICAL OXIDATION

THEME 4.1. MAIN CATABOLIC PATHWAYS:

PYRUVATE OXIDATIVE DECARBOXYLATION. TRICARBONIC ACID
CYCLE. ENZYMES OF RESPIRATORY CHAIN. OXIDATIVE
PHOSPHORYLATION (SEMINAR)

INTRODUCTION
The catabolism occurs in all living cells of the body in the form of oxidation.
This results in multiple transfer of protons and electrons or only electrons from a do-
nor to an acceptor. The final products of this oxidation process are water and carbon
dioxide (CO2 and H2O). The main function of biological oxidation is to provide the
body with energy for life processes. The form of energy in human body is adenosine
triphosphate (ATP).
Some substances, such as drugs (barbiturate), and toxic (cyanides, carbon mon-
oxide) are able to inhibit the oxidative phosphorylation and ATP synthesis.

THE AIM OF PRACTIC IS

The study of pyruvate dehydrogenase complex reactions and citric acid cycle,
the structure of the respiratory chain and mechanisms of mitochondrial oxidative
phosphorylation.

SELF-STUDY QUESTIONS
1. Plastic (anabolism) and energy (catabolism) metabolic functions.
2. Stages of catabolic reactions in the body related to the free energy release.
What is the release and storage of energy at each stage?
3. The structure and function of mitochondria.
4. ATP chemical formula, the role of ATP? Significance of cycles ATP-ADP
and NADPH-NADP+.
5. The main energy-rich substances in cell: ATP, 1.3-diphosphoglycerate, phos-
phoenolpyruvate, creatine, phosphate, acetyl-S CoA. Substrate phosphorylation.
6. Sources of key metabolic products – acetyl-S CoA and pyruvic acid. Further
fate of substances.
7. The structure of the multienzyme pyruvate dehydrogenase complex, its en-
zymes and coenzymes. The overall reaction of oxidative decarboxylation of pyruvic
acid. The chemical reactions of five separate reactions. Its regulation.
8. Reaction tricarboxylic acid cycle (Krebs cycle, citric acid cycle). The mecha-
nism of the acetyl group oxidation. Enzymes and coenzymes participation in this pro-
cess. The biological significance of the TCA cycle. Role of oxaloacetate, and NADH
of TCA cycle in metabolic rate regulation.
9. Connection of TCA with catabolism of carbohydrates, lipids, proteins.

35
 10. Characterize of the oxidative phosphorylation following the plan:
• molecular organization and sequence of the electron transport chain enzyme
complexes, draw a scheme of the respiratory chain enzymes,
• transport of electrons in the respiratory chain complexes, the role of coenzyme
(FMN, FeS-proteins, the Q coenzyme, cytochrome heme group),
• the role of oxygen – the final electron acceptor substrates recovered biological
oxidation,
• pumping protons from the matrix of the mitochondria – areas of transmem-
brane transport (area of coupling oxidation and phosphorylation), the electrochemical
gradient formation,
• ATP synthase structure, the role of an electrochemical gradient in the ATP syn-
thase.
11. The ratio of phosphorylation – P/O. Its significance for NADH and FADH2
forming. Calculation of ATP produced by oxidation of several substrates (alanine,
aspartic and glutamic acid).
12. Complexes of respiratory enzymes, which could act as inhibitors. How is the
process of oxidative phosphorylation being inhibited?
13. Uncoupling of oxidation and phosphorylation. The mechanism of this phe-
nomenon. Uncouplers.
14. Brown adipose tissue: its function, localization. The function of the protein
thermogenin. Its role in thermogenesis.
15. Causes of hypo energetic states.
16. The oxidative phosphorylation regulation. Respiratory control. Role of ATP
and ADP ratio in the regulation of the respiratory chain.
17. Examples of the nucleotides application as medicines (ATP, ADP, AMP,
FMN).

TESTS
Choose the correct answer.

1. THE PYRUVATE DEHYDROGENASE REACTION RATE IS INHIBITED

BY
1) ATP, calcium, NADH
2) calcium, acetyl-S CoA, NAD+
3) ADP, FADH2, NADH
4) acetyl-S CoA, NADH, ATP

2. FADH2 MOLECULE IS FORMED IN THE CITRIC ACID CYCLE BY

ACTION OF
1) malate dehydrogenase
2) isocitrate
3) succinate
4) α-ketoglutarate dehydrogenase

36
3. THE RATE OF THE CITRIC ACID CYCLE IS DETERMINED BY
1) α-ketoglutarate
2) oxaloacetate
3) succinic acid
4) citrate

4. THE ELECTRONS FLOW THROUGH THE RESPIRATORY ENZYMES

CHAIN IS DEPENDENT ON
1) the energy of ATP breakdown
2) the pumping the hydrogen protons through the membrane
3) the work of iron-sulfur centers
4) a different electronegativity of carriers

5. THERE IS A DEPENDENCE OF ELECTRONS FLOW RATE THROUGH

THE RESPIRATORY CHAIN ON
1) the ratio of ADP and ATP concentration
2) the NADH concentration
3) the amount of consumed oxygen
4) the ATP synthase activity

6. THE INCREASE OF A ELECTROCHEMICAL GRADIENT IS LEAD TO

1) an increase the protons pumping speed
2) an acceleration of ATP synthesis
3) an improvement the electron transfer rate
4) an increased CO2 and H2O release

7. ENERGY RELEASED BY ELECTRON TRANSFER ALONG THE RES-

PIRATORY ENZYMES CHAIN IS USED FOR
1) the pumping of H+ ions through the membrane
2) the oxidation of the iron-sulfur centers
3) the formation of water molecules
4) the ATP synthesis

8. THE FORMING OF A PROTON GRADIENT ON THE MITOCHONDRI-

AL MEMBRANE IS DETERMINED BY
1) the ATP breakdown
2) the oxidation of NADH
3) the flow of electrons
4) the pumping out of H + ions in exchange for Na+

9. THE UNCOUPLERING AGENTS LEAD TO

1) the reduction of NADH oxidation
2) the activation of ATP synthesis
3) the reduction of respiratory chain electron transport
4) an increase of the proton gradient

37
 10. REDUCED EQUIVALENTS FORMED IN THE CITRIC ACID CYCLE
ARE USED
1) in the respiratory chain enzymes
2) in the synthesis reactions of glucose, fatty acids, etc.
3) for the ATP synthase
4) for the acetyl-S CoA synthesis

CASE STUDIES
1. The intake of uncoupling agents leads to sweating and increased body tem-
perature. Give an explanation of this fact and describe the molecular mechanism.
Explain the change in the ratio P/O in the presence of uncoupling agents.

2. The brown adipose tissue is well developed in some animals hibernate or

adapted to living in cold areas. The ATP yield is less than one molecule per 1 atom of
oxygen. It is produced 2-3 ATP molecules per 1 atom of oxygen in other tissues.
Identify physiological function of thee low P/O ratio in brown adipose tissue of
babies. Mark a possible mechanism of a low P/O ratio in mitochondria of brown adi-
pose tissue.

38
 UNIT 5
AMINO ACIDS AND PROTEINS METABOLISM

THEME 5.1. EXTERNALMETABOLISM OF PROTEINS.

PROTEINSDIGESTION AND ABSORPTION

INTRODUCTION
Food sources of proteins are animal and vegetable products. The modification of
digestive juice composition or the appearance of its pathological components leads to
development of digestive diseases. Abnormalities of protein digestion and amino ac-
ids absorption benefit in a lack of protein synthesis in the body and in a metabolism
disturbance development.
The measuring of the gastric juice composition is of great importance, especially
the investigation of its digestive ability in normal state and in pathologies.

THE AIM OF THE PRACTICAL CLASS IS

To study the enzymes and mechanisms of protein digestion in the stomach and
intestine.
To learn the techniques of gastric juice qualitative analysis for measuring the
stomach secretory function in normal state and pathologies.

SELF-STUDY QUESTIONS
1. Structure of amino acids and proteins, the peptide bond role in the organiza-
tion of the protein molecules.
2. Characteristics of the enzyme class of "hydrolases".
3. The term "nitrogen balance" and the reasons for its change (balance of posi-
tive and negative nitrogen balance). Features of nitrogen balance in children.
4. Dietary sources of protein. The daily protein requirements for children accord-
ing to their age and for adults.
5. The biological value of proteins. The concept of the reference protein. Clinical
manifestations of protein lack in children. "Kwashiorkor" disease.
6. The mechanism of the hydrochloric acid synthesis in gastric juice and its bio-
logical role. "Hyperchlorhydria", "hypochlorhydria", "achlorhydria", "achylia".
7. Digestion of proteins in the stomach and intestine. Characterize gastric en-
zymes (pepsin, gastrixin, chymosin (rennin)), pancreatic juice (trypsin, chymotrypsin,
elastase, carboxypeptidase) and intestinal juice (aminopeptidase, dipepti-
dase) according to the plan:
• place of synthesis,
• mechanism of activation,
• optimal conditions for work,
• substrate specificity.
8. Secondary active transport of amino acids through cell membranes.

39
 9. Age characteristics of protein digestion and amino acid absorption in children.
Causes of the normal digestion and absorption in children and the relationship of
these disorders with the development of allergic reactions. Causes and clinical fea-
tures of "celiac disease".
10. General characteristics of the "protein putrefaction" in the large intestine.
The causes and consequences of this process. Substances formed due to the protein
decay.
11. Reactions of amino acid conversion by the enzymes of the intestinal micro-
flora:
• formation reaction of phenol and cresol,
• formation reaction of scatole and indole,
• formation reaction of cadaverine and putrescine,
• sources of methyl mercaptan and hydrogen sulfide.
12. Disposal of toxic products in the liver: microsomal oxidation and conjugation
system. Enzymes involved in the oxidation of microsomal. The structure of UDP-
glucuronic acid (UDPGA) and phosphoadenosine phosphoric acid (PAPA). Reactions
of indicant formation.
13. Qualitative reaction of free hydrochloric acid. The principle of methods. The
normal pH of gastric juice, clinical and diagnostic significance of pH determination
in gastric juice.
14. Qualitative reaction of lactic acid in the gastric juice. The principle of the
method and normal values. Clinical and diagnostic significance.
15. Hemoglobin detection in blood and in gastric juice. The principle of the
method. Normal values. Clinical and diagnostic significance.
16. Tubeless method of determining the gastric juice acidity (acidotest). Clinical
and diagnostic significance.

TOPICS FOR REPORTS

1. Diseases of the gastrointestinal tract, accompanied by disturbances of protein
digestion and amino acid absorption.
2. Specific changes in protein deficiency and in "kwashiorkor"disease.

Practical 1
QUALITATIVE REACTION OF FREE HYDROCHLORIC ACID IN GAS-
TRIC JUICE

Gastric acid analysis methods are used for diagnosis and monitoring of diseases
treatment in clinical practice.
Material of investigation
Test samples of gastric juice N 1, 2, 3 with different activity.

40
 With Congo red
Principle
In the presence of free hydrochloric acid in the gastric juice Congo red changes
color to blue. At weakly acidic, neutral or alkaline medium the color of stain remains
red (transition zone 5.2 pH 3.0).
Reagents
Indicator paper "Congo red".
Procedure and observation
Apply 1 drop of gastric juice samples on the test paper strip with a glass rod.
With methyl orange
Principle
Methyl orange indicator in the presence of free hydrochloric acid is red, in the
alkaline medium – orange-yellow (the transition zone pH 3.1-4.4).
Reagents
Indicator methyl orange.
Procedure and observation
Take 10 drops of gastric juice in test tubes. Then add 2 drops of methyl orange.

Normal values
Gastric juice pH 1.5-1.8
Clinical and diagnostic significance
High acidity of gastric juice is observed in duodenal ulcer, and in some cases of
stomach ulcers. It is known the stress development is associated with vagus-mediated
enhancement of acid secretion.
Reducing the acidity of gastric juice is found in atrophic gastritis, pernicious
anemia, gastric carcinoma. At the pathological conditions the acidity of gastric juice
may be zero, increased or decreased.
Hyperchlorhydria (the increased free HCl and total acidity content) occurs in
case of hyper acidic gastritis and often is accompanied by gastric ulcer and duodenal
ulcer.
Hypochlorhydria (low acidity) occurs in hypo acidic gastritis, sometimes in
stomach ulcers. As a consequence, in this case the absorption of B vitamins, iron ab-
sorption reduces and iron deficiency anemia develops. Then processes of proteins pu-
trefaction are activated in the intestine.
Achlorhydria (complete absence of hydrochloric acid) and is observed in a case
of a significant reduction of total acidity. This condition is found in atrophic gastritis,
pernicious anemia, gastric carcinoma. Achlorhydria is diagnosed only after the test
with stimulation of secretion.
Since in the absence of hydrochloric acid in the stomach under the influence of
microbial fermentation processes develop, achlorhydria is accompanied by the ap-
pearance in the stomach fermentation products – dairy, oil, acetic acid, as a result, pa-
tients may be bad breath.
Achylia (lack of hydrochloric acid and pepsin) is associated with cancer of the
stomach, pernicious anemia.
41
 Design of laboratory work
Note the principle, laboratory procedures, and results of analysis, its clinical and
diagnostic significance. Make a conclusion about the presence of pathologies pres-
ence.

Sample of gastric Changes of indicator color

pH value
juice Congo red Methyl orange
1
2
3

Practical 2
QUALITATIVE REACTION OF LACTIC ACID PRESENCE IN GASTRIC
JUICE

Principle
Lactic acid converts phenolate iron (III) of violet color in iron lactate salt of yel-
low-green color.
Reagents
1) 1% phenol solution, 2) 1% FeCl3 solution, 3) 40% lactic acid solution.
Material of investigation
Normal gastric juice and gastric juice with lactic acid.
Procedure and observation
Prepare the solution of iron phenolate (III). Mix the solution of iron with 2.0 ml
of 1% phenol solution, then add 3 drops of 1% FeCl3 solution.
Pour the mixture into 4 tubes:
• take a lactic acid solution in first tube dropwise;
• take a sample of normal gastric juice and gastric juice with lactic acid in other
tubes.
In the presence of lactic acid, the violet color of solution is changed to a yellow-
green.
Normal values
Lactic acid Absence
Clinical and diagnostic significance
The accumulation of lactic, butyric, acetic acid in the stomach is the result of hy-
po- or achlorhydria and achylia due to lack of hydrochloric acid protective function.
It develops in cases of chyme stagnation and bacterial fermentation of food compo-
nents.
Design of laboratory work
Note the principle, laboratory procedures, results of analysis, its clinical and di-
agnostic significance. Make a conclusion about the presence of pathologies.

42
 Solution color Presence of lactic acid
Lactic acid solution
Normal gastric juice
Gastric juice with lactate
Practical 3
DETECTION OF BLOOD AND HEMOGLOBIN IN GASTRIC JUICE BY
DIAGNOSTIC STRIPS "HEMOPHAN"
Principle
The "Hemophan" test strip contains stable organic hydroperoxide, an acid buffer
and a chromogen. There is an enzymatic activity in the blood presence in the sample.
The peroxidase catalyzes the oxidation of chromogen hydroperoxide to form a blue
colored product. In the presence of free (dissolved) hemoglobin band zone turns to
blue color. In presence of red blood cells in gastric juice bright blue dots on a light
background are revealed.
Material for investigation
Normal gastric juice and gastric juice with blood.
Procedure and observation
In each test tube dip an indicative gastric band of diagnostic strip, then take out
rapid and compare with the color scale on the label after 30 seconds. On a scale of
comparison on the packaging test strip the concentration of hemoglobin and red
blood cells is determined.
Normal values
Blood Absence
Clinical and diagnostic significance
Bleeding in the stomach cavity is observed in gastritis, ulceration of the stomach
wall, in malignant tumors. Thus, under the action of hydrochloric acid the blood is
converted to dark brown color hematin reminding coffee grounds. It is detected either
by probing or coloring feces black. Bleeding gums may also give a positive result.
Design of laboratory work
Note the principle, laboratory procedures, results of analysis, its clinical and di-
agnostic significance. Make a conclusion about the presence of pathologies.

Practical 4 (in theory)

TUBELESS METHOD OF ACIDITY DETECTION IN GASTRIC JUICE
(TEST OF ACIDITY)

The tubeless method of the gastric acidity detection is convenient, reliable, spar-
ing the patient. It is recommended for patients with contraindications to fractional
probing (hypertension, neuropathy), as well as for young children.
Principle
Inputted dye per os (2.4-diamino-4-etoksiazobenzol) is exempt from pellets and
absorbed in the stomach due to hydrochloric acid action (pH<3). The amount of dye
is detected in 1.5 hours in the urine after its intake. The hydrochloride salt of a red
color is formed in the case of the urine acidifying to 25% level. The degree of urine
43
coloration (amount of dye) is directly proportional to the acidity of gastric juice. The
comparison of the color intensity with the scale is a quantitative measurement of
acidity.
Reagents
1) 25% hydrochloric acid, 2) dragee of 2.4-diamino-4-ethoxybenzol dye,
3) tablets of sodium caffeine benzoate.
Material for investigation
"Control" urine portion and urine sample collected in 1.5 hours after the dye in-
take.
Procedure and observation
Patient preparation: patient intakes sodium caffeine benzoate (in 100 ml of wa-
ter) after starvation for 8 hours and bladder empting. This medicine stimulates gastric
secretion and diuresis.
The "control" urine is collected in 1 hour after sodium caffeine benzoate intake.
The patient swallows dye pellets not chewing and in1.5 hours after its intake urine is
collected again.
The analysis is performed simultaneously with both urine portions: adjust to
200.0 ml with water, take 5.0 ml of dilute urine and add 5.0 ml of a 25% solution HCl
and compare with the scale.
Design of laboratory work
Note the principle, laboratory procedures, clinical and diagnostic significance.

THEME 5.2. INTRACELLULAR AMINO ACID METABOLISM

INTRODUCTION
Proteins perform a number of unique functions, maintaining a dynamic state be-
tween the organism and the environment. There are over 20 amino acids, some of
them are essential and are included both in the general and in specific metabolism
that explains the specific features in amino acid metabolism.
A variety of amino acid metabolism disorders are described in medicine.

THE AIM OF THE PRACTICAL CLASS IS

To study the general amino acid metabolism and amino acid transport system
through the cellular membrane.
To study the amino acids basic reactions in intracellular metabolism (deamina-
tion, transamination, decarboxylation).
To learn the method of transaminases activity determination in serum.

SELF-STUDY QUESTIONS
1. Transport of amino acids through cell membranes.
2. Sources and ways of amino acid transformations in the tissues (amino acid
metabolism). Metabolism of glucogenic and ketogenic amino acids.

44
 3. Types of amino acid deamination (reductive, hydrolytic, intramolecular, oxi-
dative).
4. Oxidative deamination. The difference between direct and indirect oxidative
deamination.
5. Reaction of direct oxidative glutamic acid deamination.
6. Indirect oxidative deamination – trans deamination.
7. The mechanism of transamination reactions. The role of vitamin B6. The vita-
min B6 structure and coenzyme forms.
8. The significance of transamination reactions. Characteristics of aspartate ami-
notransferase (AST) and alanine aminotransferase (ALT). Reactions catalyzed by
these enzymes.
9. Features of indirect deamination in muscle tissue (a IMP-AMP cycle).
10. The fate of the α-keto acid formed in the process of deamination by the ex-
ample of pyruvate, oxaloacetate, α-ketoglutarate.
11. The reactions of the biogenic amine synthesis (γ-amino butyric acid, hista-
mine, serotonin, dopamine). The role of these biogenic amines.
12. Methods of biogenic amine disposal. Reactions involved in deamination of
monoamine oxidase (MAO) and methylation reactions.
13. Anabolic role of amino acids, formation of creatine as an example. The struc-
ture of creatine and creatine phosphate, the reaction of their synthesis, the process of
localization. The biological role of creatine phosphate. The cause of physiological
creatinurea in children?
14. Determination of AST and ALT activity in serum. The principle of the meth-
od, clinical and diagnostic significance. Normal values.

TOPICS FOR REPORTS

1. Anabolic processes in which amino acids are involved. Application of amino
acids in the medical practice.
2. Aminoaciduria, its types, etiology and pathogenesis, clinical manifestations,
treatment bases.
Practical
DETERMINATION OF AMINOTRANSFERASES ACTIVITY IN SERUM

Principle
The aspartic acid and alanine are formed in transamination reactions of
2-oxoglutarate and pyruvate. The enzymes are aminotransferase (AST, L-aspartate :
2-oxoglutarate aminotransferase, EC 2.6.1.1. and alanine aminotransferase (ALT,
L-alanine : 2-oxoglutarate aminotransferase, EC 2.6.1.2). The 2-oxoglutarate, under-
going spontaneous decarboxylation, is converted into pyruvate. Adding
2.4-dinitrophenylhydrazine in enzymatic reaction leads to reaction stoppage with
formation of hydrazine of pyruvic acid. This product gives brown color in alkaline
medium. The color intensity is proportional to the amount of formed pyruvic acid.

45
 Reagents
1) AST substrate solution: mixture of α-ketoglutarate and aspartic acid, 2) ALT
substrate solution: mixture of α-ketoglutarate and alanine, 3) 2.4-
dinitrophenylhydrasine in 1.0 М HCl, 4) 0.4 М NaOH solution.
Standardized pyruvic acid solution, 0.1 mmol/l.
Material of investigation
Serum.
Procedure and observation
Tube 1, Tube 2, Tube 3,
standard, ml test for ALT, ml test for AST, ml
ALT substrate solution 0.25 0.25 ––
AST substrate solution –– –– 0.25
Standardized pyruvic acid
0.05 –– ––
solution
Serum –– 0.05 0.05
Incubate 30 min at 37°С
2.4-dinitrophenylhydrasine 0.25 0.25 0.25
Incubate 20 min at room temperature
NaOH 2.5 2.5 2.5
Incubate 10 min at room temperature. Measure the op-
tical density of standard and test tubes at 540 nm
against the water (green filter).
Calculation
Е test2
ALT activity, mmol/l·h = Е standard ×Сstandard× 2
Е test3
AST activity, mmol/l·h= Е standard ×Сstandard× 2,
Еstandard, Еtest2, Еtest3 – optical density of standard and test samples for ALT and
AST activity measurement, Сstandard – concentration of standard solution, 2 – coef-
ficient of 30 min in 1 hour conversion.

Normal values
Serum ALT activity 0.10-0.68 mmol/l·h
AST activity 0.10-0.45 mmol/l·h
The de Ritis ratio 1.33±0.40

46
 Clinical and diagnostic significance
The definition of AST and ALT activity is used usually in clinical practice to
identify the pathological processes in the myocardium and liver.
The myocardium has a high activity of AST than ALT. Increased activity in the
blood of both enzymes, AST especially, is a marker in acute myocardial infarction
and is available in 95% of cases. AST activity reaches a peak after 24-36 hours (usu-
ally increased by 4-5 times) and in a case of adequate treatment is reduced in 3 or 7
day. The activity of enzymes in blood varies slightly in stenocardia (angina).
Lesions of liver (toxic, infectious hepatitis) lead to increase in both enzymes ac-
tivity. ALT level is more expressed than AST in this case. The enzyme activity in-
creases before the appearance of jaundice in infectious hepatitis. In half of the cases
of cirrhosis a AST activity is higher than ALT activity.
The de Ritis ratio (the ratio of AST/ALT) is significantly increased in myocardi-
al infarction, decreased in hepatitis.
Design of laboratory work
Note the principle, laboratory procedures, normal values, its clinical and diag-
nostic significance and make a conclusion of possible pathological processes.

THEME 5.3. AMMONIA METABOLISM AND ITS DISPOSAL

INTRODUCTION
The formation of ammonia in the body determines the need for its detoxification
and disposal. Inherited and acquired disorders of ammonia disposal processes cause
serious clinical complications. The knowledge of these processes is necessary for the
treatment of liver and kidney diseases.

THE AIM OF THE PRACTICAL CLASS IS

To study the basic ways of ammonia disposal with the formation of the protein
metabolism end products.
To learn the method of urea and creatinine determination in serum and urine.

SELF-STUDY QUESTIONS
1. The main sources of ammonia in the tissues. Reactions of biogenic amine dis-
posal, the direct deamination of glutamic acid.
2. The main ways of ammonia formation in the cells:
• reductive amination reaction (reamination), an enzyme, and the significance of
reactions,
• amide formation reaction of glutamic and aspartic acid, mark their biological
importance, describe organs where these reactions occur,
• carbamoyl phosphate synthesis.
3. The ammonia transport form in blood (glutamine, asparagine, alanine). Glu-
cose-alanine cycle.
4. The role of the liver, kidney and intestines in the formation and disposal of
ammonia.
47
 5. Ornithine urea cycle reactions, its localization, enzymes, significance. Con-
nection of the ornithine cycle with the TCA cycle.
6. Presentation of hyperammonemia, their causes and consequences. Normal and
maximum permissible levels of ammonia concentration in blood. Causes of ammonia
toxicity.
7. Ammoniagenesis, reactions, location, significance.
8. Creatine and phosphocreatine synthesis reaction. The biological role of crea-
tine phosphate.
9. Creatinine, formation reaction, excretion.
10. Quantitative determination of urea in blood serum and urine. The principle of
the method, its clinical and diagnostic significance, normal values.
11. Quantitative determination of creatinine concentration in the serum and
urine. The principle of the method, its clinical and diagnostic significance, normal
values.
TOPICS FOR REPORT
1. Hyperammonemia: causes, pathogenesis, clinical manifestations, treatment.
Neonatal hyperammonemia. Molecular mechanisms of ammonia toxicity.

Practical 1
DETERMINATION OF UREA CONTENT IN SERUM AND URINE
Principle
Urea by the action of urease hydrolyzes to ammonia and CO2. Ammonium ions
in an alkaline medium react with the nitroprusside salicylate hypochlorite reagent to
form a green color complex of indophenol. The color intensity is proportional to the
urea amount.
Reagents
1) Urease stabilized solution, 2) nitroprusside salicylate reagent, 3) hypochlorite,
4) urea standardized solution (8.33 mmol/l).
Material of investigation
Serum, Urine (diluted in 1:100).
Procedure and observation
Test 1, ml Test 2, ml Standard, ml
Urease stabilized solution 0.1 0.1 0.1
Serum 0.01 –– ––
Urine (diluted in 1:100) –– 0.01 ––
Urea standardized solution –– –– 0.01
Mix well and incubate 5 min at room temperature
Nitroprusside salicylate rea-
1.0 1.0 1.0
gent
Hypochlorite 1.0 1.0 1.0
Mix well and incubate 5 min at 37°С.
Measure the optical density at 620nm against the
water (red filter).

48
 Calculation
Е test
Serum urea concentration, mmol/l = Е standard ×Сstandard
Е test
Urine urea concentration, mmol/day = Е standard ×Сstandard× 100 ×D,
Еtest and Еstandard – optical density test and standard samples, Сstandard – urea concen-
tration in standard sample, 100 – urine dilution, D – diuresis amount (daily urea
formation) (1.3-1.5 l/day).
Normal values
Serum Children 1.8-6.4 mmol/l
Adults 2.5-8.3 mmol/l
Urine 330-580 mmol/day
Clinical and diagnostic significance
The urea level in serum and urine is dependent on its synthesis rate in the liver
and on its excretion by kidneys, on the protein metabolism.
Serum
The increased serum urea levels are observed in diseases of the kidneys (disor-
ders of kidney excretory functions), modifications of renal perfusion (congestive
heart failure), depletion of water in the body by vomiting, diarrhea (relative increase
in concentration), in cases of increased protein catabolism (fever, starvation) and at a
diet with high protein consumption.
The reduced urea concentrations are found in cases of a diet with low protein
consumption, in increased protein metabolism in tissues (children, pregnancy), severe
liver disease associated with impaired urea synthesis (parenchymal jaundice, hepati-
tis, cirrhosis).
Urine
The determination of urea in urine allows monitoring processes of body proteins
anabolism and catabolism (nitrogen balance).
The increasing concentration of urea in the urine is observed in cases of negative
nitrogen balance, in the excess protein in diet in the postoperative period, in hyper-
thyroidism, in fevers and starvation.
The reducing urea excretion is a sign of a positive nitrogen balance, and could be
observed during pregnancy, during the growth.
Design of laboratory work
Note the principle, laboratory procedures, normal values, clinical and diagnostic
significance and make a conclusion of possible pathological processes.

49
 Practical 2
DETERMINATION OF CREATININE CONTENT IN SERUM AND URINE

Principle
The creatinine in an alkaline medium reacts with picric acid and forms creatinine
picrate of orange color. The color intensity is proportional to the solution concentra-
tion of creatinine in a biological fluid.
Reagents
1) 10% NaOH solution, 2) saturated solution of picric acid, 3) 10% trichloroace-
tic acid solution (ТCA), 5) standardized creatinine solution, 177 µmol/l.
Material of investigation
Serum, urine (dilution in 1:50).
Procedure and observation
Test 1, ml Test 2, ml Standard, ml

Serum 0.5 –– ––
Distilled water 1.0 –– ––
10% TCA solution 0.5 –– ––
Mix well, then centrifuge at 1500 RPM
(revolutions per minute) or filtrate through-
pre-moistened with distilled water filter
Filtrate 1.0 –– ––
Distilled water –– 0.5 0.5
Urine (dilution in 1:50) –– 0.5 ––
Standardized creatinine solution –– –– 0.5
10% NaOH solution 0.5 0.5 0.5
Saturated solution of picric acid 0.5 0.5 0.5
Mix and after 20 min measure the optical
density at 540 nm against water (green fil-
ter).
Calculation
Е test
Serum creatinine concentration, µmol/l = Е standard ×Сstandard× 2,
Е test
Urine creatinine concentration, mmol/day = Е standard × 1000 ×С standard× 50 × D, where

Еtest and Еstandard – optical density test and standard samples, 1000 – coefficient of
calculation micromol in mmol; Сstandard – creatinine concentration in standard sam-
ple, 2 – dilution of serum, 50 – dilution of urine, D – diuresis amount (daily urea
formation) (1.3-1.5 l/day).

50
 Normal values
Serum Children up to 1 year 18-35 µmol/l
Children from 1 year to 12 27-62 µmol/l
years
Women 44-97 µmol/l
Men 52-132 µmol/l
Urine 4.4-17.7 mmol/day
Clinical and diagnostic significance
Serum
The concentration of creatinine in the blood of healthy people is relatively con-
stant and depends on the muscles weight.
The increase of serum creatinine level in 2-7 times is observed in an acute renal
failure, in most severe cases in 15-25 times. In addition, the creatinine level may be
not dependent on muscle word. Its concentration is found to be elevated in hyperthy-
roidism, diabetes, muscular dystrophy, extensive burns, fevers, frequent intramuscu-
lar injections.
The decrease of creatinine in the blood does not have a diagnostic value.
Urine
The increase of creatinine concentration in urine is observed in persons with in-
creased physical activity, in fevers. It is found to be elevated in liver diseases, in dia-
betes mellitus and diabetes insipidus, with crush syndrome, acute infections.
The decrease of creatinine in urine is found in chronic nephritis and other kidney
diseases, muscle atrophy, leukemia and starvation.

Design of laboratory work

Note the principle, laboratory procedures, normal values, clinical and diagnostic
significance and make a conclusion of possible pathological processes.

THEME 5.4. METABOLISM OF SOME AMINO ACIDS. THEIR FEATURES

AND DISORDERS (SEMINAR)

INTRODUCTION
Apart from common reactions of amino acid metabolism, there are specific ones,
which are associated with unique amino acid function. The derivatives of amino acid
metabolism could play an important and sometimes key role in metabolic processes
and determine the physiological state of the body. There are more than 100 diseases
caused by inherited defects of amino acid metabolism.

THE AIM OF THE PRACTICAL CLASS IS

To study the metabolism of glycine, serine, cysteine, methionine, phenylalanine,
tyrosine, tryptophan, and dicarboxylic amino acids and their disorders.

SELF-STUDY QUESTIONS
1. The structure of proteinogenic amino acids.
51
 2. Sources and common pathways of amino acid metabolism in tissues.
3. Ways of dicarboxylic amino acids and their amide use (glutamate and aspar-
tate) in metabolic reactions. Connection between the metabolism of dicarboxylic
amino acids with citric acid cycle.
4. The synthesis of glucose from serine, alanine, aspartic and glutamic acids
5. Ways of cysteine and sulphur use. Reactions of taurine synthesis. Characteris-
tics of "cystinosis", its cause, the clinical features. Cystinuria and its causes.
6. The use of glycine and serine in the body. Reactions of interconversion of gly-
cine and serine, the role of tetrahydrofolic acid.
7. The correlation of glycine, serine, methionine and cysteine metabolism:
• S-adenosylmethionine synthesis reaction from S-adenosylhomocysteine, its
role in transmethylation processes and the synthesis of definite substances,
• the reaction of homocysteine formation and pathways of its further transfor-
mation,
• participation of vitamin B9 (folic acid), vitamin B6 (pyridoxine) and B12 (cya-
nocobalamin.
8. Causes of homocysteinemia and homocystinuria. Comorbidities. Treatment.
9. Ways of phenylalanine and tyrosine use. Anabolic and catabolic pathways of
tyrosine transformations. The reaction of converting phenylalanine to tyrosine.
10. Characteristics of phenylketonuria type I (classical) phenylketonuria disease
and type II (variant). Defective enzymes, clinical manifestations, treatment bases
11. Reactions of tyrosine catabolism. Enzymes, a defect which lead to the char-
acteristic features of the disease. The basis of treatment.
12. Changes of tyrosine anabolic function – albinism and Parkinson’s disease.
Molecular mechanism, special features and the basis of treatment.

TOPICS FOR REPORT

1. A method of substances separating. Chromatography, its types. Application of
separating substances in medicine.
2. The artificial kidney. Hemodialysis. The principle of blood purification and its
use in the correction of pathological conditions.
3. Phenylketonuria and its types (classical type I, II and III variant types). Mater-
nal phenylketonuria. The molecular cause of disease pathogenesis, clinical manifesta-
tions, treatment bases.
4. Glycine catabolism to ammonia, carbon dioxide, or low molecular weight or-
ganic acids (formic acid, oxalic acid). Glycosylate cycle. Modifications of catabolism
– hyperglycinemia, hyperoxalateuria.
5. The catabolism of amino acids with branched radical (leucine, isoleucine, va-
line). Diseases "with the urine odor of maple syrup" and isovaleric acidemia. Clinical
features, basis of treatment.
6. Tryptophan metabolism. Causes of pellagra in tryptophan metabolism disor-
ders. Hartnus’a disease. Clinical features, the basis of treatment.

52
 TESTS
Choose the correct answer.

1. BIOLOGICAL SIGNIFICANCE OF PROTEINS IS DETERMINED BY

1) the order of amino acids in the protein molecule interlace
2) the amino acid composition
3) the molecular weight of proteins
4) the charge of the protein molecule

2. TO NEUTRALIZE TOXIC SUBSTANCES FORMED IN THE INTES-

TINE, WHICH ENZYME IS USED IN THE LIVER
1) hexokinase
2) aminotransferase
3) glucuronyl transferase
4) sucrase

3. ENTEROPEPTIDASE IS AN ACTIVATOR OF ENZYME

1) pepsinogen
2) trypsinogen
3) chymotrypsinogen
4) proelastase

4. AN ACTIVE ENZYME GLUTAMATE DEHYDROGENASE CONTAINS OF

1) nicotinamide adenine dinucleotide (NAD)
2) flavin adenine dinucleotide (FAD)
3) flavin mononucleotide (FMN)
4) pyridoxal phosphate (PLP)

5. THE SEROTONIN IS FORMED IN THE DECARBOXYLATION REAC-

TION OF
1) cysteine
2) tryptophan
3) tyrosine
4) 5-hydroxytryptophan

6. THEFORMATION OF γ-AMINOBUTYRIC ACIDS IS CATALYZED BY

1) histidine decarboxylase
2) tyrosine monooxygenase
3) glutamate decarboxylase
4) ornithine

7. THE COENZYME OF AMINOTRANSFERASES IS

1) nicotinamide
2) flavin adenine dinucleotide
3) thiamine diphosphate
4) pyridoxal phosphate

53
 8. THE HYDROCHLORIC ACID SYNTHESIS IS STIMULATED BY
__________ IN THE STOMACH
1) tyramine
2) histamine
3) dopamine
4) tryptophan

9. THE HYPOVITAMINOSIS C IS ASSOCIATED WITH DISORDERS OF

___________ METABOLISM
1) tyrosine
2) leucine
3) methionine
4) cysteine

10. THE BINDING OF AMMONIA OCCURS IN

1) synthesis of glutamate from 2-oxoglutarate
2) synthesis of creatine
3) transamination of alanine
4) synthesis of serotonin

CASE STUDIES
1. The patient complains of a pain in the stomach, belching smelly "rotten eggs",
rumbling and flatulence.
Identify the processes that may cause these symptoms. Give recommendations
for the digestion improvement.

2. It is found that the addition of the glutamic acid in solution has a positive im-
pact on the physiological function of the heart muscle, particularly under low oxygen
supply.
Explain the mechanism of the amino acid positive impact on the heart activity.

3. The urea content in blood is about 2 mmol/l. About 180 mmol of urea is ex-
creted with urine per day.
Identify the impaired body function. Name the enzymes that should be investi-
gated to verify the diagnosis.

54
 UNIT 6
STRUCTURE AND METABOLISM OF PURINE AND
PYRIMIDINE NUCLEOTIDES

THEME 6.1. STRUCTURE AND METABOLISM OF PURINE AND

PYRIMIDINE NUCLEOTIDES

INTRODUCTION
Purine and pyrimidine nucleotides perform several important functions in the
cell, one of which is the nucleic acid synthesis. Nucleic acids are not essential nutri-
tional factors, and therefore the majority of cells in the body is capable for nucleotide
synthesis. It determines the nucleotide metabolism rate.
The disorders of purine nucleotide metabolism are gout, kidney disease and uro-
lithiasis (formation of uric acid stones), Lesch-Nyhan syndrome. The orotate aciduria
is a disease associated with pyrimidine nucleotide metabolic disorders.
Nucleotides are also medicines which could be used in sport medicine and as the
therapeutic agents (for example, methyluracil, potassium orotate).

THE AIM OF THE PRACTICAL CLASS IS

The study of biosynthesis and catabolism of purine and pyrimidine nucleotides,
introduction to metabolic disorders associated with these processes.
Acquiring the skills for determining the uric acid concentration in the serum and
urine.

SELF-STUDY QUESTIONS
1. The digestion of nucleoproteins in the gastrointestinal tract, enzymes. The fur-
ther fate of purine and pyrimidine nucleotides and bases.
2. The synthesis of purine nucleotides:
• reaction of 5-phosphoribosylamine formation,
• sources of carbon and nitrogen atoms of the purine ring,
• synthesis of AMP and GMP from IMP,
• AMP conversion reaction into ATP and GTP conversion reaction into GMP.
3. The regulation of the purine nucleotide synthesis according to the negative
feedback mechanism. Its cross positive regulation with the participation of ATP and
GTP.
4. Catabolism of purine nucleotides:
• the reaction of AMP decay,
• the reaction of GMP decay,
• the reaction of uric acid formation from hypoxanthine and xanthine, the role of
xanthine oxidase.
5. Primary and secondary hyperuricemia:
• urolithiasis, its causes, basis of treatment,

55
 • gout, its causes, clinical manifestation, basis of treatment. The mechanism of
allopurinol action in the gout treatment.
6. Lesch-Nyhan syndrome, its causes, basis of treatment and prognosis.
7. Synthesis of pyrimidine nucleotides:
• reaction of UMP and UTP synthesis,
• reaction of CTP synthesis from UTP.
8. Regulation of the pyrimidine nucleotides synthesis by the mechanism of nega-
tive feedback.
9. Synthesis of deoxyribonucleotides. Role of NADPH and thioredoxin.
10. Synthesis of dTMF. The role of tetrahydrofolic acid. The cause of megalo-
blastic anemia with folate deficiency. The mechanism of sulfonamides antibacterial
activity.
11. The catabolism of pyrimidine nucleotides. The end products of the process.
12. Diseases associated with disorders of pyrimidine metabolism. Orotatate
aciduria, causes, clinical manifestation, basis of treatment.
13. Application of synthesis inhibitors of purine and pyrimidine nucleotides in
medicine. Methotrexate, 5-fluorouracil, azidothymidine.
14. Quantitative determination of the uric acid concentration in serum and urine.
The principle of the method, clinical and diagnostic significance, normal values.

TOPICS FOR REPORTS

1. Gout: etiology, molecular mechanisms of development, clinical manifesta-
tions, treatment.
2. Urolithiasis: etiology, types of urinary stones, diagnosis and clinical manifes-
tations, treatment.
3. Enzymes of ribonucleotides or deoxyribonucleotides synthesis as targets for
antiviral and anticancer drugs.

Practical
DETERMINATION OF URIC ACID CONCENTRATION IN SERUM AND
URINE
Principle
The uric acid is cleaved by the enzyme uricase to allantoin with simultaneous
formation of hydrogen peroxide. The last reacts with dihydroxybenzol sulfate and 4-
aminoantipyrinedue to the action of peroxidase forming products of pink color. The
color intensity is proportional to the amount of uric acid.
Reagents
1) Working reagent with phenol, uricase, peroxidase, dihydroxybenzol sulfate
and 4-aminoantipyrine in potassium phosphate buffer, 2) standardized solution of uric
acid, 500 µmol/l.
Material of investigation
Serum. Urine (dilution 1:5).

56
 Procedure and observation
Test 1, ml Test 2, ml Standard, ml
Serum 0.025 –– ––
Urine (dilution 1:5) –– 0.025 ––
Standardized solution of uric acid –– –– 0.025
Working reagent 1.0 1.0 1.0
Wait 10 minutes at 37ºC.
Measure the optical density against water at 540
nm (green filter).
Calculation
Е test
Serum uric acid concentration, mmol/l = Е standard ×Сstandard,
Е test × 5 × D
Urine uric acid concentration, mmol/ day = Е standard × 1000 ×Сstandard,
Еtest and Еstandard – optical density test and standard samples, Сstandard – uric acid
concentration in standard sample, 50 – dilution of urine, 1000 – coefficient of cal-
culation micromol in mmol; D – diuresis amount (daily urea formation) (1.3-1.5
l/day).
Normal values
Serum children 0.12-0.32 mmol/l
Adult 0.16-0.45 mmol/l
Urine 1.46-4.43 mmol/day
Clinical and diagnostic significance
Serum
Blood urate (monosodium salt in combination with the protein) is determined by
the intensity of uric acid synthesis, and the rate of its removal in the body. Serum
urate stabilizes proteins, but at lower pH urate crystallizes in tissues.
Primary hyperuricemia divides into metabolic and renal one. Metabolic type is
the result of increased purine nucleotides synthesis, such as increased activity of ri-
bose-phosphate diphosphokinase (or phosphoribosyl pyrophosphate synthetase) or
insufficient activity of hypoxantine-guaninephosphoribosyl tranferase. Renal type
may be conditioned at genetic disorders associated with reducing excretion of uric
acid by the kidneys.
Secondary hyperuricemia is observed in all conditions associated with enhanced
decay of nucleoproteins: leukemia, treatment with cytostatics, irradiation, extensive
psoriasis, pernicious anemia, hemolytic anemia. The most common cause of kidney
failure is an altered filtration rate and tubular secretion of uric acid. The slowing urate
excretion rate in the body is found in myxedema, hyperparathyroidism, diabetes
mellitus, preeclampsia.
Detection of hypouricemia is diagnostically insignificant, and sometimes is ob-
served in anemia, after in taking salicylates, in an excess of corticotropin.
57
 Urine
Increased uric acid in the urine is observed in hyperuricemia of nonrenal origin.
Salicylates, lithium salt also increase the excretion of urate.
The concentration of uric acid is reduced in cases of alcohol abuse, poisoning by
salts of heavy metals and in kidney disease.
In gout uric acid is deposited in the tissues, joint capsules, cartilage, tendons, its
daily amount may sometimes decrease in urine.
Design of laboratory work
Note the principle, laboratory procedures, normal values, its clinical and diag-
nostic significance and make a conclusion of possible pathological processes.

TESTS
Choose the correct answer.
1. THE DONOR OF NITROGEN IN THE SYNTHESIS OF PYRIMIDINE
NUCLEOTIDES IS
1) glycine
2) asparagine
3) aspartate
4) glutamate

2. THE DONOR OF CARBON IN THE SYNTHESIS OF PURINE NUCLE-

OTIDE IS
1) aspartic acid
2) formyl-THFA
3) glutamine
4) tryptophan

3. THE REGULATORY ENZYME IN THE SYNTHESIS OF PYRIMIDINE

NUCLEOTIDES IS
1) xanthine oxidase
2) carbamoyl phosphate synthase II
3) dihydroorotase
4) carbamoyl phosphate synthase I

4. ALLOPURINOL IS AN ANTI-GOUT DRUG. ITS EFFICANCY IS AS-

SOCIATED WITH
1) competitive inhibition of xanthine oxidase
2) reduction hypoxanthine concentration in urine
3) increase of uric acid rate excretion by kidneys
4) decrease of purine bases formation

5. ANTIVITAMINS OF FOLIC ACID BLOCK REACTION

1) formation of dTMP from dUMP
2) formation of orotic acid
3) synthesis of carbamoyl phosphate
4) formation of CTP from UTP
58
 6. THE INHIBITOR OF CARBAMOYL PHOSPHATE SYNTHASE IS
1) CTP
2) GMP
3) dATP
4) 5-phophoribosyl-1-pyrophosphate

7. THE OROTATE ACIDURIA DEVELOPMENT IS ASSOCIATED WITH

ENZYME DISORDER
1) carbamoyl phosphate synthase I
2) carbamoyl phosphate synthase II
3) xanthine oxidase
4) OMP-decarboxylase

8. THE END PRODUCT OF PURINE NUCLEOTIDES DEGRADATION IS

1) urea
2) uric acid
3) lactic acid
4) malonic acid

9. DURING THE DECAY OF PYRIMIDINE NUCLEOTIDES IS FORMED

1) acetyl-SCoA
2) sodium urate
3) xanthine
4) β-aminoisobutyric acid

10. PATIENTS WITH LESCH-NYHAN SYNDROME HAVE THE GENET-

IC DEFECT OF
1) xanthine oxidase
2) hypoxanthine-guanine phosphoribosyl transferase
3) galactose-1-phosphate uridylyl transferase
4) phosphoribosyl diphosphate synthase

CASE STUDIES
1. 1-year-old child was admitted to the emergency room with symptoms of
physical and mental retardation. It was revealed a high concentration of uric acid in
the urine.
Identify the source of uric acid in the urine.

2. A lack of folic acid in the diet leads to megaloblastic anemia development,

leukopenia, impaired state of the mucous membranes and skin.
Indicate the biochemical cause of described disturbances.

59
 UNIT 7
BIOSYNTHESIS OF NUCLEIC ACIDS AND PROTEINS

THEME 7.1. NUCLEIC ACIDS SYNTHESIS AND

ITS REGULATION

INTRODUCTION
Nucleic acids are responsible for the storage and transferring the genetic infor-
mation. Errors that occur during DNA replication and repair, protein biosynthesis
lead to the appearance of the abnormal products and disruption of biochemical pro-
cesses in the cell. The development of many diseases is caused by the presence of
such hereditary or acquired errors.

THE AIM OF THE PRACTICAL CLASS IS

To study the main stages of nucleic acids synthesis.
To learn the extraction of yeast nucleoprotein components and to perform the
qualitative reactions for their detection.

SELF-STUDY QUESTIONS
1. The structure of nucleic acids DNA and RNA. The structure of the nucleopro-
tein. Types of histones, features of their structure and their role. Non-histone proteins
and their function.
2. The structure of ribosomes, their role in the cell.
3. DNA biosynthesis (replication) in eukaryotes according to the following plan:
• the total equation,
• connections with phases of the cell cycle,
• the location of process,
• components of DNA synthesizing system,
• main stages, the sequence of reactions, substrates and enzymes,
• end products,
• energy sources for the DNA synthesis,
• scheme of replication fork, specify the location of the Okazaki fragments and
each replication enzyme in view of its function.
4. The DNA repair process, its significance.
5. The biosynthesis of RNA (transcription) in eukaryotes according to the fol-
lowing plan:
• the total equation,
• connections with phases of the cell cycle,
• the location of process,
• components of the RNA-synthesizing system,
• main stages, the sequence of reactions, substrates and enzymes,
• end products,
60
 • energy sources for the biosynthesis,
• scheme of transcriptional folk, select the position of the promoter, the TATA
box, and the terminator of RNA polymerase.
6. The regulation of transcription in prokaryotes by synthesis induction (Jacob-
Monod scheme), the lactose operon as an example, and by synthesis repression, the
tryptophan operon as an example.
7. Main methods of transcription regulation in eukaryotes.
8. The processing of messenger RNA: splicing, capping, attaching poly A se-
quence.
9. The secondary transfer of RNA structure, the concept of tRNA processing.
Localization of tRNA and the role of modified nucleotides (pseudouridine, dihy-
drouriine). An adapter role of tRNA.
10. The concept of ribosomal RNA processing. Types of rRNA in eukaryotes.
Function rRNA.
11. The application of DNA and RNA biosynthesis inhibitors as drugs. Doxoru-
bicin, melphalan, novobiocin, rifamycin. What is the mechanism of their action?
12. The analysis of the complex protein chemical composition (nucleoproteins).
The principle of the methods.

TOPICS FOR REPORT

1. The gene engineering, the principle of method and its significance. The appli-
cation of gene engineering for the medication production.
2. Genetically modified foods. Prospects, challenges and solutions.
3. Cloning, the principle of method and significance. The potential application in
medicine.
4. The hybridization of nucleic acids, principles and significance. The applica-
tion in medicine and biology.
5. Ribozymes: structure, classification, properties. Ribozymes as medicaments.

Practical
ANALYSIS OF CHEMICAL COMPOSITION OF NUCLEOPROTEINS
Nucleoproteins comprise a protein part, purine or pyrimidine bases, ribose and
deoxyribose carbohydrates, and phosphoric acid. All these components are deter-
mined during the practical lesson.
Reagents
1) 1% thymol solution in ethanol, 2) 10% NaOH solution, 3) concentrated
NH4ОН (ammonium), 4) ammonium molybdate acidic, 5) concentrated H2SO4,
6) 1% CuSO4 solution, 7) 1% AgNO3 ammonia solution.
Material for investigation
Yeast hydrolysate.
Procedure and observation

61
 Principle
The Biuret reagent (copper sulfate in a strong base) reacts with peptide
bonds in proteins to form a violet complex known as the "Biuret com-
Biuret
plex".
Test
Procedure and observation:
Add 10 drops of 10% sodium hydroxide solution and 1 drop of 1% copper
sulfate solution to 5 drops of yeast hydrolysate.
Principle
Purine bases (adenine and guanine) react with silver nitrate in 5-10
Silver test minutes to form a light fluffy brown precipitate of silver salts.
for purine Procedure and observation:
bases Add 10 drops of concentrated ammonium solution, 10 drops of 1% of
ammonium silver nitrate solution to 5 drops of yeast hydrolysate. After
waiting specific precipitate is formed.
Principle
Molisch's
After dehydration of pentoses the hydroxymethyl sulfuric acid is formed.
test (for the
Its condensation with thymol hydroxymethyl furfural is associated with
presence
the development of red color, and pink rings appear in vitro.
of carbohyd
Procedure and observation:
rates– β-D-
Add 2-3 drops of thymol solution to 10 drops of yeast hydrolysate. Mix
ribose)
gently and add concentrated H2SO4.
Principle
Phosphoric acid presented in the precipitate interacts with ammonium mo-
Molyb-
lybdate in nitric acid, forms a lemon-yellow color ammonium phosphomo-
denum test
lybdate complex compound.
for phos-
Procedure and observation
phoric acid
Add 20 drops of a molybdenum reagent to 10 drops of yeast hydrolysate.
Heat the tube in water bath. Ammonium phosphomolybdate precipitates
after cooling.

Design of laboratory work

Note results of work and fulfil the table. Make a conclusion of chemical compo-
sition of nucleoproteins:
Object of in- Complex pro- The revealed Color Conclusion
vestigation teins component
Yeast Nucleoproteins Protein
Purine bases
Pentoses
Phosphoric acid

62
 THEME 7.2. PROTEIN BIOSYNTHESIS AND ITS REGULATION

INTRODUCTION
Proteins, as well as other cellular components are in a state of dynamic equilibri-
um, that is continuously being updated. Knowledge of the mechanism of protein bio-
synthesis and principles of its regulation are necessary for understanding the molecu-
lar basis and for the rational application of therapeutically agents in practical medi-
cine.

THE AIM OF THE PRACTICALCLASS IS

To study the main stages of protein biosynthesis and mechanisms of its regula-
tion.
To introduce the method of protein determination in the serum.

SELF-STUDY QUESTIONS
1. The structure of proteinogenic amino acids, DNA and RNA nucleic acids. The
structure of ribosomes, their role in the cell.
2. The genetic code and its properties.
3. The transfer RNA adapter role. The synthesis of aminoacyl-tRNA, the amino-
acyl-tRNA synthetase.
4. Characteristics of protein biosynthesis according to the following plan:
• the total equation,
• connections with phases of the cell cycle,
• the location of the process,
• components of the protein-synthesizing system,
• main stages, the sequence of reactions and enzymes,
• end products,
• energy sources for biosynthesis.
5. The post-translational modification of protein molecules. Examples of pro-
teins involved in these processes. What is a folding, what is the role of the chaper-
one?
6. Medicines as protein biosynthesis inhibitors. The mechanism of action (tetra-
cycline, chloramphenicol, erythromycin, streptomycin).
7. The quantitative determination of protein in the blood serum. Biuret test. The
principle of the method, clinical and diagnostic significance, normal values.

TOPICS FOR REPORTS

1. Chaperones. Their types, structure, participation in maturation and stabiliza-
tion of the protein molecule. Protein folding.
2. Prions. Their origins and properties. Prion diseases.
3. Medicines as inhibitors of matrix biosynthesis of RNA, DNA and protein. The
mechanism of action.

63
 Practical
DETERMINATION OF PROTEIN CONCENTRATION IN SERUM
Biuret test
Principle
The peptide bond in an alkaline medium forms a complex compound with cop-
per (Biuret reaction). The intensity of development of a blue-violet color is propor-
tional to the protein content.
Material for investigation
Serum.
Reagents
1) Biuret reagent: mix of CuSO4 and NaOH, 2) 0.9% NaCl solution.
Standardized albumin solution, 70 g/l.
Procedure
Test, ml Standard, ml
Serum 0.04 ––
Standardized albumin solution –– 0.04
Biuret reagent 3.0 3.0
Wait for 15 minutes.
Measure the optical density of tubes against the
water at 540 nm (green filter).
Calculation
Е test
Protein concentration, g/l =
Еstandard ×С
standard,

Еtest and Еstandard – optical density of test and standard tubes,

Сstandard – protein concentration in a standard tube.
Normal values
Serum Children from 1 year to 3 years 54-85 g/l
Children from 4 to 18 years
65-85 g/l
Adults
Clinical and diagnostic significance
Changes in the total protein concentration in the blood can be either absolute or
relative. The absolute changes are the result of protein content modification in the
blood. Relative changes depend on the volume of blood that is observed in dehydra-
tion or hyperhydration.
Hyperproteinemia
Absolute increase in protein concentration in the blood is most often associated
with an excess of globulin fractions. It occurs in acute infections (increased synthesis
of acute phase proteins), in chronic infections (γ-globulinemia), in multiple myeloma,
lymphogranulomatisis, sarcoidosis.

64
 Relative hyperalbuminemia is caused by the loss of intravascular fluid as a re-
sult of profuse diarrhea (cholera), sweating, vomiting, diabetes insipidus, severe and
extensive burns, generalized peritonitis.
Hypoproteinemia
Reducing the protein concentration in the blood is most often associated with a
decrease in albumin fraction in the blood.
The absolute hypoproteinemia is connected with:
• insufficient protein intake with food – gastrointestinal disease, narrowing of
the esophagus by tumors, total or partial starvation;
• with a decrease in protein synthesis– the unbalanced amino acid composition
of food, chronic parenchymal hepatitis, toxic, cancer, treatment with corticosteroids;
• with an enhanced dissolution of proteins – cachexia, severe infections, pro-
longed inflammation, fevers, hyperthyroidism;
• protein loss– disorders of the permeability of the capillary walls, bleeding,
burns, acute and chronic bleeding, nephrotic syndrome.
Relative hypoproteinemia is associated with the changes in water balance – hy-
perhydration with hyperaldosteronism, renal failure with decreased excretion of salts,
when sea water with inadequate infusions of saline solutions was used for drinking.
Design of laboratory work
Note the principle, laboratory procedures, normal values, its clinical and diag-
nostic significance and make a conclusion about possible pathological processes.

TESTS
Choose one or more correct answers.

1. PROPERTIES OF GENETIC CODE COULD BE CHARACTERIZED BY

THE FOLLOWING STATEMENT
1) each codon corresponds to three amino acids
2) one amino acid can encode several triplets
3) each amino acid corresponds to only one codon
4) The mRNA codons are read in a direction from the 3' end 5'

2. MATRIX FOR TRANSCRIPTION PROCESS IS

1) DNA
2) mRNA
3) tRNA
4) rRNA

3. IT IS NECESSARY FOR TRANSLATION PROCESS

1) lysosomes
2) RNA-polymerase
3) mRNA
4) ATP

65
4. THE FORMATION OF PEPTIDE BOND IS DEPENDENT ON
1) aminoacyl-tRNA synthase
2) peptidyl transferase
3) translocase
4) carboxypeptidase

5. POST-TRANSLATIONAL CHANGES OF PROTEINS ARE

1) partial proteolysis
2) polyadenylation
3) covalent attachment of a prosthetic group
4) carboxylation

6. THE PROTEIN BIOSYNTHESIS IS STIMULATED BY THE HORMONE

1) insulin
2) glucagon
3) adrenalin
4) vasopressin

7. THE REASON OF PHENOTYPIC DIFFERENCES OF ORGANS AND

TISSUES IN MULTICELLULAR ORGANISMS IS
1) persistent repression of individual genes
2) the allosteric inhibition of various enzymes
3) the differences in the set of DNA
4) The differences in posttranslational modification of proteins

8. THE PROTEIN SYNTHESIS IN THE PROMOTION STAGE IS INHIB-

ITED BY
1) penicillin
2) streptomycin
3) erythromycin
4) chloramphenicol

9. THEPEPTIDYLTRANSFERASE REACTION IS INHIBITED BY

1) chloramphenicol (levomycetin)
2) ampicillin
3) rifampicin
4) erythromycin

10. THETRANSLOCASE REACTION IS INHIBITED BY

1) erythromycin
2) tetracycline
3) ampicillin
4) chloramphenicol

66
 CASE STUDIES
1. It is found that substitution of last adenine for uridine in the mRNA codon 5'-
GAA-3' leads to the impaired formation of β-polypeptide chain and to the disturb-
ances of hemoglobin synthesis.
Identify the reason for the substitution, and name the disease.
2. The antitumor medicine, cisplatin, is an inhibitor of topoisomerase enzymes.
Describe the state of tumor cells by its action.
3. There are molecules of lipoproteins, which are used for the transfer of lipo-
proteins in the blood. Lipoproteins contain proteins called apoB-48 and apoB-100. It
is known that these proteins are encoded by one gene. But the molecular weight of
proteins is differed approximately in 2-fold from one another (apoB-48 – 241 kDa,
apoB-100 – 512 kDa).
Consider the reason for the difference.

CHECKLIST FOR THE FINAL LESSON (UNIT 5, 6, 7)

1. The nitrogen balance in the body. The concept of nitrogen equilibrium. The
biological significance of peptides. Essential and non-essential amino acids. Normal
values of protein intake in children and adults. Protein food sources. What is the ref-
erence protein? Features of protein deficiency.
2. The protein digestion in gastro-intestinal tract. Hydrochloric acid formation,
its role. Regulation of hydrochloric acid secretion. Gastro-intestinal enzymes, exon
and endopeptidases, their location, mechanism of enzyme activation, their pH opti-
mum and specifics. The mechanism of amino acid absorption.
3. Features of protein digestion and absorption in children of different age. Rea-
sons for protein digestion and absorption disorders in children. The connection of
these disorders with the development of allergic reactions. What is a celiac disease?
Identify the causes and clinical signs of the disease.
4. Disposal of toxic products in the liver: microsomal oxidation and conjugation
system. What enzymes are involved in the microsomal oxidation? The structure of
UDP-glucuronic acid (UDPGA) and phosphoadenosine phosphoric acid (PAPA). Re-
actions of indicant formation.
5. Qualitative reaction of free hydrochloric acid in gastric juice.
6. Detection of lactic acid in gastric juice. Principle of method, procedure, nor-
mal values and clinical and diagnostic significance.
7. Detection of blood and hemoglobin in gastric juice. Principle of method, pro-
cedure, normal values and clinical and diagnostic significance.
8. Tubeless method of acidity detection in gastric juice. Principle of method.
9. Sources of amino acids in tissues. What is the principle of amino acid division
into on glucogenic and ketogenic? Application of amino acids in the medical practice.
10. Types of amino acid deamination reactions. Feature of an oxidative deamina-
tion. Feature of trans deamination– the mechanism of reactions, enzymes, coen-
zymes, the location process. The significance of transamination reactions. The role of
the cycle IMP-AMP, its reactions.
67
 11. Characteristics of aspartate aminotransferase (AST) and alanine aminotrans-
ferase (ALT), their reactions. The method of quantitative ALT AST activity determi-
nation in the serum. Note their clinical and diagnostic significance in the blood, nor-
mal values.
12. Glutamate dehydrogenase: location, structure, role, activity regulation. The
fate of nitrogen and α-keto acids formed in the deamination process.
13. The significance of the amino acid decarboxylation. Role of biogenic amines
– histamine, serotonin, γ-aminobutyric acid dopamine. The reactions of synthesis of
biogenic amines – chemistry, enzymes, coenzymes, products, process location. Reac-
tions of biogenic amine inactivation.
14. Reactions of formation and binding of ammonia in tissues (scheme). The role
of the liver, kidney and intestines in the removal of ammonia. What is the acceptable
level of ammonia concentration in the blood? The main causes of ammonia toxicity.
Hyperammonemia, note their causes and consequences. The glucose-alanine cycle,
its significance.
15. The urea synthesis reaction, its location, significance. Changes in urea syn-
thesis. Quantitative determination of urea in the blood serum and urine. The principle
of the method, the procedure, normal values, clinical and diagnostic values.
16. Ammoniagenesis, reactions, their location, value.
17. Synthesis of creatine and phosphocreatine, reaction. The biological role of
creatine phosphate. Physiological creatinurea in children.
18. Synthesis of creatinine, reaction, location. Determination of the creatinine
concentration in the serum and the urine. The principle of the method, the procedure,
normal values, clinical and diagnostic significance.
19. Metabolism of glutamic and aspartic acid (scheme). Glutamic and aspartic
metabolic reactions. Connection of amino acid metabolism with the citric acid cycle.
20. Metabolism of cysteine and its sulfur fate (scheme). Reactions of taurine syn-
thesis. Causes and consequences of disorders in cystinosis and cystinuria.
21. Metabolism of serine and glycine (scheme). Reactions of glycine and serine
interconversion, glycine catabolism reaction. The role of tetrahydrofolic acid.
22. Reactions reflecting exchange relationship between glycine, serine, methio-
nine, and cysteine. Participation of folic acid and vitamin B12. Role of adenosylme-
thionine in transmethylation processes. Homocysteinemia and homocystinuria, its
causes and consequences.
23. Substances in the synthesis reactions involving THFA (dTMP, serine, methi-
onine). The mechanism of the sulfonamides antibacterial activity.
24. Metabolism of tyrosine and phenylalanine. Ways of tyrosine use (Scheme).
Reaction of tyrosine synthesis and phenylalanine catabolism.
25. Phenylketonuria, types I and II: causes, clinical features, basis of treatment.
26. Tyrosinemia, types I, II and III, homogentisuria, parkinsonism, albinism:
causes, characteristics of diseases, the basis of treatment.
27. Nucleoprotein structure: proteins, nucleic acids. The structures of nitroge-
nous bases, nucleosides, nucleotides. Enzymes of nucleoproteins digestion in the
stomach. The metabolism fate of the purines and pyrimidines.
68
 28. Purines structure, sources of carbon and nitrogen atoms in the purine ring.
The first two reactions of purine nucleotide synthesis, the synthesis reaction of AMP
and GMP, AMP conversion reaction into ATP, GTP conversion reaction into GMP.
Regulation of the purine nucleotide synthesis.
29. Purine catabolism, uric acid. Reutilization of guanine and hypoxanthine.
30.Disorders of the purine catabolism:
• hyperuricemia, its causes, types and consequences, basis of treatment,
• urolithiasis, its causes, types and consequences, basis of treatment,
• gout, its causes, types and consequences, basis of treatment,
• Lesch-Nyhan syndrome, its causes, types and consequences, basis of treatment.
31. Synthesis of UTP and CTP pyrimidine nucleotides, reactions, location, its
regulation. Orotate aciduria.
32. Synthesis of deoxyribonucleotides. Role of NADPH and thioredoxin. Reac-
tions of dTMP synthesis, participation of methylene THFA.
33. Degradation of pyrimidine nucleotides to the carbon dioxide, ammonia and
water.
34. Medicines – inhibitors of purine and pyrimidine nucleotide synthesis. The
mechanism of their action.
35. Features of the structure and the differences between RNA and DNA primary
and secondary structures. Types of RNA, their location and function. The role of his-
tones in the formation of the tertiary structure of DNA (supercoiling).
36. Replication of eukaryotic DNA. The overall equation, DNA-synthesizing en-
zyme system, basic stages and features of DNA replication. Connection with the cell
cycle phases. DNA repair.
37. RNA transcription, enzymes and components of RNA-synthesizing system.
The concept of exons and introns. The processes of tRNA, rRNA and mRNA matura-
tion. Regulation of transcription in prokaryotes by induction and repression. Methods
of transcription regulation in eukaryotes.
38. Stages of translation, components of the protein-synthesizing system, en-
zymes, regulation of processes. What is the genetic code? The properties of the genet-
ic code. An adaptive role of transfer RNA. The synthesis reaction of the aminoacyl-
tRNA.
39. The post-translational modification of proteins, examples. Folding mecha-
nism. The role of the chaperone.
40. Medicines – RNA, DNA, protein biosynthesis inhibitors. The mechanism of
their action.
41. The principle and the procedure of protein determination in the serum and
the urine. Biuret method. Normal values, clinical and diagnostic significance.
42. Determination of the uric acid concentration in the serum and the urine. The
principle of the method, the procedure, normal values, clinical and diagnostic signifi-
cance.
43. Analysis of the nucleoprotein chemical composition. The principle and the
procedure of the method.

69
 UNIT 8
STRUCTURE AND METABOLISM OF CARBOHYDRATES

THEME 8.1. STRUCTURE AND METABOLISM OF CARBOHYDRATES.

GLYCOGEN METABOLISM

INTRODUCTION
Carbohydrates play a vital role in the body of human beings and animals and
perform subsequent functions:
• supply of energy (to 67% of daily energy required for the body),
• building material for cells,
• precursor molecules for synthesis of lipids, proteins and nucleic acids,
• carbohydrate compounds are included in immunoglobulins and other mole-
cules.
Diseases associated with pathology of carbohydrate metabolism include diabetes
mellitus, glycogen storage diseases, mucopolisacharidosis, galactosemia, fructosemia,
lactose and sucrose intollerance.

THE AIM OF THE PRACTICAL CLASS IS:

To study carbohydrate digestion in the digestive tract. Glycogen metabolism as
the energy storage pool.

SELF-STUDY QUESTIONS
1. Biological role of carbohydrates. Daily needs in carbohydrates for adults and
children. Dietary products rich in carbohydrates.
2. Classification of carbohydrates depending on the number of monomers in the
molecule (mono-, di-, oligo-, and polysaccharides), depending on the number of car-
bon atoms (trioses, tetroses, pentoses, hexoses) and on the localization of a carbonyl
group (aldoses and ketoses).
3. Structure and functions of carbohydrates:
• monosaccharaides (glucose, fructose, galactose, ribose, deoxyribose, glycer-
aldehyde, dioxyacetone),
• disaccharides (maltose, lactose, sucrose),
• polysaccharides (starch, glycogen, cellulose).
4. Monosaccharide derivatives. What are the sialic acids? The chemical structure
of N-acetylneuraminic acid.
5. Compound carbohydrates – glycosaminoglycans. The structure of hyaluronic
acid and chondroitin acids, their biological significance. The structure and role of
glycoproteins.
6. Carbohydrate digestion in the mouth and intestine. Characteristics of digestive
enzymes: α-amylase of the mouth, enzymes of pancreatic juices (α-amylase, oligo-
1.6-glycosidase), enzymes of small intestine responsible for carbohydrate digestion.
70
 7. Age-dependent peculiarities of digestion and absorption of carbohydrates. Bi-
ochemical reasons for sucrose and lactose intolerance in children.
8. Reasons why cellulose can’t be digested in digestive tract of humans. Role of
cellulose in digestion.
9. Transport of monosaccharide through cellular membranes.
10. Pathways of glucose metabolism in the cell. Sources of glucose in the cell.
Phosphorylation of glucose, the significance of the reaction.
11. Conversion of fructose into glucose. Ways of fructose metabolism. What is
the role of fructose in the metabolism of the fetus and the newborn? Essential fructo-
suria.
12. The role of galactose in the body. Conversion of galactose to glucose. Galac-
tosemia, molecular causes, clinical manifestations and the basics of treatment.
13. Synthesis of glycogen from glucose-6-phosphate (glycogenesis). Breakdown
of glycogen to glucose-6-phosphate (glycogenolysis). Specific features of glycogen
metabolism in the liver and muscles (well-fed state, fast, muscle exercises).
14. Regulation of the activity of glycogen metabolism enzymes – glycogen syn-
thase:
• hormonal – the influence of adrenaline and glucagon (adenylyl cyclase mecha-
nism, role of cAMP and protein kinase A); role of insulin and participation of phos-
phodiesterase in decreasing cAMP concentration on the cell,
• allosteric regulation of glycogen phosphorylase by AMP,
• Ca2+-dependent activation of glycogen phosphorylase kinase.
15. Genetic glycogen storage diseases: liver, muscle and combined glycogen
storage disease. Glycogenosis.
16. Specific properties of carbohydrate metabolism in the liver. The role of glu-
cosidase and glucose-6-phosphatase in hepatic regulated maintenance of glucose con-
centration in the blood.
17. Carbohydrates are medicines (glucose, hyaluronic acid, chondroitin sulfate,
dextrins, heparin, cardiac glycosides).
18. Detection of glucose in urine. Principle of method. Clinical-diagnostic signif-
icance.

TOPICS FOR REPORTS

1. Sialic acids. Glycosaminoglycans (hyaluronic acid, chondroitin sulfate, der-
matan sulfate, keratan sulfate, heparin). Their characteristics: structure, biological
role. Use in medicine.
2. Fructose: its importance in fetal and neonatal exchanges. Chemistry of fruc-
tose metabolism. Disorders of fructose metabolism: essential fructosemia, hereditary
intolerance to fructose.

71
 Practical
DETECTION OF GLUCOSE IN URINE USING "GLUCOPHAN" DIAG-
NOSTIC STRIPS
Principle
The principle of glucose determination is based on enzymatic reaction of glucose
oxidase. The display area is impregnated with solutions of the enzyme glucose oxi-
dase, peroxidase and dye tetramethylbenzidine. Glucose using glucose oxidase is ox-
idized by air oxygen to gluconic acid with formation of hydrogen peroxide. Hydrogen
peroxide in the presence of enzyme peroxidase oxidizes a dye, and yellow turns into
green.

Normal values
Urine
Glucose test strips "Glucophan" test was negative
other methods 0.1-0.8 mmol/l

Clinical and diagnostic significance

The level of glucose in the urine increases in all cases of hyperglycemia over 10
mmol/l (renal threshold).
The glycosuria can be physiologic and pathologic. Physiologic ones include ali-
mentary glycosuria, glycosuria of pregnant and neurogenic stress glycosuria.
Pathological glycosuria occurs most often as a result of severe hyperglycemia
and pathological changes in the pancreas (acute pancreatitis, diabetes mellitus), ad-
renal gland ("bronze" or steroid diabetes), hyperthyroidism, acromegaly, myocardial
infarction, hemorrhages in internal organs, poisoning by morphine, phosphorus, in
acute infections.
The defeat of the renal tubules and the absence of reabsorption of glucose also
lead to glycosuria.
Design of work:
Write down the principle of the used analytical method, put all results in a clear-
ly organized table, note the clinical-diagnostic value, and make a conclusion about
the possible pathology.
The examined material Reaction Result
Normal urine
Urine with glucose

THEME 8.2. OXIDATION OF GLUCOSE IN AEROBIC CONDITIONS.

GLUCONEOGENESIS

INTRODUCTION
Glycolysis is the main pathway of glucose catabolism. Glycolysis is the only
mechanism in bodythat produces energy in anaerobic conditions. Particularly due to
glycolysis the organism can survive in hypoxic conditions. In erythrocytes anaerobic
72
metabolism of carbohydrates is the only process that produces ATP and supports
their integrity and function. Under aerobic conditions glycolysis is the initial stage of
glucose breakdown, ending in the aerobic oxidation of the resulting intermediate
products.
A stable glucose level during physical exercises or starvation is sustained by the
reactions of gluconeogenesis. This contributes to the reduction of acidosis in these
states and provides glucose for nervous tissue and erythrocytes.

THE AIM OF THE PRACTICAL CLASS IS:

To study the processes of glycolysis, gluconeogenesis, regulation of the process-
es of glucose breakdown and synthesis.
To obtain practical skills for the quantitative determination of glucose in urine
and serum as well as lactic acid in homogenate of muscle tissue.

SELF-STUDY QUESTIONS
1. Sources and ways of glucose metabolism in the cell. Role of glucose-6-
phosphate in metabolism of glucose.
2. Characteristics of glycolysis process (lactic acid fermentation):
• localization and the conditions of the process,
• the sequence of reactions and enzymes,
• the final products,
• participation of adenyl nucleotides and energy effects,
• irreversible reactions of glycolysis,
• reactions of glycolysis associated with ATP consumption,
• the reaction of substrate phosphorylation, their nature and value,
• glycolytic oxidation reduction of NAD+ and NADH, its meaning and value.
3. Characteristics of the process of gluconeogenesis according to the plan:
• localization and conditions of a reaction,
• substrates (lactic acid, glycerol, amino acids). Where do these substances arise
from?
• the sequence of reactions and enzymes,
• reactions of gluconeogenesis associated with the consumption of GTP and
ATP,
• irreversible reaction of gluconeogenesis,
• biological significance during fasting and physical work,
• energy consumption for the synthesis of one molecule of glucose.
4. The role of glycolysis and gluconeogenesis in the metabolism of fetus and
newborns.
5. Hormonal regulation of glycolysis and gluconeogenesis. The role of insulin,
adrenaline, cortisol, glucagon. The enzymes regulated by these hormones.
6. Allosteric regulation of glycolysis and gluconeogenesis, role of ATP, ADP,
AMP, citrate, fatty acids, glucose-6-phosphate, fructose-6-phosphate, fructose-1.6-
diphosphate, acetyl-S CoA. Regulated enzymes.

73
 7. Glucose-lactate cycle (Cori cycle), its value in physical work. Sources of lac-
tic acid in the body.
8. Glucose-alanine cycle, its value in physical work and fasting.
9. Energy effect of glucose oxidation in anaerobic conditions. Comparison of the
energy effect of anaerobic glucose oxidation and breakdown of glycogen to lactate.
10. Alcoholic fermentation: the localization process, the specific reaction condi-
tions, the sequence of reactions, energy effect, the end products. The similarities and
differences between alcoholic fermentation and glycolysis.
11. The metabolism of ethanol in the liver, with the participation of alcohol de-
hydrogenase and acetyl dihyddehydrogenase, sequence of reactions, the end products.
Energy effect of the oxidation of one molecule of ethanol.
12. The influence of ethyl alcohol on the metabolism of carbohydrates in the
human body. What are the causes of hyperlactatemia and hypoglycemia in alcoholic
intoxication?
13. The synthesis of glucose from alanine, aspartate and glutamate. Under what
conditions and where do these reactions take place? What is their biological signifi-
cance?
14. Quantitative determination of glucose in serum and urine. The principle of
the method. The normal levels. Clinical-diagnostic value.
15. How to detect lactic acid in the muscle tissue by reaction of Uffelman?

TOPICS FOR REPORTS

1. Molecular mechanism of alcohol intoxication development. The causes of hy-
persensitivity of children to alcohol. Chronic alcoholism. Clinical and laboratory di-
agnostic methods.
2. Alcohol dehydrogenase, catalyzed reactions, substrates. Its participation in bi-
ochemical and physiological processes. Isozymes.
3. Gluconeogenesis and its importance in the metabolism of a fetus and a new-
born. Hypoglycemia of newborns, criteria, causes.

Practical 1
THE GLUCOSE OXIDASE TEST FOR THE DETERMINATION OF GLU-
COSE CONCENTRATION IN SERUM AND URINE
Principle
Glucose using glucose oxidase is oxidized to gluconic acid with formation of
hydrogen peroxide. Hydrogen peroxide in the presence of the chlorophenol and the
enzyme peroxidase oxidizes dye 4-aminoantipyrine, transforming it in to Heinemann,
painted product raspberry pink color. The color intensity is proportional to concentra-
tion of glucose and is determined by photocolorimetry.
Material of investigation
Serum. Blood.
Reagents
1) The working reagent, containing phenol, glucose oxidase, peroxidase,
4-aminoantipyrine in potassium phosphate buffer.

74
 A standard solution of glucose, 5.5 mmol/l.

Procedure
Experiment 1, ml Experiment 2, ml Standard, ml
Blood serum 0.02 –– ––
Urine –– 0.02 ––
A standard glucose solu-
–– –– 0.02
tion
The working reagent 2.0 2.0 2.0
Incubation for 25 minutes at 37°C.
Measure the optical density against water at a wavelength
of 540 nm (green light filter).
Calculation
Е ex
Glucose concentration, mmol/l = Е st × Сst, where
Еex and Еst – optical density of examined samples and standard one,
Сst – standard solution concentration.
Normal values
Blood serum adults 3.5-5.5 mmol/l
urine 0.06-0.83 mmol/l

Clinical and diagnostic significance

Serum
The glucose content increased in the blood is observed both in physiological and
in pathological conditions.
Physiological hyperglycemia
The physiological hyperglycemia is alimentary (nutritional) one that arises when
simultaneously the large amounts of easily digestible carbohydrates – mono- and di-
saccharides are absorbed, and neurogenic one, for example, in stressful situations
through the release of large quantities of catecholamine into the blood. Physiological
hyperglycemia is transient and quickly passes.
Pathological hyperglycemia
Pathological hyperglycemia is usually caused by neuroendocrine disorders:
• diabetes mellitus is associated with absolute or relative insulin insufficiency,
• pituitary disease, accompanied by increased secretion of somatotropin and cor-
ticotropin into the blood (acromegaly, a Itsenko-Cushing disease, pituitary tumors,
etc.),
• medulla tumors of the adrenal glands, when formation of catecholamines is en-
hanced (pheochromocytoma),
• tumors of the adrenal cortex with increased production of glucocorticoids,
• hyperthyroidism, some liver diseases (infectious hepatitis, liver cirrhosis).
75
 The glucose concentration decreased in the blood can also be physiological and
pathological.
Physiological hypoglycemia
Physiological hypoglycemia includes the excess blood insulin secretion in re-
sponse to nutritional hyperglycemia; hypoglycemia after severe and prolonged mus-
cular work, at full or partial fasting. Hypoglycemia can occur in women during lacta-
tion as a result of enhanced absorption of glucose by the mammary gland. Hypogly-
cemia is often observed by newborns, caused by sudden stoppage of the "parent" glu-
cose, immaturity of the liver structure and increased sensitivity of cells to insulin.
Pathological hypoglycaemia
Pathological hypoglycemia is observed in hyperinsulinism, hyperplasia β-cells
of the islets of Langerhans (insulinoma, adenoma and cancer of the pancreas). The
most common cause of hypoglycemia – insulin overdose. In addition, hypoglycemia
causes a deficiency of the hormone cortisol by hypofunction of the adrenal cortex
(Addison's disease, adrenal tumors), hypofunction of the anterior pituitary (Sim-
monds disease), hypothyroidism. Hypoglycemia can occur because of toxic liver
damage, glycogen storage, diseases of kidneys due to the glycosuria.
Urine
(Theme 8.1.).
Design of work:
Write down the principle of the method, the experimental procedure, the normal
value and the results of the study, note the clinical and diagnostic value of the index
and draw conclusions on the possible pathology.

Practical 2
DETECTION OF LACTIC ACID IN MUSCLE TISSUE BY UFFELMAN'S RE-
ACTION
Principle
The method is based on the complex compound interaction of violet iron phenolate
with lactic acid to form yellow-green lactate of iron.
Material for investigation
Muscle tissue.
Reagents
1) Phosphate buffer, pH 7.2, 2) 1% solution of phenol, 3) 1% solution of FeCl3.
Procedure
1. Preparation of the extract of muscle tissue.
A piece of muscle tissue is triturated in a mortar with 5.0 ml of phosphate buffer
solution, and then mixed. Obtained muscle slurry is filtered through 2 layers of
cheesecloth or centrifuge at 1500 rpm.
2. Color reaction for lactic acid.
In the test tube the 1% FeCl3 solution is added to 10 drops of 1% phenol solution
until the appearance of the purple color. Then, 3 drops of extract of muscles are add-

76
ed to the vial and the color change is observed. In the presence of lactic acid, the vio-
let color of the solution becomes yellow-green due to the formation of lactate of iron.
Practical significance
Lactic acid is the end product of catabolism of glucose under anaerobic condi-
tions, a small amount of lactate in the muscle is formed under aerobic conditions.
Production of lactate is activated with the muscular exercise both physiologically
(physical work), and pathologically (disruption of blood flow and hypoxia of the
muscles, epilepsy, tetanus, tetany).
The production of lactic acid is activated in most tissues in hypoxic conditions
associated with cardiac and pulmonary failure, anemia and blood rheology disorders.
Design of work:
Describe the principle of the method, the experimental procedure and the results
of the study, note the practical value of an indicator and draw conclusions about pos-
sible pathology.

THEME 8.3. AEROBIC OXIDATION OF GLUCOSE. PENTOSE

PHOSPHATE PATHWAY

INTRODUCTION
Aerobic breakdown of glucose is a main pathway of its catabolism in aerobic or-
ganisms. Aerobic breakdown of glucose releases more energy than anaerobic glycol-
ysis. Intermediate products of the oxidative catabolism of glucose are also used as
precursors in the biosynthesis of amino acids, lipids and other biomolecules. The
brain is the most dependent on aerobic breakdown of glucose. It consumes about 120
g of glucose per day.
The pentose phosphate pathway fulfills an anabolic function. It provides the cell
with the molecules of NADPH for reductive synthesis and pentoses for synthesis of
nucleotides.

THE AIM OF THE PRACTICAL CLASS IS:

To study the reactions of aerobic breakdown of glucose to carbon dioxide and
water, reactions of pentose phosphate pathway, the nervous and hormonal regulation
of glucose metabolism, the disorders of carbohydrate metabolism.
The acquisition of practical skills for testing glucose tolerance and building gly-
cemic curves.

SELF-STUDY QUESTIONS
1. Sources of blood glucose. The normal concentration of glucose in the blood.
Possible causes of hypo- and hyperglycemia.
2. Specific and general pathways of catabolism of glucose. The overall equation
of aerobic breakdown of glucose.
3. Stages of aerobic glucose breakdown: 1 – oxidation of glucose to pyruvate, 2
– oxidative decarboxylation of pyruvate, 3 – citric acid cycle, 4 – electron transport
chain and the formation of endogenous water.
77
 4. The overall equation of oxidative decarboxylation of pyruvic acid and its indi-
vidual reaction. Components of the multienzyme pyruvate-dehydrogenase complex,
enzymes and coenzymes. The regulation of the process. What vitamins are involved
in the work of pyruvate dehydrogenase? Their characteristics. What other enzyme
complexes have similar structure?
5. The citric acid cycle, enzymes and coenzymes, biological role of the cycle.
The regulation of the process.
6. Glycerol phosphate and malate-aspartate shuttle systems. What is their value?
7. Benefits of aerobic oxidation of glucose. Pasteur's effect, its biochemical
mechanism.
8. Feature pentose phosphate pathway of glucose oxidation according to the
plan:
• distribution and the role of pentose phosphate pathway,
• the reaction of the oxidative phase,
• the idea of non-oxidative phase (schematically),
• enzymes, coenzymes, vitamins,
• the relationship of the process with glycolysis,
• the value of pentose phosphate pathway, for example, in an adipose cell, the
erythrocyte, in dividing cells.
9. The formation of ATP in aerobic and anaerobic breakdown of glucose. The
role of anaerobic and aerobic breakdown of glucose during muscular work. How does
the dependence of the metabolism of nervous tissue from the aerobic breakdown of
glucose manifest?
10. Specific features of glucose oxidation in the erythrocyte. The role of glycoly-
sis, pentose phosphate shunt, 2.3-diphosphoglycerate shunt.
11. Hereditary enzymopathy of glucose-6-phosphate dehydrogenase. The factors
causing the manifestation of insufficiency of the enzyme. Consequences.
12. Nervous regulation of carbohydrate metabolism. The role of the sympathetic
and parasympathetic systems.
13. Hormonal regulation of carbohydrate exchange. The effect of insulin,
adrenaline, glucagon, cortisol on blood glucose level and intracellular processes of
transformation of glucose. Endocrine-sensitive enzymes of carbohydrate metabolism.
14. Characteristics of diabetes type 1 and 2. What ways of carbohydrate ex-
change is broken? Biochemistry of diabetes complications.
15. Glucose tolerance test. Diagnostic value of parameters of glycemic curve –
the steepness of the ascent, the magnitude of ascent, the returning time to the original
values. Under what conditions does the type of the glycemic curve change?

TOPICS FOR REPORTS

1. Metabolic functions of NADPH. Reactions of the formation of NADPH for-
mation. Anabolic reactions with NADPH participation.
2. Molecular mechanisms of diabetes mellitus 1 and 2 types. Biochemical mech-
anisms of fast and delayed complications of diabetes mellitus.

78
 Practical
GLUCOSE TOLERANCE TEST
The glucose tolerance test (test with a sugar loading) is an informative test to de-
tect diabetes at the early stages, violations of liver glycogenolysis function and to as-
sess the function of the small intestine.
Principle
Glucose tolerance test is based on the determination of the concentration of glu-
cose in the blood after a certain period of time after ingestion of glucose.
The concentration of glucose in the blood is determined by the glucose oxidase
method (see Theme 8.2.).

The glucose tolerance test procedure

In clinical diagnostic laboratories samples of capillary blood, taken on an
empty stomach and after a certain period of time after the glucose loading is exam-
ined. The test is carried out as follows:
Patient fasting blood is taken from a finger, afterwards glucose with warm water
or weak tea is given to the patient. It is recommended to give children, aged 1.5 to 3
years, glucose at the rate of 2.0 g per 1 kg of body weight, from 3 to 12 years –
1.75 g/kg, after 12 years – 1.25 g/kg. Adults take glucose in the amount of
1.0-1.5 g/kg. Blood samples are taken repeatedly after 30, 60, 90 and 120 minutes af-
ter taking glucose. Next, the glucose concentration in samples is measured.
In practical class the method of the sugar loading is carried out with a model
serum samples of blood taken prior to a glucose loading and after 30, 60 and 120
minutes after glucose loading. The glucose concentration in all taken samples is de-
termined by glucose oxidase method (see Theme 8.2.).
Material for investigation
Three sets of model samples of blood serum containing normal, reduced and el-
evated concentrations of glucose.
Reagents
1) The working reagent, containing phenol, glucose oxidase, peroxidase,
4-aminoantipyrine in potassium phosphate buffer.
A standard solution of glucose, 5.5 mmol/l.
The glucose concentration determination
Experimental samples, ml
Standard
Before Time after glucose loading
sample,
glucose ml
loading 30 minutes 60 minutes 120 minutes
1 2 3 4 5
The working solu- 2.0 2.0 2.0 2.0 2.0
tion
Blood serum 0.02 0.02 0.02 0.02 ––
Glucose standard –– –– –– –– 0.02
79
 The content of the tubes is mixed, incubated at 37°C for 15
minutes. The optical density at a wavelength of 540 nm (green
filter) is measured.
Calculation
In each blood sample, the concentration of glucose is calculated according to the
following formula:
Еex
Glucose concentration, mmol/l = Еst ×СST, where
Eex and Est – optical density of experiment and standard samples, Cst – concentra-
tion of standard glucose solution.
Normal values
Fasting 3.5-5.5 mmol/l 100%
After 60 minutes 5.3-9.6 mmol/l 150-175%
After 120 minutes less than 5.3 mmol/l approximately 100%
Evaluation of the glycemic curve
There are the following types of glycemic curves:
Glucose level
Initial level of Hypoglycemic
Type of curve Maximum rise by the end of
glucose phase
the 2nd hour
Normal Normal In one hour In two hours The initial lev-
or missing el
Hyperglycemic Hyperglycemia After 1.0-1.5 missing The initial level
hours is not reached
Hypoglycemic Hypoglycemia One hour missing The initial level
In a healthy person after glucose loading the glucose level in the blood is
changed as follows:
1. 30 minutes after the intake of glucose, an increase in the glucose amount in
the blood is measured. The increase rate of glucose concentration during the first 30
minutes (the steepness of the curve) shows the reflex irritation strength of the sympa-
thetic nerve endings during the contact with glucose in the digestive tract and the ef-
ficiency of glucose absorption in the intestine.
2. By 60-th minute, there is a maximum increase in the glucose concentration in
the blood about 50-75% over the baseline. The interval from 30 to 60 minutes is as-
sociated with the speed of glucose absorption, and with the general state of the liver
and its glycogenesis function.
3. In 90-120 minutes, glucose content in the blood returns to normal. The de-
crease in the level of blood glucose in this period is due to enhanced release of insulin
from the pancreas. The degree of reduction reflects the functional activity of para-
sympathetic nervous system, the glycogenesis function of the liver, insulin sensitivity
of muscle and adipose tissue. In some cases, the glucose concentration may fall be-
low the initial value, since it is usually secreted more insulin than it is required to re-
store normal glucose levels in the blood, which leads to hypoglycemia.
In a healthy person the glucose loading does not cause glycosuria.
80
 The results of the survey are usually expressed graphically and can be reflected
in relative or absolute units:
1. Normal curve,
2. Hyperglycemic curve,
3. Hypoglycemic curve.

Clinical-diagnostic significance
Hyperglycemic curves are presented during the damage of the parenchyma of
the liver, diseases of the central nervous system, hidden forms of diabetes, hyperthy-
roidism and adrenal cortex, infectious diseases (rheumatism, diphtheria, typhoid, dys-
entery, sepsis, pneumonia), pancreatitis, glycogen storage diseases.
Hypoglycemic curves are observed in the adenoma of the islets of Langerhans,
hypothyroidism, Addison's disease, encephalitis, bowel disease, dysbiosis, and hel-
minthiasis.
Design of work:
Write down the principle of formation of the glycemic curves, determine the ob-
tained values, build on them glycemic curve in absolute and relative units.
Glucose Concentration in the blood
The number of
sample Before load- Time after loading
ing 30 minutes 60 minutes 120 minutes

Note the clinical diagnostic significance of the method. Make a conclusion about
the possible causes of changes in the shape of glycemic curves.

81
 TESTS
Choose one or more correct answers.
1. MALTASE IS SYNTHESIZED BY
1) pancreas cells
2) mucous cells of gastric
3) mucous cells of small intestine
4) mucous cells of large intestine

2. CARBOHYDRATE WITH GLUCOSE-FRUCTOSE STRUCTURE, CON-

NECTED WITH α-1.2-GLYCOSIDIC BIND IS
1) lactose
2) maltose
3) sucrose
4) residue of starch

3. DIGESTION IS ACCOMPANIED BY
1) decomposition of disaccharides to CO2 and water
2) splitting of polysaccharides to oligo- and monosaccharides
3) hydrolysis of cellulose
4) decomposition of glucose with the formation of lactate

4. THE KEY ENZYME OF GLYCOGEN MOBILIZATION IS ________

1) glycogen synthase
2) amylase
3) hexokinase
4) glycogen phosphorylase

5. THE ANAEROBIC TRANSFORMATION OF GLUCOSE INTO LAC-

TATE IS TAKEN PLACE IN
1) cells of nerve tissue
2) cells of the cortical layer of the kidneys
3) erythrocytes
4) myocarditis

6. LACTATE – THE GLYCOLYSIS FINAL PRODUCT - CAN BE RESYN-

THESIZED IN GLUCOSE IN _________
1) muscle tissue
2) nervous tissue
3) the liver
4) kidney tissue

7. SHORT-TERM FASTING LEADS TO ACTIVATION OF _________

1) glycolysis in muscles
2) glycogenolysis in cardiac tissue
3) glycogenolysis in the liver
4) synthesis of glycogen in the liver
82
 8. THE PROLONGED STARVATION LEADS TO ACTIVATION OF ___
1) glycolysis in muscles
2) glycogenolysis in the liver
3) gluconeogenesis
4) synthesis of glycogen in the liver

9. THE RATE OF GLUCONEOGENESIS INCREASES DUE TO THE AC-

TION OF_________
1) cortisol
2) increased concentration of ADP and AMP
3) insulin
4) high concentration of NAD and FAD

10. THE PENTHOSE PHOSPHATE PATHWAY FUNCTION IS ________

1) the formation of glucose
2) generation of NADPH
3) ATP supplement of tissues
4) formation of lactate

CASE STUDIES
1. A 7-year-old child needs to determine blood sugar to detect diabetes mellitus.
The child cried before the sample in the laboratory. It was found that the child's blood
sugar level is higher than normal.
Note the cause of hyperglycemia.

2. One athlete ran at a distance of 100 m, the other – 5000 m.

What is the level of lactic acid in sportsmen’s blood?

3. Suggest a change in the ratio between the pentose-phosphate and glycolytic

pathways of carbohydrate metabolism in the bone marrow of a patient who had
bleeding.
Identify enzymes that are useful for the hypothesis evaluation.
CHECKLIST FOR FINAL LESSON (UNIT 8)
1. The role of carbohydrates in the body. Classification of carbohydrates accord-
ing to their structure and functions. The structure of the main representatives of car-
bohydrates: monosaccharides (triose, pentoses, hexoses), di- and polysaccharides.
The role and structural formula of glycosaminoglycans: neuraminic and N-
acetylneuraminic, chondroitin and hyaluronic acid. Examples of the use of carbohy-
drates as the drugs.
2. The carbohydrates presented in the food. Where and what enzymes participate
in their digestion? The mechanism of glucose absorption. The role of cellulose in di-
gestion. The reasons for intolerance to sucrose and lactose.
3. Detection of glucose in urine. The principle of the method. Clinical-diagnostic value.

83
 4. The role of the liver in the metabolism of carbohydrates in different situations.
Specific features of the functioning of the enzyme glucokinase and glucose-6-
phosphatase. The reaction of interconversion of carbohydrates: metabolism of galac-
tose and fructose in the body.
5. Reactions of biosynthesis of glycogen and glycogenolysis, the physiological
significance of the processes. The energy effect of glycogen usage in aerobic and an-
aerobic conditions. Regulation of activity of phosphorylase and glycogen synthase
(the role of cAMP, calcium ions and calmodulin). Differences in glycogen metabo-
lism in the liver and in the muscles. Characteristics of the glycogenoses and agly-
cogenoses, defects of enzyme and consequences of such defects.
6. Sources and ways of transformation of glucose in tissues (scheme). Character-
istics of oxidation of glucose under anaerobic conditions: the sequence of reactions of
glycolysis, the net reaction, the energy effect, regulation, method of ATP formation,
the localization of the process. Subsequent fate of lactic acid. Specify the role of an-
aerobic breakdown of glucose in red blood cells and in the muscle tissue.
7. The sequence of reactions of alcoholic fermentation, its net reaction, energy
effect, the method of ATP formation, the localization of the process. Similarities and
differences of glycolysis and alcoholic fermentation
8. The metabolism of ethanol in humans. Localization of enzymes. What is the
cause of hypoglycemia and lactic acidosis in alcohol poisoning?
9. The determination of glucose concentration in blood serum with glucose oxidase
method. The principle of the method, definition, clinical-diagnostic value, normal values.
10. Detection of lactic acid in the muscles by the reaction of Uffelman. The prin-
ciple of the method and steps of analysis, practical significance.
11. The oxidation of glucose in aerobic conditions: the sequence of reactions,
energy effect. Pasteur's effect, its biochemical mechanisms. Reactions of glycerol
phosphate and malate-aspartate shuttle system functioning, the source of NADH. The
role of the aerobic breakdown of glucose in the brain.
12. The pentose phosphate pathway (PPP) conversion of glucose, its localization. The
reaction of the oxidative phase of the pentose formation. Reactions of non-oxidative phase
(scheme). The role of the 1st and 2nd stages of the PPP in adipose tissue and red blood cells,
in dividing cells, its relationship to glycolysis. The regulation of the process. The conse-
quences of glucose-6-phosphate dehydrogenase enzymopathies.
13. The sequence of reactions of gluconeogenesis, possible precursors, its value.
The regulation of gluconeogenesis. Glucose-lactate cycle (Cori cycle) and glucose-
alanine cycle (scheme), their role. Reaction of synthesis of glucose from amino acids
on the example of alanine, aspartate and glutamate.
14. Reactions of carbohydrate metabolism, accompanied by the formation of
carbon dioxide and reactions which use it.
15. What is allosteric regulation of enzymes? What enzymes are affected by in-
termediate metabolites of carbohydrate metabolism, NADH, ATP and AMP?
16. Regulation of glucose concentration in the blood. Sources and ways of use of
blood glucose. The impact of insulin, glucagon, adrenaline and cortisol. Change in
carbohydrate metabolism when fasting, during exercise and after meal.
84
 17. Types of diabetes. What are the changes of carbohydrate metabolism in dia-
betes type 1 and type 2?
18. Test of tolerance to glucose. The principle of the method, of procedures.
Clinical-diagnostic value of the test. Normal values of the glycemic curve. The form
of normal, hypo- and hyperglycemic curves. What determines the shape of the curve?
19. The stages of metabolism and their relationship. What, besides ATP, are the
high energy compounds? ATP-ADP cycle. The main methods of phosphorylation of
ADP and the use of ATP. The general scheme of the catabolism of proteins, fats and
carbohydrates in the body, specific and general ways of catabolism, their value.
20. NAD-dependent dehydrogenase, the reactions catalyzed by them in metabo-
lism of carbohydrates. Structural formula of oxidized and reduced forms of NAD+.
Characteristics of vitamin that is included in NAD+: the biological name, signs of de-
ficiency, daily requirement, dietary sources.
21. FAD-dependent dehydrogenases, reactions catalyzed by them in metabolism
of carbohydrates. Structural formula of oxidized and reduced forms of FAD. Charac-
teristics of vitamin that is included in the structure of FAD: the biological name, signs
of deficiency, daily requirement, dietary sources.
22. The sequence of oxidative decarboxylation of pyruvate, the link with the respira-
tory chain. The regulation of the process. Participation of vitamins in the process and their
characteristics: biological name, signs of deficiency, daily requirement, dietary sources.
23. The sequence of reactions of the citric acid cycle, the link with the respirato-
ry chain. Regulation of reactions. Participation of vitamins in the process, their char-
acteristics, energy effect.
24. The principle of oxidative phosphorylation. Scheme of the structural organi-
zation of the respiratory chain. The coupling of oxidation with phosphorylation. The
structure of the H+-ATP synthase. The ratio P/O for NADH and FADH2. The mecha-
nism of respiratory control. How does ATP influence the oxidative phosphorylation?
25. The uncoupling of respiration and phosphorylation. What determines the
function of heat-producing brown adipose tissue? Inhibitors of the respiratory chain.
Causes of hypoenergetic states. The ratio P/O and the number of produced ATP
molecules during complete oxidation of glucose.

85
 UNIT 9
STRUCTURE AND METABOLISM OF LIPIDS

THEME 9.1. STRUCTURE AND EXTERNAL METABOLISM OF LIPIDS

INTRODUCTION
Lipids, low-molecular organic substances, are diverse in chemical structure and
functions, insoluble in water, but soluble in organic solvents. Lipids include triacyl-
glycerols, complex lipids (phospholipids, glycolipids, sphingolipids), cholesterol (in-
cluding, as a precursor of bile acids, hormones, vitamin D). The multiplicity of lipid
biological functions determines the need for their study.

THE AIM OF THE PRACTICAL CLASS IS:

To study the structure and functions of the major lipids in human tissues, pro-
cesses of their digestion and absorption, as well as resynthesis in enterocytes and
transport of fats.
To obtain practical skills for the determination of phosphatidyl choline composi-
tion from egg yolk.

SELF-STUDY QUESTIONS
1. Characteristics of fatty acids according to the following plan:
• classification according to the number of carbon atoms, double bonds and their
position,
• sources of saturated and polyunsaturated fatty acids for the body,
• use of saturated and polyunsaturated fatty acids in the cell,
• biological role.
2. Fatty acids of ω-6 family (linoleic, γ-linolenic, arachidonic acid) and ω-3 fam-
ily (α-linolenic, eicosapentaenoic, docosa hexaenoic acid). The length and position of
double bonds. Biological role of polyunsaturated fatty acids.
3. Сharacteristics of derivatives of eicosatrienoic (ω-6), arachidonic (ω-6) and
eicosapentaenoic (ω-3) acids – eicosanoids (prostaglandins, prostacyclin, leukotri-
enes, thromboxanes). Biological role of certain types of eicosanoids. Scheme of the
initial reactions of synthesis on the example of arachidonic acid. Role of enzymes:
phospholipase A2, cyclooxygenase, lipoxygenase. What hormones and pharmaceuti-
cal substances influence on the synthesis of eicosanoids?
4. Structure of triacylglycerols, and fatty acids included in their composition.
Characteristics of the class of triacylglycerols (neutral fats), their biological role and
functions.
5. Chemical formula of cholesterol, its biological role and functions. Derivative
of cholesterol.
6. Characteristics of complex lipids:
• glycerophospholipids (phosphatidyl serine, phosphatidyl ethanolamine, phos-
phatidyl choline, phosphatidyl inositol), their chemical formula, biological functions,
86
 • sphingophospholipids (sphingomyelins), their structure. Biological functions of
sphingolipids,
• glycolipids (cerebrosides, gangliosides, sulfolipids), and their structure. Bio-
logical functions of glycolipids.
7. External lipid metabolism. Food sources of lipids, the daily need of children
and adults in liquid and solid fats.
8. Composition of bile and its role for body in digestion of lipids. Types of bile
acids, their functions, structure. Reaction of the synthesis of bile acids, for example
cholic acid, vitamins involved in this process. Chemical formula of taurocholic and
glycocholic acids. Causes and consequences of disorders of bile production and bile
secretion.
9. What is the emulsion? What are the characteristics of emulsions? Role of bile
and bile acids in the emulsification of dietary fat. Scheme of emulsified fat droplet.
10. Enzymes engaged in the digestion of triacylglycerols, phospholipids and cho-
lesterol esters in small intestine. Place of formation and activation of these enzymes.
11. Micelle products of fat digestion. Scheme of micelle structure formed after
the digestion of lipids. What is their role in the absorption of lipids?
12. Characteristic of lipid digestion in infants.
13. Possible causes of digestion and absorption disorders of dietary fat. Causes
of hypovitaminosis and steatorrhea in disorders of lipiddigestion.
14. Resynthesis of lipids in enterocytes and its role. Reaction of resynthesis of
triacylglycerols, cholesterol esters and phospholipids in the intestinal wall.
15. Composition of chylomicrons, and their functions. Apoproteins. Scheme of
chylomicron structure. Utilization of chylomicrons in tissues. Role of lipoprotein li-
pase. What hormones influence on its activity?
16. Characteristics of very low density lipoproteins: their composition, the ratio
of lipid fractions, their value. Apoproteins, their function. Scheme of VLDL struc-
ture. Where and when are these lipoproteins formed? Utilization of VLDL in tissues.
Role of lipoprotein lipase.
17. Characteristics of triacylglycerol transport disorders in tissue – dyslipopro-
teinemia I and V types. Their cause and clinical implications.
18. Determination of the composition of egg-yolk phosphatidylcholine. Principle
for determining the constituent elements.

TOPICS FOR REPORTS

1. The use of lipids as drugs and biologically active additives (essential, lecithin,
choleic acid, ω-3 fatty acids, etc.).
2. Unsaturated and polyunsaturated fatty acids. Types, structural role, participa-
tion in metabolism and behavioral reactions.
3. Liposomes in medicine. The structure and characteristics of liposomes. Possi-
bility of using medicines in the blood as a transport form.
4. Glycolipids, the structure of lipid and carbohydrate components. Functions of
glycolipids. Disturbance of glycolipid metabolism.

87
 Practical
THE STUDY OF PHOSPHATIDYL CHOLINE COMPOSITION
Principle
Method is based on the hydrolysis of egg-yolk phosphatidylcholine (lecithin) by
heating it in NaOH solution with subsequent determination of its structural compo-
nents in the extract: fatty acids, glycerol, choline, phosphoric acid.
Material for investigation
Dry egg yolk.
Reagents
1) 10% NaOH solution, 2) 10% HCl solution, 3) conc. HNO3, 4) molybdenum
reagent, 5) 1% CuSO4 solution.
Procedure
1. Hydrolysis of phosphatidylcholine
Place a piece of egg yolk into a test tube, add 3.0-4.0 ml of 10% NaOH solution,
boil in water bath for 15 minutes.
During heating in alkaline environment choline is converted
Detection of choline into trimethylamine N(CH3)3, which is detected at the end of
hydrolysis by the smell of herring brine
2. Hydrolysate is divided into 3 tubes:
Detection of fatty In the 1-st test tube: add dropwise 10% HCl solution until the
acids appearance of flakey suspension of fatty acids.
To the 2-nd part of hydrolysate: carefully add 5-7 drops of
concentrated HNO3 and drops of molybdenum reagent until the
appearance of yellow precipitate of ammonium phosphomo-
Detection of phos-
lybdate.
phoric acid
If it is necessary, heat in a boiling water bath. After cooling
tubes with running water, phosphomolybdate ammonium pre-
cipitates.
Add 5 drops of 10% NaOH solution and 1 drop of 1% CuSO4
Detection of glyc-
solution to the 3-rd part of hydrolysate, mix. Chelated copper
erol
compound with glycerol of bright blue color get formed.

Design of work:
Record results of reactions and conclusions in the form of a table:

Products of hydrolysis of
Result of reaction Conclusions
phosphatidylcholine
Choline
Fatty acids
Phosphoric acid
Glycerol

88
 THEME 9.2. INTRACELLULAR METABOLISM OF FATTY ACIDS AND
TRIACYLGLYCEROLS

INTRODUCTION
Triacylglycerols (neutral fats) are esters of glycerol and higher fatty acids.
Knowledge of the neutral fat metabolism is necessary to understand the metabolic
switch between fasting and muscular work, to study the pathogenesis of diseases, for
example, obesity, diabetes, atherosclerosis.
Acquaintance with the method for determining the triacylglycerols blood content
allows to use this information to detect diseases associated with lipid metabolism dis-
orders.

THE AIM OF THE PRACTICAL CLASS IS:

To study the processes of lipogenesis, lipolysis, oxidation of fatty acids, fatty ac-
id synthesis from glucose and synthesis of ketone bodies; regulation of lipid break-
down and synthesis.
To obtain practical skills for the quantitative determination of triacylglycerol in
serum.

SELF-STUDY QUESTIONS
1. Reactions of tricarboxylic acid cycle and their connection with the respiratory
chain and energy yield. Process of oxidative phosphorylation. Concept of respiratory
control.
2. Characterization of fatty acid synthesis from glucose according to the follow-
ing plan:
• localization and conditions of the process,
• reaction of acetyl-S CoA formation from glucose,
• role of citrate to transfer acetyl groups in cytosol, and its further transfor-
mations,
• synthesis of malonyl-S CoA,
• structure of the multienzyme complex, chemistry of reactions occurring in the
complex,
• the final product of synthesis,
• participation of vitamins and coenzymes, sources of NADPH, role of "malic"-
enzyme,
• regulation of the process with the participation of hormones: insulin, adrena-
line, glucagon,
• influence of ATP, acyl-S CoA, malonyl-S CoA, citrate on fatty acid synthesis.
3. Synthesis reaction of glycerol-3-phosphate from glucose. Localization and
role of the process.
4. Reaction of phosphatidic acid synthesis from fatty acids and glycerol-3-
phosphate according the following plan:
• localization in the cell,
• sources of glycerol-3-phosphate, fatty acids and energy,
89
 • sequence of reactions,
• relationship with the metabolism of carbohydrates,
• further use of phosphatidic acid.
5. Structure of triacylglycerols, their fatty acid composition. Reactions of tri-
acylglycerol synthesis (lipogenesis). Conditions for the occurrence of lipogenesis in
the liver and adipose tissue. Communication of triacylglycerol synthesis with carbo-
hydrate metabolism.
6. Sources of TAG in the liver. Characteristics of very low density lipoproteins:
their composition, Apoproteins, function. Scheme of VLDL structure. Conditions for
the formation of these lipoproteins. Utilization of VLDL in tissues. Role of lipopro-
tein lipase.
7. Similarities and differences in the biosynthesis of triacylglycerol in adipose
tissue and liver.
8. Characteristic reactions of lipolysis according to the following plan:
• localization and conditions of the process,
• sequence of reactions and enzymes,
• the final products,
• hormonal regulation of the process,
• transportation and utilization of free fatty acids, formed during lipolysis.
9. Hormone sensitive lipase, role of adenylate cyclase system in the regulation of
its activity. Regulation of lipase by hormones: adrenaline, glucagon, cortisol and in-
sulin. Allosteric regulation of activity of lipolysis and lipogenesis enzymes.
10. Oxidation of glycerol to pyruvate. Possible metabolic pathways of pyruvate.
Energy yield of glycerol oxidation in aerobic and anaerobic conditions.
11. Oxidation of fatty acids to carbon dioxide and water:
• role of carnitine in fatty acid oxidation,
• localization and flow conditions of β-oxidation,
• sequence of β-oxidation reactions and enzymes,
• participation of vitamins and coenzymes,
• the final products,
• connection with the TCA and the respiratory chain,
• energy yield of the process,
• calculation of the energy value β-oxidation of palmitic acid.
12. Features of triacylglycerol metabolism during certain physiological condi-
tions (food intake, starvation, muscle activity). Features of brown adipose tissue.
13. Causes of disorders of triacylglycerol metabolism – obesity, hyperlipopro-
teinemia type I (hyperchylomicronemia) and type V.
14. Synthesis of ketone bodies. Conditions, localization and role of the process.
Reaction of ketone bodies utilization in tissues. Causes of ketoacidosis in starvation
and diabetes. Role of oxaloacetate deficiency in the activation of ketogenesis.
15. Determination of the concentration of triacylglycerol in the blood serum.
Clinical-diagnostic significance and normal values.

90
 TOPICS FOR REPORT
1. White and brown adipose tissue. Adipose tissue distribution in the body and
metabolic features. Its role in the body.
2. Primary and secondary acetonemic syndromes in children.
3. Modern ideas about the role of L-carnitine in the body. Participation of car-
nitine in the pre- and postnatal development of the child. The assumed role of car-
nitine in "death in the cradle" syndrome development (syndrome of sudden child
death).

Practical
THE DETERMINATION OF TRIACYLGLYCEROL CONCENTRA-
TION IN SERUM
Principle
The method is based on the use of connected enzymatic reactions, carried out:
1) lipase, catalyzing the hydrolysis of triacylglycerol to glycerol and fatty acids,
2) glycerol kinase, catalyzing the phosphorylation of glycerol, 3) glycerol phosphate
oxidase, oxidizing glycerol-3-phosphate in the presence of O2 to dioxyacetone phos-
phate with the formation of H2O2, 4) peroxidase, catalyzing oxidation of 4-
aminoantipyrine by hydrogen peroxide with the formation of khinonimine, painted
the product in raspberry pink color, in the presence of chlorophenol.
Material for investigation
Serum.
Reagents
Working reagent: lipase, glycerol kinase, glycerol phosphate, peroxidase, chlo-
rophenol, 4-aminoantipyrine in a buffer solution.
A standard solution of triacylglycerol (triolein): 2.29 mmol/l.

Procedure
Experienced test, ml Standard, ml
Serum 0.03 ––
Standard solution of –– 0.03
triacylglycerols
Working reagent 3.0 3.0
Incubate for 20 minutes at 20-25°C, measure the opti-
cal density of experimental and standard samples
against water at a wavelength of 540 nm (green light
filter).
Calculation
Concentration of triacylglycerols, mmol/l = Eex/Est ×Cst
Where Eex and Est – optical density of experiment and standard samples, Cst –
concentration of standard solution.

91
 Normal values
Serum Children 0.15-1.56 mmol/l
Adults: 0.45-1.71 mmol/l

Clinical-diagnostic significance
Determination of triacylglycerol concentration of is the most informative for typ-
ing primary defects of its exchange – hyperlipoproteinemia.
Increase in the level of triacylglycerols is observed in obesity, diabetes, hyper-
tension, coronary heart disease, pancreatitis, chronic renal failure and nephrotic syn-
drome, hypothyroidism, atherosclerosis, alcoholism.
Decrease in the concentration of triacylglycerols is observed in hyperthyroidism,
chronic obstructive pulmonary disease, end-stage liver disease, malabsorption syn-
drome.
Design of work:
Write down the principle of the method, the experimental procedure, the normal
value and the results of the study, note the clinical and diagnostic value of the index
and draw conclusions on the possible pathology.

THEME 9.3. INTRACELLULAR METABOLISM OF PHOSPHOLIPIDS AND

CHOLESTEROL. TRANSPORT OF LIPIDS IN BLOOD

INTRODUCTION
Phospholipids and cholesterol are the part of the cell membranes. They partici-
pate in the formation of lipoproteins. If the synthesis of phospholipids is disrupted,
the normal metabolism of cells and the formation of transport lipoproteins will
change.
Disorders of cholesterol metabolism cause many diseases: atherosclerosis and
cholelithiasis.
The drugs applied in cardiology affect the reactions of cholesterol synthesis in
tissues, as well as the low and high density lipoprotein metabolism in blood.

THE AIM OF THE PRACTICAL CLASS IS:

To study the processes of biosynthesis and catabolism of phospholipids and gly-
cosphingolipids, synthesis of cholesterol, the role of lipoproteins in transport of free
cholesterol and its esters in blood plasma.
To obtain practical skills for the quantitative determination of cholesterol con-
centration in blood serum.

SELF-STUDY QUESTIONS
1. Structure of phospholipids: phosphatidyl serine, phosphatidyl ethanolamine,
phosphatidyl choline, phosphatidyl inositol. Their biological role.
2. Catabolism of phospholipids. Enzymes, splitting phospholipids in the intestine
and tissues. Role of phospholipase A2 and C.

92
 3. Characterization of phosphatidic acid synthesis from fatty acids and glycerol
according to the following plan:
• localization in the cell,
• sources of glycerol, fatty acids and energy,
• the sequence of reactions,
• relationship with carbohydrate metabolism,
• further use of phosphatidic acid.
4. Interconnection reactions of glycine, serine and methionine, role of vitamins
B6, B12 and folic acid in the metabolism. Synthesis reaction of S-adenosyl methionine
from S-adenosyl homocysteine, its role in the transmethylation in the synthesis of a
number of substances, including phosphatidyl choline.
5. Reactions of phospholipid biosynthesis in tissues. Two pathways of phospho-
lipid biosynthesis. Role of amino acids and vitamins in the process. Lipotropic sub-
stances. Causes of change in phospholipid synthesis. Causes and consequences of fat-
ty liver.
6. Chemical structure and biological role of cholesterol. Food sources of choles-
terol. Ways of removing cholesterol from the body.
7. Transport of free cholesterol and its esters in blood plasma. Composition and
structure of lipoproteins of low and high density. Types of apoproteins and their func-
tions. Metabolism of LDL and HDL in blood plasma. The reaction catalyzed by
LCAT (lecithin cholesterol acyl transferase).
8. Localization and role of apoB-100-receptor. Importance of receptor-mediated
endocytosis of LDL and pathway components after endocytosis. Role of ACAT (acyl
cholesterol acyl transferase).
9. Scheme of cholesterol synthesis stages. Interconnection of cholesterol synthe-
sis and metabolism of carbohydrates. Regulation of the synthesis. Hormonal and allo-
steric regulation mechanisms. Medicinal regulation of cholesterol synthesis.
10. Characterization of cholesterol metabolic disorders – hyperlipoproteinemia
type IIA (family hypercholesterolemia), atherosclerosis (in stages), gallstone disease.
Causes, consequences, basics of treatment.
11. Characteristics of acetyl-S CoA formation: catabolism of glucose, amino ac-
ids, fatty acids and ketone bodies. Using of acetyl-S CoA: TCA, fatty acid synthesis,
cholesterol, ketone bodies.
12. Lipidoses or lipid storage diseases. Niemann-Pick's, Gaucher’s and Tay-
Sachs diseases.
13. Determination of cholesterol concentration in blood serum. Principle of the
method, its clinical-diagnostic value, normal levels.

TOPICS FOR REPORT

1. Atherosclerosis, causes, pathogenesis, consequences. Modern approaches to
the treatment of atherosclerosis.
2. Metabolic syndrome (syndrome X). Its connection with obesity, atherosclero-
sis, type 2 diabetes, hypertension. Causes. Basics of treatment.

93
 3. Disease of lipid accumulation: Niemann-Pick, Thea-Sachs, Gaucher,
Schueller-Christian, Wolman. Molecular causes, pathogenesis. Basics of treatment.

Practical
THE DETERMINATION OF CHOLESTEROL CONCENTRATION IN
SERUM
Principle
The method is based on conjugated enzymatic reactions. Cholesterol esterase
carries out the hydrolysis of cholesterol esters; then cholesterol oxidase turns choles-
terol into cholestenone with the formation of H2O2. Hydrogen peroxide, in the pres-
ence of phenol with the participation of peroxidase, oxidizes 4-aminoantipyrine with
the formation of khinonimine, paints the product in raspberry pink color. The color
intensity is proportional to the concentration of cholesterol and is determined by pho-
to colorimetric method.
Material for investigation
Serum.
Reagents
1) Working reagent: solution of cholesterol esterase, cholesterol oxidase, peroxi-
dase, phenol, 4-aminoantipyrine in 0.1 M potassium-phosphate buffer.
A standard solution of cholesterol: 4.65 mmol/l.

Procedure
Test, ml Standard, ml
Serum 0.02 ––
Standard solution –– 0.02
Cholesterol 2.0 2.0
Incubated for 20 minutes at 37°C. Measure the optical
density of experienced and standard samples against wa-
ter at a wavelength of 540 nm (green light filter).
Calculation:
Concentration of cholesterol, mmol/l = Etest/Est ×Cst
Where Etest and Est – optical density of experiment and standard samples, Cst –
concentration of standard solution.

Normal values
Serum Children 1.2-5.2 mmol/l
Adults 3.0-5.2 mmol/l

Clinical and diagnostic significance

Cholesterolemia, more than 5.2 mmol/l, is a high-risk factor of atherosclerosis,
and coronary heart disease and stroke are its clinical complications. The high concen-
tration of cholesterol in blood is observed in hyperlipoproteinemia IIA and IIB, III,
nephrotic syndrome, diabetes mellitus, hypothyroidism, kidney damage, intra- and
extrahepatic cholestasis.
94
 Reduction of cholesterol concentration in the blood (hypocholesterolemia) is ob-
served in starvation and malabsorption syndrome, hyperthyroidism, acute pancreati-
tis, liver cirrhosis, malignant tumors.
Design of practical:
Write down the principle of the method, the experimental procedure, the normal
value and the results of the study, note the clinical and diagnostic value of the index
and draw conclusions on the possible pathology.

TESTS
Choose one or more correct answers.
1. STEROIDS ARE
1) bile acids
2) gangliosides
3) sphingomyelins
4) pituitary hormones

2. THE NUTRITION ESSENTIAL FACTOR IS__________

1) cholesterol
2) phosphatidylcholine
3) linolenic acid
4) oleic acid

3. THE HIGH FATTY ACID METABOLISM IS_________

1) decarboxylation
2) glycogenolysis
3) β-oxidation
4) lipolysis

4. THE RESIDUE OF FATTY ACID TRANSFERS THROUGH THE MITO-

CHONDRIAL MEMBRANE IS THE MOLECULE OF ________
1) carnosine
2) carnitine
3) creatine
4) keratin

5. THE NADPH SOURCE FOR FATTY ACID SYNTHESIS IS ________

1) pentose phosphate pathway
2) catabolism of triacylglycerols
3) oxidative decarboxylation of pyruvate
4) the reaction of glycolysis

6. THE CHYLOMICRON CONSISTS OF __________

1) 90% of triacylglycerols and 2% of proteins
2) 50% cholesterol and its esters
3) 50% of proteins and 20% of cholesterol and its esters
4) 10% proteins and 50-55% triacylglycerols

95
 7. ___________ DOES NOT USE KETONE BODIES IN STARVATION
1) nerve cell
2) skeletal muscle
3) heart
4) liver

8. _______________ IS ACTIVATED IN LONG-TERM STARVATION

1) the lipogenesis in adipose tissue
2) the β-oxidation in the liver
3) the synthesis of cholesterol in nerve tissue
4) the cycle of tricarboxylic acids in the liver

9. THE KEY ENZYME OF CHOLESTEROL SYNTHESIS IS__________

1) hydroxymethylglutaryl-SCoA reductase
2) acyltransferase
3) pyruvate kinase
4) acetoacetyl-SCoA synthase

10. CHOOSE THE HORMONES, STIMULATING THE LIPID SYNTHESIS

IN ADIPOSE TISSUE
1) adrenaline
2) glucagon
3) the hormone of growth
4) insulin

CASE STUDIES
1. A delay in the outflow of bile from the gallbladder was found in patients after
an endoscopy investigation.
Suggest the consequences of this pathology.

2. The concentration of triacylglycerols and phospholipids is 1.5-2.0 times high-

er in the cardiac muscle as compared to skeletal one.
Identify the biochemical value of this difference.

3. Parents are worried about an overweight child. Without consulting a doctor,

they limited the amount of sugar in the baby's food and increased the protein content,
without non-reduced fat amount. The child's health worsened in a few weeks. The
vomit appeared.
Identify the metabolism disturbance and its cause.
Recommend the set of biochemical investigations for metabolic disorder detec-
tion.

CHECKLISTS FOR FINAL LESSON (UNIT 9)

1. Structural formula and characteristics of the main classes of lipids.

96
 2. Types of fatty acids, their physic-chemical properties and biological role, food
sources. Structure of palmitic, stearic and oleic acids, polyunsaturated fatty acids of
ω-6 family (linoleic, γ-linolenic, arachidonic acid) and ω-3 family (α-linolenic,
eicosapentaenoic, docosahexaenoic acid). Transport of fatty acids in the blood.
3. Derivatives of polyunsaturated fatty acids of ω-6 and ω-3 families, biological
role of certain types of eicosanoids. The initial reaction of arachidonic acid synthesis.
Role of phospholipase A2, cyclooxygenase, lipoxygenase. What hormones and phar-
maceutical substances influence on the synthesis of eicosanoids?
4. Triacylglycerols: chemical structure, fatty acids included in the composition,
physic-chemical properties, biological role. Transport of triacylglycerols in the blood.
5. Phospholipids: chemical structure (phosphatidyl serine, phosphatidyl ethano-
lamine, phosphatidyl choline, phosphatidyl inositol), fatty acids contained in phos-
pholipids, physical and chemical properties, biological role. What is the role of phos-
pholipids in lipid transport in the blood?
6. Chemical structure of sphingolipids (sphingomyelins) and glycolipids (cere-
brosides, sulfolipids, gangliosides), their biological role and functions.
7. Structure of cholesterol and its esters, and its biological role. Transport of cho-
lesterol in the blood.
8. Daily needs and food sources of fat. Role of enzymes and components of bile
in the digestion of dietary lipids in the digestive tract. Synthesis of bile acids, role of
vitamins in the process. Chemical structure of taurocholic and glycocholic acids. Re-
synthesis of lipids in the intestinal wall. Consequences of violations of digestion and
absorption.
9. Characteristics of chylomicrons and very low density lipoproteins, their com-
position, scheme of structure, function, reactions of metabolism in the bloodstream.
Utilization of chylomicrons and VLDL in tissues. The role of lipoprotein lipase.
What hormones are activated?
10. Detection of egg yolk phosphatidyl choline. Principle of the method and
course of the measurement.
11. Lipolysis, chemical reactions, the role of lipolysis. Regulation of lipolysis. In
what physiological situations does it happen? Transport and utilization of fatty acids
formed during lipolysis. Adenylate cyclase mechanism of TAG-lipases activation.
The influence of insulin, adrenaline, glucagon on lipolysis.
12. Reaction of β-oxidation of fatty acids, connection to the TCA and the res-
piratory chain. Energy yield of the process on the example of palmitic, stearic and
palmitoleic acids.
13. Ways of formation and use of acetyl-S CoA in the body (schema).
14. Reactions of synthesis and degradation of ketone bodies. Causes of ketone-
mia and ketonuria in starvation and diabetes.
15. Reactions of fatty acid synthesis from glucose. The key stage and localiza-
tion of the process, role of citrate. Regulated enzymes. Composition of the multien-
zyme complex (fatty acid synthase).
16. Synthesis reaction of glycerol-3-phosphate from glucose and oxidation of
glycerol to pyruvate. Energy yield of the process.
97
 17. Chemistry of triacylglycerol synthesis. In what conditions and where does li-
pogenesis take place? Differences in biosynthesis of fats in adipose tissue and in the-
liver. Regulation of lipogenesis. Sources of glycerol, fatty acids and energy. Relation-
ship of triacylglycerols synthesis with glucose metabolism.
18. Metabolism of triacylglycerols and saturated fatty acids in certain physiolog-
ical conditions (food intake, hunger, muscular activity, diabetes type 1 and 2).
19. Determination of triacylglycerols concentration in the blood serum. Principle
of determination, clinical-diagnostic and normal values.
20. Pathways of the synthesis of phospholipids, difference between them. Chem-
istry of reactions. Role of amino acids and vitamins. Energy sources for the synthesis
of phospholipids. What is lipotropic substances? Their functions. Consequences of
the lack of lipotropic substances.
21. Metabolism and functions of cholesterol: synthesis to mevalonic acid, the
view about further stages, regulation of synthesis, interconnection of cholesterol syn-
thesis and metabolism of carbohydrates. Ways of cholesterol excretion from the
body.
22. Determination of concentration in the blood. Principle of the method, defini-
tion, clinical-diagnostic value, normal values.
23. Characterization of the lipoproteins of high and low density: role in the cho-
lesterol metabolism and its esters, the main apoproteins of lipoproteins. Interaction of
LDL and HDL in blood plasma. Role of lecithin-cholesterol-acyltransferase. Utiliza-
tion of LDL in cells. Intracellular use of cholesterol and removal of its excess from
the cell. Role of acyl-S CoA cholesterol acyltransferase.
24. Interconnection of lipid and carbohydrate metabolism. The conversion of
glucose into fatty acids, cholesterol and triacylglycerol (scheme). Role of pentose
phosphate pathway in the synthesis of fats.
25. Hormonal and allosteric regulation of lipid metabolism. The effect of insulin,
glucagon, adrenaline, glucocorticoids on lipolysis and lipogenesis, synthesis and β-
oxidation of fatty acids, synthesis of cholesterol.
26. Biochemical mechanism of lipid metabolism disorders: atherosclerosis, gall-
stone disease, obesity, fatty liver, diabetes type 2, hyperlipoproteinemia type I (chy-
lomicronemia), type V and IIa type (family hypercholesterolemia). What is Tay-
Sachs, Niemann-Pick’s, Gaucher’s diseases?
27. Stages of metabolism and their relationship. What other high-energy connec-
tions are there apart from ATP? A cycle of ATP ADP. The main ways of phosphory-
lation of ADP and ways of using ATP. The general scheme of catabolism of proteins,
fats and carbohydrates in the body, specific and common ways of catabolism, their
significance.
28. NAD-dependent dehydrogenases, carbohydrate metabolism reactions cata-
lyzed by them. Structural formulas of oxidized and reduced forms of NAD+. Charac-
teristics of the vitamin, which is a part of NAD+: biological name, signs of insuffi-
ciency, daily need, food sources.
29. FAD-dependent dehydrogenases, carbohydrate metabolism reactions cata-
lyzed by them. Structural formulas of oxidized and reduced forms of FAD. Charac-
98
teristics of the vitamin, which is a part of the FAD: biological name, signs of insuffi-
ciency, daily need, food sources.
30. The sequence of reactions of the oxidative decarboxylation of pyruvate, the
connection with the breathing chain. Regulation of the process. The participation of
vitamins in the process and their characteristics: biological name, signs of insuffi-
ciency, daily need, food sources.
31. Sequence of reactions of the tricarboxylic acid cycle, connection with the
respiratory chain. Regulation of reactions. The participation of vitamins in the pro-
cess, their characteristics, energy effect.
32. Principle of oxidative phosphorylation. Scheme of the structural organization
of the respiratory chain. Conjugation of oxidation with phosphorylation. Structure of
H+-ATP synthase. P/O ratio for NADH and FADH2. The mechanism of respiratory
control. How does ATP affect oxidative phosphorylation?
33. Dissociation of respiration and phosphorylation. What determines the heat-
forming function of brown adipose tissue? Inhibitors of the respiratory chain. The
causes of hypo energetic states. P/O ratio and the number of ATP molecules formed
upon complete oxidation of palmitic acid.

99
 UNIT 10
HORMONAL REGULATION OF METABOLISM AND
FUNCTIONS IN THE HUMAN BODY

THEME 10.1. THE MECHANISMS OF HORMONAL SIGNAL

TRANSDUCTION. CLASSIFICATION OF HORMONES. HORMONES OF
PITUITARY GLAND. (SEMINAR)

INTRODUCTION
One of the characteristic features of living organisms is their ability to maintain
the constancy of homeostasis by means of self-regulatory mechanisms in which a ma-
jor role belongs to hormones. The effect of hormones on cells is carried out through
special mechanisms, the violation of which leads to the failure or change of the hor-
monal effect. In order to correctly evaluate the causes of hormonal diseases it is nec-
essary to know the mechanisms by which the hormonal signal is transmitted into the
cell.
The changes in the endocrine regulation with insufficient or excess synthesis of
hormones lead to disorders of metabolic processes in the body. In clinical practice
hormones and hormonal drugs are widely used for the treatment of endocrine and no
endocrine diseases.

THE AIM OF THE PRACTICAL CLASS IS:

To study the mechanisms of action of protein and peptide hormones, hormones –
derivatives of amino acids, steroid hormones.

SELF-STUDY QUESTIONS
1. Principles of regulation of metabolic processes. The hierarchy of the regulato-
ry systems of the body. The role of the hypothalamus and pituitary.
2. The mechanism of a negative feedback in the regulation of hormone produc-
tion and action.
3. Common biological characteristics of hormones. Classes of hormones accord-
ing to their chemical structure, biological functions and belonging to endocrine
glands.
4. Characterization of three membrane mechanisms by which the hormonal sig-
nal is transmitted in the target cells:
• a receptor with enzymatic activity (schematically on the example of the insulin
receptor),
• the receptor that forms an ion channel (schematically on the example of the ac-
etylcholine receptor),
• transmission of the hormonal signal using G proteins (cAMP-mediated, calci-
um-phospholipid mechanism). Specify the components of the signal transmission
system, note the role of activating and inhibitory α subunit of the G protein. What
hormones use these mechanisms?
100
 5. cGMP-mediated mechanism of signal transmission. General characteristics of
this mechanism.
6 The structure and sources of secondary mediators of hormonal signal transmis-
sion: cAMP, cGMP, inositol triphosphate (IP3), diacylglycerol (DAG), Ca2+ions.
7. Characterization of the cytosolic mechanism of hormonal signal transmission
into the target cells. Hormones which operate according to this mechanism.
8. Hormones of the hypothalamus.
9. Hormones – derivatives of proopiomelanocortin.
10. Characterization of pituitary hormones (growth hormone, vasopressin, oxy-
tocin, lipotropic hormone, melanocyte stimulating hormone) according to the plan:
• name,
• chemical nature,
• regulation of hormone synthesis and secretion
• the target organs,
• the localization of receptors in the cell and mechanism of action,
• impact on the metabolism of carbohydrates, proteins, lipids, minerals, water –
reactions and enzymes that are sensitive to the hormone action,
• hyper- and hypofunction of hormones, the reasons and main clinical manifesta-
tions.

TOPICS FOR REPORT

1. Influence of cholera and diphtheria toxins on the activity of adenylate cyclase
mechanism in the affected cell.
2. Mechanisms and levels of regulation of endocrine gland functions, violation
of these mechanisms, consequences.
3. Oxytocin – a hormone of interhuman relationships?
4. Guanylate cyclase, types, regulation, ways of transmission of the hormonal
signal. Hormones using the guanylate cyclase mechanism.
5. The role of nitric oxide (NO) in the regulation of a cell activity under normal
and pathological conditions. The participation of nitric oxide in the action of drugs.
6. Hormone-dependent induction and repression of protein synthesis. Features of
the receptor and hormone receptor complex, their effect. The hormone sensitive ele-
ment of DNA.

THEME 10.2. HORMONES OF HYPOTHALAMUS, PITUITARY, THYROID,

PANCREAS AND PARATHYROID GLANDS

INTRODUCTION
Hormones of the hypothalamus and pituitary gland regulate the growth and func-
tion of other endocrine glands or influence of metabolic responses in target tissues.
The posterior pituitary secretes hormones that regulate water balance and milk ejec-
tion from lactating mammary gland. Loss of function of the anterior pituitary (hypo-
pituitarism) leads to the atrophy of thyroid, adrenal and sex glands.

101
 Thyroid hormones play an important role in the regulation of metabolism, devel-
opment and differentiation of tissues. The calcium homeostasis regulating hormones
are produced in the parathyroid and thyroid glands and maintain the plasma calcium
concentration in very narrow limits.
Insulin and glucagon, pancreatic hormones, play a major role in the blood glu-
cose homeostasis, affecting the metabolism of carbohydrates and lipids in the liver
and adipose tissue.

THE AIM OF THE PRACTICAL CLASS IS:

To study the protein and peptide hormone influence on the metabolism of carbo-
hydrates, lipids, proteins, water and minerals.
To conduct qualitative reactions for the detection of insulin and the concentra-
tion of prolactin in serum.

SELF-STUDY QUESTIONS
1. Classes of hormones according to their chemical structure, biological func-
tions and belonging to endocrine glands.
2. Hormones of the hypothalamus (releasing hormones).
3. Characterize the pituitary hormones – growth hormone, vasopressin, oxytocin,
adrenocorticotropic, lipotropic and melanocyte stimulating hormones, lactotropic,
follicular-stimulating and luteinizing hormones according to the plan:
• name,
• chemical nature,
• regulation of hormone synthesis and secretion
• the target organs,
• localization of the receptors in the cell and mechanism of action,
• impact on the metabolism of carbohydrates, proteins, lipids, minerals, water –
reactions and enzymes that are sensitive to the hormone action.
4. Causes and metabolic consequences of the antidiuretic hormone hypofunction
(diabetes insipidus). What clinical manifestations of a disease are observed? What is
the vasopressin hyperfunction?
5. Characteristics of disorders associated with growth hormone: pituitary dwarf-
ism, acromegaly, gigantism. What are the causes and metabolic disorders? The clini-
cal manifestation of a disease.
6. Characteristics of thyroid hormones: tyroliberin. thyroid stimulating hormone,
tri- and tetra iodothyronine. The thyroxine and triiodothyronine chemical structure,
their target organs, localization of the receptors in the cell and mechanism of action.
Impact on the carbohydrates, proteins, lipids metabolism. Enzymes that are sensitive
to hormone. Hypo- and hyperthyroidism causes. Metabolic disturbances and clinical
manifestations of the diseases.
7. Characterization of calcitonin and parathyroid hormone according to the plan:
• name,
• the chemical nature,
• the hormone synthesis and secretion regulation,
102
 • the target organs,
• localization of the receptors in the cell and mechanism of action,
• the influence on the exchange of mineral substances.
8. How does the calcitonin and parathyroid hormone effect together with the cal-
citriol action (a vitamin D derivate)?
9. Characterize the pancreas hormones – glucagon and insulin according to the
plan:
• the name,
• the chemical nature,
• the regulation of hormone synthesis and secretion,
• the target organs,
• localization of the receptors in the cell and mechanism of action,
• the impact on carbohydrates, proteins, lipids metabolism – hormone sensitive
reactions and enzymes.
10. Types of diabetes. The causes of absolute and relative insulin deficiency.
Metabolic disturbances in different types of diabetes, their clinical manifestations,
basic treatment.
11. Conducting the insulin qualitative reactions.
12. Enzyme linked immunoassay for the prolactin determination in serum. The
principle of the method. Normal values. Clinical-diagnostic value.
13. Make a table: hormones of pituitary gland, thyroid, parathyroid and pancreat-
ic glands according to the following diagram:

Hypo- and hyperfunction of

The name and the chemical

Regulation of hormone ac-

Receptors localization,

The impact on metabolism

mechanism of action
Place of synthesis

The target organs

Minerals and water

hormones
nature

Carbohydrates
tion

Proteins

Lipids

TOPICS FOR REPORT

1. Melatonin, a place of synthesis, chemical nature, influence on metabolism un-
der the normal and pathological conditions. The possibility of melatonin use in the
clinical practice.
2. Dysfunction of the growth hormone. Molecular causes. Clinical manifesta-
tions of diseases, their diagnosis. Basics of treatment.
3. Diabetes mellitus, its types. Molecular mechanisms. Metabolic disorders.
Clinical symptoms. Basics of treatment.

103
 4. Causes of deficiency of thyroid function. Symptoms, clinical manifestations in
children and adults. Basics of treatment. The role of hypothyroidism in the reproduc-
tive health of women.

Practical 1
INSULIN QUALITATIVE REACTIONS
The principle
Insulin is a simple protein and gives characteristic qualitative reactions on pro-
tein: Biuret, xanthoprotein, Fole etc. These reactions are not specific.
Material for investigation
Insulin solution.
Reagents
1) The Fole’s solution containing 5% solution Pb(CH3COO)2 and 30% NaOH
solution, 2) 0.5% solution of ninhydrin, 3) 30% solution NaOH, 4) 10% solution
NaOH, 5) 5% solution Pb(CH3COO)2, 6) 5% solution sodium nitroprusside, 7) strong
HNO3, 8) 5% CuSO4 solution.
Procedure
5 drops of insulin solution are poured in a test-tube and qualitative reactions on
protein are made.
Biuret test for Proteins
The Biuret Test is done to show the presence of peptide bonds, which are the
basis for the formation of proteins. These bonds will make the blue Biuret reagent
turn purple.
The principle
In alkaline medium the peptide group forms with ions Cu2+ complex compound
of violet color with a red or blue hue depending on the number of peptide bonds. The
color intensity is proportional to the number of peptide groups.
Procedure
3 drops of 10% solution NаОН and 1 drop of 5% solution CuSO4 are added to 5
drops of insulin solution in a test tube.
Reaction for α-amino group detection
The ninhydrin reaction is used to detect α-amino groups contained in the ami-
no acids and the final insulin of α-amino groups.
The principle
The oxidative α-amino group cleavage and recovery of ninhydrin occur while
the protein is heated with ninhydrin. The restored ninhydrin reacts with ammonia and
another oxidized ninhydrin molecule to form ninhydrin complex of blue-violet color.
Procedure
5 drops of insulin solution are mixed with 5 drops of 0.5% solution of ninhydrin.
The tube is heated and boiled until the appearance of blue-purple staining.
Aromatic amino acids reaction
For the detection of aromatic amino acids (phenylalanine, tyrosine, trypto-
phan) xanthoprotein reaction is used.

104
 The principle
2 drops of concentrated HNO3 are added to 5 drops of 1% insulin solution and
carefully heated. Observe the appearance of yellow staining, in the absence of yellow
color 1-2 more drops of conc. HNO3 are added. While adding an excess 30% NаОН
solution the color changes to orange
Sulfur-containing amino acids reactions
The principle
Insulin sulfhydryl groups are subjected to alkaline hydrolysis, resulting in the
cleavage of sulfur in the form of sodium sulfide Na2S, entering into the further reac-
tions:
• Fole’s reaction – Na2S with lead acetate Pb(CH3COO)2 gives black or brown
lead sulfide sediment,
• nitroprusside reaction– Na2S with nitroprusside sodium gives the red-brown
compound.
Procedure
5 drops of insulin solution and 5 drops of 30% NaOH solution are boiled for 1-2
minutes. The content is split into 2 parts for reactions "a" and "b".
a) Fole’s reaction
1 drop of acetic acid lead is added to 5 drops of hydrolysate and heated to boil-
ing. The appearance of brown or black sediment is determined.
b) Nitroprusside reaction
2-3 drops of 5% sodium nitroprusside solution are added to 5 drops of hydroly-
sate. The appearance of red-brown staining is noted.
Design of practical:
Explain the principle of methods, record the analysis results and make a conclu-
sion about the insulin presence in the test material.

Practical 2
IMMUNOASSAY METHOD FOR PROLACTIN DETERMINATION IN
SERUM
The principle
The solid-phase enzyme immunoassay is based on specific binding of monoclo-
nal prolactin antibodies adsorbed on the wells of immunological tablet, with the sub-
sequent formation of the conjugate.
Material for investigation
Serum.
Reagents
1) Conjugate monoclonal prolactin antibodies with horseradish peroxidase, a
solution for dilution of serum, 2) phosphate-saline buffer solution with tween, 3) a
solution of tetramethylbenzidine, 4) stop reagent 5) the control sample with a known
prolactin content, 6) calibration samples containing a known prolactin amount.

105
 Procedure
1. The introduction of the samples. Make in duplicate, starting from the top
wells of the first two strips with 100 µl calibration samples. Add 100 µl of control
sample and 100 µl of the analyzed serum samples in the rest of the wells.
2. The introduction of monoclonal antibody conjugate. The conjugate is ready to
be used. 50 µl of conjugate are added into the wells.
3. Incubation. Sealed film strips are incubated at the temperature of 37°C for 60
minutes in a thermostatic shaker with a frequency of 650 rpm.
4. Washing. At the end of incubation, the sticky bar is removed and placed in a
disinfectant solution container. With the help of the flushing device the tablet is
washed 5 times with washing solution, alternating aspiration and immediate filling up
the holes of each strip. In each well at least 350 µl of liquid are contributed in each
wash cycle. After rinsing the remaining moisture from the wells is thoroughly re-
moved by tapping the inverted tablet on the filter paper.
5. The introduction of tetramethylbenzidine (TMB). A TMB solution is ready
for use. Insert 100 µl of TMB to all wells.
6. Incubation. Sealed film strips are incubated in the dark at a temperature of
37°C for 15 minutes in a thermostatic shaker with a frequency of 650 rpm.
7. The introduction of stop reagent. 100 µl of stop reagent are added into all
wells. Shake the plate on a shaker for 10-15 seconds; the content of the wells turns
yellow.
8. Measuring. The optical density in the wells of the tablet is measured by the
spectrophotometer in dual-wave mode: when the main wavelength is 450 nm and the
comparison length is in the range 620-655 nm.
Normal levels
Men's 2.5-17 ng/ml (53-360 mlU/l);
Women – follicular phase of 4.5-33 ng/ml (98-784 mlU/l), middle of a cycle
6.3-49 ng/ml (134-975 mlU/l), luteal phase 4.9-40 ng/ml (104-848 mlU/l).
Clinical-diagnostic value
The normal prolactin increase occurs during sleep, exercise, sexual intercourse.
During pregnancy the hormone increases from the 8-th to the 25-th week and during
lactation. Before birth there is a decrease in prolactin.
Concentration increase Concentration decrease
Prolactinoma Acute porphyria
Acute and chronic physical and mental
Neurogenic and psychiatric disorders,
stressful situation (depression, surgery,
menstrual problems
painful periods)
Acromegaly Hypoglycemia
Hirsutism (hyperandrogenism)
Design of practical:
The principle of the method, the working procedure, the normal values and the
results of the study are indicated, clinical and diagnostic value of the index is noted
and conclusions on the possible pathology are drawn.

106
 THEME 10.3. HORMONES OF HYPOPHYSIS, ADRENAL AND SEXUAL
GLANDS

INTRODUCTION
In the human body corticotropin and adrenal hormones perform functions related
to the activities of the body in a state of acute and chronic stress, providing resistance
to damaging environmental influences. Hormones of reproductive organs are in-
volved in the maintenance of sexual behavior and reproduction.
In clinical practice, glucocorticoids are used as anti-inflammatory and anti-
allergic drugs. Sex hormones and their analogues are used in cancer patients, hor-
mone replacement therapy, hormonal contraception.

THE AIM OF THE PRACTICAL CLASS IS:

To learn the structure and biological effects of hormones of the adrenal glands
and the gonads.
To determine the testosterone level in serum.

SELF-STUDY QUESTIONS
1. Classes of hormones according to their chemical structure, biological func-
tions and belonging to endocrine glands.
2. Chemical formula of adrenaline and noradrenaline. Characteristic features of
adrenaline according to the plan:
• chemical nature,
• place and the chemistry of synthesis reactions,
• regulation of synthesis and secretion of the hormone,
• the target organs,
• localization of the receptors in the cell and mechanisms of action,
• impact on the metabolism of carbohydrates, proteins, lipids – reactions and en-
zymes that are sensitive to the hormone action,
• the concept of pheochromocytoma, clinical manifestations, basic treatment.
3. Types of adrenergic receptors and their actions. Biochemical effects of the
hormone in stressful situations. What is the mechanism of therapeutic action of epi-
nephrine during cardiac arrest, asthma attacks?
4. The characteristics of the following hormones: corticoliberin, corticotropin
(ACTH), cortisol according to the plan:
• name,
• the chemical nature and structure,
• place of synthesis, transport in the blood,
• regulation of hormone synthesis and secretion,
• the target organs,
• localization of the receptors in the cell and mechanism of action,
• impact on the carbohydrates, proteins, lipids, mineral substances metabolism,
reactions and enzymes that are sensitive to the hormone action,
• hypo- or hyperfunction of the hormone, metabolic disorders, symptoms.
107
 5. Altered metabolism in adipose, muscle, lymphoid, epithelial tissue under hy-
po- and hypercortisolism. What does the expression "steroid diabetes" mean?
6. The main stages of the steroid hormone synthesis. The role of pregnenolone
and progesterone – the key compounds in the synthesis pathway. A specific hydrox-
ylase, determining the formation of mineralocorticoids and glucocorticoids. The role
of aromatase in the synthesis of estrogens.
7. Characteristic of mineralocorticoids (aldosterone) according to the plan:
• the chemical nature and structure,
• place of synthesis, transport in blood,
• regulation of hormone synthesis and secretion,
• the target organs,
• localization of the receptors in the cell and mechanism of action,
• influence on the exchange of mineral substances and water – reactions and en-
zymes that are sensitive to the hormone action,
• hypo- or hyperfunction of the hormone, metabolic disorders, symptoms.
8. The role of the renin-angiotensin system in the regulation of aldosterone syn-
thesis and secretion. The biochemical mechanism of renal hypertension development
9. Oxytocin, prolactin, follicle-stimulating and luteinizing hormones of the pitui-
tary gland, progesterone and estradiol, testosterone. Their characteristics according to
the plan:
• name,
• the chemical nature and chemical formula (for steroid hormones),
• place of synthesis,
• regulation of the hormone synthesis and secretion,
• target organs, transport in blood,
• localization of the receptors in the cell and mechanism of action,
• impact on the metabolism of carbohydrates, proteins, lipids, mineral substanc-
es; the biochemical processes that are sensitive to hormones,
• hypo- or hyperfunction of the hormone, metabolic disorders, symptoms.
10. Cyclical changes in the concentration of gonadotropins, progesterone and es-
trogen in a woman's body (menstrual cycles).
11. Immunoassay method for testosterone determination in serum. The principle
of the method. Normal values. Clinical-diagnostic value.
12. Make a table for adrenocortical hormones and sex hormones, according to
the given scheme:

108
 Hypo- and hyperfunction of
The name and the chemical

tors, mechanism of action

Localization of the recep-
Regulation of hormone
Effect on metabolism

Place of synthesis

Target Organs

Minerals and water

hormones
nature

Carbohydrates
action

Proteins

Lipids
TOPICS FOR REPORT
1. Glucocorticoids as medicines. The mechanism of anti-inflammatory and anti-
allergic action of glucocorticoids. Side effects.
2. The role of glucocorticoids in adaptive reactions under stress, in the develop-
ment of the adaptation syndrome. The works of H. Selje, F. Meerson, and others.
3. Use of synthetic analogues of estrogens and progestins for hormonal contra-
ception. Their mechanisms. Side effects.
4. Synthetic analogues of testosterone as medicaments. Use of anabolic steroids
and other hormones in sports medicine. Side effects.
5. Hormonal changes in the body of a woman during pregnancy. Influence of
hormonal diseases of the mother on fetal development.

Practical
IMMUNOASSAY METHOD FORTESTOSTERON DETERMINATION
IN SERUM
The principle
The method based on a solid-phase enzyme immunoassay is specific binding of
monoclonal antibodies to testosterone, adsorbed on the wells of immunological tab-
let, with the subsequent formation of the conjugate.
Material for investigation:
Serum.
Reagents
1) Conjugate of the monoclonal antibodies to testosterone with horseradish pe-
roxidase, a solution for dilution of serum, 2) phosphate-saline buffer solution with
tween, 3) a solution of tetramethylbenzidine, 4) stop reagent 5) the control sample
with a known testosterone content, 6) calibration samples containing known testos-
terone amounts.
Procedure
1. The introduction of the samples. Make in duplicate, starting from the top
wells of the first two strips with 100 µl of calibration samples. In the rest of the wells
add 100 µl of a control sample and 100 µl of the analyzed serum samples.
2. The introduction of monoclonal antibody conjugate. The conjugate is ready to
be used. 50 µl of conjugate are added into the wells.

109
 3. Incubation. Sealed film strips are incubated at the temperature of 37°C for 60
minutes in a thermostatic shaker with a frequency of 650 rpm.
4. Washing. At the end of incubation, the sticky bar is removed and placed in a
disinfectant solution container. With the help of the flushing device the tablet is
washed 5 times with washing solution, alternating aspiration and immediate filling up
the holes of each strip. In each well at least 350 µl of liquid are contributed in each
wash cycle. After rinsing the remaining moisture from the wells is thoroughly re-
moved by tapping the inverted tablet on the filter paper.
5. The introduction of tetramethylbenzidine (TMB). A TMB solution is ready
for use. Insert 100 µl of TMB to all wells.
6. Incubation. Sealed film strips are incubated in the dark at a temperature of
37°C for 15 min in a thermostatic shaker with a frequency of 650 rpm.
7. The introduction of stop reagent. 100 µl of stop reagent is added into all
wells. Shake the plate on a shaker for 10-15 seconds; the content of the wells turns
yellow.
8. Measuring. The optical density in the wells of the tablet is measured by the
spectrophotometer in dual-wave mode: when the main wavelength is 450 nm and the
comparison length is in the range of 620-655 nm.
Normal levels
Men above 14 years: 5.76-28.14 nmol/l;
Women over 10 years: 0.45-3.75 nmol/l.
Clinical-diagnostic value
Insufficient secretion of testosterone causes the development of hypogonadism,
wherein the clinical picture is directly related to the age of hormonal deficiency de-
velopment onset. Related disorders are Klinefelter or Turner syndromes and cryptor-
chidism or anorchia, and climacteric period and menopause in women. Hypergonad-
ism characterized by excessive testosterone secretion is diagnosed in women or men
with androgen-producing tumor of the testicles, ovaries or adrenal cortex. Elevated
testosterone levels in women are the confirmation of diseases such as hirsutism,
virilization and polycystic ovaries.
The testosterone level increase is one of the factors contributing to the prostate
cancer development in men older than 60 years, and is used as a treatment efficiency
control in this category of patients.
Design of practical:
The principle of method, the working procedure, the normal values and the study
results are indicated, the clinical and diagnostic value of the index is noted and con-
clusions on the possible pathology are drawn.
TESTS
Choose one or more correct answers.
1. THEEXCHANGE OF POTASSIUM, SODIUM AND CHLORINE IONS IS
REGULATED BY HORMONE ______________
1) insulin
2) aldosterone
110
 3) glucagon
4) adrenaline

2. THECONTENT OF CALCIUM AND PHOSPHORUS IS REGULATED

BY _______________
1) adrenaline
2) thyroxine
3) parathyroid hormone
4) calcitonin

3. GLUCOCORTICOID SYNTHESIS IN ADRENAL GLANDS IS STIMU-

LATED BY ___________
1) corticotropin
2) corticoliberin
3) cortisol
4) calcitonin

4. THEINSULIN FUNCTION IS ____________

1) lipolysis stimulation
2) activation of glycogenolysis
3) increased glycemia
4) increased lipogenesis

5. THEGLUCOCORTICOID FUNCTION IS______________

1) enhancement of gluconeogenesis
2) an increase in the proteins synthesis
3) stimulation of glucose uptake by tissues
4) activation of glycolysis

6. HYPOTHALAMUS HORMONES PROVIDE DIRECT ACTION ON

1) the thyroid gland
2) the pancreas
3) the pituitary gland
4) the adrenal glands

7. THEGLUCONEOGENESIS STRENGTHENS ACTION OF THE-

HORMONE____________
1) aldosterone
2) insulin
3) cortisol
4) testosterone

8. THE EFFECT OF CALCITONIN INVOLVES

1) a decrease in the level of Ca2+ ions in the blood
2) increase in the level of Ca2+ ions in the blood
3) increase in the level of K+ ions in the blood
4) decrease in the level of K+ ions in the blood
111
 9. THEINCREASE IN SOMATOTROPYN CONTENT LEADS TO
1) acromegaly
2) Itsenko-Cushing syndrome
3) Basedov's disease
4) diabetes mellitus

10. THEPARATHYROID HORMONE AFFECTS THE CALCIUM ION EX-

CHANGE DUE TO THE AFFECTON__________
1) kidney and bone tissue
2) pancreas and intestine
3) liver and muscles
4) adipose tissue and bone tissue

CASE STUDIES
1. A five-year old boy was examined by the doctor. A mental and physical retar-
dation, a slowdown in growth, a decrease in body temperature were revealed. The
child was non-active and lack in emotions. In the blood, the cholesterol content was
reduced.
Specify the gland whose function was altered. Explain the cause of symptoms.

2. The patient had dry mouth, thirst, copious and frequent urination, weakness,
sleep disturbance, weight loss.
Identify a disease characterized by these symptoms. Suggest a complex of bio-
chemical research to clarify the diagnosis and evaluate the state of metabolism.

3. The patient complained of severe weakness, increased fatigue. There was of-
ten hypoglycemia. Pigmentation of the skin was intensive. There was anemia, lym-
phocytosis, eosinophilia, reduced reabsorption of sodium from urine.
Indicate hormones, the insufficiency of which can be assumed.

112
 UNIT 11
BIOCHEMISTRY OF BLOOD

THEME 11.1. NITROGEN-CONTAINING SUBSTANCES OF THE BLOOD:

PROTEINS, ENZYMES, FRACTIONS OF RESIDUAL NITROGEN

INTRODUCTION
There is a close relationship between blood and all tissues of the body. The study
of various nitrogen-containing blood components and their role in metabolism allows
diagnosing metabolic disorders in the body, monitoring the development of the
pathological process and to evaluating the therapy efficacy. The ratio of nitrogen-
containing compounds in the blood varies depending on the lifestyle and age of the
person.

THE AIM OF THE PRACTICAL CLASS IS:

To study the composition of blood, nitrogen-containing substances of blood, def-
inition of a total blood protein and the main protein fractions.
To obtain practical skills for the thymol test of colloid-resistance of serum pro-
teins, as well as the determination of protein fractions of blood serum by the electro-
phoresis.

SELF-STUDY QUESTIONS
1. Organic and inorganic components of blood. Formed elements, plasma, se-
rum.
2. Sources of glucose, triacylglycerols and cholesterol in the blood. Clinical-
diagnostic value of their determination in blood.
3. Nitrogen-containing substances of blood.
4. Definition of a total blood protein. Physiological functions of blood proteins,
normal concentrations of total blood protein. Causes of hypo- and hyperproteinemia.
5. The main protein fractions of blood serum. Normal values of their concentra-
tion in the blood. Give 1-3 examples of proteins for each fraction. Dysproteinemia
and paraproteinemia. Age dynamics of protein fractions. Embryospecific proteins and
their diagnostic value.
6. Definition of the proteinogram. Change in the ratio of protein fractions in
acute and chronic inflammation; pathology of kidneys, tumor, liver diseases.
7. The key enzymes of plasma and serum. What is enzyme diagnostics? True
plasma enzymes. Two groups of organ specific enzymes: enzymes of cellular metab-
olism and excretory (secreted) enzymes.
8. Residual nitrogen of blood. List all components and their quantitative compo-
sition. Identify reasons and types of azotemia.

113
 9. Reactions of creatine and creatinine synthesis. Normal concentrations in the
blood. Clinical-diagnostic value of determination of creatinine concentration in blood
and urine.
10. Reactions of urea synthesis. Normal value of its concentration in the blood.
Clinical-diagnostic value of urea concentration in blood and urine.
11. Reaction of uric acid synthesis. Normal concentrations in the blood. Clinical-
diagnostic value of uric acid concentration in the blood and urine.
12. Hyperammonemia, causes and consequences. Normal and maximum permis-
sible concentrations of ammonia in the blood. The reasons for toxicity of ammonia.
13. Thymol test for colloid-resistance of serum proteins. Principle of the method.
Normal values and clinical-diagnostic value.
14. Protein fractions of blood serum. Principle of the electrophoresis. Normal
values and clinical-diagnostic value.
15. With a help of additional material 2 make a table of individual globulins in
serum, noting their functions:
α1-globulins α2-globulins β-globulins γ-globulins

TOPICS FOR REPORT

1. Age dynamics of protein fractions. Embryospecific proteins and their diagnos-
tic significance.
2. Residual nitrogen: its main components. Dynamics of the level of residual ni-
trogen fractions in the postnatal period.

Practical 1
THYMOL TEST FOR COLLOID-RESISTANCE OF SERUM PROTEINS

The stability of proteins depends on their charge and the presence of hydration
shell. The violation of colloidal stability of proteins under the influence of various
agents is manifested firstly by bonding (coagulation) protein molecules, and then by
precipitation. Thus, at first, large and less charged proteins are deposited – globulins.
Thymol test
As all coagulation tests, thymol test is a nonspecific reaction. However, it is
more acceptable for functional studies of the liver, than other colloidal samples.
The principle
In serum β-, γ-globulins and lipoproteins are precipitated by thymol reagent at
pH 7.55 due to the formation of globulin-thymol-lipid complex.
Reagents
Thymol buffer, pH 7.55-7.60.
Material for investigation
Serum.

114
 Procedure
Test tube, ml
Serum 0.05
Thymol buffer 3.0
Mix and incubate for 15 minutes at room temperature. Mix
again and compare with calibration samples.
Result is expressed in units of turbidity S-H (authors:
Shank-Haagland).
Calibration scale
Solutions with different intensity of turbidity are used as calibration samples.
Samples must be thoroughly mixed before use.

N of sample Units of turbidity, S-H U

1 5
2 10
3 15
4 20

Normal values
Serum 0-4 S-H U
Clinical-diagnostic significance
Test is used for the differential diagnosis of liver diseases. In such cases as dam-
age of liver parenchyma (infectious and toxic hepatitis), even at early stage, thymol
test is above normal values in 90 100% of cases. Thymol test has normal value in
dysfunction of other organs, in other liver diseases.
Design of practical
Write down the principle of the method, the experimental procedure, the normal
value and the results of the study, note the clinical and diagnostic value of the index
and draw conclusions on the possible pathology.

Practical 2 (in theory)

ELECTROPHORESIS OF PROTEINS ON PAPER AND ACETATE
CELLULOSE FILMS
The principle
Protein molecules, negatively charged at pH 6.8, are moved towards the anode
in the electric field of direct current. The fastest protein is albumin, then α1-, α2-, β-
and γ-globulins.
The course of electrophoresis is influenced by the following factors:
• charge (usually depends on pH), size and shape of molecules;
• electrical field – speed of ion movement is directly proportional to the amper-
age and voltage; inversely proportional to the resistance (depending on type and size
of supporting medium and ionic strength of the buffer),

115
 • buffer – composition, concentration, pH and ionic strength (depending on the
concentration of ions and their charge),
• supporting medium – its hydrophilicity, adsorption of substances on supporting
medium molecules.
Material for investigation
Serum.
Equipment
Electrophoresis apparatus, densitometer.
Procedure (basic steps)
Serum samples are applied equally at the start line on supporting medium (pa-
per, acetate cellulose film). Medium is placed in the apparatus for electrophoresis,
and then the electric current serves. Buffer solution, moving in the electric field, cap-
tures protein molecules. Molecules with the most negative charge and smaller size,
i.e. albumins, move faster than others. The largest and most neutral molecules (γ-
globulins) are the last. After some time (set for each device individual-
ly) electrophoresis ends.
Strips of paper or acetate cellulose film are washed from the buffer solution and
stained. As a result, areas, containing the protein, stain; area and intensity of the color
depend on content of protein fractions. Currently, the quantification of colored zones
on the electrophoretogram is performed using densitometer. Operation of the densi-
tometer is based on the transmittance of light through a moving strip of medium. If
we have change of the color intensity, there is a "splash" and the presence of a col-
ored zone (protein fraction) is recorded. In parallel, modern devices calculate auto-
matically the percentage of protein fractions.

116
 Clinical-diagnostic value
Albumin:
Decrease in the content of albumin fraction occurs in cases, characterized by:
• reduced synthesis of albumin – congenital analbuminemia, protein starvation,
malabsorption, severe liver damage (cirrhosis, degeneration, necrosis, active hepati-
tis, amyloidosis),
• increased catabolism of albumin – fever, cachexia, severe infections, pancreati-
tis, collagen diseases, thyrotoxicosis, Itsenko-Kushing disease (hypoadrenalism),
• loss of albumin through the burn surface, kidneys, gastrointestinal tract,
• inflammation due to the release of albumin from the bloodstream into the inter-
cellular space.
The albumin level <20 g/l is accompanied by edema.
α-globulins:
Increase in the content of α1- and α2-globulin fractions is associated with acute
and subacute inflammatory processes, and some malignant tumors, injuries, as these
fractions include the majority of acute phase proteins (C reactive protein, α2-
macroglobulin, α1 glycoprotein α1 antitrypsin, ceruloplasmin, haptoglobin).
β-globulins:
The large share of β-globulin fraction is β-lipoproteins (VLDL and LDL), there-
fore, increase in this fraction is the most often associated with hyperlipoproteinemia.
In addition, transferrin, hemopexin and components of the complement system have
the influence on dynamics of β-globulin fraction.
γ-globulins:
The content of γ-globulin is increased in pathological conditions associated with
chronic inflammatory processes (immunoglobulins G, A and M).

THEME 11.2. IRON EXCHANGE. HEMOPROTEINS. THE SYNTHESIS

AND BREAKDOWN OF HEME

INTRODUCTION
A wide variety of biologically important functions of hemoglobin and other
hemoproteins (e.g., cytochromes) requires studying the structure and role of these
proteins in metabolism. Conditions associated with the heme synthesis and its degra-
dation violations lead to the development of blood and liver diseases.

THE AIM OF THE PRACTICAL CLASS IS:

To study heme synthesis and degradation reactions, bilirubin metabolism.
To determine the bilirubin concentration in the blood serum, bile pigments in the
urine.

SELF-STUDY QUESTIONS
1. Iron exchange in the organism: the need, absorption, transport, iron-binding
proteins, storage form. Food sources. The iron deficiency symptoms and clinical
manifestations. The hemochromatosis concept.

117
 2. The function of these erythrocyte proteins – spectrin, glycophorin, band 3 pro-
tein. Glucose oxidation features in the erythrocyte. How are the oxygen active forms
neutralized?
3. The structure of heme, reactions and main stages of its synthesis. Regulation
of heme and hemoglobin synthesis.
4. Causes of porphyry, clinical manifestations, the basics of porphyry treatment.
5. Thalassemia, their types and causes.
6. The structure of the most abundant hemo containing proteins in the body (he-
moglobin, myoglobin, cytochromes, catalase, peroxidase). Their functions and locali-
zations.
7. Pathological and physiological types of hemoglobin (met hemoglobin, sickle
cell, glycosylated hemoglobin, carboxyhemoglobin, oxygenated hemoglobin, carbo-
hemoglobin). The importance of determining the concentration of glycosylated he-
moglobin, oxyhemoglobin and carbohemoglobin.
8. The scheme of reactions occurring in the erythrocytes in the lungs and tissue
capillaries.
9. Mechanisms of carbon dioxide transport. In what form is carbon dioxide trans-
ferred? How is it connected with hemoglobin? The role of carbonic anhydrase. The
erythrocyte role in the plasma bicarbonate ions concentration changes.
10. The binding of hemoglobin with oxygen. The oxygen normal saturation of
hemoglobin. The oxygen transport mechanism. The effect of temperature, pH, CO2
concentration on the hemoglobin affinity to oxygen, regulation of the process. Coop-
eration of protomers, the Bohr effect, role of 2.3-diphosphoglycerate.
11. Curves of saturation of hemoglobin with oxygen, or the dissociation of he-
moglobin. What does the S-shaped nature of the hemoglobin dissociation curve ex-
plain?
12. The decomposition reactions of hemoglobin and heme in the reticuloendo-
thelial system.
13. Indirect (free) bilirubin, its structure, reaction formation. The further fate of
the indirect bilirubin.
14. Direct (linked) bilirubin, its structure, reaction formation, its fate. The role of
the enzyme UDP-glucuronyltransferase. How is the final breakdown of heme prod-
ucts excreted?
15. Conditions associated with the hemoglobin excessive breakdown. Causes of
hemolytic jaundice and its laboratory criteria.
16. Conditions associated with impaired bile flow. Causes of obstructive jaun-
dice and its laboratory criteria.
17. Conditions associated with failure of hepatocytes function. Causes of paren-
chymatous jaundice and its laboratory criteria.
18. Physiological jaundices of the newborn.
19. Pathological jaundices of newborn:
• hemolytic jaundice, their causes. The physiological basis of the phenobarbital
using,

118
 • inherited disorders of bilirubin excretion – Gilbert's, Meulengracht, Dubin-
Johnson, Crigler-Nayar syndrome,
• the idea of acquired disorders of bilirubin excretion – the excess of estrogens in
milk, infectious and toxic causes,
• the idea of the mechanical jaundice due to cystic fibrosis, Niemann-pick dis-
ease, hypoplasia of the biliary tract.
20. Estimation of total bilirubin and its fractions in the blood serum. The method
principle, normal values, clinical-diagnostic value.
21. Detection of bilirubin and urobilinogen in the urine. The principle of the
method, normal values, clinical-diagnostic value.

TOPICS FOR REPORT

1. Iron deficiency states, their causes. Diagnostics. Effects. Treatment.
2. The congenital and acquired met hemoglobinemia. Causes. Insufficiency of
NADH-met hemoglobin reductase. Clinical signs.
3. Porphyria. Classification, causes, pathogenesis, clinical manifestations, the ba-
sis of treatment.

Practical 1
DETERMINATION OF THE TOTAL BILIRUBIN AND ITS FRACTIONS
CONCENTRATION IN BLOOD SERUM
The principle
The interaction of sulfanilic acid with nitrous sodium acid gives diasoftnisurance
acid, and then bilirubin together with it forms a colored azopigment (Ehrlich's diazo
reaction). Bound (direct) bilirubin reacts quickly, so its concentration is judged by the
initial color intensity. Unbound bilirubin reacts only after addition of an accelerator
(caffeine). The last releases bilirubin from complex with proteins and thereby accel-
erates the reaction of associate (azo coupling).
Material for investigation
Serum.
Reagents
1) Sulfanilic acid in HCl (reagent 1), 2) sodium nitrous acid NaNO2 (reagent
2), 3) caffeine reagent (reagent 3), 4) buffer solution (reagent 4), 5) 0.9% NaCl so-
lution, 6) the albumin solution, 20 g/l.
A standard solution of bilirubin 5 μmol/l.

119
 Procedure
Common Direct Standard, Control,
bilirubin, ml bilirubin, ml ml ml
Sulfanilic acid 0.2 0.2 0.2
––
(reagent 1)
NaNO2 (reagent 2) 1 drop 1 drop 1 drop ––
Caffein reagent (re-
1.0 –– 1.0 1.0
agent 3)
NaCl –– 1.0 –– 0.2
Blood serum 0.2 0.2 –– 0.2
Standard-bilirubin –– –– 0.2 ––
Mix and leave for 10 minutes
Buffer solution
1.0 1.0 1.0 1.0
(reagent 4)
Measure the optical density of standard sample for direct
bilirubin against control at a wavelength of 540 nm
(green filter).
After 10 minutes measure the optical density of the sam-
ple on total bilirubin against control at a wavelength of
540 nm (green filter).

Calculation
According to the formula the concentration of total and direct bilirubin is calcu-
lated, the concentration of indirect bilirubin is found as the difference between the
concentration of total and direct bilirubin.
Е ex
The concentration of bilirubin, µmol/l = Е st ×Сst, SST, where
Eex and Est – optical density of experiment and standard samples, Cst – concen-
tration of standard solution.
Normal values
Serum
Total bilirubin
Children 3.4-17.1 µmol/l
Adults 8.5-20.5 µmol/l

Direct bilirubin
Children lack
Adults 2.2-5.1 µmol/l
Clinical and diagnostic significance

120
 The table shows the shifts of the content of the main pigments in the blood se-
rum, the urine and feces of healthy people and in different types of jaundices (↑ in-
crease, ↓ the lower, N – normal values):
Types of jaundice
Types of jaundice
Hemolytic Parenchymal Mechanical
Blood bilirubin
Common ↑ ↑ ↑↑
Indirect ↑↑ ↑ N or ↑
Direct N or ↑ ↑ ↑↑
Bilirubin in urine N N or ↑ ↑
The urobilin of urine ↑↑ ↑ ↓
Feces stercobilin ↑↑ N or ↓ Missing
Blood serum:
The accumulation of bilirubin in the blood (more than 43 mmol/l) leads to its
binding by the skin and conjunctiva elastic fibers, which manifests as jaundice. For
the differential diagnosis of jaundice, it is necessary to define the fraction responsible
for bilirubinemia:
1. Hemolytic (suprarenal) jaundice is the accelerated formation of bilirubin as a
result of hemolysis Hyperbilirubinemia develops due to the indirect fraction of bili-
rubin. The content of urobilin in the urine increases dramatically, bilirubin is absent,
sterkobilin is found in feces. This type of jaundice can develop with B12 deficiency
anemia, hemolytic anemias of different origin (porphyria, medications, blood incom-
patibility, defect in glucose-6-phosphate dehydrogenase).
2. Parenchymal (liver cell) jaundice – the extraction of bilirubin in the liver
cells, its conjugation and excretion are broken. Hyperbilirubinemia develops due to
both fractions: the number of indirect bilirubin increases due to the functional insuffi-
ciency of hepatocytes or mitigating of them, and direct bilirubin – through cytolysis
of hepatocytes. There is bilirubin in the urine, moderately increased concentrations of
urobilin, the level of feces stercobilin is normal or reduced.
This type of jaundice is observed in viral and other forms of hepatitis, cirrhosis
and liver tumors, fatty degeneration, and other conditions.
3. Mechanical (obstructive) jaundice develops as a result of the bile outflow vio-
lations by the blockage of the bile duct. As a result of bile stagnation the bile capillar-
ies are stretched, their permeability is increased. Not having the outflow into the bile,
direct bilirubin enters the blood and it results in the development of hyperbiliru-
binemia. In severe cases, due to the direct bilirubin overflow of hepatocytes the con-
jugation with glucuronic acid is disrupted and the amount of unbound bilirubin in
blood increases. Levels of bilirubin in the urine are sharply increased; there is virtual-
ly no feces stercobilin.
In addition to cholelithiasis, obstructive jaundices are identified in tumors of the
pancreas and helminthiasis.

121
 Design of practical
Write down the principle of the method, progress and results of research, note
clinical-diagnostic value, and make the conclusion about the possible pathology.

Practical 2
DETECTION OF BILIRUBIN AND UROBILINOGEN IN THE URINE
USING "ICTOPHAN" DIAGNOSTIC STRIPS
The principle
Strips contain two areas of indication for bilirubin and urobilinogen. The test is
based on the reaction of bilirubin combination with stabilized diazoreagent. The reac-
tion zone contains p nitrophenyldiazonium-p-toluensulfonate, sodium bicarbonate
and sulfosalicylic acid. The lilac-beige (lilac-pink) colour appears after 30 seconds
upon contact with the conjugated (direct) bilirubin. The intensity of coloring depends
on the determined bilirubin concentration.
The urobilinogen determination is based on the reaction of urobilinogen azo-
combination with the diazonium stabilized salt. In the urobilinogen presence the reac-
tion zone changes color to pink or red.
Material for investigation
Normal urine and urine with bilirubin.
Normal values
Urine
Bilirubin The test is negative
Urobilinogen to 17.0 mmol/l
Clinical-diagnostic value
Urine
Bilirubinuria is a characteristic of obstructive and parenchymatous jaundice with
the increasing level of direct bilirubin in serum but hemolytic form is not character-
ized by it. In hepatitis, the bilirubin may be detected in the urine before the appear-
ance of jaundice.
The increasing concentration of urobilinogen in the urine is observed in paren-
chymal liver disease (hepatitis, cirrhosis, poisoning), hemolytic conditions, intestinal
diseases, associated with the excessive absorption of stercobilinogen by the intestine
mucous membrane (enterocolitis, constipation).
Design of practical
Write down the principle of the method, and the results in the table, note the
clinical-diagnostic value, and made the conclusion about the possible pathology.

The material of study Reaction The result

Normal urine
Urine with bilirubin

122
 THEME 11.3. INORGANIC SUBSTANCES OF THE BLOOD.
ACID-BASE STATUS

INTRODUCTION
Blood occupies a special place in metabolism due to a number of specific func-
tions belonging to its chemical components. Irreplaceable role of blood in gas ex-
change and regulation of the acid-base state of the body, violations of which are often
found in clinical practice.

THE AIM OF THE PRACTICAL CLASS IS:

To study the parameters of acid-base status, their normal values, chemical and
physiological mechanisms of acid-base status regulation, disorders of acid-base bal-
ance.
To obtain practical skills for the quantitative determination of the concentration
of inorganic phosphate and chloride ions in serum.

SELF-STUDY QUESTIONS
1. Electrolytes of blood plasma:
• macronutrients: sodium, potassium, calcium, phosphorus, iron, chlorine. What
are their distribution and importance in the body? Normal concentrations in blood
plasma. What does their concentration in blood plasma depend on?
• micronutrients: iodine, copper, zinc, cobalt and selenium. Examples of their
participation in metabolism.
2. Mechanism of carbon dioxide transport. In which form is carbon dioxide
transferred? The role of carbonic anhydrase. The role of erythrocytes in the change in
concentration of bicarbonate ions in plasma.
3. Mechanism of oxygen transport. How does oxygen bind to hemoglobin? He-
moglobin saturation curve or oxygen-hemoglobin dissociation curve.
4. Scheme of reactions occurring in erythrocytes in capillaries of lungs and capil-
laries of tissues.
5. Parameters of acid-base status and their normal values.
6. Chemical mechanisms of ABS regulation. How do blood buffer systems work
(phosphate, protein, bicarbonate, hemoglobin)? Chemical reactions.
7. Physiological systems of ABS-disorder compensation – role of lungs, kidneys,
liver and bone. How do they work?
8. Influence of gastric and pancreas secretion on acid-base status of the body.
Role of theliver.
9. The main types of ABS-disorders – respiratory acidosis and alkalosis, meta-
bolic acidosis and alkalosis, and their causes. The change of acid-base status indica-
tors in these disorders. Methods of compensation for violations.
10. Causes of ABS shifts in the following conditions, their chemical and physio-
logical compensation:

123
 • diabetes; • chronic renal insufficiency (de-
• pneumonia; creased kidney function);
• tissue hypoxia; • traumatic brain injury with stimula-
• alcohol poisoning; tion of the respiratory center;
• uncontrollable vomiting; • high lifting into the mountains;
• diarrhea; • right ventricular heart failure.
• attack of asthma;
• chronic bronchitis;
11. Principle of the quantitative measurement of inorganic phosphate in the se-
rum and urine. Clinical-diagnostic value and normal levels.
12. Determination of the concentration of chloride ions in the serum and urine.
Clinical-diagnostic value, normal levels
TOPICS FOR REPORT
1. Combined disorders of the acid-base state. Methods for determining acid-base
balance parameters in clinical and laboratory practice.
Practical 1
DETERMINATION OF THE CONCENTRATION OF INORGANIC
PHOSPHATE IN THE SERUM
The principle
In acidic environment phosphoric acid reacts with ammonium vanadate and am-
monium molybdate with the formation of phospho-vanado-molybdic acid, which has
a yellow color. The color intensity is directly proportional to the concentration of in-
organic phosphorus in the sample and is determined photometrically.
Material for investigation
Serum. Urine, diluted in 1 to 5.
Reagents
1) 10% trichloroacetic acid, 2) working solution, containing 1 mmol/l ammoni-
um molybdate and 1 mmol/l ammonium vanadate.
A standard solution of KH2PO4, 2.5 mmol/l
Procedure
Test 1, ml Test 2, ml Standard, ml
Serum 0.2 –– —
Urine, diluted in 1 to 5 –– 0.2 ––
Standard solution –– –– 0.2
distilled water 0.6 0.6 0.6
Trichloroacetic acid 0.8 0.8 0.8
Filtration in 5-8 minutes or centrifuge 10 minutes in
1500 rpm/minute.
Supernatant 0.8 0.8 0.8
Working solution 1.0 1.0 1.0
Mix well and measure the optical density of test tubes
and standard in 670 nm (red filter) against the water.
124
 Calculation
Е ex
Phosphate concentration in serum, mmol/l = Е st ×Сst ×5 × D,
Where Eex – optical density of experiment samples, Est – optical density of
standard samples, Cst – concentration of standard solution; 5 – urine dilution, D –
value of diuresis (1300-1500 ml per day).
Normal values
Serum 0.81-1.48 mmol/l
Urine 25.8-48.4 mmol/per day
Clinical-diagnostic significance
The concentration of phosphate in serum and urine primarily depends on the
function of parathyroid and thyroid glands, kidney function, regulatory influence of
calcitriol.
Serum
Hyperphosphatemia is observed in renal failure, hyperthyroidism, hypoparathy-
roidism, acromegaly, healing bone fractures, bone metastases, overdose of vitamin D,
and acute respiratory acidosis in multiple myeloma.
Decrease in the concentration of phosphates is observed during infusion of glu-
cose and hyperinsulinism (as insulin promotes transport of phosphorus in cells); dia-
betic ketoacidosis (as glucose increases the excretion of phosphates with urine);
hypokalemia, hyperparathyroidism in rickets and osteomalacia, acute alcoholism,
malabsorption syndrome.
Urine
The release of phosphates increases with the acceleration of catabolic processes
in the body – hyperthyroidism, meningitis, diabetic ketoacidosis, leukemia, impaired
renal function.
The decrease in urinary concentration is observed with tuberculosis, hypothy-
roidism of the parathyroid glands.
Design of practical
Write down the principle of the method, the experimental procedure, the normal
value and the results of the study, note the clinical and diagnostic value of the index
and draw conclusions on the possible pathology.

Practical 2
COLORIMETRIC METHOD FOR THE DETERMINATION OF CHLO-
RIDE IONS IN THE SERUM
The principle
In the presence of chloride ions in the acidic environment, mercury thiocyanate
produces thiocyanate-ions forming the colored complex with Fe3+. The color intensity
is proportional to the concentration of chloride ions in the sample and is determined
colorimetrically.
The material for investigation
Serum. Urine diluted in 2 times.
125
 Reagents
1) Working reagent containing 2.0 mmol/l of mercury thiocyanate Hg(SCN)2,
30.0 mmol/l of iron nitrate Fe(NO3)3, 4.0 mmol/l of nitric acid HNO3.
A standard solution of NaCl, 100 mmol/l.
Procedure
Test 1, ml Test 2, ml Control, ml
Serum 0.01 —
Urine diluted in 1 to 2. — 0.01 ––
NaCl solution –– –– 0.01
Working reagent 2.0 2.0 2.0
Mix thoroughly. After 5 minutes measure the optical
density of experienced and standard samples against wa-
ter at a wavelength of 490 nm (blue light filter).
Calculation
Е ex
Concentration of chloride ions in serum, mmol/l = Е st ×Сst × 2× D,
Where Еex — optical density of experiment samples, Еst — optical density of
standard sample, Cst – concentration of standard solution, 2 – urine dilution, D –
value of diuresis (1300-1500 ml/per day).
Normal values
Serum 97-108 mmol/l
Urine 120-240 mmol/per day
Clinical-diagnostic significance
Serum
Increase in the concentration of chloride ions is observed during dehydration,
caused by insufficient fluid intake, kidney disease, decompensation of heart, hyper-
ventilation (respiratory alkalosis), hypofunction of the adrenal cortex.
Decrease is detected in the dehydration as a result of loss of fluid (vomiting, di-
arrhea, intense sweating), stenosis of the pylorus, renal diabetes, gastric hypersecre-
tion, insufficiency of the adrenal cortex, increase in the amount of extracellular fluid,
infectious diseases and other pathological conditions. Any significant hypochloremia
may lead to a compensatory increase in residual nitrogen fractions due to desire of
the organism to maintain constancy of osmotic pressure.
Urine
The concentration of chloride ions increases with insufficiency of the adrenal
cortex, nephritis, the use of diuretics.
Reduction of their concentration is noted with a large loss of chlorine through the gas-
trointestinal tract, starvation, Itsenko-Cushing syndrome, with severe sweating.
Design of practical
Describe the principle of the method, the experimental procedure and the results
of the study, note the practical value of an indicator and draw conclusions about pos-
sible pathology.

126
 TESTS
Choose one or more correct answers.

1. THEHYPOALBUMINEMIA DEVELOPS IN _________

1) dehydration of the body
2) a case of infectious diseases
3) poisoning with toxins
4) violations of the assimilation of protein

2. THEALBUMIN FUNCTION IS__________

1) transport of endogenous metabolites
2) participation in immune reactions
3) participation in blood clotting
4) regulation of protein metabolism

3. THE GREATEST QUANTITY OF IRON IN THE HUMAN BODY IS IN

THE STRUCTURE OF
1) hemoglobin
2) ferritin
3) hemosiderin
4) transferrin

4. THE GREATEST CONCENTRATION OF FERRITIN IS OBSERVED IN

1) the liver
2) erythrocytes
3) the stomach
4) the kidneys

5. THE MOST TOXIC EFFECT OF BILIRUBIN PROVIDES ON

1) hepatocytes
2) nerve cells
3) muscle cells
4) splenocytes

6. ________ IS A SIGN OF HEMOLYTIC JANDISE IN BLOOD

1) the increased concentration of direct bilirubin
2) an increase in the number of bile acids
3) the accumulation of indirect bilirubin
4) the increased hemoglobin concentration

7. THEARTERIAL BLOOD OF THE HUMAN HAS A PH LEVEL OF

_________
2) 7.37-7.45
3) 7.00-7.25
4) 6.85-7.15

127
 8. THE CAUSE OF METABOLIC ACYDOSIS IS_________
1) increase of blood viscosity
2) decrease in the concentration of carbon dioxide
3) ammonia enhancement
4) hyperventilation of the lungs

9. THEMETABOLIC ACYDOSIS DEVELOPMENT MAY BE DUE TO

____________
1) severe vomiting
2) use of hypokalemic diuretics
3) the accumulation of bicarbonate ions
4) intensive muscular work

10. METABOLIC ALKALOSIS DEVELOPMENT IS MAY BE DUE TO

__________
1) hemolytic anemia
2) intense vomiting
3) diabetes mellitus
4) hyperventilation of the lungs

CASE STUDIES
1. There were two biochemical reports of the protein level in the blood. 30 g/l
and 100 g/l, which were made in two patients – a child with extensive burns and men
with hypoacid gastritis, pancreatitis (inflammation of the pancreas).
Indicate the patients who own these tests. Justify the conclusion.

2. The patient had a significant increase in residual blood nitrogen.

Propose a complex of biochemical studies to clarify the cause of the increase in
residual nitrogen.

3. The patient had difficulties in breath, it became superficial, the blood pH was
7.31, pCO2 was 52 mmol/l, the [HCO3¯] was 37 mmol/l, the alkaline reserve was in-
creased.
Note the type of violation of the acid-base state in the patient. Assume mecha-
nisms for compensation violations.

128
 UNIT 12
BIOCHEMISTRY OF KIDNEYS

THEME 12.1. WATER-SALT EXCHANGE. NORMAL AND

PATHOLOGICAL COMPONENTS OF URINE

INTRODUCTION
Kidneys are involved in the regulation of water-salt balanсе, the maintenance of
acid-base status, osmotic pressure of body fluids, blood pressure, stimulation of
erythropoiesis.
The amount and composition of urine secreted in the kidneys can vary within
wide limits, reflecting the state of water-salt metabolism and other aspects of the
body metabolism. The examination of every patient, not only in hospital but also in
ambulatory conditions should be accompanied by mandatory urine analysis, as this
study can help in diagnosis, and often completely change the initial diagnostic as-
sumptions, to assess the effectiveness of the therapy.

THE AIM OF THE PRACTICALCLASS IS:

To study the urine formation mechanisms, common properties, and chemical
composition of urine in health and disease, the role of kidneys in the maintenance of
acid-base status.
To determine the physic-chemical properties and quantitative composition of
urine.

SELF-STUDY QUESTIONS
1. Metabolism of the kidneys. The metabolism differences in the cortex and me-
dulla layers. In what part of the kidney does aerobic and anaerobic oxidation of glu-
cose occur, what is gluconeogenesis? Metabolism features of proteins and lipids in
the kidneys. The kidneys role in the synthesis of biologically active substances (crea-
tine, erythropoietin, 1.25-dioxyholecalciferol).
2. The role of enzymes in the kidney function – glycine amidine transaminase,
Na /K+-ATPase, glutamate dehydrogenase, glutaminases, alkaline phosphatase, the
+

isoenzymes of lactate dehydrogenase.

3. Nephron structure scheme. Processes of urine formation: filtration, reabsorp-
tion and secretion. Action localizations and effect of mineral and water-salt metabo-
lism regulating hormones.
4. Characteristics of filtration, factors influencing its speed and magnitude. Fil-
tration rate evaluation in clinical practice. Clearance. Substances that are used to de-
fine clearance.
5. Reabsorption, the biochemical reactions occurring in the tubule lumen and in
the cells of the proximal and distal portions of the nephron. The transport maximum
for glucose. Counter current multiplying mechanism of the urine concentration.

129
 6. Sources of water in the body and ways of its elimination. The role of skin,
lungs, digestive tract and kidneys in the excretion of water. Recirculation of water be-
tween the blood and the gastrointestinal tract, blood and kidneys. Children and adults
clean water requirements. Water metabolism peculiarities in children.
7. Describe factors affecting the water amount in the body – blood osmolality,
blood volume, blood pressure, concentration of sodium and potassium.
8. Regulation of reabsorption of water. The role of antidiuretic hormone. The
factors stimulating its synthesis and release. Metabolic consequences of antidiuretic
hormone hypofunction, symptoms.
9. The regulation of sodium reabsorption. Activation and functioning of the ren-
in-angiotensin-aldosterone system. The diagram showing the role of renin-
angiotensin system in sodium reabsorption. The hypertension mechanism while blood
circulation disorders in the kidneys, the causes of such violations.
10. Regulation of calcium reabsorption. The role of 1.25-dioxyholecalciferol,
parathyroid hormone and calcitonin in calcium homeostasis.
11. The role of the kidneys in maintaining acid-base balance – reabsorption of
bicarbonate, acids, ammonia, the excretion of organic acids.
12. Urine general properties of a healthy man are: quantity, color, transparency,
odor, relative density, pH. How are these indicators changed in pathological condi-
tions?
13. Urine organic and inorganic components of a healthy person.
14. The reasons of urine pathological components – protein, glucose, bile pig-
ments, ketone bodies, blood, enzymes.
15. The principle of the methods for the determination of urine physic-chemical
properties (density, pH). Clinical-diagnostic value of these indicators. The normal
values.
16. The laboratory determination of urine components – protein, glucose, ketone
bodies, bilirubin, urobilinogen, hemoglobin, red blood cells. Clinical-diagnostic rele-
vance, the normal values.
17. Assessment of glomerular filtration rate in clinical laboratory diagnosis.
What is the definition for creatinine clearance?

TOPICS FOR REPORT

1. Physiological and pathological proteinuria and creatinuria.
2. Mechanism of action of diuretics. Use of diuretics in the clinical practice.

Practical 1
DETERMINATION OF URINE PHYSICO-CHEMICAL PROPERTIES
Material for investigation
Normal urine and urine samples N 1, 2, 3.
Determination of relative density
Equipment
Urometer, a high cylinder for urine.
Procedure
If there is some foam on the urine surface it is removed with filter paper.
130
 If there is a little urine, then prior to testing it can be diluted in 2-3 times with
distilled water, and the result is multiplied by the degree of dilution.
The urine is poured along the wall into a high, narrow cylinder and the urometer
is carefully immersed in it not touching the walls and bottom of the cylinder. A read-
ing on the scale of urometer is taken using the lower meniscus of the liquid.
In case of a large relative density of urine the second type of urometer (scale
1.030-1.060) is used. If the urine has a temperature that does not meet the conditions
noted on the urometer, then for every 3OC above or below the needed temperature,
respectively, 0.001 of the indications on the urometer scale are added or taken away.
Normal value
Urine 1.010-1.025
Clinical-diagnostic significance
The relative density of normal urine is directly dependent on the concentration of
soluble substances and is in the inverse relationship with the urine amount.
The increase in the urine relative density is observed in diabetes mellitus (glyco-
suria), injury of the glomerular filter (proteinuria).
The decrease in density is associated with polyuria of any etiology.
Determination of pH
The principle
The principle is based on the indicator color change in accordance with the pH of
the solution.
Procedure
A strip of indicator paper is dipped into a test tube with urine and the color
change is compared with the reference chart on the package, the pH value of the test
urine is set.
Normal value
Urine 5.0-6.5
The influencing factors
With predominantly protein food the reaction of urine is acidic, while plant-
based diet – alkaline. The acid reaction of urine is due primarily to the ions Н2РО4–
and NH4+, and alkaline – to ions НСО3–.
Clinical-diagnostic value
The prevalence of food animal protein determines the urine pH shift to the acid
side, the predominance of plant food – to alkaline.
Strongly acidic reaction of urine is observed during fever, diabetes, starvation,
etc. Alkaline reaction of urine is noted with cystitis and pielitis, severe vomiting, di-
arrhea (diarrhea), administration of sodium bicarbonate and the use of alkaline min-
eral waters.
The urine acidity determines the possibility of urinary stone formation. Uric acid
stones (the urate) tend to occur at pH below 5.5, the oxalate – at a pH of 5.5-6.0, the
calcium-phosphate – at pH 7.0 and 7.8.
Design of practical:
Write down the principle of methods and the study results. The results of this
work are used for conclusions in practical 2.
131
 The density
The density The color Transparency pH
The norm
Sample N 1
Sample N 2
Sample N3

Practical 2
QUANTITATIVE DETERMINATION OF THE URINE COMPOSITION
Material for investigation
Normal urine and urine samples N 1, 2, 3.
Equipment
Test strips "Glucophan", "Ketophan", "Albuphan", "Ictophan", and «He-
mophan".
Procedure
Without touching the display area by hands, take a strip from the packing box
and immerse it for 1-2 seconds in the test liquid. The urine drops from the strip are
removed by the edge of the vessel. The strips are left in a horizontal position. After 1
minute the target substance concentration is determined by the color scale on the
package.
Determination of glucose by "Glycophan" test strips
The principle
The principle of glucose determination is based on enzymatic reaction glucose
oxidase. The display area is impregnated with solutions of the enzymes glucose oxi-
dase, peroxidase and a dye tetramethylbenzidine. Using glucose oxidase glucose is
oxidized by air oxygen to gluconic acid with formation of hydrogen peroxide. In the
presence of enzyme peroxidase hydrogen peroxide oxidizes a dye, and there is the
transition of yellow coloring to green.
Normal value
Urine
Glucose test strips "Glycophan" test is negative
other methods 0.1-0.8 mmol/l
Clinical-diagnostic significance
The glucose level in the urine increases over 10 mmol/l (renal threshold) in all
cases of hyperglycemia.
The glycosuria can be physiologic and pathologic.
Physiological glycosuria includes alimentary glycosuria, glycosuria of pregnant
and neurogenic glycosuria on the basis of stressful conditions.
Pathological glycosuria is detected:
• by hyperglycemia – diabetes mellitus, thyrotoxicosis, acromegaly, adrenal hy-
perplasia, myocardial infarction, poisoning with morphine, phosphorus, hemorrhage
in internal organs, acute infection and neurological disease,

132
 • when renal tubules are damaged – pielo- and glomerulonephritis, toxic injury
of the kidney, renal diabetes (familial renal glycosuria), kidney disease.
Definition of ketone bodies by test strips "Ketophan"
The principle
The test is based on display with test strips that contain an alkaline buffer in the
mixture with nitroprusside of sodium, giving acetone and acetoacetic acid red, cherry
or violet coloration. The sample is more sensitive to acetoacetic acid than to acetone.
With hydroxybyturic acid, the indicator does not respond. Thus, the color intensity
reflects only the concentration of acetoacetic acid in the urine.
Normal values
Urine
Ketone bodies test strips "Ketophan" test is negative
other methods 20-30 mg/per day

Clinical-diagnostic significance
Ketone bodies in urine (ketonuria) appear by ketonemia that occurs in starvation,
diabetes, with increasing concentrations of lipid-mobilizing hormones in the blood, in
acetonemia conditions in children.
Determination of protein by test strips "Albuphan"
The principle
The test is based on the color change from yellow to blue-green acid-base indi-
cators (tetrabromphenol blue and ether tetrabromophenolphthalein) under the influ-
ence of proteins. The test is most sensitive to albumin, is much less sensitive to glob-
ulins, mucoproteins, hemoglobin. At strongly alkaline pH of the urine sample it may
give false positive results.
Normal values
Urine
Protein test strips "Albuphan" test is negative
other methods 10-140 mg/l

Clinical-diagnostic significance
A small amount of protein in daily urine is detected in healthy individuals, but in
a single urine sample it is impossible to detect such concentration by conventional
methods. Part of these proteins has serum origin, the other part is the product of the
urinary tract cells.
Proteinuria is usually subdivided, depending on the place of occurrence:
• prerenal, associated with increased tissue protein breakdown or severe hemolysis,
• renal, caused by disorders of the kidneys glomeruli or tubules,
• postrenal associated with inflammation of the urinary tract.
Determination of bilirubin and urobilinogen by test strips "Ictophan"
The principle
Strips contain two areas of indication for bilirubin and urobilinogen.
The test is based on reaction of bilirubin combinations with stabilized diazorea-
133
gent (see Unit 11.2."Determination of the total bilirubin and its fractions concentra-
tion in blood serum"). The reaction zone contains n- nitrophenyldiazonium-St-n-
toluensulfonate, sodium bicarbonate and sulfosalicylic acid. Upon 30 seconds contact
with the conjugated (direct) bilirubin the purplish beige (purplish pink) color appears,
the intensity of which depends on the defined bilirubin number.
The determination of urobilinogen is based on the reaction associated with the
diazonium stabilized salt. The reaction zone changes color in the urobilinogen pres-
ence to pink or red.
Normal values
Urine
The urobilinogen Test is negative
The bilirubin up to 17.0 μmol/l
Clinical-diagnostic significance
The appearance of bilirubin in urine is associated with parenchymal and mechan-
ical jaundice, in which direct bilirubin is filtered into the urine from blood.
Increasing the concentration of urobilinogen in the urine is observed in paren-
chymal liver disease (hepatitis, cirrhosis, poisoning), hemolytic conditions, intestinal
diseases, associated with excessive absorption of stercobilinogen the mucous mem-
brane of the intestine (enterocolitis, constipation).
Determination of hemoglobin and erythrocytes with"Hemofan"test strips
Blood in the urine can be in two types – red blood cells (hematuria, erythrocy-
turia) or blood pigment (hemoglobinuria).
The principle
The indication zone of the test strips contains an organic hydroperoxide and the
indicator is tetramethylbenzidine. Hemoglobin catalyzes the oxidation of the indica-
tor by the hydroperoxide with the formation of dyed blue-green products.
In the presence of free hemoglobin (hemoglobinuria or hemolysis of red blood
cells were present initially), the entire touch-sensitive area is colored in more or less
homogeneous blue-green color.
Unmodified (whole) red blood cells (microscopic hematuria) appear intensely
colored blue-green dots or specks on the undyed reagent zone or a uniform blue-
green color of the whole area (gross hematuria).
Negative reactions display area remains yellowish (no green tint).
Normal values
Urine
Red blood cells and children test is negative or slightly positive
hemoglobin adults test is negative
Clinical-diagnostic significance
Isolated red blood cells found in the urine of even absolutely healthy people. Up
to 1 million red blood cells are allocated every day in healthy people, which corre-
sponds to the content in 1.0 μl of urine 1 red blood cell. Hematuria is detected in le-
sions of the parenchyma of the kidneys (glomerulonephritis, pyelonephritis, and tu-
mors), intensive physical exertion, and urinary tract infections.
134
 Normal values
Urine
Protein test strips "Albuphan" test is negative
other methods 10-140 mg/l
Ketone bodies test strips "Ketophan" test is negative
other methods 20-30 mg/per day
Glucose test strips "Glycophan" test is negative
other methods 0.1-0.8 mmol/l or 1-15 mg (mg%)
Red blood cells and test is negative or slightly positive
children
hemoglobin
adults test is negative
рН 5.0-6.5
Design of practical:
Write down the principle of the methods, the results based on the study of urine
samples and on the results of "Laboratory 1", draw conclusions about possible pa-
thologies of the body.

TESTS
Choose one or more correct answers.
1. THENECESSARY QUANTITY OF CLEAN WATER IN THE DAILY
RATION OF THE CHILD OF THE FIRST YEAR OF LIFE IS
1) 15 ml per kg of body weight
2) 30 ml per kg of body weight
3) 100 ml per kg of body weight
4) 500 ml per kg of body weight

2. THEMINIMUM EXCRETION OF WATER IN ADULT TO THE DAY IS

COMPOSED BY
1) 100 ml
2) 200 ml
3) 700 ml
4) 1400 ml

3. THEWATER-SALT BALANCE IS REGULATED BY ______________

1) antidiuretic hormone
2) the renin-aldosterone system
3) atriopeptin system
4) hypothalamic-pituitary-adrenal system

4. A REGULATOR OF THE SMOOTH MUSCLE VESSELS TONUS IS

SYNTHESIZED IN THE KIDNEYS
1) bradykinin
2) norepinephrine
135
 3) oxytocin
4) vasopressin
5. THE REGULATED REBASORPTION OF CALCIUM IONS IS OC-
CURRED IN
1) proximal tubules
2) distal tubules
3) the ascending part of the loop of Henle
4) collecting tubes

6. THE REGULATED REBASORPTION OF WATER IS OCCURRED IN

1) proximal tubules
2) throughout the nephron
3) the ascending part of the loop of Henle
4) distal tubules and collecting tubes

7. THE INCREASEIN PH BLOOD LEVEL BY KIDNEYS IS CARRIED

OUT BY ACTIVATION OF
1) ammoniogenesis
2) reabsorption of sodium
3) reabsorption of water
4) secretion of uric acid

8. THE SATURATED YELLOW COLOR OF URINE IS A SIGN OF

1) pregnancy
2) orotate aciduria
3) dehydration
4) intoxication

9. IT IS EXCRETED WITH THE BILE OF THE LIVER

1) hemoglobin
2) oxycalciferol
3) triacylglycerols
4) cholesterol

CASE STUDIES
1. The patient was bedridden for a long time. He had a cardio-vascular disease.
The analysis of urine showed an increase in the content of Са2+ salts.
Find out the reason of salts Са2+ growth in the patient.

2. Several skiers made a big transition in cold weather conditions. The protein in
urine was found out in some of them.
Specify the cause of the appearance of protein in the urine of healthy athletes.

3. The woman had sudden pains in the liver, rapidly developed icteric staining
of sclera, skin, and feces acquired a light color, urine acquired the color of dark beer.
Indicate disorders of pigment metabolism and type of jaundice.

136
 CHECKLIST FOR FINAL-GOAL ASSESSMENT
(UNITS 10, 11, 12)

1. The hierarchy of regulatory systems. Place of hormones in the regulation of

metabolism and function of organs.
2. The difference in membrane and cytosolic mechanisms of transmission of the
hormonal signal into the cell.
3. Membrane mechanisms of transmission of the hormonal signal into the cell.
Three types of receptors: with enzymatic activity, ion-conductive activity and associ-
ated with G-proteins.
4. Describe the receptors associated with G-proteins:
• the system of secondary mediators and their interaction,
• adenylate cyclase the mechanism of action of hormones,
• calcium-phospholipid mechanism of action of hormones.
5. General characteristics guanylate cyclase mechanism of action of hormones.
6. Cytosolic mechanism of action of hormones.
7. Classification of hormones by chemical structure, biological functions and be-
longing to endocrine glands. The role of releasing hormones and tropic hormones.
What is negative feedback in the regulation of synthesis and actions of hormones?
8. Characterization of growth hormone: chemical nature, site of synthesis, target
organs, localization of receptors and mechanism of action, influence on metabolism
and water exchange. How are synthesis and secretion of the hormone regulated?
Condition associated with impaired action of the hormone.
9. Characteristics of antidiuretic hormone (vasopressin): the chemical nature, site
of synthesis, target organs, localization of receptors and mechanism of action, influ-
ence on metabolism and water exchange. Regulation of synthesis and secretion of
hormone. Condition caused by impaired action of the hormone.
10. Characterization of oxytocin: the chemical nature, site of synthesis, target or-
gans, localization of receptors and mechanism of action, effects. How are synthesis
and secretion of the hormone regulated?
11. Characterization of parathyroid hormone and calcitonin: chemical nature, site
of synthesis, target organs, localization of receptors and mechanism of action, influ-
ence on exchange of calcium and phosphates. Regulation of synthesis and secretion
of hormone. What is their role in the metabolism of calcium and phosphate? What is
the role of vitamin D3?
12. The pancreatic hormones glucagon and insulin: their chemical nature, site of
synthesis, target organs, localization of receptors and mechanism of action, influence
on the metabolism of carbohydrates, proteins, lipids (enzymes regulated by the hor-
mone). Regulation of synthesis and secretion of hormone. Condition caused by ab-
sence or surplus of the action of the hormone.
13. Concepts about the mechanisms of development of insulin-independent dia-
betes. The most important changes in hormonal status and metabolism in diabetes,

137
the biochemical mechanisms of development of diabetes complications and diabetic
coma.
14. Characterization of thyroid-stimulating hormone: chemical nature, site of
synthesis, target organs, localization of receptors and mechanism of action, effects.
How are synthesis and secretion of the hormone regulated?
15. The thyroid hormones- thyroxine and triiodothyronine: chemical nature, site
of synthesis, target organs, localization of receptors and mechanism of action, influ-
ence on the metabolism of carbohydrates, proteins, lipids. Regulation of synthesis
and secretion of the hormone. Condition caused by absence or surplus of the action of
the hormone.
16. Adrenaline: chemical nature, site of synthesis, target organs, localization of
receptors and mechanism of action, influence on the metabolism of carbohydrates,
proteins, lipids (enzymes regulated by the hormone). Regulation of synthesis and se-
cretion of hormone. Condition caused by impaired action of the hormone. What is the
role of adrenaline in the adaptive reactions of the body under the stress conditions?
17. Characteristics of adrenocorticotropic hormone: chemical nature, site of syn-
thesis, target organs, localization of receptors and mechanism of action, influence on
metabolism. How are synthesis and secretion of the hormone regulated? Condition
caused by absence or surplus of the action of the hormone.
18. Glucocorticoids: chemical nature, the place and the stages of synthesis, target
organs, localization of receptors and mechanism of action, influence on the metabo-
lism of carbohydrates, proteins, lipids (enzymes regulated by the hormone). Regula-
tion of synthesis and secretion of hormone. Condition caused by absence or surplus
of the action of the hormone. Use of glucocorticoids as anti-inflammatory and anti-
allergic drugs. The role of glucocorticoids in the adaptive reactions of the organism
under the stress conditions.
19. Mineralocorticoids: their chemical nature, the place and the stages of synthe-
sis, target organs, localization of receptors and mechanism of action, influence on the
exchange of electrolytes and water. How are the synthesis and secretion of hormones
regulated? The role of the renin-angiotensin system. Condition caused by absence or
surplus of the action of the hormone. Biochemical mechanisms of development of re-
nal hypertension.
20. Lactotropic hormone: chemical nature, the place and the stages of synthesis,
target organs, localization of receptors and mechanism of action, effects on metabo-
lism. How are synthesis and secretion of the hormone regulated?
21. Characterization of gonadotropic hormones: follicle-stimulating and luteiniz-
ing hormones: chemical nature, the place and the stages of synthesis, target organs,
localization of receptors and mechanism of action, effect on female monthly cycle.
Regulation of synthesis and secretion of hormones.
22. Androgens and estrogens: chemical nature, the place and the stages of syn-
thesis, target organs, localization of receptors and mechanism of action, influence on
the metabolism of carbohydrates, proteins, lipids. How are synthesis and secretion of
hormones regulated? The use of analogues of androgens and estrogens as drugs.

138
 23. Qualitative reactions to detect insulin. The principle of method for the deter-
mination of prolactin and testosterone in serum. Clinical-diagnostic value.
24. What is the total blood protein, the components included in its composition.
Physiological functions of proteins of the blood, normal levels of concentration. The
reasons for the change in the concentration of total protein in the blood.
25. Protein fractions of blood serum. Characteristics of albumin, causes of Hypo-, hy-
per- and analbuminemia. Globulins and their main faction. The main representatives
of the globulin fractions. The biological role of albumins and globulins. Normal lev-
els of protein fractions. Dysproteinemia and paraproteinemia. The reasons for the
changes in the concentration of protein fractions in the blood. The change in the ratio
of protein fractions in diseases of the liver, kidney, acute and chronic inflammation,
tumors (proteinogram).
26. Non protein nitrogenous components of blood – fractions of residual nitro-
gen, their characteristics. The role and metabolism of urea, creatinine, uric acid. Clin-
ical diagnostic value of determination of these substances in the blood, their normal
performance. Causes and consequences of hyper ammoniemia and nitrogenemia.
27. Characteristics of enzymes the blood – plasma specific, indicator, excretory
enzymes. Examples. The use of blood enzymes for diagnosis of diseases.
28. The principle of separation of serum proteins by electrophoresis.
29. Characteristics of metabolism of the erythrocyte. The role of glycolysis and
pentose-phosphate pathway.
30. Exchange of iron. Food sources, consumption, transportation, deposition and
mobilization, the role of transferrin and ferritin. What are clinical and laboratory
signs of iron deficiency?
31. The structure of the hemoglobin molecule. The structure of heme. Normal
and pathological forms of hemoglobin. The mechanism of the regulation of hemoglo-
bin affinity for oxygen – cooperative effect, Bohr effect, role of 2.3-
diphosphoglyceric acid.
32. The reaction of synthesis of heme and hemoglobin. Regulation of the synthe-
sis processes. Characteristic of disorders of hemoglobin – porphyria, thalassemia,
pathological forms of hemoglobin.
33. The reaction of degradation of heme, formation of bilirubin and bilirubin
glucuronide, their localization. The main stages of transformation of bile pigments in
the body. Way of excretion of bilirubin and other bile pigments.
34. Metabolic disorders of bile pigments. Laboratory criteria for the various
types of jaundice (suprahepatic, hepatic, subhepatic).
35. Violations of pigment metabolism in children: 1) hemolytic jaundice;
2) physiologic jaundice of newborn and premature; 3) hereditary disorders of biliru-
bin metabolism – Gilbert's syndrome, Dubin-Johnson’s syndrome, Crigler-Naya’s
syndrome.
36. The principle of the method the detection of bilirubin and urobilinogen in the
urine. Clinical-diagnostic value.
37. Methods for the quantitative determination of total bilirubin and its fractions
in the blood serum clinical-diagnostic value.
139
 38. Respiratory function of blood. Mechanisms of transport of oxygen and car-
bon dioxide.
39. Indicators of acid-base status. The chemical and physiological mechanisms
of regulation of acid-base status. The relationship between transport of oxygen and
carbon dioxide with the mechanisms of maintaining acid-base balance.
40. The role of kidneys in regulation of acid-base balance: bicarbonate reabsorp-
tion, acidogenesis, ammoniagenesis.
41. Disorders of acid-base status, its causes, the change of indicators in acid-base
status. Methods of compensation for various violations of ABS.
42. The principle of quantitative measurement of inorganic phosphate, and chlo-
ride ions in serum and urine. Clinical-diagnostic value normal values.
43. Characterization of the biochemical processes in the nephron. Counter cur-
rent multiplier mechanism of formation of urine. Features of reabsorption of electro-
lytes and water in different parts of the nephron. The role of hormones in the process-
es of reabsorption.
44. Composition and physical-chemical properties of urine. Normal and patho-
logical components of urine, their clinical-diagnostic value normal values.
45. The principle techniques and process of determining the physical-chemical
properties of urine:
• determination of relative density of urine,
• determination of urine pH using indicator paper.
46. Principle of the method and the course of determination of pathological
components of urine:
• determination of the concentration of protein, glucose, ketone bodies, bile pig-
ments, hemoglobin with test strips.

140
 ANSWERS TO TEST TASKS

Unit 1. "Structure, features and functions of proteins"

Question number Number of an answer Question number Number of an answer
1 2 6 4
2 3 7 1
3 3 8 2, 3, 5
4 5 9 1
5 1, 2, 3, 4, 5 10 4

Unit 2. "Structure, classification and role of vitamins"

Question number Number of an answer Question number Number of an answer
1 3 6 2
2 3 7 3
3 2 8 3
4 1 9 1
5 1 10 3

Unit 3."Enzymology"
Question number Number of an answer Question number Number of an answer
1 4 6 1
2 1 7 5
3 3 8 2
4 1 9 1
5 3 10 2

Unit 4. "Biological oxidation"

Question number Number of an answer Question number Number of an answer
1 4 6 2
2 3 7 1
3 2 8 3
4 4 9 2
5 1 10 1

Unit 5. "Amino acid and protein metabolism"

Question number Number of an answer Question number Number of an answer
1 2 6 3
2 3 7 4
3 2 8 2
4 1 9 1
5 4 10 1

141
 Unit 6. "Structure and metabolism of purine and pyrimidine nucleotides"
Question number Number of an answer Question number Number of an answer
1 3 6 1
2 2 7 4
3 2 8 2
4 1 9 4
5 1 10 2
Unit 7. "Matrix biosynthesis"
Question number Number of an answer Question number Number of an answer
1 2 6 1
2 1 7 1
3 3 8 2
4 2 9 1
5 1, 3, 4 10 1

Unit 8. "Structure and metabolism of carbohydrates"

Question number Number of an answer Question number Number of an answer
1 3 6 3
2 3 7 3
3 2 8 3
4 4 9 1
5 3 10 2

Unit 9. "Structure and metabolism of lipids"

Question number Number of an answer Question number Number of an answer
1 1 6 1
2 3 7 4
3 3 8 2
4 2 9 1
5 1 10 4

Unit 10. "Hormonal regulation of human metabolism and functions"

Question number Number of an answer Question number Number of an answer
1 2 6 3
2 3, 4 7 3
3 1 8 1
4 4 9 1
5 1 10 1

Unit 11. "Biochemistry of blood"

Question number Number of an answer Question number Number of an answer
1 4 6 3
2 1 7 2
3 1 8 1
4 1 9 4
5 2 10 2
142
 Unit 12. "Biochemistry of kidneys"

Question number Number of an answer Question number Number of an answer

1 3 6 4
2 4 7 1
3 1, 2, 3 8 3
4 1 9 4
5 2

ANSWERS TO CASE STUDIES

Unit 1. "Structure, features and functions of proteins"

1. It will move to cathode at pH 30 and will move to anode at pH10.5; the
charge of this peptide is negative at water solution. It will be positive at pH 3.0 and
move to the cathode at electric field. The charge of this peptide will increase at pH
10.5 and it will move to the anode.

2. Protein: A – will move to the anode; B – it will move to the cathode at pH 5.0
and will move to the anode at pH 7.0; it will stay at the pH 9.5; it will move to the
anode at pH 11.0;

3. The first peptide is similar to the hydrocarbon-containing compound, it has

hydrocarbon radicals. This protein is better soluble in nonaqueous medium, because
the radicals are hydrophobic. The first peptide reacts with Biuret and ninhydrine rea-
gents. The second one cloud participates in salt bridges formation due the presence of
free NH2-group of lysine and СООН-group of glutamic acid.

Unit 2. " Structure, classification and role of vitamins"

1. Pyridoxine participates in amino acids transamination reactions. Initially the
formation of essential amino acids from these four happens. Then it is not needed.

2. Competitive inhibition of microbe’s enzymes occurs, which are required for

folic acid synthesis. The disorders of tetrahydrofolate by bacteria can be seen as a re-
sult of the process. It is known that the treatment after bacterial infection consists of
vitamin B medicines and probiotics for microflora restoration

3. It is prescribed to take vitamin A participating in tissues regeneration, vitamin K for

reducing the possibility of bleeding and vitamin D for providing the body with calcium.

Unit 3. "Enzymology"
1. The regulatory center breakdown happens due to a heat dissociation of protein
subunits. At the same time an active center preserves its activity.

143
 2. The dry preparation and dissolution with distilled water ensures that no impu-
rities and heavy metals can be contacted with the functional group of the protein.
Gentle stirring is necessary to avoid protein’s mechanical denaturation. A low tem-
perature provides low activity of enzyme as well as escape of spontaneous unwinding
of the protein chain. It also reduces the possibility of bacterial contamination. The
drug drying and storage in special evacuated vial protects the enzyme from exposure
to oxygen and prevents oxidation of the sulfur-containing groups

3. The effect of adrenaline is the activation energetic metabolism. Its action is

intracellular disintegration of lipids metabolism during muscular work. It is also
known that the hormone increases the phosphorylation of enzymes. So, the lipase is
activated by phosphorylation

Unit 4. "Biological oxidation"

1. The speed of electrons transfer in the respiratory chain is strictly relevant
with the ATP requirements. If ATP consumption is small (ATP concentration is
high), the rate of electron transfer is small. The level of electrochemical gradient
could be reduced. The uncoupling agents (uncouplers) change the rate of electron
transfer due a decrease of proton gradient without effect on ATP synthase. The ratio
of P/O is reduced. High speed of electron transfer in the respiratory chain causes a
decrease of the free energy, part of which is dissipated as heat. Its effects are sweat-
ing and high body temperature. Application of uncouplers in vivo leads to reduce of a
total amount of ATP especially in cells with great number of mitochondria. Nervous
cells belong to them. Consequently, disturbances of the nervous system are main sign
of unclouplers action.

2. Low coefficient of P/O means that most of the energy is dissipated as heat, in-
stead of being spent on the synthesis of ATP. This allows the animals to maintain
body temperature at the right level. The mechanism responsible for low coefficient of
P/O is the uncoupling of oxidation and phosphorylation. It provides thermogenin -
protein mitochondrial membrane of brown fat cells

Unit 5. "Amino acid and protein metabolism"

1. The low acidity of gastric juice reduces pepsin activity and leads to the devel-
opment of intestine microflora. The rotting processes with the formation of hydrogen
sulfide are results of this process. To normalize digestion, it can be recommend, the
intake of hydrochloric acid with pepsin and gastric juice.

2. Glutamic acid is dicarboxylic mono amino acid. It is able to bind ammonia to

form a non-toxic glutamine. When the reamination process occurs it turns into ke-
toglutaric acid. It is an important substrate of TCA, which is able to oxidize and
forms a 4 molecules of ATP. Glutamic acid has an antioxidant effect, reducing the
content of lipid peroxides, damaging cell membranes.

144
 3. Patient has a reduced urea formation in liver. So he has a decreased level of
urea in blood and its excretion is also low. The urea enzymes activity should be in-
vestigated: ornithine carbamoyl transferase and arginase.

Unit 6. "Structure and metabolism of purines and pyrimidines"

1. High content of uric acid in the urine could be found in hyperuricemia of dif-
ferent origin. It is observed in adults with gout or in a case of purine containing food-
stuff intake. Another cause is a defect of hypoxanthine-guanine-phosphoribosyl trans-
ferase that leads to the alteration of purine nucleotudes salvage pathway (The Lesch-
Nyhan syndrome). The excess of uric acids is a result of this defect.

2. Folic acid active form is N5,N10-methylene-THFA. It participates in thymi-

dylate monophosphate synthesis (TMP), the required component of DNA replication
and cell division. The lack of erythrocytes division leads to the reduction of erythro-
cytes number (megaloblastic anemia is developed). The same trend is found in leuco-
cytes (leucopenia is developed) and in epithelial cells (the division of mucosa mem-
brane and skin cells is decreased).

Unit 7. "Matrix biosynthesis"

1. Sickle cell anemia is a disease of glutamic acid substitution on valine in he-
moglobin β-chain. It is a CTT change on CAT in sequence of mRNA.

2. Topoisomerase is an enzyme that participates in the over winding or under

winding of DNA in replication. If the enzyme is blocked, the over winding and under
winding could not be happened, the replication will be stopped and cell will not be
divided. This is the basic of enzyme inhibition application in medicine.

3. It is well-known that the proteins in human body differ from one another.
They have different amino acids sequence, nucleic acid number in genes (DNA struc-
ture). In this case one gene is responsible for functioning of two proteins. So, there is
another regulation level. For example, it may be at processing stage of mRNA for-
mation. The terminator codon will be included in processing stage in enterocytes, that
stops protein synthesis despite the translation is not finished. The apoB-48 protein
will be developed. The mRNA processing is not modified in hepatocytes, the protein
translation will not be changed, and the apoB-100 protein is produced.

Unit 8. "Structure and metabolism of carbohydrates"

1. The level of glucose in the blood can increase due to a stress reaction, which
is characterized by an increase in the adrenaline level in the blood and tissues.

2. The tissues are provided with oxygen due to an increase in the rate of blood
flow in running at a distance of 5000 m better than a 100-meter, the oxygen debt of
the body is less, therefore, the aerobic metabolic processes are activated, the level of
lactic acid in the blood and tissues is lower than that of the athlete who ran 100 m.
145
 3. One of the functions of the pentose-phosphate cycle is the supply of pentose
phosphates for the synthesis of nucleic acids. After bleeding, the regeneration of
blood elements is intensive, for the synthesis of which nucleic acids are needed.
Therefore, the processes of the pentose phosphate cycle will be intensified, accord-
ingly, its enzymes, in particular glucose-6-phosphate dehydrogenase, transketolase
and others, are activated, which it is have to investigate to check the conclusion.

Unit 9. "Structure and metabolism of lipids"

1. When the bile enters the duodenum, the activation of pancreatic lipase is dis-
turbed (inhibited), the digestion of fats, the absorption of fatty acids and fat-soluble
vitamins deteriorates, the motor function of the intestine worsens, and hypercholes-
terolemia develops.

2. The myocardium is better provided with energy reserves than the skeletal
muscle. It contains more amount of lipids, with oxidation of which gives a lot of en-
ergy (1 g of carbohydrates – 4.1 kcal (17.2 kJ), fats – 9.3 kcal (38.9 kJ). But the
amount of oxygen molecules in a fatty acid is less than in carbohydrates. The oxida-
tion of 1 g of fat requires 2019 ml of oxygen, compared to the oxidation of 1 g of
glycogen. It is only required there are only 829 ml of oxygen. In the energy balance
of cardiac metabolism, the more effective aerobic processes are leading, but they
make it very sensitive to hypoxia (oxygen starvation). In a case heart attack the skele-
tal muscle is active in the oxygen deficiency.

3. Reducing the carbohydrates in a child's diet leads to a violation of the chain

"Glucose → pyruvate → oxaloacetate" and slowing down the speed of the CTA.
The former intake of fat and the oxidation of fatty acids caused a relative excess of
acetyl-SCoA, which cannot now burn to the CTA ("fats burn in the flame of carbo-
hydrates"). This leads to ketonemia, the metabolic acidosis is developed. Simultane-
ously, the increase in gluconeogenesis from amino acids intensified deamination pro-
cesses and accumulation of ammonia. But urea synthesis is inhibited due to the lack
of ATP energy in case of CTA alteration and the hyperammonemia develops. It is
expedient to determine the content of glucose and urea in the blood, ketones in the
urine, to study the state of acid-base balance before the treatment.

Unit 10. "Hormonal regulation of human metabolism and functions

1. The slowing down of physical and mental development, a decrease in the in-
tensity of metabolic processes is characteristic of hypothyroidism of the thyroid
gland. In a pronounced degree the disease is called "cretinism".

2. These complaints are characteristic of diabetes mellitus. To diagnose the

pathological states and to evaluate the metabolic changes in diabetes it is expedient to
determine the following most informative indicatives. In the blood it should be de-
tected the level of: glucose on an empty stomach, ketone bodies, cholesterol and li-

146
pids, if necessary, a test for glucose tolerance. In the urine it should be detected the
level of: glucose, urine’s density, ketone bodies.

3. The high fatigue, frequent hypoglycemic conditions, the increased skin pig-
mentation and other symptoms are characteristic of the primary chronic insufficiency
of the adrenal cortex (Addison's disease).

Unit 11. "Biochemistry of blood"

1. In extensive burns due to fluid loss by tissues, the dehydration develops.
There is a thickening of the blood, hyperproteinemia (100 g/l). If the function of the
gastrointestinal tract is impaired, the intake of amino acids is decreased, followed by
hypoproteinemia (30 g/l) development.

2. To clarify the diagnosis, it is necessary to determine the blood content of urea

nitrogen, which is normally half of the residual nitrogen. If the excretory function of
the kidneys is disturbed, there is an increase in residual nitrogen, mainly due to urea
nitrogen. The urea-forming function of the liver is disturbed and it leads to decrease
in urea nitrogen content.

3. The pH is lowered; the acidosis is detected. An increase in the partial pressure

of СО2 indicates the cause of acidosis – the accumulation of carbonic acid due to a
violation of its excretion. The excess of НСО3– ions is a reflection of the accumula-
tion and dissociation of carbonic acid, and, first of all, compensatory reabsorption of
carbonates by the kidneys. Based on the clinical picture, the diagnosis of respiratory
acidosis is confirmed. Compensation occurs through "metabolic alkalosis", i.e. excre-
tion by the kidneys of the Н+ ion by activation of ammoniogenesis and acidogenesis.
The alkaline reserve corresponds to the sum of all the buffer bases of the blood and is
increased by the excess of carbonate ions.

Unit 12. "Biochemistry of kidneys"

1. The reason for increasing excretion of Са2+ ions in the urine may be hypo-
dynamic osteoporosis – the "washing away" of Са2+ from the bones into the blood
and excretion of it into the urine.

2. In a healthy person, the protein in the urine is absent (its content is so small
that it is not detected by laboratory methods). Proteinuria can appear at very high
physical exertion – "marching", "cold" proteinuria.

3. These symptoms are typical of mechanical (obstructive) jaundice, probably

caused by a blockage of the common bile duct by a stone. The blood content of bili-
rubin is increased due to direct (bilirubin glucuronide), since the outflow of bile in the
intestine is disturbed. Therefore, feces are colorless (acholia), does not contain ste-
ricobilinogen and the urine does not contain stericobilinogen and urobilinogen. The
dark color of urine is due to the penetration of direct bilirubin from the blood, it is al-
so possible the foaming of urine due to the presence of bile acids.
147
 RECOMMENDED LITERATURE

List of main literature:

1. Harper's illustrated biochemistry [Text] : textbook / V.W. Rodwell [et al.]. –
30th ed. – New York : McGraw-Hill, 2015. – 817 p.
2. Zurabyan, S. E. Fundamentals of bioorganic chemistry [Электронный
ресурс] : учебное пособие / S. E. Zurabyan. – Электрон. текстовые дан. – M. :
GEOTAR-MED, 2015. – 304 p. : access mode: http://studentlibrary.ru

List of additional literature:

1. Zurabyan, S. E. Fundamentals of bioorganic chemistry [Текст] : textbook for
foreign students of Medical Higher Educational Institutions / S. E. Zurabyan. – 2th.
ed. – M. : GEOTAR-MED, 2004. – 320 p.

List of internet resources:

1. Clinical Key: Access: www.clinicalkey.com.
2. Electronic database of Scientific medical library of SSMU Ac-
cess: http://medlib.tomsk.ru.

148
 ANNEXES
ANNEX 1

CLASSIFICATION AND NOMENCLATURE OF ENZYMES

In 1961 in Moscow, the Commission on Enzymes of the International Biochem-
ical Union (IUBM) adopted a modern systematic classification of enzymes.
In accordance with the systematic classification, the reaction and substrate spec-
ificity of the enzymes are taken into account. Enzymes are divided:
• on classes – according to the type of catalyzed reaction,
• each class is divided into subclasses – by the nature of the attacked chemical
group,
• subclasses are divided into sub-subclasses – by the nature of the attack or by
the nature of the acceptor or coenzyme.
There are 6 classes of enzymes:
I. Oxidoreductases,
II. Transferases,
III. Hydrolases,
IV. Lyases,
V. Isomerases,
VI. Ligase.
Each enzyme is assigned a
four-digit classification number.
For example, alcohol dehydro-
genase has a number of EC 1.1.1.1. – this oxidoreductase, acts on the OH group of
the donor with NAD+ as an acceptor with the first ordinal number in its subclass; lac-
tate dehydrogenase – EC 1.1.1.27.
Enzymes can have a trivial or systematic name:
1. Systematic name – according to modern classification
(http://www.chem.qmul.ac.uk/iubmb/enzyme/). It is often difficult to use, then it is
simplified and the working name of the enzyme is introduced.
2. The trivial name is a name that has developed historically, which is more
commonly used, for example, pepsin, trypsin. Sometimes the name of the substrate
is added with the ending "-ase" – urease, amylase, lipase. Nevertheless, all such en-
zymes have a systematic name.

I. CLASS OXIDOREDUCTASE
Enzymes catalyze the oxidation-reduction reactions underlying the biological
oxidation. The class has 22 subclasses. Coenzymes of this class are NAD+, NADP+,
FAD, FMN, ubiquinone, glutathione, lipoic acid.
Subclasses are allocated to enzyme groups acting on:
1.1. on the CH-OH group of donors;
1.2. on the aldehyde or oxo group of donors;
1.3. on the CH-CH group of donors;
1.4. on the CH-NH2 group of donors;
149
 1.5. on CH-NH group of donors
1.6. on NADH or NADPH group of donors
1.8. on a sulfur group of donors;
1.9. on a heme group of donors;
1.10. on diphenols and related substances as donors;
1.11. on peroxide as an acceptor;
1.12. on hydrogen as donors;
1.13. on single donors with incorporation of molecular oxygen;
1.14. on paired donors with incorporation of molecular oxygen;
1.15. on superoxide radicals as acceptors;
1.17. on CH or CH2 groups;
1.18. on iron-sulfur proteins as donors.
Subclasses are divided into subclasses depending on the acceptor – NAD+ or
NADP+ (1.1.1., 1.2.1., 1.3.1., 1.4.1.), disulfides (1.2.4.), oxygen (1.3.3.).
Common names include:
1. Dehydrogenase – oxidoreductase, catalyzing the dehydrogenation of a sub-
strate using as a hydrogen acceptor any molecules other than oxygen.
2. If the transfer of hydrogen from the donor molecule is difficult to prove, then
such oxidoreductases are called reductases.
3. Oxidase – oxidoreductase, catalyzing the oxidation of substrates with mo-
lecular oxygen as an electron acceptor without the inclusion of oxygen in the sub-
strate molecule.
4. Monoxygenase – oxidoreductase, catalyzing the introduction of one oxygen
atom in a substrate molecule with molecular oxygen as an oxygen donor.
5. Dioxygenase – oxidoreductase, catalyzing the introduction of two oxygen at-
oms in a substrate molecule with molecular oxygen as an oxygen donor.
6. Peroxidase – oxidoreductase, catalyzing reactions with hydrogen peroxide as
an electron acceptor.
The systematic name of oxidoreductases is formed as follows:
The electron donor : the electron acceptor – oxidoreductase

Example 1

Enzyme characteristic
The systematic name Alcohol : NAD-oxydoreductase
The working name Alcoholdehydrogenase
Class 1. Oxydoreductase
Subclass 1.1. acting on the CH-OH group of donors;
150
Subsubclass 1.1.1. with NAD+ or NADP+ as an acceptor
Serial number EC 1.1.1.1.
Cofactors Nicotinamide adenine dinucleotide. Iron or zinc.

Example 2

Enzyme characteristic
The systematic name Succinate : FAD-oxydoreductase
The working name Succinatedehydrogenase
Class 1. Oxydoreductases
Subclass 1.3. acting on the CH-CH group of donors
Subsubclass 1.3.99. with FAD as an acceptor
Serialnumber EC 1.3.99.1.
Cofactor Flavin adenine dinucleotide

Example 3

Enzyme characteristic
Phenylalanine. Tetrahydrobiopterin : oxygen-
The systematic name
oxydoreductase
The working name Phenylalanine-4-monooxygenase
Class 1. Oxydoreductase
1.14. Acting on paired donors with incorporation of mo-
Subclass
lecular oxygen
1.14.16. With the reduced pteridine as a donor and the
Subsubclass
inclusion of one oxygen atom
Serialnumber EC. 1.14.16.1.
Cofactors Tetrahydrobiopterin. Iron.
151
 II CLASS. TRANSFERASE
Transferases catalyze the transport reactions of various groups from one sub-
strate (donor) to another (acceptor), participate in the reactions of interconversion of
various substances, neutralization of natural and foreign compounds. Coenzymes are
pyridoxal phosphate, coenzyme A, THFA, methylcobalamin. The class is divided in-
to 9 subclasses depending on the structure of the groups carried by them:
2.1. single-carbon groups;
2.2. aldehyde or ketone groups;
2.3. acyl groups or groups that become alkyl groups during transfer;
2.4. glycosyl groups, as well as hexoses and pentoses;
2.5. alkyl or aryl groups, other than methyl groups;
2.6. nitrogenous groups;
2.7. phosphorus-containing groups;
2.8. sulfur-containing groups;
2.9. selenium-containing groups.
The division into subclasses depending on the nature of the group being trans-
ferred, for example: subclass 2.1.1. – methyl, subclass 2.1.2. – carboxymethyl or
formyl, subclass 2.6.1. – amino, amidino, hydroxyamino.
The systematic name of transferases is formed as follows:
Group donor : group acceptor – transferable transferase group

Example 1

Enzyme characteristic
The systematic name ATP : D-hexose-6-phsopho-transferase
The working name Hexokinase
Class 2. Transferase
Subclass 2.7. Transferingphosphorus-containinggroups
Subsubclass 2.7.1. With an alcohol group as an acceptor
Serialnumber EC 2.7.1.1.
Cofactor Magnesium

152
Example 2

Enzyme characteristic
The systematic name L-aspartate : 2-oxoglutarate aminotransferase
The working name Aspartate aminotransferase
Class 2. Transferase
Subclass 2.6. Transferring nitrogenous groups
Subsubclass 2.6.1. Fminotransferase
Serialnumber EC 2.6.1.1.
Cofactor Pyridoxal phosphate

Example 3

Enzyme characteristic
5-methyltetrahydrofolate : L-homocysteine
The systematic name
S-methyltransferase
The working name Methionine synthase
Class 2. Transferase
Subclass 2.1. Transferring single-carbon groups
Subsubclass 2.1.1. Methyltransferases
Serialnumber EC. 2.1.1.13.
Cofactors Cobalamin. Zinc.

153
 III CLASS. HYDROLASE
Hydrolases are enzymes that break the intramolecular bonds in the substrate
(with the exception of the C-C bonds) by the addition of H2O molecules. They are di-
vided into 13 subclasses. Enzymes retain trivial names due to the complexity of many
substrates: pepsin, trypsin. Coenzymes are absent.
Hydrolases are mainly concentrated in the gastrointestinal tract and lysosomes
of tissue cells. They carry out the decomposition of macromolecules, forming easily
adsorbed monomers.
Hydrolases can be further classified into several subclasses, based upon the
bonds they act upon:
3.1. ester bonds;
3.2. О-glycosides;
3.3. simple ether bonds;
3.4. peptide bonds;
3.5. non-peptide carbon-nitrogen bonds;
3.6. acid anhydrides;
3.7. carbon-carbon bonds.
There are the subsubclasses, for example, the carboxylic acid hydrolases
(3.1.1.), the phosphono-ester hydrolases (3.1.3.)
The most common are the following hydrolases:
1. Esterases – hydrolysis of ester bonds.
2. Lipases – hydrolysis of neutral fats.
3. Phosphatase – hydrolysis of monoesters of phosphoric acid.
4. Glycosidases – hydrolyses O- and N-glycosidic bonds.
5. Proteases, peptidases – hydrolysis of proteins and peptides.
6. Nuclease – hydrolysis of nucleic acids.

The systematic name of hydrolases is formed:

Hydrolyzable substrate : hydrolase release group
Example 1

Enzyme characteristic
The systematic name Triacylglycerol : acyl hydrolase
The working name TAG-lipase
Class 3. Hydrolase
154
Subclass 3.1. acts on esters
Subsubclass 3.1.1. Hydrolases of carboxylic acids
Serialnumber EC 3.1.1.3.
Example 2

Enzyme characteristic
The systematic name L-glutamine : amide hydrolase
The working name Glutaminase
Class 3. Hydrolase
3.5. acts on non-peptide carbon-nitrogen
Subclass
bonds
Subsubclass 3.5.1. Operating in linear amides
Serialnumber EC 3.5.1.2.
Example 3

Enzyme characteristic
The systematic name α-D-glucoside : glucohydrolase
The working name Maltase
Class 3. Hydrolases
Subclass 3.2. Glycosidase
Subsubclass 3.2.1. acts on O-glycosidic bonds
Serialnumber EC 3.2.1.20.

IV CLASS. LYASE
Liases are enzymes that catalyze the rupture of C-O, C-C, C-N and other bonds.
They carry out the reversible reactions of the non-hydrolytical cleavage of substrates
with various groups. There are 7 subclasses. These reactions are accompanied by the
formation of a double bond or by the attachment of groups to the double bond site.
155
The class has about 230 enzymes. Lyases are complex enzymes, pyridoxal phosphate,
thiamine diphosphate, magnesium, cobalt serve as coenzymes.
Lyases can be further classified into seven subclasses according the bond being
broken:
4.1. lyases that cleave carbon-carbon bonds;
4.2. lyases that cleave carbon-oxygen bond;
4.3. lyases that cleave carbon-nitrogen bonds;
4.4. lyases that cleave carbon-sulfur bonds;
4.5. lyases that cleave carbon-halide bonds.
There are the subsubclasses, for example, carboxylases (4.1.1.), hydrolases
4.2.1.)

The systematic name of lyases is formed:

Cleavable substrate : detachable group – lyase
Example 1

Enzyme characteristic
The systematic name 2-oxo acid : carboxylase
The working name Pyruvate decarboxylase
Class 4. Lyase
Subclass 4.1. Lyases that cleave carbon-carbon bonds
Subsubclass 4.1.1. Carboxyl aliases
Serial number EC 4.1.1.1.
Cofactor Thiamine diphosphate
Example 2

Enzyme characteristic
The systematic name Histidine : carboxylase
The working name Histidine-decarboxylase
Class 4. Lyase
Subclass 4.1. Lyases that cleave carbon-carbon bonds
156
Subsubclass 4.1.1. Carboxylase
Serial number EC 4.1.1.22.
Cofactor Pyridoxalphosphate
Example 3

Enzyme characteristic
The systematic name (S)–Malate : hydrolase
The working name Fumarase
Class 4. Lyase
Subclass 4.2. Lyases that cleave carbon-oxygen bond
Subsubclass 4.2.1. Hydrolyase
Serialnumber EC 4.2.1.2.
Example 4

Enzyme characteristic
The systematic name ATP : Lipase diphosphate (cyclization)
The working name Adenylate cyclase
Class 4. Lyase
Subclass 4.6. Lyases that cleave phosphorus-oxygen bonds
Subsubclass 4.6.1. Phosphorus-oxygen lyase
Serial number EC 4.6.1.1.

157
 V CLASS. ISOMERASE
Isomerases are enzymes that catalyze isomeric transformations within a single
molecule. The class has more than 80 enzymes, in which 6 subclasses are distin-
guished. Isomerases are complex enzymes. Their coenzymes include pyridoxal, de-
oxyadenosylcobalamin, peptide (glutathione), monosaccharide phosphates (glucose-
1.6-diphosphate), and others.
Isomerases are further classified into six subclasses:
5.1. Racemases and epimerases.
Racemases are responsible for the interconversion of L- and D-isomers, S- and
R-isomers. Epimerases change the configuration at one of the chiral carbon atoms,
for example: the interconversion of the α- and β-isomers, the conversion of ribulose
↔ xylulose, galactose ↔ glucose, mannose ↔ galactose.
5.2. Cis-trans isomerase;
5.3. Intramolecular oxidoreductases;
5.4. Intramolecular transferase – mutases, makey the transfer of chemical groups
within the molecule;
5.5. Intramolecular lyases.
There are the subsubclasses, for example: acting on amino acids and their deriv-
atives (5.1.1.), carbohydrates and their derivatives (5.1.3.), transfering the double
(C=C) bonds (5.3.3.).
The systematic name of isomerases is formed:
Substrate – [ ] – reaction,
where [ ] – a designation reflecting the essence of the reaction, for example, "the
number of the altered carbon atom", the change in "cis-trans," the change in "keto-
enol," the change in "aldose-ketose."
Example 1

Enzyme characteristic
The systematic name D-ribulose-5-phosphate-3-epimerase
The working name Ribulose phosphate-3-epimerase
Class 5. Isomerase
Subclass 5.1. Racemases and epimerases
5.1.3. acting upon on carbohydrates and
Subsubclass
their derivatives
Serial number EC 5.1.3.1.

158
Example 2

Enzyme characteristic
D-glyceraldehyde-3-phosphate-aldose-
The systematic name
ketose isomerase
The working name Triosephosphate isomerase
Class 5. Isomerase
Subclass 5.3. Intramolecular oxidoreductases
5.3.1. Catalyzing interconversion of aldose
Subsubclass
and ketoses
Serial number EC 5.3.1.1.
Example 3

Enzyme characteristic
The systematic name α-D-glucose-1.6-phosphomutase
The working name Phosphoglucomutase
Class 5. Isomerase
Subclass 5.4. Intramolecular transferases
Subsubclass 5.4.2. Phosphotransferases
Serial number EC 5.4.2.2.
Coenzyme Glucose-1.6-diphosphate

VI CLASS. LIGASE
Ligases (synthetases) are enzymes that catalyze the attachment of two molecules
to one another using the energy of high-energy ATP bonds (or other macroergs). Lig-
ases – complex enzymes, contain nucleotide, biotin, folic coenzymes.
Itisisolatedsixenzymesubclasses, dependingontypeoftheformingbond:
6.1. Carbon-oxygen.

159
 6.2. Carbon-sulfur.
6.3. Carbon-nitrogen.
6.4. Carbon-carbon.
6.5. Phosphorus-oxygen.
6.6. Nitrogen-metal.
There are subsubclasses, for example forming the aminoacyl-tRNA bond
(6.1.1.), synthesizing compounds acid-thiol (6.2.1.), amides (6.3.1.).
The systematic name of the enzyme:
Substrate 1 : substrate 2 – ligase
Example 1

Enzyme characteristic
The systematic name L-glutamate : ammonia ligase
The working name Glutamine Synthesis
Class 6. Ligases
Subclass 6.3. Forming carbon-nitrogen bonds
Subsubclass 6.3.1. Amide synthase
Serial number EC 6.3.1.2.
Example 2

Enzyme characteristic
The systematic name Pyruvate : carboxyl ligase (ADP-forming)
The working name Pyruvate carboxylase
160
Class 6. Ligases
Subclass 6.4. Forming carbon-carbon bonds
Subsubclass 6.4.1. Forming carbon-carbon bonds
Serialnumber EC 6.4.1.1.
Cofactors Biotin. Magnesium. Zinc.
Example 3

Enzyme characteristic
The systematic name Succinate : CoA ligase
Succinyl-CoA synthetase
The working name
Succinate-thiokinase
Class 6. Ligases
Subclass 6.2. Forming bonds of carbon
Subsubclass 6.2.1. Ligase acid
Serial number EC 6.2.1.4.

161
 ANNEX 2

PROTEINS OF BLOOD PLASMA

Protein Key members of Acute-phase

fractions protein fractions protein
Albumins Pre- and postalbumins.
Albumin
α1-Lipoprotein
α1-Acid seromucoid
α1-Glycoprotein
Transcortin α1-Glycoprotein
α1
Prothrombin α1-Antitrypsin
Antiplasmin
α1-Antitrypsin
Vitamin B12 binding protein
C-reactive protein
Haptoglobin (Hp-1, Hp-1-2 Hp-2-2)
Ceruloplasmin
α2-Lipoprotein С-reactive protein
α2-HS-Glycoprotein α2-Macroglobulin
α2
α2-Macroglobulin Haptoglobin Cerulo-
Cholinesterase plasmin
Глобулины Alkaline phosphatase
Proaccelerine
The Christmas factor
β1А-globulin
β-Lipoprotein
β1В-globulin
Transferrin Plasminogen
β Plasminogen Complement compo-
nents C1 C4, C9
Proconvertin
Fibrinogen
Complement components C1 C4, C9
Hemopexin
G-immunoglobulin
A-immunoglobulin
γ
D-immunoglobulin
E-immunoglobulin

162
 CHARACTERISTICS OFSOME BLOOD PROTEINS

Fibrinogen
Fibrinogen is synthesized in the liver. It is a protein of blood clotting.
Normal values
Serum 2.0-4.0 g/l
Clinical and diagnostic significance
Increased concentrations cause acute inflammatory processes and cardiovascular
diseases (atherosclerosis). Decrease – hyperfibrinolysis (Disseminated Intravascular
Coagulation, DIC) or inherited insufficiency.

α1-GLOBULINS

Acid α1-glycoprotein
Acid α1-glycoprotein (orosomucoid) has acidic properties and contains high
amounts of carbohydrates. The protein has a high affinity for polyanions (for example
heparin) and probably regulates the amount of free heparin in the plasma. α1-
Glycoprotein binds medicines (propranolol and lidocaine), steroids (progesterone,
testosterone). It is synthesized in the liver.
Normal values
Serum 0.55-1.40 g/l
Clinical and diagnostic significance
An increase in the level of protein is observed in acute and chronic inflammato-
ry processes, rheumatoid arthritis, malignant tumors, fevers, injuries, myocardial in-
farction, exercise training, pregnancy, nephrotic syndrome. Increase in the protein
level in blood is observed in acute and chronic inflammatory processes, rheumatoid
arthritis, malignant tumors, fever, trauma, myocardial infarction, physical exertion,
pregnancy, nephrotic syndrome.
α1-Antitrypsin
α1-Antitrypsin is a glycoprotein, is formed in the liver, the acute-phase protein.
It is an inhibitor of proteinases (trypsin, chymotrypsin, kallikrein, plasmin) and ac-
counts for 92-94% of the total blood antiproteolytic function. Its autosomal recessive
inherited disorder is one of the factors of the emphysema pathogenesis in lungs,
bronchiectasias and chronic bronchitis, early cirrhosis of the liver. Obviously, the ab-
sence of an inhibitor leads to unrestricted proteolysis of cells in the inflammation
zone, which lengthens and deepens the destructive processes in the tissues.
Normal values
Serum 2.0-2.4 g/l
Clinical and diagnostic significance
α1-Antitrypsin level in the blood increases in acute infections, inflammatory
processes, malignant formations, hormones (pregnancy, steroid therapy), systemic
lupus erythematosus and cancers.

163
 α1-Antichymotrypsin
α1-Antichymotrypsin is one of the first reacting acute-phase proteins (the serum
level can be doubled for several hours), it is a weak specific inhibitor of chymotryp-
sin, but it acts to other proteases is also noted.
Normal values
Serum 0.3-0.6 g/l
Clinicalanddiagnosticsignificance
The increase in protein content is due to acute phase reactions: inflammation,
trauma after surgery, myocardial infarction, bacterial infections.

α2-GLOBULINS

С-reactive protein
C-reactive protein (CRP) is a mesenchymal protein that has undergone partial
denaturation due to tissue disintegration in inflammatory and destructive processes. It
takes part in the activation of the classical complement pathway, immune reactions, is
an inhibitor of platelet aggregation, binds lipids, carbohydrates, and participates in
catalase activity.
Normal values
Serum < 50 mg/l (absense)
Clinicalanddiagnosticsignificance
The level of this acute-phase protein rises rapidly in 15-25 times in acute and
chronic infections, cell necrosis, myocardial infarction, rheumatoid arthritis, gout.

Haptoglobin
Haptoglobin is an acute-phase protein synthesized in the liver. It has the follow-
ing functions: binds free hemoglobin of plasma and protects the body from loss of
iron, this complex is destroyed in cells of RES and liver; performs a nonspecific pro-
tective function, integrating with protein and non-protein substances that appear dur-
ing the decay of cells; is a natural inhibitor of cathepsin B; participates in the
transport of vitamin B12. Haptoglobin in low concentrations is present in many body
fluids: cerebrospinal fluid, lymph, synovial fluid, bile.
Normal values
Serum 0.8-2.7 g/l
Clinical and diagnostic significance
The protein level non-specific increases in response to tissue damage, inflam-
mation, and the oncogenesis (especially to metastasis development). High indicators
are observed in diabetes mellitus, nephrotic syndrome, pyelonephritis, burns, acute
and chronic inflammatory conditions, tissue necrosis, myocardial infarction, active
autoimmune diseases, systemic rheumatoid diseases.
A decrease in the protein amount is noted in the lesions of liver, hemolytic ane-
mia. The level of haptoglobin is considered to be a sensitive indicator of hemolytic
conditions: the release of hemoglobin causes a decrease in the haptoglobin level.

164
 α2-Macroglobulin
α2-Macroglobulin is a high-molecular zinc-containing protein, consists of 4
identical subunits and includes a carbohydrate component, is synthesized in the liver.
It is an inhibitor of proteinases (both the blood coagulation system and others) -
plasmin, pepsin, trypsin, chymotrypsin, endopeptidases, cathepsin D, thrombin, kal-
likrein. It transports enzymes and hormones, the receptor of lymphocytes, participates
in the interaction of the mother and fetus, has an immunomodulating effect, the inhib-
itor of the complement component.
Normal values
Serum children (1-3 years) about 4.5 g/l
men 1.50-3.50 g/l
women 1.75-4.20 g/l
Clinical and diagnostic significance
Protein controls the development of infections and inflammatory processes. An
increase in its level is revealed in cirrhosis of the liver, acute and chronic hepatitis,
pregnancy, congenital heart diseases, endocrine diseases (diabetes mellitus, myxede-
ma), pneumonia, nephrotic syndrome. A decrease – in rheumatic polyarthritis, loss of
protein or its deficiency in nutrition, disseminated intravascular coagulation, fibrino-
lytic therapy, acute pancreatitis, myocardial infarction, stomach and duodenal ulcers.

Ceruloplasmin
Ceruloplasmin contains 8 copper atoms. This is an acute-phase protein, the cop-
per metabolism regulator in the body (it aggregates 90% of all plasma copper) -
transports copper ions from the liver to other organs. Ceruloplasmin is an oxidase of
polyphenols and diamines, promotes iron saturation of apotransferrin, participates in
the exchange of biogenic amines (adrenaline, norepinephrine, serotonin) and ascorbic
acid, regulates the level of sympathetic brain mediators, as a serum antioxidant elimi-
nates superoxide radicals of oxygen, restores O2 to water and prevents oxidation of
unsaturated fatty acids.
Normal values
Serum 0.15-0.50 g/l
Clinicalanddiagnosticsignificance
Elevated level of protein is determined in rheumatoid arthritis, systemic lupus
erythematosus, chronic inflammatory processes, cholestasis, hepatitis, liver cirrhosis,
myocardial infarction, acute infections, malignant tumors with metastases, melano-
ma, schizophrenia.
A decrease in the protein content is revealed in reduction in the synthesis of the
enzyme (Wilson Konovalov's disease), increased loss (gastrointestinal disease, ne-
phrotic syndrome), a decrease in absorption in the intestine (impaired absorption,
malnutrition).

165
 β-GLOBULINS

Transferrin family
The protein called transferrin belongs to the transferrin family, as well as ovo-
transferrin, lactoferrin, melano- transferrin, and others.
Proteins of this family, binding iron ions (III) and preventing their recovery, are
an important component of the body's antioxidant defense. In addition, the binding of
iron by transferrin prevents its use by microorganisms, which determines the bacteri-
ostatic activity of these proteins.

Transferrin
Transferrin is synthesized in the liver and reticuloendothelial system. Transferrin
transports trivalent iron along with the anion of bicarbonate from the duodenum and
spleen to all tissues.
Normally, only 1/3 of the total amount of transferrin is saturated with iron.
Normal values
Serum Childern 2.0-3.6 g/l
Men 2.1-3.6 g/l
Women 2.5-3.8 g/l
Clinical and diagnostic significance
Its level increases in a lack of iron in the body, pregnancy, estrogen, lipoid ne-
phrosis.
Reduction is observed in inherited synthesis failure, testosterone intake, neph-
roses, malaria, hemochromatosis, malnutrition, tumors.

Lactoferrin
Protein is widely represented in blood plasma, secretory fluids: milk, saliva,
tear, bile, secrets of nasal and bronchial glands.
The main biological function of lactoferrin is the binding and transport of iron
ions, but also the protein has broad antibacterial, antiviral and antifungal activity.
Normal values
Serum 0.2-0.6 mg/l
Human milk to 7.0 g/l
Clinical and diagnostic significance
An increase in the protein content in the blood is noted in pregnancy, gestosis,
skin diseases, cancers of the gastrointestinal tract.

166
 ANNEX 3

NORMAL VALUES OF STUDIED

BIOCAMICAL INDICATORS

Serum
Indicator Sex, age, etc Normal values
Amylase 16-30 g/l·h
ALT activity 0.10-0.68 mmol/l·h
AST activity 0.10-0.45 mmol/l·h
The De Ritis Ratio 1.33±0.40

Residual nitrogen 14.3-28.6 mmol/l

Urea Children 1.8-6.4 mmol/l
Adults 2.5-8.3 mmol/l
Creatinine Children
up to 1 year 18-35 µmol/l
from 1 year to 12 years 27-62 μmol/l
Adults
women 44-97 μmol/l
men 52-132 μmol/l
Uric acid 0.12-0.32 mmol/l
Meat Diet 0.16-0.45 mmol/l
Protein total Children
newborns 51-60 g/l
children up to 1 year 51-73 g/l
children from 1 to 3 years 54-85 g/l
from 4 years 65-85 g/l
Adults 65-85 g/l
Fractions of proteins
albumins 30-50 g/l 50-70%
α1-globulins 1-3 g/l 3-6%
α2-globulins 6-10 g/l 9-15%
β-globulins 7-11 g/l 8-18%
γ-globulins 8-16 g/l 15-25%
The albumin / globulin ratio 1.2-1.8 1.2-1.8
The albumin /α1+α2-glibulins coefficient 3.9-6.1 3.9-6.1
Timole sample 0-4 S-H units

167
Glucose 3.5-5.5 mmol/l
Glucose Tolerance Test
On an empty stomach 3.5-5.5 mmol/l 100%
After 60 min 5.3-9.6 mmol/l 150-175%
After 120 minutes below 5.3 mmol/l about 100%

Triacylglycerols Children 0.15-1.56 mmol/l

0-5 years 0.2-1.1 mmol/l
6-11 years 0.3-1.3 mmol/l
12-15 years 0.4-1.6 mmol/l
16-19 years 0.5-1.8 mmol/l
Adults
20-29 years 0.5-2.1 mmol/l
30-39 years 0.5-3.2 mmol/l
40-49 years 0.6-3.4 mmol/l
50-59 years 0.6-3.4 mmol/l
Cholesterol total Children
newborns 1.2-2.7 mmol/l
0-19 years 2.9-5.2 mmol/l
Adults
20-29 years 3.70-6.51 mmol/l
30-39 years 4.25-7.04 mmol/l
40-49 years 4.37-7.70 mmol/l
over 50 years 4.55-8.24 mmol/l

Prolactin Adults
follicular phase
4.5-33 ng/ml
(98-784 micro Units/l)
middle of cycle
women 6.3-49 ng/ml
(134-975 micro Units/l)
luteal phase
4.9-40 ng/ml
(104-848 micro Units/l)
2.5-17 ng/ml
men
(53-360 micro Units/l)
Testosteron Adults
women over 10 years old 0.45-3.75 nmol/l
men over 14 years old 5.76-28.14 nmol/l

168
Hemoglobin Children 100-140 g/l
Adults
women 120-140 g/l
men 130-160 g/l
Total bilirubin Adults 8.5-20.5 μmol/l
Fullterm babies
blood from the umbilical cord < 34.2 μmol/l
age up to 5 days < 205.2 μmol/l
age up to 5 days 3.4-17.1 μmol/l
Preterm babies
blood from the umbilical cord < 34.2 μmol/l
age up to 5 days < 273.6 μmol/l
Direct Bilirubin Children absense
Adults 2.2-5.1 μmol/l

Potassium Children
newborns 3.7-5.9 mmol/l
up to 2 years 4.1-5.3 mmol/l
over 2 years old 3.4-4.7 mmol/l
Adults 3.5-5.1 mmol/l
Sodium Children
newborns 134-146 mmol/l
children 138-146 mmol/l
Взрослые 136-146 mmol/l
Ferrum Children
newborns 17.9-44.8 μmol/l
up to 2 years 7.1-17.9 μmol/l
over 2 years old 8.9-21.4 μmol/l
Adults
men 8.9-28.6 μmol/l
women 7.1-26.8 μmol/l
Phosphates Children
newborns 1.13-2.78 μmol/l
young age 1.45-2.16 μmol/l
school-age 1.46-1.76 μmol/l
Adults 0.81-1.48 μmol/l
Calcium 2.0-2.6 μmol/l
Chlorides 97-108 mmol/l

рН Newborns 7.21-7.38
Children and adults
arterial blood 7.37-7.45
venus blood 7.35-7.43

169
рСО2 Newborns and children 27-41 mmHg
Adults
35-48 mm Hg or
men
4.66-6.38 кilo Pa
32-45 mm Hg or
women
4.26-6.00 kilo Pa
Buffer bases 44-48 mmol/l
Bicarbonates Newborns 17-24 mmol/l
Childern 19-24 mmol/l
Adults
arterial blood 21-28 mmol/l
venus blood 22-29 mmol/l
Residual anions 12-16 mmol/l
Excess buffer bases Newborns from –10 to –2 mmol/l
Children up to 2 years old from –7 to +1 mmol/l
Children from –4 to –2 mmol/l
Adults from –2 to +3 mmol/l
Adults 83-108 mm Hg or
рО2
11.04-14.36 kilo Pa
Oxyhemoglobin (HbО2) Adults 94-97%
Saturation of hemoglobin Newborns
with oxygen (HbOSAT, 40-90%
SО2)
Adults 94-98%
Urine
Amylase 28-160 g/l·h
Urea 330-580 mmol/day
Creatinine 4.4-17.7 mmol/day
Uric acid regular diet 1.46-4.43 mmol/day
meat diet 2.36-5.90 mmol/day
Protein 50-150 mg/day
Glucose 0.06-0.83 mmol/l
pH 5.0-6.5
Phosphates 25.8-48.4 mmol/day
Calcium 2.5-7.5 mmol/day
Chlorides 120-240 mmol/day

Gastric juice
Hydrochloric acid Total acidity 40-60 mmol/l
Free HCl 20-40 mmol/l
Bound HСl 10-20 mmol/l

170
 Clearance of endogenous creatinine
Men Women
Children up to 1 year old 65-100 ml/min 65-100 ml/min
from 1 to 30 years 88-146 ml/min 81-134 ml/min
Adults
from 30 to 40 years 82-140 ml/min 75-128 ml/min
from 40 to 50 years 75-133 ml/min 69-122 ml/min
from 50 to 60 years 68-126 ml/min 64-116 ml/min
from 60 to 70 years 61-120 ml/min 58-110 ml/min
over 70 years old 55-113 ml/min 52-105 ml/min

171
 CONTENTS

Unit 1. Structure, features and functions of proteins ....................................... 3

Theme 1.1. Amino acid structure and classification. Protein structure. ... 3
Theme 1.2. Structure, physical and chemical properties of proteins ........ 6
Theme 1.3. Protein classification. Protein structure and functions in
human body. Complex proteins ................................................................ 10
Unit 2. Vitamins structure, classification and role ............................................ 15
Theme 2.1. Fat soluble vitamins ............................................................... 15
Theme 2.2. Water soluble vitamins........................................................... 18
Unit 3. Enzymology ..............................................................................................23
Theme 3.1. Enzyme structure and properties. ........................................... 23
Theme 3.2. Enzyme activity regulation .................................................... 28
Theme 3.3. Enzyme classification and nomenclature (seminar) ............. 30
Unit 4. Biological oxidation..................................................................................35
Theme 4.1. Main catabolic pathways: pyruvate oxidative
decarboxylation. Tricarbonic acid cycle. Enzymes of respiratory
chain. Oxidative phosphorylation (seminar) ............................................. 35
Unit 5. Amino acids and proteins metabolism ................................................... 39
Theme 5.1. External metabolism of proteins. Protein digestion and
absorption ..................................................................................................39
Theme 5.2. Intracellular amino acid metabolism ...................................... 44
Theme 5.3. Ammonia metabolism and its disposal .................................. 47
Theme 5.4. Metabolism of some amino acids. Their features and
disorders (seminar) ....................................................................................51
Unit 6. Structure and metabolism of purine and pyrimidine nucleotides ...... 55
Theme 6.1. Structure and metabolism of purine and pyrimidine
nucleotides .................................................................................................55
Unit 7. Biosynthesis of nucleic acids and proteins ............................................. 60
Theme 7.1. Nucleic acid synthesis andregulation ..................................... 60
Theme 7.2. Protein biosynthesis and regulation ....................................... 63
Unit 8. Structure and metabolism of carbohydrates......................................... 70
Theme 8.1. Structure and metabolism of carbohydrates. Glycogen
metabolism ................................................................................................70
Theme 8.2. Oxidation of glucose in aerobic conditions.
Gluconeogenesis ........................................................................................72
Theme 8.3. Aerobic oxidation of glucose. Pentose phosphate pathway... 77
Unit 9. Structure and metabolism of lipids ........................................................ 86
Theme 9.1. Structure and external metabolism of lipids .......................... 86
Theme 9.2. Intracellular metabolism of fatty acids and triacylglycerols .. 89
Theme 9.3. Intracellular metabolism of phospholipids and cholesterol.
Transport of lipids in blood ....................................................................... 92

172
Unit 10. Hormonal regulation of metabolism and functions in human
body ........................................................................................................................100
Theme 10.1. The mechanisms of hormonal signal transduction.
Classification of hormones. Hormones of pituitary gland. (seminar)....... 100
Theme 10.2. Hormones of hypothalamus, pituitary, thyroid, pancreas
and parathyroid glands ..............................................................................101
Theme 10.3. Hormones of hypophysis, adrenal and sexual glands .......... 107
Unit 11. Biochemistry of blood ............................................................................113
Theme 11.2. Iron exchange. Hemoproteins. The synthesis and
breakdown of heme. ..................................................................................117
Theme 11.3. Inorganic substances of the blood. Acid-base status ........... 123
Unit 12. Biochemistry of kidneys ........................................................................ 129
Theme 12.1. Water-salt exchange. Normal and pathological
components of urine ..................................................................................129
Answers to test tasks ............................................................................................141
Answers to case studies ........................................................................................143
Recommended literature .....................................................................................153
Annexes ..................................................................................................................149

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 Educational edition

LABORATORY MANUAL FOR PRACTICAL BIOCHEMISTRY

Tutorial

Authors:
NOSAREVA Olga L.
STEPOVAYA Elena A.
FEDOROVA Tatyana S.
TIMIN Oleg A.
SHAKHRISTOVA Evgenia V.
SPIRINA Liudmila V.
SEREBROV Vladimir Yu.

Editorial and publishing department Siberian State Medical University

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Tel. 8(382–2) 51–41–53
Е-mail: [email protected]
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Signed in print 23.01.2019 г.
Format 60х84 116 . Offset paper.
Print the risograph. Headset «Times». Printed paper 11.
Edition 100 экз. Оrder № 1
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Printed in the laboratory of operative polygraphy of SibGMU
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