DANYLO HALYTSKY
LVIV NATIONAL MEDICAL UNIVERSITY
DEPARTMENT OF BIOLOGICAL CHEMISTRY
BIOLOGICAL CHEMISTRY
METHODICAL INSTRUCTION FOR PRACTICAL EXERCISES
Part ІI
Lviv – 2008
Methodical instruction prepared by:
Prof. Sklyarov A.Ya., M.D., Ph.D.
Prof. Lutsik M.D., Ph.D.
Fomenko I.S., Ph.D.
Mayor Ch., MSc
Editor: Prof. Sklyarov A.Ya., M.D., Ph.D.
2
MODUL 4. MOLECULAR BIOLOGY. BIOCHEMISTRY OF
INTERCELLULAR COMMUNICATIONS
Thematic plan of practical lessons in modul 4
№
1.
2.
3.
4.
5.
6.
7.
8.
9.
Theme of the lesson
Investigation of biochemical composition and biosynthesis of
purine and pyrimidine nucleotides. Biochemical function of
nucleotides and nucleic acids.
Catabolism of purine nucleotides, determination of end
products of their metabolism. Hereditary disorders of
nucleotide metabolism.
Replication of DNA and transcription of RNA. Analysis of
mutation mechanisms and DNA reparations. Principles of
recombinant DNA technology, transgenic proteins.
Biosynthesis of proteins in ribosomes, initiation, elongation
and termination in synthesis of polypeptide chain. Inhibiting
effect of antibiotics. To learn principles of gene engineering
and gene cloning, their application in modern medicine.
The investigation of molecular mechanisms of the effect of
hormones of protein and amino acid origine upon target cells.
Humoral regulation of calcium homeostasis.
Investigation of molecular and cellular mechanisms of steroid
and thyroid hormones action upon target cells.
Investigation of nerve tissue. Pathochemistry of psychotic
disorders.
Biochemistry of muscle tissue. Investigation of mechanism of
muscle contraction.
Independent individual students work
Summary leson, control work
Totally
Hours
Points
number
3
14
3
14
3
14
3
14
3
14
3
14
3
14
3
14
3
27
8
80
200
3
Topics for independent students work
№
1.
Topic
Hours
Preparation for the practical study:
To get a practical skills in molecular biology and genetics:
To draw the schemes of the successive stages of processes of
1
replication, transcription and translation.
1.1 To explain the molecular mechanisms of regulation in the
1
realization of genetic information.
To estimate the innate vices of metabolism (molecular illnesses) as
1
a result of genetic damages and point mutations.
To get a practical skills in biochemistry and molecular biology of the hormonal
regulation:
To write the structural formulas of hormones – derivatives of
1
amino acids and steroid hormones.
To explain molecular and cellular mechanisms of actions peptide,
1.2
1
steroid, thyroid, amine-derived hormones.
To estimate violation of metabolism at insufficient and surplusive
1
formation of hormones.
To estimate the changes of homeostasis of calcium at a hormonal
1
disbalance.
Individual independent students work (individual task) – after the
2.
1
themes of abstracts.
3. Preparation to summary control of mastering of the module 4.
2
Totally
10
The assumption of theme is evaluated according to traditional system as
follows: “5” 14 points, “4” 10 points, “3” 5 points, “2” 0 points.
Maximal points number for current learning activity of student 120.
Student obtains permission for participance in summary module control after
fulfillment of educational program and in a case when he (she) achieved for current
learning activity points number not less then 40 =(5 x8=40) points.
The summary module control is accepted if the student shows the knowledge of
practical methods of investigation and achieved in control of theoretical level points
number not less then 50.
4
Sense module № 12. Principles of molecular biology.
Topic № 1. Investigation of biochemical composition and biosynthesis of purine
and pyrimidine nucleotides. Biochemical function of nucleotides and nucleic
acids.
Objective: to learn chemical structure of nucleoprotein compounds, structure
and function of nucleic acids, their significance in template synthesis. To perform
qualitative reactions for detection of nucleoprotein compounds.
Actuality of the theme: Nucleoproteins are complex proteins in which
nonprotein part is presented by nucleic acids (DNA and RNA). DNA which is
localized in a nucleus stores genetic information about the features of structure of
whole organism. The different types of RNA play an important role in the protein
biosynthesis.
Specific objectives:
 To interpret chemical structure of nucleoprotein compounds, structure and
function of nucleic acids, their role in protein biosynthesis.
 To know methods of isolation of nucleoproteins from tissues and qualitative
reactions for detection of their components: a) biuret test for polypeptides, b)
Trommer test for sugars (pentoses), c) silver probe for purine bases, d)
molybdenum probe for phosphates.
1.
2.
3.
4.
5.
6.
7.
8.
Theoretical questions
Biochemical functions of nucleic acids and nucleotides. Formation of nucleic acid
chain from nucleotides.
Constituents of nucleotides and nucleosides. Minor nitrogenous bases and
nucleotides.
Free biologically active nucleotides and their biochemical functions: participance
in metabolic reactions (ATP, NAD, NADP, FAD, FMN, CTP, UTP) and in their
regulation (cyclic nucleotides – cAMP, cGMP).
Nucleic acids: structure, properties, stages of investigation. Primary structure of
nucleic acids, polarity of polynucleotides, specific features of DNA and RNA
structure.
Structure, properties and biological significance of DNA. Experimental proves of
DNA significance in heredity (phenomenon of transformation). Molecular mass,
dimensions and nucleotide composition of DNA molecules of viruses, prokaryots
and eukaryots.
Secondary structure of DNA, role of hydrogen bonds in stabilization of secondary
structure (Chargaff rules, Watson-Crick model), antiparallelism of chains.
Tertiary structure of DNA. Physico-chemical properties of DNA: interaction with
cationic ligads; hyperchromic effect ; denaturation and renaturation of DNA.
Structure, properties and biological functions of RNA. Types of RNA: mRNA,
tRNA, rRNA, snRNA; specific features of structure (secondary and tertiary) of
different RNA types.
5
9. Molecular organization of nuclear chromatin and ribosomes of eukaryots.
Chromatin: nucleosomes, histones and non histone proteins. Ribosomes: subunit
structure, protein and RNA composition.
Practical part
Qualitative reactions on structural components of nucleoproteins.
Principle of the method. For investigation of chemical composition of
nucleoproteins yeast cells are a convenient object. After hydrolysis the components
of nucleoproteins can be detected in hydrolysate with reactions, which are specific to
each constituent of nucleoproteins.
Reagents and materials. 500 mg of fresh yeasts or 100 mg of dried yeasts, 10%
solution of sulfuric acid, pipettes, filter paper, water bath.
Method. Into large tube (15 x 1.5 cm) 500 mg of baker yeast or 100 mg of dried
yeast are placed into the tube, 4 ml of 10% H2SO4 are added. The tube is closed with
plug with glass tube of 25-30 cm length, which serves as a cooler and placed on
boiling water bath. After 1-1.5 h of boiling the tube is cooled and the liquid is
filtered. In the filtrate products of nucleoproteins are detected, i.e: polypeptides,
purine and pyrimidine nitrogenous bases, ribose, deoxyribose, phosphoric acid.
Draw a conclusion.
Experiment 1. The biuret probe for polypeptides.
Principle of the method. Peptides and proteins make a complex with copper
sulfate in alkaline medium, which has red-violet color.
Performance. To 5 drops of hydrolysate 10 drops of 10% NaOH and 1 drop of
1% CuSO4 are added. The liquid becomes pink-violet.
Experiment 2. The silver probe for purine bases.
Principle of the metod. Purine bases are precipitated with silver nitrate in
alkaline medium, sediment possess light brown color.
The chemical equation for this reaction is:
Performance. 10 drops of the hydrolysate is neutralized with concentrated
ammonia and 5 drops of 1% AgNO3 are added. After3-5 min a small light-brown
precipitate of silver salts of purine bases (adenine, guanine) is formed.
Draw a conclusion.
Experiment 3. The Trommer probe for ribose and deoxyribose.
Principle of the method. Sugars with free hemiacetal group possess reductive
properties and reduce some metal ions with changes in color or other effects. In
Trommer test copper ion (blue) is reduced to cuprous ion (orange red) in presence of
reductive sugars (ribose, deoxyribose).
6
Performance. To 5 drops of hydrolyzate 10 drops of 30% NaOH and 2-5 drops
of 7% CuSO4 are added up to the appearance of Cu(OH)2. sediment. The liquid is
mixed and warmed to boiling. Red precipitate of cuprous oxide falls down (in
consequence of ribose oxidation and reduction of cupric hydroxide to cuprous
oxide).
Draw a conclusion.
Experiment 4. Diphenylamine reaction for ribose and deoxyribose.
Principle of the method. Diphenylamine interacts with pentoses and forms
compounds of blue-green color. Sugars with free hemiacetal possess reductive
properties and reduce some metal ions with changes in color or other effects.
Explain the results.
Performance. To 5 drops of the hydrolyzate 20 drops of 1% diphenylamine are
added and boiled in water bath during 15 min. Appear of blue-green color indicates
on a presence of pentoses (ribose and deoxyribose in case of nucleic acids).
Experiment 5. Molybdenum probe for phosphoric acid.
Principle. Phosphates in acidic medium
forms with molibdates
phosphomolibdenum complexes of intense yellow color.
A molybdenum test on phosphatidic acid:
H3PO4 + 12 (NH4)2MoO4 + 21 HNO3
(NH4)3PO4MoO2 + 21 NH4NO3 + 12 H2O
Performance. To 10 drops of molybdenum reagent (a solution of ammonium
molybdate in nitric acid) 5 drops of the hydrolyzate are added and boiled several
minutes on the fire. In presence of phosphoric acid the liquid becomes lemonyellow. After the cooling yellow crystals of ammonium phosphomolybdate complex
appears as yellow precipitate. In a conclusion attention is accented on formation of
complex connection of ammonium of ammonia phosphate molybdenum.
Explain the results.
Clinical diagnostic significance. Analysis of DNA is a routine investigation in
diagnostics of hereditary diseases. It may be used for genotyping of fetal tissue in
prenatal diagnostics, as well as in paternity determination.
Derivatives of nitrogenous bases are widely used in practical medicine.
Mercaptopurine posses an antileukemic activity, as it serves as purines structural
analog and antimetabolite. Fluorouracil and fluorofur also has antitumor activity , as
they are transformed into 5-fluoro-2-deoxyuridine 5’-monophosphate, which is a
strong inhibitor of thymidylate synthase.
Control of laboratory work fulfillment
1.
What is complete and incomplete hydrolysis of nucleoproteins?
2.
Indicate the products of nucleoproteins hydrolysis.
3.
What products of hydrolysis could be reveald by Tromer test?
4.
What products of hydrolysis can show a positive biuret reaction?
5.
The new-born in the period of suckling do not get nucleic acids with milk, but
they grow intensively, the amount of cells is increased, continuously goes
hemopoiesis, synthesis of proteins, other processes which need iРNA, tRNA, rRNA,
DNA. What process provides rise in nucleic acids countent?
7
6. In two preparations of DNA the content of adenine is accordingly 25 and 12 %
from general amount of nitrogenous bases. Calculate subsequent quantity of thymine,
cytosine and guanine in these DNA preparations.
Examples of tests „Crock-1”
1. RNA of AIDS virus penetrated into leucocyte and using the enzyme revertase
caused a synthesis of viral DNA in the cell. This process occures due to...
А. Reversal transcription
B. Derepression of operon
C. Repression of operon
D. Convariant replication
E. Reversal translation
2. From nitrates, nitrites and nitrosamines in organism is formed nitrous
acid which causes
oxidative
deamination
of
nitrogene
bases
of
nucleotides. This induce a point mutation by replacement of cytosine to...
A. Thymine
B. Uracil
C. Adenine
D. Guanine
E. Inosine
3. Nitrosamines belong to deaminating mutagens. From what nitrogene base does
uracyl appears as a result of their action?
A. Cytosine
B. Adenine
C. Guanine
D. Thymine
E. Methyluracil
Independent individual students work
1.
Structure, properties and biological significance
of nucleoproteins,
phosphoproteins, lipoproteins, glycoproteins
2.
Peculiarities in synthesis and in degradation of nucleoproteins, glycoproteins
and proteoglycans.
1.
2.
3.
4.
5.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
8
6. Biological chemistry. Methodical instructions for practical exercises. Lviv-2002,
P.1, P.29-33.
Topic № 2. Catabolism of purine nucleotides, determination of end products of
their metabolism. Hereditary disorders of nucleotide metabolism.
Objective: To learn reactions of synthesis and degradation of purine and
pyrimidine nucleotides in normal conditions and in hereditary enzymopathias of this
metabolic pathways. To learn the method of determination of uric acid in biological
fluids and to interpret obtained results.
Actuality of the theme: Disorders in processes of biosynthesis and catabolism
of purine or pyrimidine bases and nucleotides can cause a development of such
diseases as a Lesch-Nyhan syndrome, gout, orotate aciduria. Knowledge of basic
metabolites and enzymes of these processes is necessary for diagnostics and
monitoring of treatment.
Specific objectives:
 To analyze the sequence of reactions of biosynthesis and catabolism of purine
nucleotides, disorders in uric acid metabolism and biochemical principles of gout
development.
 To analyze the sequence of reactions of biosynthesis and catabolism of pyrimidine
nucleotides.
 To conduct quantitative determination of uric acid in biological fluids and to
interpret obtained results.
1.
2.
3.
4.
5.
Theoretical questions
Biosynthesis of purine nucleotides; scheme of reactions of IMP synthesis;
synthesis of AMP, GMP, ATP, GTP. Regulation of purine nucleotides
synthesis on a princip of feedback inhibition.
Biosynthesis of pyrimidine nucleotides: reactions, regulation.
Biosynthesis of deoxyribonucleotides. Formation of thymidyl nucleotides;
inhibitors of dTMP biosynthesis as antitumor drugs. (structural analogs of
dTMP, pterine derivatives).
Catabolism of purine nucleotides; hereditary disorders of uric acid metabolism.
Biochemical background of hyperuricemia, gout, Lesch-Nyhan syndrome.
Catabolism of pyrimidine nucleotides.
Practical part
Experiment 1. Quantitative determination of uric acid in blood serum.
Principle of the method. Uric acid reduces phosphotungsten reagent with
formation of blue color compound. The absorbance of light in 640 nm wavelength
(optical density) is proportional to the concentration of uric acid in the tested
specimen.
Reagents. Blood serum, 10% solution of sodium dihydrogentungstate, 10%
solution of sodium carbonate, 0,35 M sulfuric acid, phosphotungstic reagent (Folin
reagent), 30 μM solution of uric acid.
10
Performance. 0,5 ml of blood serum is put into centrifuge tube and 4 ml of
water are added. Then is added 0,25 ml of 0,35 M sulfuric acid and 0,25 ml 0f 10%
solution of sodium dihydrogentungstate. After 5 min the tube is centrifuged 10 min at
3000 rpm. A supernatant is collected and the concentration of uric acid is determined
according to the scheme, as shown in a table.
Determination of uric acid in blood serum
Control
Standard sample
Test sample
Supernatant (ml)
-
-
2
Standard solution of uric acid
(ml)
-
2
-
Distilled water (ml)
2
-
-
Sodium carbonate solution
(ml)
1
1
1
Phosphotungstic reagent
0,5
0,5
0,5
After 30 min the optical density of probes is measured in colorimeter on
wavelength 640 nm (red filter, 590-700 nm).
Calculation of uric acid concentration is conducted according to the following
formula:
C= Dt/ Ds x 30 x 10 = Dt/ De x 300, where:
C – concentration of uric acid in a blood serum sample, in mmoles/L
Dt – optical density of the test sampleDs – optical density of uric acid standard
30 – concentration of uric acid in standard , μ moles/L
10 –dilution of serum sample
Explain the tesults.
Normal values of uric acid content in blood serum are 240-500 μ moles/L in males
and 160-400 μ moles/L in females.
Experiment 2. Quantitative determination of uric acid in urine.
Principle of the method is based on the property of uric acid to reduce
phospho- tungstic complex anion to a blue color compound. The quantity of phosphotungstic blue compound is determined by titration with potassium ferricyanide,
which oxidizes phosphotungstic complex and decolorize it.
Reagents. Urine, phosphotungstig reagent (Folin reagent), 20% solution of
sodium carbonate, 0,01 n solution of potassium ferricyanide ( K3[Fe(CN)6]), stadard
solution of uric acid (0,5 mg/ml), microburette.
Performance. To 1.5 ml of urine and to 1,5 ml of standard solution of urate in
small flasks 1 ml of 20% sodium carbonate (Na2CO3) solution and 1 ml of Folin
reagent are added to each flask. The content of flasks is titrated with 0.01 n
K3[Fe(CN)6] solution up to disappearance of blue color.
11
The content of uric acid (in mg) in daily urine is calculated according to
formula:
X = 0,75 x B x D / 1.5 x C (mg/day),
where: 0,75 – amount of uric acid in standard, mg; B - volume of K3[Fe(CN)6] used
for titration of urine sample, ml; C - volume of K3[Fe(CN)6] used for titration of
standard sample, ml; D – - daily diuresis, ml.
Draw a conclusion.
Clinical and diagnostic significance. Normal value of uric acid excretion with
urine is 1.60 - 3.54 mmoles/day (270 - 700 mg/day). The normal values of uric acid
concentration in blood serum is 0,05-0,06 mg/ml in males and 0,04-0,05 mg/ml in
females. The increase of excretion of uric acid is observed in cases:of diseases with
an enhanced breakdown of nucleoproteins (leukoses, treatment with cytostatic drugs,
action of ionizing radiation, combustion, rheumatism, hemolytic anemia, lead
intoxication, toxicosis etc.). consumption of nutrients with high content of purines .
A decrease of uric acid excretion is observed in kidney diseases (nephritis, renal
insufficiency), progressive myodystrophie.
An increase in uric acid concentration in blood is called hyperuricemia. It is
observed in gout, a disease , caused by hyperproduction of urates and their deposition
in tissues, first of all in synovial sheets of joints. Deposition of uric acid is due to its
poor solubility in water media. In treatment of gout are used drugs, which inhibit
production of uric acid (allopurinol) or stimulate its excretion with urine (anturan,
cinhophen). In patients suffering from gout the concentration of uric acid in blood is
higher then 0,075-0,08 mg/ml, in acute phases of disease its concentration is not less
then 0,08-0,09 mg/ml .
Control of laboratory work fulfillment
1. What method is used for determination of uric acid in urine?
2. What is the normal content of uric acid in blood and in urine of healthy adult?
3. Uridine derivative – fluorouracil- is transformed in the cell to the
fluorodeoxyuridildiphosphate, which is strong inhibitor of thymidylate
synthase. How can be explained a suppression of rapid division of cancer cells
by fluorouracil in experimental animals?
Examples of tests „Crock-1”
1. In urine of monthly child was detected the enchanced quantity of orotic acid. A
child gains body mass badly. What substances should be used for correction of
metabolism?
A. Uridine
B. Adenosine
C. Guanosine
D. Thymine
E. Histidine
2. Formation of thymidine nucleotides, which are used for the biosynthesis of
DNA, begins from dUDP, which on the first stage is hydrolised to dUMP, and
thereafter methylated. What compound serves as the donor of methyl groups?
A. Lecithin
12
B. Choline
C. Methylenetetrahydrofolate
D. Methionine
E. Carnitine
3. In the reaction of transformation of ribose into deoxyribose during formation of
deoxyribonucleotides, which are used for synthesis of DNA, takes part the low
molecular weight protein thioredoxin. It contains two SH-groups which can be
oxidised to disulphide form. What coenzyme is used for reducing thioredoxin?
A. Coezyme Q
B. Pyridoxal phosphate
C. NADPH
D. NADH
E. AMP
4. A person in age of age 60 had surgery on prostate cancer. In 2 months he was
treated by course of chemotherapy. In the complex of medical preparations 5fluorodeoxyuridine was used as an inhibitor of thymidine synthetase. Synthesis of
what compound is blocked by drug?
A. iRNA
B. rRNA
C. tRNA
D. DNA
E. Protein
1.
2.
1.
2.
3.
4.
5.
6.
Individual independent student’s work
Peculiarities of biosynthesis and degradatyion of purine and pyrimidine
nucleotides in health and disease.
Role of adenyl nucleotides in regulation of enzymatic activity.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey.
Lippincott′s Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv2004, P.III, p. 67-68.
2
Topic № 3. Replication of DNA and transcription of RNA. Analysis of mutation
mechanisms and DNA reparations. Principles of recombinant DNA technology,
transgenic proteins.
Objective: To learn principles of template synthesis of nucleic acids, stages of
these processes, mechanisms of mutations and reparations of damaged DNA,
development of hereditary diseases. To interpret mechanism of action of antibiotics
and other inhibitors of nucleic acids synthesis. To conduct quantitative determination
of DNA in biological material.
Actuality of the theme: In the process of nucleic acids biosynthesis may
occure various disorders in nucleotide sequence under the action of physical
(ionizing and corpuscular irradiations), chemical (mutagens) and biological (viruses)
agents. Pharmaceutical preparations are widely used in medical practice that can
inhibit biosynthesis of nucleic acids in eukaryots and retard tumour cells proliferation
in oncologic patients.
Specific objectives:
 To learn the method of quantitative determination of DNA in biological material.
 To interpret molecular mechanisms of storage and transfer of genetic information,
role of enzymatic systems, which prowide semiconservative mechanism of DNA
replication in prokaryots and in eukaryots.
 To explain mechanism of action of enzymatic system of RNA transcription.
 To interpret mechanisms of regulation of genes expression on level of operon
transcription, which include structural and regulatory genes, promotor and
operator.
 To interpret biochemical mechanisms of genes recombination, genes
amplification, specific features of regulation of gene expression in eukaryots.
 To analyze consequences of genom, chromosome and gene mutations, mechanism
of action of the most known mutagenes, biological significance and mechanisms
of DNA reparations (reparation of UV-induced gene mutations).
Theoretical questions
1. Biological significance of NDA replication. The sense of J.Watson and F.Crick
discovery (1953). Semiconservative mechanism of replication, the scheme of
Meselsohn and Stahl experiment.
2. General scheme of DNA synthesis. Enzymes of DNA replication in prokaryots
and eukaryots. Molecular mechanisms of DNA replication: topoisomerases,
helicases, the significance of antiparallelism of DNA strains, Okazaki fragments.
Stages of synthesis of dauther chains of DNA.
3. General scheme of transcription. Coding and noncoding DNA chains. RNA
polymerases of prokaryots and eukaryots. Stages and emzymes of RNA
synthesis. Markers of transcription: promotor, initiator, tetrmination segments of
genome.
4. Processing as posttranscriptional modification of RNA. Antibiotics, which
inhibit transcription.
5. Regulation of gene expression in prokaryots: scheme of regulation according to
F.Jacob and J.Monod. Structure of Lac-operon of E.coli structural and regulatory
genes, promotor, operator, regulatory gene and production of protein repressors,
repression and induction of Lac-operon function.
14
6.
7.
8.
9.
10.
11.
12.
13.
Peculiarities of structure and function of eukaryot genom. Molecular
organization of DNA in eukaryots (exones, intrones, repeated sequences).
Nuclear chromatin and chromosomes of eukaryots, human karyotype.
Genetic recombinations; transpozones. Recombinations in prokaryot genom
(transformation, transduction, conjugation). Recombination processes in
eukaryots on an example of formation of genes of H- and L immunoglobulin
chains.
Gene amplification (genes of metallothioneins, dihydrofolate reductase).
Regulation of gene expression on transcriptional level; systems of transcriptional
markers – promotor sequences, enhancers, attenuators, silensers. Covalent
modification of histones and non histone proteins as one of the mechanisms of
gene expression.
Phases of cell cycle of eukaryots. Biochemical mechanisms of control of cell
mitosis; cyclins and cyclin dependent kinases.
Mutations; genomic, chromosomal, gene (point mutations), their significance in
appearance of enzymopatias and human hereditary diseases.
Biochemical mechanisms of action of chemical mutagenes – analogs of
nitrogenous bases, deaminating, alkylating agentsultraviolet and ionizing
radiation.
Biological significance and mechanisms of DNA reparation. Reparation of UVinduced mutations, xeroderma pigmentosum.
Practical part.
Experiment 1. Determination of DNA on basis of phosphorus content in a
sample.
Principle of the method. The method consists on determination of phosphorus
content as phosphate anion produced after mineralisation of DNA specimen with
concentrated sulfuric acid. The quantity of phosphorus is determined by ammonium
molybdate in the presence of reducer (ascorbic acid). The product of reaction has a
blue color, its intensity is proportional to quantity of phosphorus in a sample.
Performance. The isolation of DNA. 100 mg of liver are heated with 1 ml of 1
M sol of NaOH during 15 min in a boiling water bath. The solution is cooled to O oC
and 0,5 ml of saturated NaCl in 20% acetic acid is added for precipitation of protein.
The sediment is eliminated by centrifugation. To a supernatant 6 ml of alcohol are
added and the mixture is placed in a cold for an hour in order to precipitate DNA.
Sediment is collected by centrifugation. The pellet of DNA is washed by
trichloroacetic acid, thereafter it is quantitatively transferred to Kjeldahl flask, 1,5 ml
of conc. sulfuric acid are added and the mixture is heated on a sand bath for several
hours . At the end of mineralization several drops of 30% H 2O2 are added for
decolorisation of solution. The resulting solution is neutralized with 30% solution of
NaOH and adjusted to a final volume 50 ml in a graduated cylinder.
Determination of phosphorus.
From obtained mineralizate 5 ml of sample are transferred to a tube, 0,5 ml of
5% sol of (NH4)2MoO4 and 0,5 ml of 1% sol of ascorbic acid are added. Five min
later to the tube are added 2 ml of carbonate-sulfite mixture and the volume is
adjusted to 10 ml. After 10 min the optical density (extinction) is measured on a
15
photocolorimeter at a red filter in 5 mm cuvette. The content of phosphorus is
determined according to calibration curve..
Concentration of DNA in mg% is calculated according to formula:
CDNA = a x 10 mg%,
where a –is a concentration of P in mg/ml. The normal content of DNA in rat liver
is 25-35 mg%
Explain the results.
Diagnostic and practical application. Determination of DNA content in tumor
tissues has some prognostic significance in treatment of patients. More important is
investigation of DNA in samples of biological origin (tissue bioptates, blood cells,
spermatozoa, sediment obtained from urine, human hair, etc.) by the method of
polymerase chain reaction (PCR) in diagnostics of viral, hereditary diseases and in
identification of person (DNA diagnostics). The content of DNA is used also for
control the process of purification of subcellular fractions (microsomes, lysosomes,
Golgi vesicles etc.).
Control of laboratory work fulfilment
1. Mineralized DNA interacted with solution of ammonium molybdate and gave a
positive reaction – molybdenum blue. What component of DNA gives a positive
reaction?
A. Purine bases
B. Pyrimidine bases
C. Purine nucleosides
D. Pyrimidine nucleosides
E. Phosphate anion
2. On one of the stages of determination of DNA upon phosphorus are used 1 %
solution of ascorbic acid. In what type of chemical reaction does it takes part?
A. Oxidation
B. Reduction
C. Hydrolysis
D. Mineralization
E. Formation of complexes
3. Uridine derivative – fluorouracil, which transforms in a cell to fluorodeoxiuridylat
is strong irreversible inhibitor of thymidine synthetase. How to explain the inhibition
of rapid proliferation of cancer cells in experimental animals by fluorouracil?
4. In a patient was recognized a xeroderma pigmentosum. His skin is extraordinarely
sensitive to sunlight. Explain the cause of appearence of this pathology. The
consequence of inherited defect in synthesis of what enzyme is this disease?
5. What is the method of DNA purification based on?
6. What is the sense of DNA mineralization?
7. What is a principle of DNA determination by estimation of phosphorus?
8. What reagents are used for protein precepitation from cooled hydrolyzate?
16
9. What reagent and in what conditions the complete sedementation of DNA takes
place?
10. Indicate a final product of reaction, which is formed in a DNA detection by
phosphorus?
11. What is a clinical and diagnostical significance of DNA determination in
bioptate material?
Examples of tests „ Crock–1”
1. From nitrates, nitrites and nitrosamines in organism is formed nitrous acid which
induces oxidative deamination of nitrogenous bases nucleotides. It can result in a
point mutation by changing of cytosine into..
A. Thymine
B. Uracil
C. Adenine
D. Guanine
E. Inosine
2. A patient suffers from AIDS. RNA of AIDS virus invades leucocyte and using the
enzyme revertase cause synthesis of viral DNA in the cell. This occurs due to the
next process...
А. Reversal transcription
B. Derepression of operon
C. Repression of operon
D. Convariant replication
E. Reversal translation
3. For the treatment of urogenital infections are used quinolones – inhibitors of
enzyme DNA-gyrase. What process is altered first of all under the action of
quinolones?
A. Gene amplification
B. Replication
C. Reverse transcription
D. Reparation
E. Gene recombination
4. Salts of mercury were acidentally introduced to human body. It resulted the
increase in frequency of transcription of gene, providing heavy metals detoxication.
Gene amplification of of what protein takes place in this case?
A. Metallothionein
B. Ceruloplasmin
C. Interferon
D. Transferrin
E. Ferritin
9
5. The structural analysis of haemoglobin in patient suffering from anaemia revealed
a replacement of 6–Glu to 6–Val in β–chain. What is the molecular mechanism of
this pathology?
A. Gene mutation
B. Chromosome mutation
C. Genom mutation
D. Gene amplification
E. Gene transduction
1.
2.
3.
4.
5.
6.
Individual independent work of students
1. Modern methods of DNA and RNA investigation, their clinical significance.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.III, p. 70-78.
Sense module № 13. Bases of molecular genetics.
Topic № 4. Biosynthesis of proteins in ribosomes, initiation, elongation and
termination in synthesis of polypeptide chain. Inhibiting effect of antibiotics. To
learn principles of gene engineering and gene cloning, their application in
modern medicine.
Objective: To learn general principles of protein synthesis, stages of this
process, possible mechanisms of appearance and development of hereditary diseases.
To interpret the mechanisms of antibiotics action as well as other inhibitors of protein
synthesis. To know principles of gene engineering and gene cloning, its application in
modern medicine. To learn principles of the method of polymerase chain reaction
(PCR) ,its application in express diagnostic.
Actuality of the theme: Proteins are genetically determined systems which is
genetically programed with a specific set of individual protein molecules. While
studing this theme special attention should be payed on recent achievements of gene
engineering, on gene cloning, that it is important for the study of both nucleotide
sequence of probed gene and sequence of mRNA and protein, which are encoded by
this gene. Due to gene engineering technology the productionof interferon, human
insulin, somatotropin, somatostatin, protein preparations for diagnostics of AIDS is
achieved. In particular, in recent years in diagnostics of many diseases and detection
of bacilli-cariers is used an special method – polymerase chain reaction.
Specific objectives:
 To interpret the conception of protein synthesis system in ribosomes.
20
 To explain mechanisms of action of protein synthesis system by involvement of
enzymes activating amino acids, initiation, elongation and termination of
polypeptide chain biosynthesis.
 To explain biochemical processes of posttranslational modification of proteins.
 To explain the influence of physiologically active substances and antibiotics on
translation.
 To explain biochemical and molecular biology principles of methods used in
recombinant DNA technologies, transplantation of genes and formation of hybrid
DNA molecules.
 To explain principles of gene cloning for production of drugs with biotechnologies
methods.
Theoretical questions
1. The genetic code, triplet structure, its properties. Table of genetic code.
2. Protein synthesis system of ribosomes. Components of protein synthesis system.
3. Transfer RNA and amino acid activation. Aminoacyl-tRNA synthetases, second
genetic code.
4. Stages and mechanisms of translation: initiation, elongation, termination.
Initiating and terminating codons of mRNA. The role of protein factors of
ribosomes in translation.
5. Post-translational modification of polypeptide chains. Regulation of translation.
Molecular mechanisms of translation control on example of globin synthesis.
6. The influence of biologically active compounds on translation. Antibiotics as
inhibitors of transcription and translation in prokaryots and eukaryots, their
biomedical application.
7. Biochemical mechanisms of antiviral activity of interferons. Block of protein
synthesis by diphteria toxin (ADP-ribosylation of translation factors).
8. Gene engineering or recombinant DNA technology: general principles, biomedical
significance.
9. Technology of gene transplantation and formation of hybrid DNA molecules. The
employment of restrictases. Gene cloning for obtaining of medicinals and
diagnostic tools using methods of biotechnology (hormones, enzymes, antibiotics,
antigens, interferons etc.).
10.Polymerase chain reaction, its biomedical application in diagnostics of contagious
and hereditary diseases , identification of person (DNA-diagnostics).
Practical part
Experiment № 1. The polymerase chain reaction (PCR)
A major difficulty in gene manipulation has been that any given gene makes up
an exceedingly small part of an organism’s total genome. It is estimated that a human
being has between 50,000 and 100,000 genes. Early techniques used to find a
particular gene and clone it were very time consuming for routine use:
oligonucleotides often hybridize with DNA sequences other then their complements;
the use of restriction endonucleases to cleave DNA also required the use of
electrophoretic separation of the fragments to achieve some degree of purification of
20
the desired gene; and gene cloning using plasmids and dideoxysequencing also
required large amount of time.
In contrast, the polymerase chain reaction technique (refered now simply as
PCR) developed in the mid-1980s by Kary B. Mullis and colleagues has overcome
these difficulties. In the space of hours, this cell free reaction can make copies of an
target DNA sequence in quantities that would take days or weeks using the earlier
amplification techniques such as gene cloning. For example, with 25 cycles of the
PCR, the target sequence can be amplified about 8 million times (Table 1) and within
a few hours billions of copies can be made.
An important feature of the PCR is that the target DNA need not be isolated
from the rest of its genome. But, once completed, the PCR yields sequences that can
easily be separated from the rest of DNA by gel electrophoresis. Also important is
that the starting material containing the desired sequence can be as little as the
amount of DNA contained in a single cell. Essentially, all that is required to begin the
PCR procedure is to know or determine the nucleotide sequences flanking either side
of the double-stranded target region. Primers of about a dozen nucleotides
complementary to the 3’ ends of the flanking sequences are made. (Remember, DNA
is synthesized in the 5’ to 3’ direction, and DNA polymerase requires a primer).
The PCR uses the normal enzyme of DNA synthesis – the DNA polymerase –
but the enzyme is derived from an unusual source. The PCR polymerase is isolated
from the bacterium Thermus aquaticus, which lives in water at temperatures of about
75 oC, a temperature that would detature most proteins. This enzyme, called Taq
polymerase has an optimum temperature of 72 oC and reasonably stable at 94 oC.
(Keep this temperatures in mind as we describe the PCR). The PCR is a cyclical
reaction; at the completion of the reaction the system automatically returns to the first
step and repeats itself. The cycling continues until a substrate is completely used up
or the reaction is
stopped.:
Table 1. PCR ampilification of DNA fragment
Cycle number
Number of double
stranded molecules
1
()
2
()
3
2
4
4
5
8
6
16
7
32
8
64
9
128
10
256
11
512
12
1024
13
2048
14
4096
15
8192
21
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,544,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
Samples of DNA required for the PCR are obtained from specimens of tissues
(bioptates, nucleated blood cells, epithelial cells from skin or mucosa etc.). The
isolation of DNA is performed with the use of phenol or guanidine method. Reagents
are supplied with special kits.
The steps of the PCR are as follows:
1. The target DNA sequence, two primers, DNA polymerase, and a mixture of the
triphosphate deoxynucleotides are mixed in a total volume of about 100 μl
2. The mixture is heated to 94 oC for about 5 minutes to separate the strands of
the double-stranded DNA. The single strands become templates for the
primers.
3. The temperature is lowered to between 30o and 65 oC to allow the primers to
bind to the templates. The temperatures and times used in this step, the
annealing step, vary. They depend on the sequence to be amplified. Some
sequences require higher temperatures and longer times then others.
4. Once the DNA single strands and primers are bound, the temperature is raised
to 72 oCto allow synthesis of new strands by Taq polymerase starting at each
primer. This step requires 2-5 minutes.
5. The temperature is raised to 94 oC once again to separate the strands of the
newly synthesized double-stranded DNA. And the step 3 begins again.
Note, that at the completion of each cycle the end products of the reaction become
the substrates or templates for the next cycle. As more and more cycles are
completed, more and more end product or substrate is generatedso that each cycle
doubles the amount of amplified DNA (Table 1), which is then the template for
the next round of DNA synthesis.
After the completion of the reaction the synthesized DNA is detected by
electrophoresis in agarose gel, bands of DNA are detected with a specific fluorescent
22
dye - ethidium bromide- which greatly enhances its fluorescence after binding with
DNA. The gels are irradiated with UV light of 310 nm.
Clinical and diagnostic significance. The PCR in medical investigations is
used for diagnoscics of contagious diseases by identification of pathogenic bacteria
and viruses in human biological fluids. For every infection are required primers,
specific to a distinct strain of bacteria or virus. The PCR permits to detect one distinct
molecule of DNA among millions of othe DNA molecules. The PCR is widely used
in early diagnostics of AIDS, viral hepatitis, as well as for evaluation of the
efficiency of treatment and for monitoring of disease.
The PCR is employed in detection of hereditary diseases (e.g. phenylketonuria,
hemophilia etc.), in “molecular dactyloscopy” in forensic medicine (determination of
paternity), determination of the sex of fetus on the 10 week of pregnancy, in
oncology etc.
Control of laboratory work fulfilment
1. In modern biochemical investigations for diagnostics of inherited diseases, detection of
certain viruses (for example HIV), authentification of individual (gene dactylography in
forensic medicine) so called "DNA-diagnostics" is imployed. What method is used in these
investigations?
A. Electrophoresis
B. Chromatography
C. Polymerase chain reaction
D. Rentgen-structural analysis
E. Electron microscopy
2. Tetracyclines are antibiotics of wide spectrum of action which are inhibitors of
protein synthesis on 70 S ribosomes of prokaryot, not influencing on 80 S ribosomes
in eukaryots. Ribosomes of eukaryotic mitochondria have similar structure to
prokaryotic ribosoms (70 S). Using these information, explain the toxic effect of
tetracyclines.
3. After the longterm exhausting illness and surgery interference patient has a sharp
loss of body mass and delay of convalescence process. Doctor prescribed him
anabolic preparation. What biochemical process are stimulated by such preparations?
4. What does it mean ”clinical sample” for PCR?
5. What methods are usually used for DNA isolation?
6. What is a principle of polymerase chain reaction?
7. Name the composition of reaction mixture for PCR.
8. Describe three stages of cycle of synthesis ( amplification) of specific segment of
DNA–target.
9. For what purpose is primer used in PCR?
10. On what the selection of the primer system for diagnostics of every infection is
based?
11. What method is used for identification of products of amplification?
12. On what is based the analysis of PCR results?
13. What is the clinical, diagnostic and practical significance of PCR as a method of
express diagnostics?
23
Examples of tests „ Crock–1”
1. Diphtheria causes inhibition of translation process in human cells due to a loss the
factor of elongation еEF-2 ability to carry out translocationu of peptide residue
from A- on the P-site of ribosoms. What enzyme is a reason of еEF-2 blocking?
A. ADP-ribosyltransferase
B. eIF-2-proteinkinase
C. Peptidyl transferase
D. Peptidyl translocase
E. Hypoxantine-guanine phosphoribosyltransferase
2. For treatment of infectious bacterial diseases are used antibiotics (streptomycin,
neomycin, kanamycin). What stage in the synthesis of protein of bacterial cell do
they inhibit?
A. Replication
B. Transcription
C. Translation
D. Processing
E. Splicing
3. What enzyme is involved in the pathway of synthesis of different genes carried out
by from mRNA and DNA in the gene engineering (this enzyme catalyze a process,
discovered in some RNA–containing of viruses)?
A. Reverse transcriptases
B. Exonucleases
C. Endonucleases
D. DNA-ligases
E. Helicases
4. Patiet who lives on specific geochemical territory has a diagnosis – endemic goitre.
What type of posttranslational modification of thyroglobulin is violated in the
organism of patient?
A. Phosphorylation
B. Methylation
C. Acethylation
D. Iodation
E. Glycosylation
5. The inherited information is saved in DNA, though directly in the synthesis of
protein in a cell it does not participate. What process does provide the realization of the
inherited information in a polypeptide chain?
A. Transcription
B. Translation
C. Translocation
D. Replication
E. Transformation
24
Topics for independent individual student’s work
1. Transcription processes in normal and pathological conditions. Programmed cell
death- apopthosis. Biochemical mechanisms of apopthosis.
2. Gene engineering. Cloning. The aplication of gene engineering methods in
modern medicine.
Refernces
7. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
8. Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
9. Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
10.Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
11.Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
12.Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.III, p. 70-78...
13.Paolella P. Introduction to molecular biology. The McGraw- Hill Co., 1998, 241
pp.
Sense module № 14. Molecular mechanisms of hormone action on target cells.
Topic № 5. The investigation of molecular mechanisms of the effect of hormones
of protein and amino acid nature on target cells. Humoral regulation of calcium
homeostasis.
Objective: To learn biochemical and physiological function of hormones in a
system of intercellulat integration in the organism. To learn structure of hormones of
protein and amino acid nature, derivatives of amino acids and steroid hormones,
mechanisms of their action upon the target cells, role of secondary messengers in cell
response to the hormones of protein or amino acid nature.
Actuality of the theme: Understanding of biochemical mechanisms of
realization of hormones effect on functioning of cell systems allows to explain
mechanisms of development of pathological states, caused by disorders in
functioning of endocrine glands and target cells as well as it also forms in students
considerations about the correction of hypo- or hyperfunction of endocrine glands.
Specific objectives:
 To interpret the biochemical and physiological functions of hormones and
bioregulators in a system of intercellular integration of vital functions of human
organism.
 To analyse and to explain correspondence between the structure of protein and
peptidehormones, derivatives amino acid derived and steroid hormones to their
function and mechanism of action up on targets cells.
16


To interpret the molecular mechanisms of action of hormones of protein and
peptide nature, derivatives of amino acids (catecholamines) up on target cells
with involvement of signaling mediator molecules.
To interpret the molecular mechanisms of direct regulatory effect of steroid
hormones on the genome of targets cells.
Theoretical questions.
1. Hormones in a system of intercellular integration of physiological functions in
human organism. Classification of hormones.
2. Synthesis and secretion of hormones. Cyclic nature of hormone secretion,
transport of hormones in blood. Factors, influencing the secretion and effect of
hormone action.
3. Targets of hormonal action; types of cell response to the hormone. Membrane and
cytosol receptors of hormones in the cell.
4. Mechanisms of hormonal action - amino acid derivatives, peptide and protein
hormones, steroid hormones. Regulatory sites in DNA, which interacts with
hormone-receptor complexes.
5. Messenger function of cyclic nucleotides, Ca/calmodulin system
phosphoinositides. Serine, threonine and tyrosine proteinkinases in effectory
response of the cell.
6. Hormones of thyroid gland. Structure and function of thyroid hormones.
Pathology of thyroid gland, metabolic disorders in hypo- and hyper- thyreosis.
Endemic goiter and its prevention.
7. Regulation of calcium turnover by parathyroid hormone (PTH) and calcitonine
(CT). Calcitriol: biosynthesis, the effect on intestinal absorption of calcium and
phosphates. Calcitonine – structure, the effect upon calcium and phosphate
turnover.
8. Biochemical characterization of disorders in calcium metabolism (rickets,
osteoporosis). Hypo- and hyper- parathyroidism. Distribution of calcium in the
body, molecular forms of calcium in blood plasma. Role of bone tissue, intestines
and kidneys in support of calcium homeostasis.
9. Hormones of pancreatic gland. Insulin – structure, biosynthesis and secretion.
Role of insulin in carbohydrate, lipid and protein metabolism. Growth factors and
oncoproteins.
10.Glucagone. Chemical structure and biological activity of hormone.
11.Role of epinephrine in regulation of storage and mobilization of glycogen.
12.Eicosanoids:
general
characteristics,
classification
(prostaglandines,
thromboxanes, leucotrienes, lipoxins). Biosynthesis of different classes of
eicosanoids, cyclooxygenase and lipoxygenase.
13. Biological and pharmacological activity of eicosanoids. Acetyl-salicylic acid
and other non steroid anti-inflammatory drugs as inhibitors of prostaglandine
biosynthesis.
Practical part
Experiment 1.Qualitative reactions of insulin. Insulin is a protein and gives all
characteristic reactions of proteins.
27
Biuret probe. To 10 droplets of insulin solution 5 drops of 10 % solution of
NaOH and one drop of CuSO4 solution are added. A red-violet colour appears,
which is characteristic for proteins and peptides.
Fohl’s reaction. To 5 droplets of insulin solution 5 droplets of Fohl’s reagent
(alkaline solution of lead acetate) are added and the mixture is boiled for several
minutes. Aftyer 1-2 min of standing a dark precipitate of lead sulfide is formed.
Millon’s reaction. To 5 droplets of insulin solution 1-2 ml of Millon’s reagent
are added and the mixture is heated on a flame. A red precipitate forms.
Explain the results.
Experiment 2. Qualitative reactions of adrenalin.
2.1. Principle of the method. Adrenalin is readily oxidized by air oxygen to
adrenochrome which gives a characteristic green color with ferric chloride or red
color with diazotisized sulfanilic acid (diazoreagent).
Performance. To 3 drops of adrenalin solution 1 droplet of iron chloride
solution is added. The green color appears, which after addition of ammonia changes
to red, and thereafter - to brown.
Diazoreaction of adrenalin.
Performance. To 3 drops of 1% solution of sulfanilic acid in a clean tube 3
droplets of sodium nitrite are added, 5 droplets of 0,1 % adrenalin solution and 3
droplets of 10 % solution of sodium carbonate. The color changes to red.
Explain the results.
Experiment 3. Qualitative reaction of thyroxine.
Principle. During destruction of the molecule of thyroxine iodide is formed,
which after interaction with potassium iodate releases elementary iodine. The last
gives a blue color with starch solution.
Performance. To 20-25 drops of cooled thyroxine hydrolysate several drops of
sulfuric acid are added to acidic reaction. Then 3 drops of 10% solution of potassium
iodate are added and produced iodine is detected with the aid of starch solution.
Explain the results.
Clinical and diagnostic significance. The investigation of hormones and
mediators metabolism has an important significance in diagnostics of endocrine
disorders, as well as for evaluation of body status in different psthological
conditions, connected with alteration of function of central and vegetative nerve
systems, heart, liver, kidneys and other parenchimatous organs.
Whatever disorders in a system hypothalamus-hypophysis-cortical adrenals lead
to a change in corticosteroid secretion.
In interpretation of results it is useful to remember, that adrenalin secretion in
females and males is almost equal , exception is in children in age of 12-15 years (in
boys excretion is higher than in girls) and in 41-50 years old persons (in men higher
then in women).
Secretion of noradrenalin is eqwual in boys and girls up to 8-11 years of life, but
thereafter its secretion in women is higher, then in men.The secretion of adrenalin
and noradrenalin is not constant during the day. In day time it is respectively 7,5and 30,9 mg/min, in night it corresponds to 1,9 and 11,3 mg/min.
Smoking, physical load, emotional stress cause increase in excretion of
catecholamines in urine. Enhanced catecholamines excretion is observed in liver
28
cirrhosis, acute phase of ulcer disease of stomach and duodenum, Disorders in
excretion have significance in pathogenesis of uremia.
Control of laboratory work fulfillment
1. What is the prinsiple of color change in reaction mixture in course of biuret test,
reactions of Fohl’s and Millon’s reactions?
2. What qualitative reactions for adrenalin detection do you know? What is the
difference between these reactions?
3. How to explain the appearance of blue colouring of solution in a qualitative
reaction on thyroxine?
4. Lifetime of most hormones in blood is comparatively short. For example, if to
inject an animal the radiolabeled insulin, half of the injected hormone is inactivated
in blood during 30 min. Why is it important the fast inactivation of circulating
hormones? How can be supported constant level of hormone in blood taking into
account its rapid inactivation? How are achived the rapid changes in concentration of
circulating hormones in the body?
5. An amount of adrenalin in medulla of adrenals is 0,08 % from mass of glands.
What is amount of adrenalin in glands, if it is known that an amount of noradrenalin
is 0,008 % (0,8 mg)?
6. A 33-years-old patient is addressed to Consultation of Thyroid Pathology by the
family physician. During 6 months she felt an anxiety, rapid fatigue, enchanced
sensitivity to the heat, loss of body weight in normal appetite, palpitation and
promoted perspiration. After investigation it was revealed: pulse 130 beats/min,
warm and moist palms, trembling, retarted closure of eyelids and exophtalmia, also
soft and enlarged. Testing results: thyroxin – 260 нмоль/л (normal 50 – 150).
7. A man is in a stress situation. What influence will have this state on the function
of endocrine glands?
8. What advantages for the organism gives the production of hormones in form of
prehormones and prohormones?
Examples of tests „ Crock–1”
1. A patient appealed to the doctor with complaints about tremor and hypokinesia.
The biochemical analysis of blood showed the reduced amount of dopamine. Name
its methabolite-precursor.
A. Dioxyphenylalanine
B. Tyrosine
C. Tyramine
D. Phenylalanine
E. Phenylpyruvate
2. Prostoglandins take part in the development and regulation of inflammation as a
general pathological process in the human organism. Name the compound able to
inhibit cyclooxygenase activity?
A. Acetylsalicylic acid
B. Aceton acetic acid
C. -Ketoglutaric acid
29
D. Propionic acid
E. Butyric acid
3. Some hormones increase the permeability of internal membranes of mitochondria
for Н+, that results in uncoupling of respiration and phosphorylation processes and
stop in synthesis of ATP. Name this compound:
A. Adrenalin
B. Vasopressin
C. Thyroxine
D. Insulin
E. Oxytocin
4. In pregnancy is increased requirement for cholecalciferol; one of its metabolites is
a powerful antagonist of parathyreoid hormone, which stimulates the process of bone
resorbtion and output of calcium and phosphates into blood. Indicate this metabolit.
A. 1,25-Dihydroxycholecalciferol
B. 1-hydroxycholecalciferol
C. Cholecalciferol
D. Ergocalciferol
E. 25-Hydroxycalciferol
5. Protein hormones regulate biochemical processes by an increase of calcium ions
concentration in a cell. Indicate the compound which increases the concentration of
calcium ions.
A. Phosphatidylinositol 4,5-biphosphate
B. Inositol-1,4,5-triphosphate
C. Inositol-3,6-diphosphate
D. Inositol-6-phosphate
E. Inositol
Individual independent student’s work
1. Transformations of arachidonic acid in human organism and the influence of
products on metabolic processes.
1.
2.
3.
4.
5.
6.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.IV, p. 70-78.
30
Sense module № 15. Biochemistry of hormonal regulation.
Topic № 6. Investigation of molecular and cellular mechanisms of steroid and
thyroid hormones action upon target cells.
Objective: To analyze changes in carbohydrate, lipid and protein metabolism
which occur in endocrine glands dysfunction and to interpret prognostic significance
of distinct disorders. To learn mechanisms of hormonal regulation of calcium
homeostasis: distribution of Ca+2 in organism, fractions of calcium in human blood
plasma, the role of bone tissue, intestines and kidneys in support of calcium
homeostasis. To know biochemical mechanisms of pathological processes
development and typical manifestations in endocrine diseases, with special attention
to thyroid gland dysfunction.
Actuality of the theme: Hormones play an important role in mechanisms of
homeostasis. These substances regulate the activity of enzymes in cells, influence the
expression of cell genom and change the intensity of metabolism in target cell and in
the body as well. The knowledge of mechanisms of the neurohumoral regulation of
metabolism gives basis for diagnostics and rational therapy at endocrinological
disorders.
Specific objectives:
 To analyze metabolic changes and biochemical indexes, which characterize the
carbohydrate metabolism, metabolism of proteins and lipids in disorders of
endocrine glands and to summarize the prognostic estimation of these disorders.
 To interpret the mechanisms of hormonal regulation of calcium homeostasis:
distribution of calcium in the body, fractions of calcium in human blood plasma,
the role of bone tissue, small intestine and kidneys in support of calcium
homeostasis.
 To explain the biochemical mechanisms of development and manifestation of
pathological processes and typical disorders of endocrine system.
1.
2.
3.
4.
5.
6.
7.
8.
Theoretical questions.
Hormones of hypothalamus and pineal gland, releasing hormones and statins.
Hormones of pituitary gland, their role in regulation of endocrine glands
function.
Hormone family
“growth hormone-prolactin-gonadotropins”, pathology
connected with GH, somatomedine, prolactin dysfunction.
Glycoprotein hormones of hypophysis – TSH, FSH, ICSH, prolactin.
Proopiomelanocortine – products of processing of this hormone precursor,
(ACTH, lipotropins, endorphins).
Hormones of neurohypophysis – oxitocine and vasopressin (ADH), pathology
caused by disorder in ADH secretion.
Steroid hormones, classification. Scheme of biosynthesis of steroid hormones
from cholesterol.
Steroids of suprarenal glands. Glucocorticoids:
cortisol, cortisone,
corticosterone, their role in regulation of gluconeogenesis, anti-inflammatory
32
effects. Itsenko-Cushing syndrome. Mineralocorticoids, role of aldosterone in
regulation of water and mineral metabolism, Adison disease, aldosteronism.
9. Hormones of sexual glands. Estrogens – estradiol, estriol, estrone, ,
physiological and biochemical effects, regulation of synthesis and secretion,
connection with female month cycle.
10. Androgens – testosterone, dihydrotestosterone, physiological and biochemical
effects, regulation of biosynthesis and secretion.
11.Hormones of thyroid gland. Structure and function of thyroid hormones.
Pathology of thyroid gland, metabolic disorders in hypo- and hyperthyreosis.
Endemic goiter and its prevention.
12.Regulation of calcium turnover by parathyroid hormone (PTH) and calcitonine
(CT). Calcitriol: biosynthesis, the effect on intestinal absorption of calcium and
phosphates. Calcitonine – structure, the effect upon calcium and phosphate
turnover. Hypo- and hyperparathyroidism
13. Biochemical characterization of disorders in calcium metabolism (rickets,
osteoporosis).. Distribution of calcium in the body, molecular forms of calcium
in blood plasma. Role of bone tissue, intestines and kidneys in support of
calcium homeostasis.
14. Biogenic amines with hormonal and mediatory activity: structure, biosynthesis,
physiological significance, mechanisms of action. Receptors of biogenic amines,
medical application of histamine receptors antagonists.
Practical part
Experiment 1. Qualitative determination of 17-ketosteroids in urine.
Principle of the method. 17-ketosteroids after reaction with m-dinitrobenzene
in strong alkaline medium give a condensation product with a violet-pink color
(maximum of absorption at 530 nm). The intensity of color is proportional to a
quantity of 17-ketosteroids in urine.
Performance. 5 drops of urine are added into the tube, thereafter 5 drops of 30
% of NaOH solution (caution, hazard!) and 5 drops of m-dinitrobensene solution are
added. Mix the content and wait for 2-3 min., a characteristic cherry red color appears
in presence of sufficient quantity of 17-ketosteroids.
Explain the results.
Clinical significance. Maximal excretion of 17-ketosteroids is observed at 25
years age, thereafter the quantity of excreted steroids diminishes. During stress
situation quantity of 17-ketosteroids in blood and urine is increased. In pathology the
content of ketosteroids is changed depending from the function of suprarenal glands.
At hypofunction of suprarenal glands (Addison disease) excretion of 17-ketosteroids
consists of 20-30 % of normal value. The excretion of ketosteroids is also decreased
at hyperthyreosis, hepatic cyrrhosis, suprarenal gland tumors. 17-ketosteroids are
excreted mainly in conjugated form (with sulfuric or glucuronic acid). Simultaneous
determination of free and conjugated 17-ketosteroids in urine permits more precisely
evaluate functional state of suprarenal glands.
Testosterone is the most active androgene, which is produced in men.
Determination of its diurnal excretion permits to evaluate function of male sexual
33
glands. Diurnal excretion of testosterone in males of 20-40 years old is round 70 μg,
in females of the same age – 8 μg.
In kidney diseases during development of renal insufficiency the concentration
of total 17-ketosteroids in blood plasma is in normal values, but fraction of free 17ketosteroids excreted in 24 hours is greatly increased.
Experiment 2. Quantitative determination of adrenaline (with Folin’s
reagent).
Principle of the method. The quantity of adrenaline is determined
colorimetrically after interaction with Folin’s reagent.
Performance. 1 ml of test solution, containing adrenaline, is introduced into the
tube, thereafter 4 ml of 10 % of NaHCO3 and 0,5 ml of Folin reagent are added.
After 5 min the intensity of blue colour is measured in a colorimeter at red filter.The
concentration of adrenaline is calculated with the help of calibration curve.
Optical density
of solution, А
1,5
1,0
0,5
0,01
0,02
0,03
0,04
conc. g/l
Control of laboratory work fulfilment
1. What functional group of ketosteroids interracts dinitrobenzol?
2. What is the value of quantitative determination of adrenalin?
3. A patient was treated with corticosteroid hormones for a long time, thereafter
abruptly stoped their acception them. What changes in the methabolism are possible?
4. One of approaches in treatment of breast cancer is surfical removal of ovaries.
Besides additionally are introduced androgenes. Explain the biochemical basis of this
treatment.
5. In adrenal cortex damage develops specific pigmentation of skin, muscular
weakness, marked disorder in water-salt turnover, as well as in protein and
carbohydrate metabolism. What is the cause of these events?
6. Vasopressine and oxytocin are accumulated in posterior pituitary. Indicate, where
are they synthesized and how they get a posterior pituitary?
34
Examples of tests „ Crock–1”
1. The main representative of mineral corticosteroids is…
A. Corticosteron
B. Hydrocortizone
C. Dihydro-corticosterone
D. Aldosteron
E. Synestrol
2. What is the effect of glucocorticoids up on carbohydrate methabolism in liver?
A. Stimulate glycogenesis from glucose
B. Stimulate gluconeogenesis
C. Stimulate glycogen hydrolysis
D. Stimulate glycogen phosphorolysis
E. Stimulate activity of glycogenphosphorylase
3. In human body some amino acids are transformed into hormones. What compound
is produced from tryptophan?
A. Histamine
B. Serotonin
C. GABA
D. Corticosteron
E. -alanine
4. What hormone regulates water balance and osmotic pressure in blood plasma, as
well stimulates contraction of smooth muscles in vessel wall?
A. Prolactin
B. Somatostatin
C. Corticoliberin
D. Vasopressin
E. Kinins
Independent individual student’s work
1.The role of hormone-receptor interaction in the development of hormonal effect.
References
1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
2. Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
3. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
4. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
5. Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.III, p. 70-78.
35
Topic № 7. Investigation of nerve tissue. Pathochemistry of psychotic disorders.
Objective: To learn the composition and peculiarities of metabolism in nerve
tissue, its functioning in normal conditions and in some diseases. To use practically a
method of cholinesterase activity determination.
Actuality of the theme: Brain and spinal cord are basic organs which povide the
function of CNS in the body. Nerve tissue has age dependent specifities of metabolism,
it influences up on the mechanisms of development of pathological states in the body.
In many diseases of nerve system and especially in stress situation it is important to
determine the content not only neurotransmitters, but also the activity of subsequent
enzymes, for example, acetylcholinesterase in the blood serum.
Specific objectives.




To explain peculiarities of chemical composition of white and grey brain matter.
To analyze differences between cerebrospinal fluid and blood plasma .
To interpret specific features of metabolism in brain tissue.
To explain the role of neurotransmitters in regulation of functions of organs and
cells.
 To analyze changes in cholinesterase activity in different diseases.
Theoretical questions
1. Peculiarities of biochemical composition and metabolism of nerve tissue:
 Chemical composition of brain tissue
 Neurospecific proteins and lipids (gangliosides, cerebrosides, cholesterol)
 Peculiarities of aminoacid composition of brain tissue
 Role of glutamic acid system.
2. Energetic metabolism in hujan brain tissue.
 The significance of aerobic oxidation of glucose
 Changes in condition of physiological sleep and narcosis.
3. Neurotransmitters (acetylcholine, noradrenalin, dopamine, serotonin, excitatory
and inhibitory amino acids).
4. Receptors for neurotransmitters and physiologically active substances.
5. Peptideergic system of brain.
6. Opioid peptides (enkephalins, endorfines, dynorphines).
7. Molecular principles of bioelectric processes on neurone membranes.
8. Disorders in metabolism of neuromediators and modulators in brain during
psychoses.
9. Neurochemical mechanisms of action of psychotrope factors (neuroleptics,
antidepressants, anxiolytics, nootropics).
10. Enzymes involved in biosynthesis and degradation of neurotransmitters.
Practical part
Experiment 1. Determination activity of cholinesterase in blood serum by
titrimetric method of Michel.
39
Principle of the method. Acetylcholine is hydrolysed enzymatically with
production of acetic acid, which is determined by titration with sodium hydroxide.
Materials and reagents. 1,5% solution of acetylcholine, 1% phenolphtaleine,
0,01 n sodium hydroxide, thermostate, tubes.
Performance: Into each of two tubes is introduced 1 ml of acetylcholine
solution, then to the test tube is added 1 ml of tested serum, to the second tube
(control) - 1 ml of inactivated by heating at 56 oC 30 min the same serum. To each tube
is added 2-3 droplets of phenolphthaleine and samples are titrated by 0,01 n NaOH.
Calculation. The difference in volumes of NaOH, expended for titration of test
and control samples is determined : V = Vt – Vc (ml), where Vt and Vc are volumes
of 0,01 n NaOH, expended for titration of test and control probes respectively. .
Normal values correspond to 2 - 4 ml of 0,01 n NaOH for titration of 1 ml of serum.
Normal ranges of activity corresponds to 45-95 μmoles/sec x l.
Draw a conclusion.
Clinical and diagnostic significance. Activity of cholinesterase in healthy
persons deviates in wide ranges, but is relatively constant in the same person.
Cholinesterase is secreted by liver cells. Contrary to the majority of other enzymes its
activity in blood is decreased in liver diseases due to alteration of biosynthesis of this
enzyme in liver cells. Severe decrease in activity is observed in acute and chronic
hepatitis, cirrhosis and malignant tumors of liver
Determination of cholinesterase activity is most frequently used as a prognostic
index in cases of acute or chronic damage of liver parenchyma with phosphororganic
toxins The degree of enzymatic activity decrease corresponds to gravity and degree of
liver cells damage.
Cholinesterase activity is slightly increased in some psychotic diseases, e.g. in
maniacal-depressive psychosis, in stress conditions, in depression, in schizophrenia,
during demielinization of nerve trunks and fibers.
Significant increase in activity of cholinesterase in amniotic fluid may indicate
on serious damage of fetus nerve system.
Control of laboratory work fulfilement
1.
What is a principle of Michel's method
of choline esterase activity
determination in blood serum?
A. Oxidation of para-phenylenediamine with formation of the coloured compound.
B. Colour reaction with picric acid in an alkaline medium
C. Formation with ferric (III) nitrate purple colored compound.
D. Produxtion of intensive blue colour, after interraction with the Folin reagent
E. Enzymatic hydrolysis of acetylcholine with formation of acetic acid which is
determined by titration with sodium
2.
The normal values of activity of cholinesterase in the blood serum is:
А. 10 – 15 μmol/(s-l)
В. 12 – 30 μmol/(s-l)
С. 45 – 95 μmol/(s-l)
40
D. 98 – 115 μmol/(s-l)
Е. 105– 145 μmol/(s-l)
3.
Patients with insufficiency of thiamine usually shows several neurological
symptoms: loss of reflexes, excitability, loss of consciousness. Explain, why does lack
of thiamine deficiency affects the brain functions?
4.
It is known that glycogen which provides the energetic metabolism in the body
is accumulated in liver and muscules, but not in such important tissue as brain, which
preferentially utilizes glucose. Explain, why glycogen is not accumulated in brain?
Examples of tests „ Crock–1”
1. Tetanus toxin causes tonic contraction of skeletal muscles because it supress release
from nerve endines the next neurotransmitter:
A. GABA
B. Noradrenalin
C. Acethylcholine
D. Glycine
E. Glutamate
2. Parkinson disease a dofaminergic transmission is altered and that in treatment is
used precursor of dofamin – L-DOPA. In order to decrease side affects and dosis, LDOPA is used in combination with:
A. Inhibitor of aromatic amino acids decarboxylase
B. Activatior of aromatic amino acids decarboxylase
C. Inhibitor of monoaminoxidase
D. Activator of monoaminoxidase
E. Blockators of dofamin receptors
3. In brain of schizophrenia patients an increase in the amount of the next receptors is
observed:
A. Dofamin receptors
B. Serotonin receptors
C. Adrenalin receptors
D. Cholin receptors
E. GABA receptors
4. Disorder in process of myelinization of nerve fibres results in heavy neurological
disorders and mental disfunction. Such clinical status is caused by inherited defects in
metabolism of the next compound:
A. Neutral fats
B. Cholesterol
C. Sphingolipids
D. Glycerophospholipids
E. Lipoproteins
41
References
1. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
2. Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
3. Biochemistry / Trudy McKee, James R. McKee.- 1999, 288 p.
4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
6. Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.III, p. 70-78.
Sense module № 16. Biochemistry of muscle tissue and mechanism of its
contraction.
Topic № 8. Biochemistry of muscle tissue. Investigation of mechanism of muscle
contraction.
Objective: To learn the composition and peculiarities of metabolism in muscle
tissue, its function in normal and pathological conditions. To perform a quantitative
determination of creatinine and creatine in urea and its diagnostic significance in some
pathology.
Actuality of theme: Muscle tissue has specific features of metabolism depending
on age of a person, pathological states, caused with both endogenous and exogenous
factors, and that is why in a clinic the special place beloungs to biochemical methods
of diagnostics. For example, at some diseases of muscles it is important to know the
excretion of creatine and creatinine in 24-hours urine.
Specific objectives:

To analyze biochemical composition of muscles and the role of proteins in
formation of muscle cell structure.

To explain biochemical mechanisms of contraction and relax of muscle fiber.

To analyze pathways of energetic support of muscle contraction, role of ATP
and creatine phosphate in these processes.
Theoretical questions
1. Fine structure and biochemical composition of myocytes; structural organization of
sarcomers. Myofibril proteins: myosine, actin, tropomyosine, troponine complex.
Molecular organization of thick and thin filaments.
2. Nitrogen containing and nitrogen free water soluble organic compounds, their
structure and functional significance. Molecular mechanisms of muscle contraction:
43
3.
4.
5.
6.
7.
8.
9.
modern data on interaction of muscle filaments. Role of Ca 2+ ions in regulation of
contraction and relax of striated and smooth muscles.
Modern ideas on energetics of muscle contraction and relaxation. Macroergic
compounds of muscles. Structure, production and role of ATP, creatine phosphate,
creatine phosphokinases, sources of ATP in muscle cell; role of creatine phosphate
in energetic supply of contraction. Pathobiochemistry of muscles-- myopathias.
Cell organization and peculiarities of metabolism in cardiac muscle, its
interrelations with metabolism in nerve, endocrine systems, in liver, lungs, vessels.
Peculiarities of bioenergetic processes in myocardiocytes and regulation of their
contraction.
Peculiarities of smooth muscle function. Molecular mechanisms in regulation of
tonus in vessels and bronchi.
Metabolic disorders of coronary vessels and myocardium in cases of myocardial
infarction. Changes in enzymatic activity pattern of blood plasma in acute
myocardial infarction: diagnostics of stenocardia (coronary insufficiency),
microinfarction, alcohol intoxication.
Heart as endocrine organ. Cardiopeptides, their functional significance.
Damage of heart muscle in some diseases (thyrotoxicosis, hypothyreosis,
hypercorticism, diabetes mellitus, dysfunction of parathyroid glands, chronic renal
insufficiency, ionizing radiation, porphyria, gout, nutritional disorders, alcohol
intoxication). Biochemical tests in diagnostics of heart and skeletal muscle diseases.
Pathobiochemistry of hypertonia, changes of biochemical data on different stages of
hypertonic disease. Symptomatic arterial hypertension. The use of biochemical data
for evaluation of endomyocardium activity. Biochemical characterization of
myocardium diseases (myocarditis, myocardiopathia). Diseases of pericardium.
Practical part
Experiment 1. Quantitative determination of creatinine in urine after Folin
method.
Principle of the method. Creatinine combine with picric acid in alkaline medium
and forms colored complex (Jaffe reaction). The intensity of color is proportional to
creatinine quantity and is measured in photocolorimeter.
Performance. Into one calibrated cylinder 0,5 ml of urine is measured (test),to
another - 0,5ml of water is added (control). To both cylinders 0,2 ml of 10 % NaOH
and 3 ml of saturated solution of picric acid are added, after five min distilled water is
added to the final volume 100 ml. Solutions are mixed and the intensity of color is
measured in photocolorimeter in 1 cm cuvette at green light.
The content of creatinine is calculated according to calibration curve. A daily
excretion of creatinine is calculated, using known values of daily volume of urine and
volume of tested specimen of urine.
Explain the results.
Clinical and diagnostic significance. Normal daily excretion of creatinine is 8,817,7 mMole (1,0-2,0 g) in males and 7,1-15,9 mMoles (0,8-1,8 g) in females. The
enhancement of creatinine excretion is observed in excessive ingestion of meat
44
(exogenous source), in breakdown of proteins in cytoplasm of cells, in intensive
physical efforts, diabetes mellitus and diabetes insipidus, nfectious diseases, etc.
Creatinine is not reabsorbed in kidney tubules, which permits to evaluate the
glomerular filtration index.
Experiment № 2. Quantitative determination ofcreatine in urine.
Creatine is transformed to creatinine by heating the specimen in acidic
medium, thereafter creatinine is determined as described above.
Performance. Into one tube 0,5 ml of urine is measured (test specimen), to
other tube 0,5 ml of water are added (control). To each tube 0.1 ml of
concentrated HC1 are added and tubes are placed onto boiling water bath for 3
min. After cooling into tubes are added 0,2 ml of 10% NaOH and 3 ml of
saturated solution of picric acid and tubes are left for 5 min. Thereafter the content
of tubes are transferred to calibrated cylinders and final volume is adjusted to 100
ml by distilled water. Color intensity is measured in photocolorimeter. Obtained
data correspond to total creatinine and creatine content, m order to obtain the
value of creatine from results of experiment 2 are subtracted values of creatinine,
obtained in exp. 1. the difference is multiplied over 1,16.
Explain the results.
Clinical and diagnostic significance. In healthy persons creatine in urine is
absent.The appearance of creatine in urine is termed creatinuria and can be
observed in enhanced physical load, during the growth in childrens, in pregnancy,
during protein or carbohydrate starvation. Creatinuria is observed at increased
tissue breakdown (combustion, neoplasma, tuberculosis), avitaminosis E, diabetes
mellitus, etc.
Control of laboratory work implementation
1.
What is the principle of the method of creatin determination in urine?
2.
In what units the amount of creatin in urine is determinated?
3.
What significance has the increase or decrease in creatinine excretion in urine ?
4. Name the cause of creatinuria.
5.
It is known that the main feature of bronchial asthma is a spasm of smooth
muscles of bronchioles. What causes manifestation of such symptoms?
6.
Patient has vitamin E hypovitaminosis. How it will influence the function of
muscles?
7.
Patients with insufficiency of thiamine usually have a series of neurological
symptoms: loss of reflexes, excitability, loss of consciousness. Explain, why does of
thiamine deficiency affect the brain functions?
Examples of tests „ Crock–1”
1. For early diagnostics of muscular distrofy is the most informative an increase in
activity of certain enzyme in blood plasma. Note it.
A. Lactate dehydrogenase
B. Alanine aminotransferase
C. Aspartate aminotransferase
45
D. Creatinkinase
E. Alfa-amylase
2. In a patient with progressive muscular dystrophy a biochemical investigation of
urine was conducted. Appearance of what compound in great amount in urine can
support diagnosis of muscle disease in this patient?
A. Hippuric acid
B. Creatin
C. Porphyrins
D. Carbamide
E. Creatinine
3. People which for a long time were in a state of hypodynamia, have intensive pains
in musclesafter the physical load. What is most reliable cause of this symptom?
A. Decrease of lipids in muscle
B. Increase of ADP in muscles
C. Accumulation of cretine in muscles
D. Increase of muscule proteins disintegration
E. Accumulation of lactic acid in muscules
4. For synthesis of ATP in skeletal muscles and myocardium are used different
substrats. One of them is utilized in myocardium, but not in skeletal muscles.
A. Glycogen
B. Glucose
C. Lactic acid
D. Fatty acids
E. Keton bodies
5. The most rapid mechanism of ATP regeneration in case of intensive physical load in
muscles is the next:
A. Generation of ATP from creatinphosphate
B. Aerobic glycolysis
C. Glycogenolysis
D. Anaerobic glycolysis
E. Oxidation of triglycerides
1.
2.
Topic № 9. Summary leson, control work. Module 4
Therretical questions:
Biochemical functions of nucleic acids and nucleotides. Formation of nucleic acid
chain from nucleotides.
Constituents of nucleotides and nucleosides. Minor nitrogenous bases and
nucleotides.
40
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Free biologically active nucleotides and their biochemical functions: involvement in
metabolic reactions ( ATP, NAD, NADP, FAD, FMN, CTP, UTP) and in their
regulation ( cyclic nucleotides – cAMP, cGMP).
Nucleic acids: structure, properties. Primary structure of nucleic acids, polarity of
polynucleotides, specific features of DNA and RNA structure.
Structure, properties and biological significance of DNA. Experimental proves of
DNA significance in heredity (phenomenon of transformation). Molecular mass,
dimensions and nucleotide composition of DNA molecules of viruses, prokaryots
and eukaryots.
Secondary structure of DNA, role of hydrogen bonds in stabilization of secondary
structure (Chargaff rules, Watson-Crick model), ant parallelism of chains.
Tertiary structure of DNA. Physico-chemical properties of DNA: interaction with
cationic ligads; hyperchromic effect ; denaturation and renaturation of DNA.
Structure, properties and biological functions of RNA. Types of RNA: mRNA,
tRNA, rRNA, snRNA; specific features of structure (secondary and tertiary) of
different RNA types.
Molecular organization of nuclear chromatin and ribosomes of eukaryots.
Chromatin: nucleosomes, histones and non histone proteins. Ribosomes: subunit
structure, protein and RNA composition.
Biosynthesis of purine nucleotides; scheme of reactions of IMP synthesis; synthesis
of AMP, GMP, ATP, GTP. Regulation of purine nucleotides synthesis on a
principle of feedback inhibition.
Biosynthesis of pyrimidine nucleotides: reactions, regulation.
Biosynthesis of deoxyribonucleotides. Formation of thymidyl nucleotides;
inhibitors of dTMP biosynthesis as antitumor drugs. (structural analogs of dTMP,
pterine derivatives).
Catabolism of purine nucleotides; hereditary disorders of uric acid metabolism.
Biochemical background of hyperurucemia, gout, Lesch-Nyhan syndrome.
Biological significance of DNA replication. The sense of J.Watson and F.Crick
discovery (1953). Semiconservative mechanism of replication, the scheme of
Meselsohn and Stahl experiment.
General scheme of DNA synthesis. Enzymes of DNA replication in prokaryots and
eukaryots. Molecular mechanisms of DNA replication: topoisomerases, helicases,
the significance of antiparallelism of DNA strains, Okazaki fragments. Stages of
synthesis of daughter chains of DNA.
General scheme of transcription. Coding and noncoding DNA chains. RNA
polymerases of prokaryots and eukaryots. Stages and emzymes of RNA synthesis.
Markers of transcription: promotor, initiator, tetrmination segments of genome.
Processing as posttranscriptional modification of RNA. Antibiotics, which inhibit
transcription.
Regulation of gene expression in prokaryots: scheme of regulation according to
F.Jacob and J.Monod. Structure of Lac-operon of E.coli. structural and regulatory
genes, promotor, operator, regulatory gene and production of protein repressors,
repression and induction of Lac-operon function.
45
19. Peculiarities of structure and function of eukaryot genom. Molecular organization
of DNA in eukaryots (exones, intrones, repeated sequences). Nuclear chromatin and
chromosomes of eukaryots, human karyotype.
20. Genetic recombinations; transpozones. Recombinations in prokaryot genom
(transformation, transduction, conjugation). Recombination processes in eukaryots
on an example of formation of genes of H- and L immunoglobulin chains.
21. Gene amplification (genes of metallothioneins, dihydrofolate reductase).
22. Regulation of gene expression on transcriptional level; systems of transcriptional
markers – promotor sequences, enhancers, attenuators, silensers. Covalent
modification of histones and non histone proteins as one of the mechanisms of gene
expression.
23. Phases of cell cycle of eukaryots. Biochemical mechanisms of control of cell
mitosis; cyclins and cyclin dependent kinases.
24. Mutations; genomic, chromosomal, gene (point mutations), their significance in
appearance of enzymopatias and human hereditary diseases.
25. Biochemical mechanisms of action of chemical mutagens – analogs of nitrogenous
bases, deaminating, alkylating agentsultraviolet and ionizing radiation.
26. Biological significance and mechanisms of DNA reparation. Reparation of UVinduced mutations, xeroderma pigmentosum.
27. The genetic code, triplet structure, its properties. Table of genetic code.
28. Protein synthesis system of ribosomes. Components of protein synthesis system .
29. Transfer RNA and amino acid activation. Aminoacyl-tRNA synthetases, second
genetic code.
30. Stages and mechanisms of translation: initiation, elongation, termination. Initiating
and terminating codons of mRNA. The role of protein factors of ribosomes in
translation.
31. Post-translational modification of peptide chains. Regulation of translation.
Molecular mechanisms of translation control on example of globin synthesis.
32. The influence of biologically active compounds on translation. Antibiotics as
inhibitors of transcription and translation in prokaryots and eukaryots, their
biomedical application.
33. Biochemical mechanisms of antiviral activity of interferons. Block of protein
synthesis by diphteria toxin (ADP-ribosylation of translation factors.
34. Gene engineering or recombinant DNA technology: general principles, biomedical
significance.
35. Technology of gene transplantation and formation of hybrid DNA molecules. The
employment of restrictases. Gene cloning for obtaining of medicinals and
diagnostic tools using methods of biotechnology (hormones, enzymes, antibiotics,
antigens, interferons etc.).
36. Polymerase chain reaction, its biomedical application in diagnostics of contagious
and hereditary diseases, identification of person (DNA-diagnostics).
37. Hormones in a system of intercellular integration of physiological functions in
human organism. Classification of hormones.
46
38. Synthesis and secretion of hormones. Cyclic nature of hormone secretion, transport
of hormones in blood. Factors, influencing the secretion and effect of hormone
action.
39. Targets of hormonal action; types of cell response to the hormone. Membrane and
cytosol receptors of hormones in the cell.
40. Mechanisms of hormonal action - amino acid derivatives, peptide and protein
hormones, steroid hormones. Regulatory sites in DNA, which interacts with
hormone-receptor complexes.
41. Messenger function of cyclic nucleotides, Ca/calmodulin system phosphoinositides.
Serine, threonine and tyrosine proteinkinases in effectory response of the cell.
42. Hormones of thyroid gland. Structure and function of thyroid hormones. Pathology
of thyroid gland, metabolic disorders in hypo- and hyper- thyreosis. Endemic goiter
and its prevention.
43. Regulation of calcium turnover by parathyroid hormone (PTH) and calcitonine
(CT). Calcitriol: biosynthesis, the effect on intestinal absorption of calcium and
phosphates. Calcitonine – structure, the effect upon calcium and phosphate
turnover.
44. Biochemical characterization of disorders in calcium metabolism (rickets,
osteoporosis). Hypo- and hyper- parathyroidism. Distribution of calcium in the
body, molecular forms of calcium in blood plasma. Role of bone tissue, intestines
and kidneys in support of calcium homeostasis.
45. Hormones of hypothalamus and pineal gland, releasing hormones and statins.
46. Hormones of pituitary gland, their role in regulation of endocrine glands function.
47. Hormone family “growth hormone-prolactin- gonadotropins”, pathology connected
with disorders of GH, somatomedine, prolactin dysfunction.
48. Glycoprotein hormones of hypophysis – TSH, FSH, ICSH, prolactin.
49. Proopiomelanocortine – products of processing of this hormone precursor, (ACTH,
lipotropins, endorphins).
50. Hormones of neurohypophysis – oxitocine and vasopressin (ADH), pathology
caused by disorder in ADH secretion.
51. Steroid hormones, classification. Scheme of biosynthesis of steroid hormones from
cholesterol.
52. Steroids of suprarenal glands. Glucocorticoids: cortisol, cortisone, corticosterone,
their role in regulation of gluconeogenesis, anti-inflammatory effects. ItsenkoCushing syndrome. Mineralocorticoids, role of aldosterone in regulation of water
and mineral metabolism, Adison disease, aldosteronism.
53. Hormones of sexual glands. Estrogens – estradiol, estriol, estrone, physiological
and biochemical effects, regulation of synthesis and secretion, connection with
female month cycle.
54. Androgens – testosterone, dihydrotestosterone, physiological and biochemical
effects, regulation of biosynthesis and secretion.
55. Clinical application of agonists and antagonists of sex hormones.
56. Biochemical principles of humoral regulation of digestion. Hormones of gastroentero-pancreatic system, gastrin, cholecystokinine, secretin.
47
57. Biogenic amines with hormonal and transmitter activity: structure, biosynthesis,
physiological significance, mechanisms of action. Receptors of biogenic amines,
medical application of histamine receptors antagonists.
58. Peculiarities of biochemical composition and metabolism of nerve tissue
- Chemical composition of brain
- Neurospecific proteins and lipids (gangliosides, cerebrosides,
cholesterol)
- Peculiarities of amino acid composition of brain tissue
- Role of glutamic acid system.
59. Energetic metabolism in human brain tissue.
- The significance of aerobic oxidation of glucose
- Changes in condition of physiological sleep and narcosis.
60. Neurotransmitters (acetylcholine, noradrenalin, dopamine, serotonin, excitatory and
inhibitory amino acids), fumctional role, mechanism of action.
61.Receptors for neurotransmitters and physiologically active substances.
62.Peptidergic system of brain. Opioid peptides (enkephalins, endorfines, dynorphins).
63.Molecular principles of bioelectric processes on neurone membranes.
64.Disorders in metabolism of neuromediators and modulators in brain during
psychoses.
65.Neurochemical mechanisms of action of psychotrope factors (neuroleptics,
antidepressants, anxiolytics, nootropics).
66.Principle of acetylcholinesterase activity determination.
67.Fine structure and biochemical composition of myocytes; structural organization of
sarcomers. Myofibril proteins: myosine, actin, tropomyosine, troponine complex.
Molecular organization of thick and thin filaments.
68.Nitrogen containing and nitrogen free water soluble organic compounds of muscules,
their structure and functional significance. Molecular mechanisms of muscle
contraction: modern data on interaction of muscle filaments. Role of Ca ions in
regulation of contraction and relax of striated and smooth muscles.
69.Modern concept on energetics of muscle contraction and relaxation. Macroergic
compounds of muscles. Structure, production and role of ATP, creatine phosphate,
creatine phosphokinases, sources of ATP in muscle cell; role of creatine phosphate
in energetic supply of contraction. Pathobiochemistry of muscles-- myopathias.
70.Cell organization and peculiarities of metabolism in cardiac muscle. , its
interrelations with metabolism in nerve, endocrine systems, in liver, lungs, vessels.
Peculiarities of bioenergetic processes in myocardiocytes and regulation of their
contraction.
71.Peculiarities of smooth muscle function. Molecular mechanisms in regulation of
tonus in vessels and bronchi. Role of NO and NO-synthase
72.Metabolic disorders of coronary vessels and myocardium in cases of myocardial
infarction. Changes in enzymatic activity pattern of blood plasma in acute
myocardial infarction: diagnostics of stenocardia (coronary insufficiency),
microinfarction, alcohol intoxication.
73.Heart as endocrine organ. Cardiopeptides, their functional significance.
48
74.Damage of heart muscle in some diseases ( thyrotoxicosis, hypothyreosis,
hypercorticism, diabetes mellitus, dysfunction of parathyroid glands, chronic renal
insufficiency, ionizing radiation, porphyria, gout, nutritional disorders, , alcohol
intoxication). Biochemical tests in diagnostics of heart and skeletal muscles diseases.
75.Pathobiochemistry of hypertonia, changes of biochemical data on different stages of
hypertonic disease. Symptomatic arterial hypertension. The use of biochemical data
for evaluation of endomyocardium activity. Biochemical characterization of
myocardium diseases (myocarditis, myocardiopathia). Diseases of pericardium.
76.To explain the mechanism of DNA double helix formation.
77.To explain the formation of hairpins in RNA molecule.
78.What supramolecular complexes produce nucleic acids? How are detected the
principal components of nucleoproteins in hydrolyzate ( proteins, nitrogenous base,
pentose, phosphoric acid).
79.Determination of uric acid in biological fluids with Folin reagent, principle of the
method.
80.To explain antitumor activity of antibiotics, does all antibiotics exhibit antitumor
activity? To explain mechanism of action of afidicoline and actinomycine D.
81.Interpret mechanism of action of antibiotics – inhibitors of initiation: streptomycine,
rifamycine, rifampicine.
82.Interpret mechanism of action of antibiotics- inhibitors of elongation step –
amicetine, chloramphenicol, erythromycine, cycloheximide, puromycine,
tetracyclins.
83.Interpret mechanism of action of antibiotics- inhibitors of. Termination
step:anisomycine, chloramphenicol, erythromycine, lincomycine, streptomycine.
84.Explain mechanism of action of interferons.
85.Explain mechanism of action of diphtheria toxin.
86.Explain molecular mechanisms of mutations. Give the example of the most known
mutagens.
87.What methods of gene engineering can be used in biology and medicine.
88.Characteristic reaction for proteins. Chemical structure of insulin, is this reaction
specific to insulin only.
89.Biuret reaction on hormones of protein and polypeptide nature . What polypeptide or
protein hormones do you know?
90.What method can be used for detection of metabolites of steroid hormones? What
hormones belong to this group?
91.Detection of adrenalin with iron chloride. Chemical nature of adrenaline, to write its
formula.
92.Detection of iodine containing hormones, what hormones belong to this group?
49
MODUL 5. BIOCHEMISTRY OF TISSUES AND PHYSIOLOGICAL
FUNCTIONS
Thematic plan of practical lessons in modul 5
№
п/п
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Theme of the lesson
Investigation of digestion and assimilation of nutritional
substances (proteins, carbohydrates, lipids) in digestive tube.
Investigation of functional role of water soluble and fat
soluble vitamins in metabolism and providement of cell
functions.
Investigation of acid-base equilibrium in blood and
respiratory function of red blood cells. Pathological forms of
haemoglobin.
Investigation of blood plasma proteins: proteins of acute
phase, genuine and indicatory enzymes. Investigation of
nonprotein nitrogen containing and nitrogen free components
of blood.
Investigation of coagulation, anticoagulation and fibrinolytic
systems of blood. Investigation of principal pathways of
immune response. Immunodeficiencies.
Investigation of end products of heme catabolism.
Pathobiochemistry of jaudices.
Investigation of biotransformation of xenobiotics and
endogenous toxic compounds. Microsomal oxidation,
cytochrome P-450.
Investigation of water and mineral metabolism.
Functional role of kidneys in urinogenesis. Normal and
pathological constituents of urine.
Investigation of biochemical constituents of connective tissue.
Individual independent student’s work
Preparation to summary control of modul-2
Totally
Hours
Points
number
3
11
3
11
3
11
3
11
3
11
3
11
3
11
3
11
3
11
3
11
10
80
200
3
33
50
Topics for independent students work
№
Topic
Hours
1. Training in the practical lessons:
To get a practical skills in biochemistry of nutrition
To explain the biochemical mechanisms of protein, carbohydrate and
1
lipid digestion by enzymes in a gastrointestinal tract
To explain the role of coenzyme vitamins in functioning of enzymes.
1
1.1
To explain the role of fat-soluble vitamins in metabolic processes and
0,5
realization of cellular functions.
To estimate vitamin provision of organism and features of
0,5
hypovitaminosis by biochemical indexes.
To get the practical skills in functional and cellular biochemistry of organs and
tissues:
To estimate the state of the blood system, its biochemical functions.
1
To explain the role of proteins and indicator enzymes of blood plasma
1
in a norm and pathology.
To estimate the indexes of nitrous metabolism and changes of
1
maintenance of nitrogenous nonprotein components of blood.
To analyse and interpret the indexes of blood coagulation and
1.2
1
fibrinolysis.
To estimate the state of the immune system of organism.
1
To explain biochemical bases of processes of xenobiotics and
1
endogenous toxins detoxication.
Using biochemical indexes to estimate the detoxicative function of
1
liver.
To estimate the indexes of maintenance of normal and pathological
1
components in urine.
Individual independent students work (individual task) – after the
2.
1
themes of abstracts.
Preparation of scientific lecture is on meeting of student group in
3. biochemistry. Participating in the final round of student olympiad et
1
al.
4. Preparation to summary control of mastering of the module 5.
2
Totally
15
Hours
50
The assumption of theme is evaluated according to traditional system as follows:
“5” 11 points, “4” 8 points, “3” 4 points, “2” 0 points.
Maximal points number for current learning activity of student 120.
Student obtains permission for participance in summary module control after
fulfillment of educational program and in a case when he (she) achieved for current
learning activity points number not less then 40 =(5 x8=40) points.
The summarymodule control is accepted if the student shows the knowledge of
practical methods of investigation and achieved in control of theoretical level points
number not less then 50.
51
.MODUL 5. BIOCHEMISTRY OF TISSUES AND PHYSIOLOGICAL
FUNCTIONS
Sense module №. Biochemistry of nutrition. Vitamins as nutritional components.
Topic № 1. Investigation of digestion and assimilation of nutritional substances
(proteins, carbohydrates, lipids) in digestive tube.
Objective: to learn biochemical processes of digestion in stomach and intestines,
methods of determination of constituents in gastric juice and to interpret obtained
results; to learn the mechanisms of pathobiochemical processes, which develop during
disorders of digestive system.
Actuality of the theme: Because the percentage of people with pathology of
digestive system organs grows annually, knowledge of mechanisms of origin of
gastroenterology diseases is a necessity for their correct and timely diagnostics and
subsequent treatment. A high-quality and quantitative estimation of secrets of digestive
glands (glands of stomach, pancreas) is an important diagnostic criterion for the
correction of pathochemical processes.
Specific objectives:
 To interpret physiological requirements and energetic value of principal nutritional
compounds: proteins, carbohydrates, lipids, vitamins, trace elements.
 To explain biochemical mechanisms of digestive processes and assimilation of
nutritional products in normal conditions and in hereditary or acquired disorders in
activity of proteolytic, amylolytic or lipolytic enzymes.
 To explain the development of the main disorders of digestion in pathology
1.
2.
3.
4.
5.
6.
Theoretical questions
General characteristic of human nutritional components: macrocomponents
(carbohydrates, lipids, proteins), microcomponents (vitamins, inorganic and trace
elements). Physiological requirement in energy and energetic value of principal
nutrients.
Requirements of human organism in nutrients – carbohydrates, lipids, proteins.
Biological value of some nutrients. Rational nutrition. The content of nutrients in
common dietary products.
Trace elements in human nutrition. Biological function of iodine, bromine, fluor,
copper, mangane, zinc, cobalt, selenium; manifestations of trace elements
insufficiency.
General characteristic of digestion and absorption of nutrients. Enzymes,
biochemistry of digestionmof proteins, carbohydrates, lipids in stomach, small and
large intestines.
Disorders of digestion of distinct nutrients in stomach and intestines; hereditary
enzymopathias.
Changes in biochemical indexes in disorders of stomach function and their
diagnostic significance.
52
7. Disorders of secretory function of pancreas in acute and chronic pancreatitis,
biochemical characterization.
8. Steatorrhea and its variants: pancreatic (deficiency of lipase), hepatogenic
(deficiency of bile in intestines), enterogenic (inhibition of lipolytic enzymes and
triacylglycerides resynthesis in intestines).
9. Hereditary enzymopathias of intestinal disaccharidases. Biochemical diagnostics of
lactose intolerance.
Practical part
Experiment 1. Determination of total acidity, free hydrochloric acid, bound
hydrochloric acid in a single sample of gastric juice.
Total acidity of gastric juice correspond to mMoles of NaOH, expended for
titration of 1000 ml of juice to pH 9-9.4 (indicator phenolphthalein).
Free hydrochloric acid of gastric juice correspond to mMoles of NaOH, expended
for titration of 1000 ml of juice to pH 3.5 (titration in the presence of 2,4dimethylaminoazobenzene, transition interval - pH 2.9-4.0.
Total hydrochloric acid corresponds to mMoles of NaOH expended for titration of
1000 ml of gastric juice from starting point up to pH 7,2.
Bound hydrochloric acid correspond to difference between total and free
hydrochloric acid
Gastric juice is titrated by 0.1 n NaOH in the presence of two indicators phenolphthalein and dimethylaminoazobenzene.
Performance. 5 ml of gastric juice are measured into the flask, two drops of
alcohol solution of 0.5% of 2,4-dimethylaminoazobenzene and of phenolphthalein are
added and the mixture is titrated by 0.1 n NaOH solution with the use of burette.
Change of the solution color is registered , i.e. pink red (A ml, starting point) →yellowpink(B ml) →pure yellow(C ml) →. Red (D ml). In points of color changes the volume
of NaOH solution is registered.
Calculation.
1. Free HCl: (B-A) ml x 20 mMoles/l . (20 is a coefficient, obtained from 1000 ml of
gastric juice x 0,1 n NaOH / 5 ml of tested sample).
2. Totall acidity: (D-A) ml x 20 mMoles/l.
3. Total HCl: [(C-A) + (D-A)] : 2 x 20, mMoles/ml.
4. Bound HCl is calculated as difference between total and free HCl.
Example. Starting point on burette was 0. First change of color (B) was achieved
at 1,6 ml, second change (C) –at 1,8 ml, the third change (D) – at 2,4 ml.
Free HCl is (1,6-0) x 20 = 32 mM/l, total HCl is [(1,8-0) + (2,4-0)]: 2 x 20 =
42 mM/l, bound HCl is 42-32 = 10 mM/l, total acidity is (2,4-0) x 20 = 48 mM/l.
Clinical and diagnostic significance. In normal condition free HCl is in ranges
20-40 mMoles/l, bound HCl – 10-20 mMoles/l, total acidity – 40-60 mMoles/l.
In patients with duodenal ulcer or with hyperacidic gastritis the values of free HCl
hyperchlorohydria) and of total acidity (hyperaciditas) are increased. Decrease in free
HCl and total acidity is characteristic to chronic atrophic gastritis. Hypochlorhydria,
achlorohydria and achylia (complete absence of HCl and pepsin is observed in cancer of
stomach.
53
More modern is method of intragastric pH-metry. According to this method pH 1,62,2 corresponds to normoacidity, pH 3,6 – 6,9 –to significant hypoacidity, pH 0,9 – 1,2 –
to a marked hyperacidity.
Experiment 2. Detection of lactic acid (Uffelmann reaction).
Principle of the reaction. Violet colored solution of iron phenolate changes his
color to yellow-green in the presence of lactic acid.
Performance. To 5 ml of 1% phenol solution 2-3 drops of 3% FeCl3 solution are
added. Violet color appears. To this solution several drops of tested gastric juice sample
are added. In presence of lactic acid color changes to yellow-green.
Clinical and diagnostic significance. The presence of lactic acid in stomach
reflexes the processes of glycolysis and lactic fermentation. (indirect prove of the
absence or low concentration of HCl). It is assumed, that lactic acid is overproduced by
cancer cells due to intensive aerobic glycolysis.
Experiment 3. Benzidine test for detection of blood.
To 1-2 ml of tested gastric juice 4-5 drops of 10% solution of benzidine in acetic
acid are added and thereafter 2-3 drops of 3% H2O2. In the presence even traces of blood
a blue or greenish color appears due to oxidation of benzidine.
Clinical and diagnostic significance. Blood in stomach may appear due to the
bleeding from gastric mucosa damage, e.g. in gastric ulcer. It also may be caused by
bleeding from oesophageal vein varicoses, in cases of bleeding in nose or in lungs.
Experiment 4. Urease method for detection of Helicobacter. Pylori in gastric
OH
juice.
a)
3
+ FeCl3
(C6H5O)3Fe + 3HCl
Principle of the method. H. pylori can be detected by presence of urease, an
enzyme, which decompose urea to ammonia and CO2. Ammonia shifts the pH of
solution to alkaline region and this is registered by appearance of pink color in the
presence of phenolphthalein.
Performance. To 2 ml of gastric juice 1-2 ml of urea solution are added and
thereafter 2-3 droplets of phenolphthalein. Tubes are left for several minutes at room
temperature and changes in color are observed. The appearance of red color indicates the
presence of urease, which may originate from H. pylori.
Clinical and diagnostic significance. H.pylori is detected in 92-100% patients
with duodenal ulcer, in 90% patients with gastritis, in 70-90% patients with gastric ulcer,
in 80 % cases of stomach cancer, in 40-70% patients with dispepsias without ulcers. The
main reason of harmful effect H.pylori is high affinity to membranes of epithelial cells
in gastric mucosa, which creates a favorable conditions for action of multiple bacterial
enzymes (urease, lipase, phospholipase, proteinases and catalase), which are excreted by
bacteria and damage the mucosa.
Experiment 5. Determination of amylase activity in urine after Caravay .
Principle of the method. Colorimetric detection of residual starch after its
cleavage by urine amylase according to the intensity of color in reaction with iodine.
54
The unit of amylase activity is accepted the amount of starch in grams, which is cleaved
by 1 L of urine in 1 hour . In normal condition it is equal to 20-160 g/L per hour.
Performance. In two tubes are prepared mixture of reagents as indicated in the
table.
Reagents
Control
Test sample
Substrate buffer solution
2 ml
2 ml
o
Incubation 5 min in a water bath at 37 C
Urine, diluted 1:5
0 ml
0,1ml
o
Mixing and incubation 5 min in a water bath at 37 C
1n HCl to stop the
3,9ml
3,9 ml
reaction
Urine, diluted 1:5
0,1 ml
0 ml
0,01 n iodine solution
0,1 ml
0,1 ml
After mixing the intensity of color is measured in colorimeter at red filtyer (630-690
nm)in 10 mm cuvette .
Calculation: D = (Ac – At) : Ac x 5 x 160, where D –activity of urine amylase (diastase)
in g/l.hour, Ac – extinction of control probe, At – extinction of test sample, 5- dilution of
urine, 160 – coefficient for transfornation to 1 ml of urine and 1 hour.
Examples of tests „Crock-1”
1. Patient has an insufficiency of enzyme producing function of stomach. Research of
activity of what enzyme would not be informative for this diseases in adults?
A. Renin
B. Pepsin
C. Uropepsin
D. Gastrixine
E. Pepsin B
2. Patient with chronic pancreas inflammation was prescribed an inhibitor of proteolytic
enzymes which are producted in a pancreas in the nonactive state as zymogens. What
mechanism lies in basis of proteolytic enzyme trypsin activation ?
A. Allosteric regulation
B. Phosphorilation
C. Proteolysis of C-end of hexapeptide
D. Dephosphorylation
E. Partly proteolysis of zymogen molecule
3. New-born child suffers from milk coagulation in stomach, which means that soluble
proteins of milk caseins are transformed into insoluble - paracaseins at participation of
calcium ions and some enzyme. What enzyme takes part in this process?
A. Secretin
B. Pepsin
C. Gastrin
55
D. Renin
E. Lipase
4. In a gastroenterology department was hospitalized a 7-year-old boy. He was
prescribed an biochemical analysis of blood and urine, in particular determination of
maintenance of urine pepsin. Uropepsin in pediatric practice is detected for the research
of...
A. Secretory activity of glands of stomach mucus shell
B. Intensities of processes of digestion in a stomach
C. Motoric and evacuatoric activity of stomach
D. Pathological processes in stomach
E. Pathological components in gastric juice
1.
2.
3.
4.
5.
6.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
Part III, P. 4-22.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.- 416p.
Topic № 2. Investigation of functional role of water soluble and fat soluble
vitamins in metabolism and providement of cell functions.
Objective: To learn the structure, principles of classification, functional
significance of vitamins, vitaminoids, antivitamins, biologically active supplements. To
recognize methods of qualitative and quantitative determination of water soluble and fat
soluble vitamins.
Actuality of the theme: water-soluble and fat-soluble vitamins take part in the
metabolism, as coenzymes and activators of many enzymatic and nonenzymatic
processes. Disorders in vitamins metabolism, reduces intensity of energetic and plastic
metabolism, which is accompanied with violations in function of several organs, decline
of immunity to the viral and infectious diseases, loss of organism ability to adapt
oneself to the different unfavorable factors.
Specific objectives:
 To interpret the role of water soluble and fat soluble vitamins and their precursors as
nutritional components in metabolic and physiological processes.
 To explain application of antivitamins as enzyme inhibitors in contagious diseases
and in disorders of homeostasis .
 To evaluate the role of water soluble and fat soluble vitamins in metabolism,
development of hypo- and hyper- vitaminoses, their prevention and treatment.
56
 To explain the role of vitaminoids in metabolic processes.
 To explain the role of biologically active supplements as nutritional components and
their effect in the organism.
Theoretical questions
1. Vitamins as essential nutritional components. History of vitamins discovery and
development of vitaminology.
2. Causes of exo- and endogenous hypo- and avitaminoses.
3. Vitamin B1 and B2, their structure, biological function, sources of supplement, daily
requirement. Symptoms of hypovitaminosis.
4. Structure and properties of vitamin H and pantothenic acid. Their participance in
metabolism, sources of supplement, daily requirement. Metabolic significance of
CoA.
5. Antianemic vitamins (B12, folic acid), their structure, biological function, sources of
supplement, daily requirement. Symptoms of hypovitaminosis.
6. Vitamins B6 and PP, their structure, biological function, nutritional sources, daily
requirement. Symptoms of hypovitaminosis.
7. Vitamin C and P, their structure, biological function, nutritional sources, daily
requirement. Functional interrelations between vitamin C and P, manifestations of
insufficiency in human organism.
8. Vitamins of D group, their structure, biological function, nutritional sources, daily
requirement. Symptoms of hypo- and hyper-vitaminosis, avitaminosis.
9. Vitamin A, its structure, biological function, nutritional sources, daily requirement.
Symptoms of hypo- and hyper- vitaminosis.
10.Vitamins E, F, their structure, biological role, nutritional sources, mechanism of
action, daily requirement. Symptoms of insufficiency, application in medicine.
11.Antihemorrhagic vitamins (K2, K3) and their water soluble forms, structure,
biological function, nutritional sources, mechanism of action, daily requirement,
symptoms of insufficiency, application in medicine.
12.Provitamins, antivitamins, mechanism of action and employment in practical
medicine.
13.Vitaminoids, their structure and biological activity.
14.Modern vitamin drugs, theirm application in treatment and prevention of diseases.
Biologically active supplements.
Practical part
Experiment 1. Reduction of methylene blue by ascorbic acid.
Principle of the method. Methylene blue is reduced by ascorbic acid to a colorless
leuko-form.
Performance. Into two tubes one droplet of 0,01 % sol. of methylene blue and one
droplet of 10% sol. of Na2CO3 are added. To the first tube several drops of an extract
from canine rose fruits are added, to the second - several drops of water. During heating
of tubes a discoloration of the first tube may be observed due to reduction of dye.
57
Experiment 2. Detection of vitamin E with ferric chloride.
Principle of the method. Alcohol solution of α-tocoferol is oxidized by ferric
chloride to tocoferylquinone, which possess a red color.
Performance. Into dry tube are placed 4-5 droplets of 0,1% alcohol solution of
tocoferol, thereafter is added 0,5 ml of 1% solution of ferric chloride. The content is
intensively mixed and heated on a flame up to the change of color.
Explain the result, draw a conclusion.
Clinical and diagnostical significance. Due to the modern ideas the main function
of tocopherols consists is to serve as antioxidants in relation to unsaturated lipids. By
the meaning hydrogen atom in its molecule α-tocopherol co-operates with the peroxide
radicals of lipids, redusing them into hydroperoxides and thus breaking the chain
reaction of peroxidation.
Most displays of tocopherol insufficiency depend from stopping of carried out by
the vitamin inhibiting action on autooxidation of the unsaturated fat acids which are
included in composition of cellular and subcellular membranes: hemolytic anemia in
prematurely born childs, atrophia of testicles and sterility; dissolving of embryo on the
early stages of pregnancy; muscular dystrophy which is accompanied with loss of
cellular nitrous components and muscle proteins. Direct reason of muscular dystrophy
is freeing of lysosomal hydrolases as a result of lysosom membrane defect.
Recommended dietary allowance for the grown man per day 20 – 30 mg,
concentration in the blood whey 3500 – 8000 nmole/l. With the membrane pathology,
presumably, are linked the areas of necrosis, which are observed at vitamin E
avitaminosis in a liver, brain tissue, especially in cerebellum.
Most enriched with the vitamin E vegetable butters are: sunflower, corn, cotton
and olive. Especially high maintenance of it is in butter, got from the wheat embryos,
oat, green a pea. Synthetic preparation of -tocopherol acetate in vegetable butter are
produced for an internal reception and for intramuscular injections. It is used as an
antioxidant at muscular dystrophy, violation of reproductive function in women and
men, to hemolytic anemia in new-born, in complex therapy of heart and vessels
diseases, eye and hepatic diseases etc.
Experiment 3. Reaction of vicasol with cysteine.
Principle of the method. Vicasol solution in presence of cysteine in alkaline
medium develops a lemon-yellow color.
Performance. In a tube are placed 5-10 droplets of 0,05% alcohol solution of
vicasol and then are added 5-10 droplets of 0,025% cysteine and 2,5 ml of 20% solution
of NaOH. The appearance of bright lemon-yellow color is observed.
Explain the result, draw a conclusion.
Clinical and diagnostical significance. The main active form of vitamin K is
menachinon MC-4, that appears from naphtochinons of vegetable and bacterial origin in
tissues.
The most studied function of vitamin K is his connection with the process of
blood coagullation. He is nessesary for a synthesis in the liver of protein factors of
coagulation: prothrombin (factor II), proconvertin (factor VII), Christmas factor (IX),
Stuart-Prower factor (X).
58
Vitamin K stimulates inserting of additional carboxyl groups in glutamate
residues in protrombin precursors. The synthesis of “complete” molecule of
protrombinu is thus completed, in other words - its posttranslational modification.
Joining of additional СОО - groups is nessesary for optimal binding of Са 2+, that
activates converting of protrombin into trombin.
Its role in this process consist in transport of НСО3--ions, that are inserted in position glutamine acid, or in activating of hydrogen -carbon atom of glutamine acid, or
in activating of one of enzymes in the reaction of carboxylation.
Modern data allows to consider that vitamin K, like other fat-soluble vitamins,
influences on the state of cell membranes and subcellular structures, being the
component part of lipoproteins of these membranes. Recommended dietary allowance
for the adult man of 1 – 2 mg per day, concentration in the whey of blood 400 – 600
nmol/l.
The lack of this vitamin more frequent develops as endogenous, caused by
violation in its biosynthesis in an intestine (sterilization of intestine by sulfonyl amide
preparations or antibiotics), or by violation of absorbtion (insufficient products of bile
or impassability of bile ducts, liver diseases). To insufficiency can lead also using of
preparations with properties of antivitamins K (for example, anticoagulants of mediocre
action). Basic signs of insufficiency are bleeding at small damages, hemorragia in newborn (before appearance of microflora in the intestine).
Naftochinons enters organism of man, mainly, with the meal of vegetable origin:
spinach, fruits, and also synthesized by the bacteria of thin bowel. Content of vitamin K
in food considerably exceeds minimum daily necessities and that is why, insufficiency
of this vitamin at a normal feed and physiology conditions of absorbtion of lipids, is
rare phenomenon.
In medical practice are used preparations of vitamin K and his synthetic watersoluble analogue – vikasol. It is prescribed at the pathological states which are
accompanied with hypotrombonemia and bleeding.
Experiment 4. Quantitative determination of carotene in a carrot.
Principle of the method. Carotene is extracted from investigated material by
organic solvents. The extract obtained has characteristic yellow-orange color. The
extinction of the extract is measured in colorimeter and compared with a standard
solution of carotene and quantity of carotene in test sample of carrot is calculated.
Performance. The weighted sample of a carrot is minced in a mortar with a sand.
To material 2-3 ml of acetone are added and the mass is grinded to uniformity.
Extraction of material. In the glass funnel a small piece of cotton is placed and
upon it a sand is poured in order to obtain a layer of about 1 cm. A mass of a minced
carrot is transfered to a funnel and is washed with an acetone to the complete
discoloration of the effluent.
To the acetone extract about 5-10 ml of petrol is added and thereafter 2-3 ml of
water. The content of the vessel is mixed and leaved for resolution of layers. Carotene is
transferred to an upper petrol layer, the lower layer is discarded.
59
The petrol extract of carotene is clarified by addition of some quantity of anhydrous
sodium sulfate. The volume of the extract is measured with a dry cylinder, after that the
extinction of the extract is measured in a colorimeter at red filter.
Quantity of carotene is calculated according to the formula:
C2= C1 x E2 / E1 , where
E1 - extinction of standard solution,
E2 - extinction of carotene extract
C1 - concentration of standard carotene solution
C2 - concentration of carotene in the extract. From the obtained value of carotene
concentration in extract a content of carotene in carrot is determined taking into account
volume of the extract and weight of the carrot sample.
Clinical and diagnostical significance. For a healthy man maintenances of
vitamin A in blood is about 15-60 мcg%. Deviation from a norm in the side of
increasing or decreasing of vitamin results in the origin of hyper- and hypovitaminosis.
The reason of origin of hypovitaminosis is an insufficient receipt of vitamin with
food; violation of processes of bile formation, that results in stopping of fat-soluble
vitamins absorbing, in particular - tocopherol, retinol calciferol etc.; decrease of activity
of thin bowel enzyme -carotinase, that predetermines violation in transformation of
carotin into retinol.
As fat-solluble vitamins have ability to accumulate in an organism after
introduction them in great amounts, it can result in development of hypervitaminosis
with special clinical features.
For the prevention of hypovitaminosis development are used pharmaceutical
preparations of vitamin A – retinol acetate, retinol palmitate as oily solutions, drops
and pills. Symptons for their prescription are an eyes disease, skin desease, infectious
diseases, and also pathological processes of the respiratory system, erosive defeats of
stomach and digestive system.
Examples of tests „Crock-1”
1. Methotrexate, used for treatment of some types of leukemia, can be considered as
antivitamin of the next vitamin:
A.Pantothenic acid
B.Folic acid
C.Ubiquinone
D. Pyridoxine
E. Thiamine
2. What is the biochemical mechanism of the effect of sulfanylamides upon bacterial
flora?
A. Activation of enzymatic system of p-aminobenzoic acid (PABA) synthesis
B. Stabilization of conformation of enzyme of PABA synthesis
C. Competition with PABA in synthesis of folic acid
D. Inactivation of enzymes of PABA synthesis
E. Induce synthesis de novo of enzymes involved in PABA synthesis
60
3. Chose from the following vitamins one which is considered as antianemic factor.
A. Thiamine
B. Pantothenic acid
C. Ascorbic acid
D. Folic acid
E. Pyridoxine
4. Select the metabolic process with which is most likely associated vitamin C.
A.Biosynthesis of DNA
B.Intestinal calcium absorption
C.Biosynthesis of collagen
D.Biosynthesis of glycogen
E.Oxidative decarboxylation of α-ketoacids
5. What water soluble drug instead of vitamin K is used for treatment of patients with
acute attack of cholelythiasis and obstruction of bile duct?
A. Ascorbic acid
B. Vikasol
C. Warfarin
D. Pyridoxine
E. Folic acid
1.
2.
1.
2.
3.
4.
5.
6.
Induvudual independent student’s work
Endogenous hypovitaminoses. Causes and mechanisms of development in diseases
of digestive or cardiovascular systems.
Biologically active dietary supplements. Their significance in pathological states
prevention and in well balanced nutrition.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2003,
Part II, P. 3-23.
Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The
Odessa State Medical University, 2003.- 416p.
61
Topic № 3. Investigation of acid-base equilibrium in blood and respiratory
function of red blood cells. Pathological forms of hemoglobin.
Objective: To learn biochemical composition of blood and to explain role of
plasma proteins and diagnostic significance of their determination; to evaluate changes
in content of final products of metabolism in blood. To perform studies of hemoglobin,
to interpret functional significance of hemoglobin and its derivatives, to know about
hemoglobinopathias and their distribution in human population.
Actuality of theme: The respiratory function of erythrocytes is carried by the
meaning of hemoglobin. Due to the ability to bind oxygen at the high meanings of
partial pressure and give back – at low, the molecule of hemoglobin executes the main
function of oxygen transporter: binds it in the capillaries of lung alveolus and gives it
back to the tissues in venous capillaries. Except for the transport of oxygen from lungs
to the capillaries of peripheral tissues, hemoglobin plays also a substantial role in
transport of carbon dioxide to the lungs.
In the diagnostics of the exigent states an important role belongs to the
determination of the acid-basic state, partial pressure of oxygen and carbon dioxide in
blood, state of the blood buffer systems, ionic composition of extracellular liquid,
osmolarity of biological liquids, dissociations of hemoglobin etc.
Specific objectives:
 To explain mechanism of oxygen and CO2 transport by hemoglobin in lung and
tissue capillaries.
 Classification of molecular forms of hemoglobin in healthy adults and existence of
pathological forms of hemoglobin, molecular mechanisms of their appearance.
 To learn the application of results of biochemical analysis of blood in evaluation of
physiological or pathological state of a person.
 To explain molecular mechanisms of appearance of pathological forms of
hemoglobin and changes in principally important indexes of blood analysis for
evaluation of functional state of a person.
1.
2.
3.
4.
5.
6.
7.
8.
Theoretical questions
Blood as internal medium of the body. Role of blood in homeostasis.
The composition of whole blood, blood plasma and serum. Physico-chemical and
biological properties of blood.
Blood volume and its pH. Cellular elements of blood: red blood cells, white blood
cells, blood platelets.
Hemoglobin, its structure, properties and molecular forms.
Pathology of hemoglobin: hemoglobinopathias and thalassemias.
Respiratory function of red blood cells in lung capillaries and in peripheral tissues.
The function of grade of hemoglobin oxygenation from partial pressure of oxygen,
dissotiation curve of oxyhemoglobin, Bohr effect.
Acid-base equilibrium of blood. Regulation of pH in biological fluids, disorders of
acid-base equilibrium: metabolic and respiratory acidosis, metabolic and respiratory
alkalosis, mechanisms of their development.
62
9. Role of kidneys and lungs in support of acid-base equilibrium.
10.Buffer systems of blood, their types, role of different buffer systems in providement
of constant pH of blood.
11.Principal types of hypoxias, mechanisms of their development, methods of
diagnostics.
12.Hypoxia, caused by lowering of oxygen pressure in air.
13.Hypoxia caused by pathological process: pulmonary type, cardiovascular type, tissue
and exhaustive types.
14.Hormones as regulators of osmotic pressure in blood. Role of renin-angiotensine
system in regulation of osmotic pressure.
15.Humoral mechanisms of regulation of acid-base status.
16. Dependence of biochemical indexes of blood from metabolic processes in organism.
Practical part
Experiment 1. Detection of oxyhemoglobin (Fe2+).
Principle of the method (for experiments 1,2,3,4). For obtaining of hemoglobin
derivatives (oxyhemoglobin, carbhemoglobin, carboxyhemoglobin, methemoglobin) the
following properties are used: HbO2 is not stable substance, MetHb is produced under
the influence of oxidants, HbCO is very stable substance. These derivatives are
identified by spectral analysis. The blood pigments selectively absorb the light at
distinct wavelength giving a characteristic spectra.
Performance. In a graduated cylinder 1,0 ml of defibrillated blood is diluted to
100 ml with distilled water. Note the color of the solution. The tube with 10 ml of HbO 2
solution is placed against the slit of the spectroscope. Two distinct dark bands of light
absorption are visible in yellow-green part of spectrum.
HbO2 solution is divided between 5 tubes in a quotes of about 10 ml for obtaining other
derivatives.
Experiment 2. Detection of hemoglobin (Hb)Fe2+.
Performance. To HbO2 solution in the second tube 2-3 pieces of sodium dithionite
Na2S2O4 are added and carefully mixed. The change of the color is registered and the
absorption spectrum is examined. A broad absorbtion band is visible between
Fraungoffer’s lines D and E characteristic for deoxy-Hb. The solution is carefully
hesitated in the tube in order to saturate Hb with oxygen. The color of the solution is
changed to scarlet red. In the absorption spectrum again appear two dark bands,
characteristic to HbO2.
Experiment 3. Detection of methemoglobin (MetHbFe3+).
Methemoglobin is hemoglobin derivative, in which Fe is in oxidized form Fe+3,
connected with a hydroxyl group.
Performance. Into the tube with HbO2 solution 1-2 drops of K3[Fe(CN)6] solution
are added and mixed. The change of the color is noted and the absorption spectrum is
checked.
63
Into MetHb solution 2-3 grains Na2S2O4 are added. The change of the color is
noted and the absorption spectrum is studied. Obtained Hb is converted to HbO 2 by
careful shaking of the solution. The spectrum of formed HbO2 is checked.
Experiment 4. Detection of hemin.
Experiment 4.1. Obtaining of acidic hemin.
To HbO2 solution 2-3 drops of 5% HCl are added. The change of colour is
observed and the spectrum of HbO2 disappears.
Experiment 4.2. Obtaining of alkaline hemin (OH).
To HbO2 solution 2-3 drops of 5% NaOH are added and heated to change of the
colour. The spectrum of HbO2 disappears.
The result of investigation of hemoglobin and its derivatives are noted in the table.
Derivative The reagent and
Iron
The color of
The characteristics of
of Hb
conditions of
valency
solution
light absorption
obtaining
spectrum
Experiment 5. Colloidal probes for blood protein condition (Weltman’s test).
Principle of the method. The method is based on the empirical observation that
after addition of CaCl2 solution to blood serum and subsequent heating, impairment of
colloidal stability of serum proteins occurs and proteins are precipitated. Of great
importance is the value of CaCl2 concentration at which precipitation takes place.
Performance. To 0.1 ml of blood serum 4.9 ml of distilled water are added, mixed,
then combined with 0.1 ml of 0.5 % CaCl2, stirred and carefully heated on a flame to a
boiling point. The tube is cooled and examined for appearance of precipitate. If no
precipitate is found, 0.1 ml of CaCl2 solution is additionally added and mixture boiled .
The procedure is repeated till appearance of visible precipitate.The results are estimated
by calculating of total volume (in ml) of 0,5% CaCl2 used in reaction. Normally
coagulation begins after addition of 0.4-0.5 ml of CaCl2 solution.
Clinical significance.The most specific for liver diseases is thymol test. It gives
positive results before the development of jaundice. Test with HgCl2 may be positive
in hepatic cirrhosis and toxic damage of liver. In Weltman’s test blood proteins
coagulation after addition of less amount of CaCl2 is observed in damage of liver
parenchyma as well as at malaria. In rheumatism or lung tuberculosis coagulation of
proteins develops in higher concentrations of CaCl2 then in healthy condition.
Experiment 6. Qualitative detection of calcium, magnesium, phosphates in blood
plasma.
Principle of the method. Calcium ions gives a precipitate with oxalate ions,
magnesium ions forms insoluble precipitate in presence of phosphate and ammonium
64
ions, phosphate anion gives with anion of molibdenic acid an insoluble precipitate of
characteristic yellow color. Subsequent reactions are shown below.
a) Са2+ + С2О4 2-→ СаС2О4 (calcium oxalate)
b) Mg+2 + NH4+ + HPO42- → MgNH4PO4
c) PO43- + MoO42- + NH4+ + n H+ → (NH4)3PO4 · 12 MoO3 · 6 H2O
Yellow crystals of phosphoromolibdenic acid can be reduced in acidic medium e.g.
by ascorbic acid and gives a mixture of molibdene oxides of blue color.
Performance.
Detection of calcium ions. To 0,5-1,0 ml of blood serum are added several drops of
saturated solution of ammonium oxalate. The white precipitate of calcium oxalate is
formed.
Detection of magnesium. Precipitate of Ca oxalate is eliminated by filtration and to
the transparent filtrate are added 3-4 droplets of conc. ammonium solution. White
precipitate of magnesium ammonium phosphate is formed.
Detection of phosphates. To 0,5-1,0 ml of blood plasma 5-6 droplets of ammonium
molibdate solution are added and formation of yellow precipitate is observed. After
addition of several drops of ascorbic acid solution an intense blue color develops.
Normal concentration of calcium in plasma is 2,1-2,6 mMoles/l; magnesium –
0,82-0,94; inorgasnic phosphate – 1,2-2,2 mMoles/l.
Examples of tests „Crock-1”
1. For transporting of СО2 from tissues to lungs is needed a participation of the next enzyme:
A. decarboxylase
B. Carboanhydrase
C. Carboxylases
D . Catalase
E. Cytochrome oxidase
2. Hemoglobinopathias are diseases caused by the next changes in hemoglobin:
A. Alteration of chains number in hemoglobin molecule
B. Changes in secondary structure of selected chains
C. Alteration in biosynthesis of α or β chains
D. Activation of genes, coding ε globin chains and appearance of embryonic isoforms
of hemoglobin
E. Substitution of distinct amino acids in globin chains α or β due to point mutations in
subsequent genes.
3. Patients blood analysis showed, that red blood cells have a half-moon form. Concentration of
hemoglobin - 78 g/l, amount of red blood cells -3,5· 1012 . What changes in the structure of
hemoglobin can result in such form of red blood cells?
A. Glutamine on glycine
B. Serine on valine
C. Methionine on Tyrosine
D. Glutamic acid on Valine
E. Leucine on Lysine
65
4. In a patient after laboratory investigation a decrease in blood pH and bicarbonate ions
was revealed, as well as increased content of lactate, pyruvate and acetoacetate in blood
and in urine. What type of disorder in acid –base equilibrium is observed?
A. Metabolic acidosis
B. Respiratory acidosis.
C. Metabolic alkalosis
D. Respiratory alkalosis
E. Hyperventilation alkalosis
5. A man was a prolong time in .high mountains. What changes in blood can be suggested?
A. Increase in leukocytes number
B. Decrease in leukocytes number
C. Increase in affinity of hemoglobin to oxygen
D. Decrease in pulse frequency
E. Decrease in affinity of hemoglobin to oxygen
Individual independent students work
1. Evaluation of condition of blood and its biochemical functions.
2. Disorders of acid-base equilibrium. Acidoses, alkaloses.
1.
2.
3.
4.
5.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Biological chemistry. Methodical instructions for practical exercises. Lviv-2002,
Part I, p. 21-28.
Topic № 4. Investigation of blood plasma proteins: proteins of acute phase,
genuine and indicatory enzymes. Investigation of nonprotein nitrogen containing
and nitrogen free components of blood.
Objective: To learn about principal proteins of blood plasma and to interpret their
quantities in diagnostics of hyper- and hypo- proteinemias, dysproteinemias,
paraproteinemias, hereditary diseases connected with deficient synthesis of distinct
proteins. To know about nonprotein nitrogen containing blood compounds, which
represents residual nitrogen, to learn methods of determination of residual nitrogen and
middle size molecules in blood and to interpret obtained results in diagnostics of
diseases.
Actuality of theme: In blood plasma was found out and described over 100
proteins which differ on their physical, chemical and functional properties. Among them
66
there are transport proteins, enzymes, enzyme inhybitors, hormones, factors of
coagulation, antibodies, anticoagulants, antioxidants et al.
Plasma proteins determine properties of plasma: colloid-osmotic pressure and
permanent blood volume, viscidity of blood, support of permanent blood pH and
function, namely: protective, regulatory, termoregulatory, respiratory, trophic and etc.
Remaining nitrogen is the important indicator of the state of protein metabolism.
The detection of nonproteinous nitrogen and its separate components is conducted with
the purpose of diagnostics of disorders kidneys and liver functions, endogenous
intoxication at the row of diseases and estimation of kidney insufficiency degree.
Specific objectives:
 To perform fractionation of blood proteins with salting out method.
 To know about diagnostic significance of quantitative determination of acute phase
proteins.
 To interpret molecular-biologic mechanisms of changes in content of secretory and
tisuue enzymes in blood.
 To characterize the principal nonprotein nitrogen containing compounds, their
metabolic origin.
 To determine residual nitrogen in blood samples.
 To learn spectrofotometric method of determination of middle size molecules.
 To interpret obtained results and their application in diagnostic purposes.
Theoretical questions
1. Principal groups of blood proteins, their composition and content in normal
conditions and in pathology.
2. Albumins and globulins. Resolution of blood plasma proteins by method of protein
electrophoresis.
3. Role of glutathion in protection from peroxide oxidation of lipids, antioxidant
vitamins.
4. Glycoproteins, their structure, biological role, changes in composition in diseases.
5. Proteins of acute phase of inflammation: C-reactive protein (CRP), ceruloplasmin,
haptoglobin, cryoglobulin, alpha-1 antitrypsin, alpha-2 macroglobulin, interferon,
fibronectin, their diagnostic validity.
6. Enzymes of blood plasma: genuine (secretory), excretory, indicatory (tissue)
enzymes. Kallicrein-kinine and renin-angiotensine systems, their biological
significance.
7. Diagnostic value of investigation of enzyme and isoenzyme activity in blood
plasma: creatine kinase (CK), LDH, AsAT, AlAT, amylase, lipase, cholinesterase.
8. Definition of total and residual nitrogen in blood. Nonprotein nitrogen containing
compounds of blood, their diagnostic significance.
9. Nitrogen free organic and inorganic compounds of blood, their metabolic origin.
Middle size molecules, their metabolic origin and diagnostic significance.
10. Nitrogenemia, its kinds and causes of development, differentiation in clinical
conditions.
11. Quantitative changes in residual nitrogen in parentheral nutrition, transfusions of
blood, blood plasma, serum or blood substituents.
67
12. Biochemical aspects of application of some medical preparations in treatment of
nitrogenemia.
Practical part
Experiment 1. Protein fractionation by precipitation with ammonium sulfate.
Principle of the method. The reversible and nondenaturating protein precipitation
by ammonium sulfate is based on the dehydration of protein molecule and neutralization
of its electric charge. The concentration of salt needed for precipitation depends from
the electrical charge and molecular weight of protein molecule. This relationship can be
used for fractionation of different protein mixtures.
Performance. To 2-3 ml of blood serum an equal volume of saturated (NH4)2SO4
is added and mixed. Globulins precipitate at 50% of the saturation. Globulins have high
molecular weight and small electric charge. The obtained sediment is collected by
filtration. The filtrate is divided in two parts which are placed in two tubes. To the first
tube crystalline (NH4)2SO4 is added up tol cumplete saturation of the solution.
Albumines are precipitatedt. The second tube with filtrate is boiled and albumines are
deneturated and form precipitate.
Experiment 2. Determination of rest nitrogen of blood (Bodansky’s method).
Principle of the method. The rest nitrogen of blood is determined in protein free
supernatant or filtrate of blood after precipitation and elimination proteins . Protein free
filtrate is mineralized by sulfuric acid. In this case nitrogen of organic compounds is
transformed into ammonia and is bound by sulfuric acid. Ammonium ion is determined
with Nessler’s reagent, which gives yellow-brown colored complex, and the intensity of
color is proportional to ammonium ion concentration.
Performance. In the tube 1,8 ml of water and 0,2 ml of blood plasma or serum are
mixed and 1 ml of trichloroacetic acid is added. After 15 min the precipitate is
eliminated by filtration. 1 ml of filtrate is taken off into the termostable tube and 3 drops
of conc.sulfuric acid are added. The content of the tube is carefully evaporated on a
sand bath and thereafter the residue is heated almost to clear solution. At the end of
combustion 1-2 drops of hydrogen peroxide are added and colorless solution is
obtained. The mineralized residue is cooled, thereafter t are added 10 ml of distilled
water, 6 droplets of 50% NaOH (be careful, hazard!) and 0,5 ml of Nessler reagent. The
control probe is prepared simultaneously. For this purpose to 10 ml of water 6 droplets
of 50 % NaOH, 1 drop of sulfuric acid and 0.5 ml of Nessler’s reagent. Both probes are
measured in a colorimeter at blue light filter in 5 mm cuvette. The quantity of nitrogen
is determined according to calibration curve. The rest nitrogen of blood is calculated
after the following formula: Ct= Cst x (At/Ast) = 21,42 x At/Ast mMoles/l, where Ct –
concentration of nitrogen in test sample, Cst – concentration of nitrogen in standard, At
– extiction of tested sample, Ast – extinction of standard sample.
Diagnostic significance. In healthy person the rest nitrogen content of blood
corresponds to 14-25 mMoles/l (20-35 mg%). Increase of rest nitrogen is observed in
glomerulonephritis, pyelonephritis, tuberculosis and amyloidosis of kidneys. Extrarenal
causes include disorders of hemodynamics and, subsequently, decrease in glomerular
68
filtration in kidneys. An increase in rest nitrogen is observed also during the
intensification of protein breakdown in malignant tumors, leukemias.
Experiment 3. Determination of middle size molecules content in blood.
Performance. In a centrifuge tube are placed 1 ml of blood serum and 0.5 ml of
10% trichloroacetic acid. After mixing the precipitate is eliminated by centrifugation at
3000 rpm during 30 min. To 0,5 ml of supernatant 4,5 ml of distilled water are added
and mixture is measured at 280 nm and 254 nm in spectrophotometer. In normal
conditions the level of extinction at indicated wavelength corresponds to about 0,246 at
254 nm and 0,296 at 280 nm.
Clinical significance. The level of middle size molecules in blood is increased in
combustions, liver and kidney insuficiency, heavy inflammatory processes, malignant
tumors.
Examples of ‘Crock -1’ tests
1. The maintenance of total protein is in patients blood plasma is within the limits of
norm. What indexes among mentioned below are relevant for this case?
А. 35-45 g/l
В. 50-60 g/l
С. 55-70 g/l
D. 65-85 g/l
Е. 85-95 g/l
2. Analysis of patients blood showed a considerable increase of activity of МВ-form of
CPK (creatine phosphokinase) and LDH1. What is the most possible pathology?
A. Rheumatism
B. Myocardial infarction
C. Cholecystitis
D. Pancreatitis
E. Hepatitis
3. Patient has Vilson-Konovalov disease, which is caractirised with ionized copper in
urine and laying of it in organs and tissues. Disorders of synthesis of what protein of
blood plasma is the most possible reason of this disease?
A. Transferrin
B. Haptoglobin
C. Ceruloplasmin
D. Properdin
E. Cryoglobulin
1.
2.
Indfividual independent student’s work
Role of proteins and indicatory enzymes of blood in normal conditions and in
pathology.
Evaluation of data of nitrogen metabolism and changes in content of nonprotein
nitrogen containing components of blood.
69
1.
2.
3.
4.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International
Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
Sense module № 18. Biochemistry and pathobiochemistry of blood.
Topic № 5. Investigation of coagulation, anticoagulation and fibrinolytic systems of
blood. Investigation of principal pathways of immune response.
Immunodeficiencies.
Objective: To learn the role of components of coagulation, anticoagulation and
fibrinolytic systems of blood in providement of physiological condition of blood as well
as their role in development of atherosclerosis, hypertonic disease. To characterize
biochemical components of immune system, to learn biochemical mechanisms of
immunodeficiencies.
Actuality of theme: Blood coagulation is a difficult physiological and biochemical
process which is the protective reaction of our organism on bleeding. Knowledge of
biochemical description of coagulatory, anticoagulatory and fibrinolytical systems of
blood is necessary for understanding of mechanisms of support of the aggregate state of
blood in norm and at numerous diseases, and also for their timely correction by drugs.
Specific objectives:
 To interpret biochemical principles of functioning of coagulation, anticoagulation
and fibrinolytic systems of blood.
 To learn methods of investigation of coagulation and fibrinolytic systems of blood,
to interpret obtained results.
 To know the role of components of coagulation, anticoagulation and fibrinolytic
systems in development of intravascular disseminated coagulation syndrome,
atherosclerosis and hypertonic disease.
 To characterize cellular and humoral factors of immune system.
 To explain mechanisms of development of immunodeficient states.
1.
2.
3.
Theoretical questions
Functional and biochemical characteristics of intrinsic and extrinsic blood
coagulation pathways.
Blood coagulation system; characteristics of coagulation factors. Cascade
mechanism of activation and function of blood coagulation; intrinsic and extrinsic
blood coagulation pathways. Role of vitamin K in reactions of hemocoagulation
(carboxylation of glutamic acid residues, its role in Ca binding). Medical
preparations as vitamin K agonists and antagonists.
Hereditary disorders of hemocoagulation.
70
Anticoagulation system of blood, functional characteristics of its components –
heparin, antithrombin III, citric acid, prostacycline. Role of vascular endothelium.
Changes in biochemical characteristics of blood in prolong treatment with heparin.
5. Fibrinolytic system of blood: stages and factors of fibrinolysis. Medicinal
influencing fibrinolytic process. Activators and inhibitors of plasmin.
6. Syndrome of disseminated intravascular blood coagulation. Formation of thrombs
and fibrinolysis in atherosclerosis and hypertonic disease.
7. General characteristic of immune system; cellular and humoral factors.
8. Immunoglobulins: structure, biological function, mechanisms of immunoglobulin
synthesis. Characteristics of distinct immunoglobulin classes of human blood.
9. Mediators and hormones of immune system; (interleukins, interferons, protein and
peptide factors of cell growth and proliferation).
10. Factors of complement system. Classical and alternative pathways of complement
activation
11. Biochemical mechanisms of immunodeficiencies: primary (hereditary) and
secondary immunodeficiencies; acquired immunodeficiency syndrome (AIDS).
4.
Practical part
Experiment 1. Determination of fibrinogen content in blood plasma by
spectrophotometric method (after Belitser V.A.).
Principle of the method. Fibrinogen in blood plasma is clotted by thrombin, the
clot is washed with saline, dissolved in acetic acid and light absorbency at 280 nm and
320 nm is estimated with spectrophotometer.
Performance. In a small tube (diameter 9 mm and height 85-90 mm) are added 0,2
ml of tested blood plasma, 1,6 ml of 0,06 M phosphate buffer pH 7,0, and tube is placed
in a water thermostat at 37oC for several minutes. Thereafter is added 0,1 ml of 0,04 M
solution of monoiodoacetate, after 3 min is introduced 0,1 ml of thrombin solution.
After each addition the content of tube is carefully mixed with a glass rod. After several
minutes fibrin clot is formed, which is winded upon a glass rod. The clot is washed in
two portions of saline, about 50 ml each, and finally in distilled water. The excess of
fluid is eliminated from the clot very carefully, and fibrin clot is dissolved in 5 ml of 1,5
% acetic acid. The light absorbency is determined at 280 and 320 nm on
spectrophotometer. The quantity of fibrin is calculated according to the following
formula:
X = (A280- A320) x 255 / 15,067 g/l, where:
A280- A320 is difference in absorbance at indicated wave length, 255 – coefficient for
expression of the result in g / l, 15,067 – light absorbance of fibrin solution 1 g/l in
acetic acid .
Example. A280 was 0,216, A320 – 0,019. Fibrin content will be
X = (0,216 - 0,019) x 255 /15,067 = 3,33 g/l.
Explain the obtained result and make a conclusion.
71
Diagnostic significance. Fibrinogen is a glycoprotein, which is synthesized in liver
and it is responsible for blood coagulation. In normal conditions its level is 2-4 g/l. The
increase of fibrinogen concentration in blood plasma (fibrinogenemia) is observed in
inflammatory processes (peritonitis, sepsis, hepatitis, nephritis, pneumonia etc.). It is
reasonable to note, that hyperfibrinogenemia does not indicate obviously on
hypercoagulation or thromboses development. In senile ages the content of fibrinogen is
increased.
Hypofibrinogenemia may be congenital or acquired in chronic hepatitis, liver
cirrhosis, traumatic or post traumatic shock due to decrease in its synthesis in liver. The
concentration of fibrinogen below 1 g/l may be a factor of hemorrhages in internal
organs.
Experiment 2. Determination of circulating immune complexes (CIC).
Principle of the method. Macromolecular immune complexes, which are
circulating in blood plasma, are precipitated with polyethyleneglycole (PEG). The
turbidity of samples is evaluated by measurement of light absorption at 450 nm.
Performance. Into control tube are introduced 2,7 ml of 0,1 M borate buffer pH
8,4 and 0,3 ml of tested blood serum, diluted three fold by borate buffer . Into test tube
are introduced 2,7 ml of 4% solution of PEG6000 in borate buffer and 0,3 ml of diluted
1:3 tested serum. Both tubes are leaved aty room temperature for 1 hour and then the
turbidity is measured in photometer at 450 nm. The difference in extinction of test and
control tubes is multiplied 1000 fold, which is accepted as arbitrary units of CIC.
Diagnostic significance. In normal conditions CIC level in blood corresponds to
30-100 arb. Units. Immune complexes has important significance in pathogenesis of
many diseases. Middle- and large sized CIC are the most dangerous, they can activate
complement system, coagulation and kallicrein systems, which exhibit harmful effect on
biological membranes and has cytopathogenic effect.
Examples of ‘Crock -1’ tests
1. Patient was transported to the clinic with suspicion on the myocardial infarction. For
the prophylaxis of thrombogenesis he was prescribed a preparation of fibrinolysine
(plazmin), which catalyse transformation...
A. Plasminogen into plasmin
B. Fibrinogen into fibrin
C. Protrombin into trombin
D. Proconvertin into convertin
E. Fibrine into peptides
2. During the test on AIDS there were got two positive results of imunofenzyme
analysis (IEA). What method must be used for the exception of pseudopositive result
got with IEA?
72
A. Immunofluorescence
B. Radioimmune analysis
C. Luminescence analysis
D. PCR
E. Molecular hybridization
3. In the patients blood there was detected certainly high activity of protrombin, that is
the threat of vessels thrombosis. What preparation should be used in this case?
A. Natrium oxalate
B. Potassium oxalate
C. Natrium citrate
D. Heparin
E. Ethylene diamine tetra acetate
4. Patient with the symptoms of the increased blood coagulation (thromboses,
thrombophlebitis) was treatet parenterally with an anticoagulant – heparin. However the
speed blood coagulation did not decreased. The deficit of what proteinous factor of the
anticoagulation system of blood is observed?
A. Antithrombin III
B. α2-macroglobulin
C. α1-inhibitor of proteinases
D. Antithromboplastin
E. Anticonvertin
5. In the excrement of new-born child which is on the pectoral feeding, was found out
high maintenance of immunoglobulin А. It is connected with...
A. High maintenance of immunoglobulin A in the mothers milk
B. Promoted synthesis of immunoglobulin A
C. Decreased synthesis of immunoglobulin A
D. Decreased synthesis of immunoglobulin C
E. Promoted synthesis of immunoglobulin A and M
1.
2.
3.
4.
References
Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
73
Sense module № 19. Functional and clinical biochemistry of organs and tissues.
Topic № 6. Investigation of end products of heme catabolism. Pathobiochemistry
of jaudices.
Objective: To learn biochemical pathways of heme metabolism. To interpret
biochemical indexes in blood and urine in sense of differential diagnostic of jaundices.
To learn the principal functions of liver in metabolism of carbohydrates, lipids and
simple and conjugated proteins.as well as in biotransformation of toxic compounds.
Actuality of theme: The metabolism of bilirubin – the product of heme
degradation – take place in a liver. Disorders in bilirubin metabolism cause a jaundice,
which are recognized and investigated with the use of biochemical tests on blood and
urine. Liver plays a central role in general metabolism, supporting the stability of
internal milieu of organism. The most important reactions of detoxification of
endogenous and exogenous harmful substances are performed in this organ.
Specific objectives:
 To interpret principal biochemical processes in liver, i.e.: metabolism of
carbohydrates and lipids, protein biosynthesis, urea biosynthesis, metabolism of bile
pigments and bile production.
 To analyze changes in biochemical indexes of blood and urine (direct and indirect
bilirubin) in recognition of jaundices types and pathogenesis.
 To explain the role of liver in regulation of glycemia (glycogenesis and glycogen
breakdown, gluconeogenesis) in normal and pathological conditions.
 To explain biochemical principles of insufficiency of liver function in chemical,
biological or radiation damage of liver tissue.
 To learn method of bilirubin determination in blood serum and to interpret obtained
results.
1.
2.
3.
4.
5.
6.
Theoretical questions
Homeostatic role of liver in whole body metabolism. Role of liver in regulation of
glycemia (glycogenesis and glycogen breakdown, gluconeogenesis), lipid
metabolism, turnover of bile and bile pigments. Biochemical composition of bile.
Biochemical characterization of liver function insufficiency in chemical, biological
or radiation damage of liver tissue; biochemistry of development of liver
encephalopathia. Disorders of biochemical processes in liver during some diseases.
Changes in biochemical indexes in acute hepatitis, caused by viruses or alcohol
intoxication, their diagnostic evaluation.
Changes in biochemical indexes in chronic hepatitis, cirrhosis, cholelythiasis,
dyskinesias and cholecystitis. Dependence of excretory function of liver and
disorders of digestion, their diagnostics.
Role of liver in turnover of bile pigments. Hemoglobin catabolism: production of
biliverdin, its transformation to bilirubin, synthesis of bilirubin diglucuronide and
excretion with bile.
Pathobiochemistry of jaundices; hemolytic (prehepatic), parenchimatous (hepatic),
occlusive (posthepatic). Enzymatic congenital jaundices:
74
 Crigler-Najjar syndrome as a consequence of insufficient activity of UDPglucuronyl- transferase.
 Gilbert disease – pathology caused by combined disorder of synthesis of bilirubin
diglucuronide and absorption of bilirubin from blood by liver cells (“absorption
jaundice).
 Dubin-Johnson syndrome – jaundice caused by disorder of transport of bilirubin
diglucuronide from liver cells to bile (“excretory jaundice”). Enzymatic jaundices
of neonates and methods of their prevention.
Experimental part
Experiment 1. Determination of bilirubin in blood serum.
Principle of the method. Bilirubin reacts with diazoreagent with formation of
pink colored azobilirubin. The intensity of color is proportional to bilirubin
concentration and can be measured in colorimeter at 530-560 nm wavelength.
Conjugated bilirubin reacts rapidly (direct reaction), non-conjugated bilirubin reacts
slowly In presence of accelerators (e.g. caffeine) non-conjugated bilirubin reacts as
rapidly, as conjugated one. For determination of total bilirubin (conjugated and free)
buffer solutions with accelerators (caffeine) are used.
Performance. Determination is conducted according to shown in table scheme:
Conjugated
bilirubin
Test
Conjugated
bilirubin
Control
Total
bilirubin
test
Total
bilirubin
control
reagent 1 without
accelerator
3 ml
3 ml
-----
-----
reagent 1 with
accelerator
-----
-----
3 ml
3 ml
reagent 2
(diazoreagent)
0.075 ml
0.075 ml
0.075 ml
0.075 ml
blood serum
0.4
-----
0.4 ml
-----
0.4 ml
-----
0.4 ml
Reagents
distilled water
ml
-----
The components are rapidly mixed and after 5 min (exactly) an optical density is
measured at green light filter (560 nm) against control probes . The quantity of bilirubin
is estimated according to calibration curve.
Explain the result, draw the conclusion.
Clinical and diagnostic significance .Normal values of total bilirubin
concentration in serum are 0,1-1,2 mg % (1,7-20,5 mol/l), non-conjugated 0,1-1,0
mg% (1,7-17,1 mol/l) , conjugated - 0,05-0,25 mg% (0,86-4,3 mol/ml. ). High
75
concentrations of bilirubin exhibit toxic effects, which are caused due to it
photosensibilization properties. As a metabolite of protoporphyrine, a very active
photosensibilizer, bilirubin transform molecular oxygen to singlet form, which is
extremely active oxidant. Singlet oxygen induce damage of biological membranes,
nucleic acids, proteins; it partially is expressed as hemolysis.
The increase in blood bilirubin concentration over 30 mol/l causes the jaundice.
The last one can be: hemolytic (prehepatic), parenchymatous (hepatic), occlusive
(posthepatic, obturation of bile ducts).
In hemolytic jaundice liver is not able to transform all the quantity of free bilirubin
in blood, which is produced due to increased hemolysis. In this case in blood is noted an
increase in concentration of free (non conjugated) bilirubin up to 100 mol/l. This form
of jaundice is characteristic to hemolytic and pernicious anemias.
In parenchimatous jaundice the conjugating capacity of liver cells is suppressed
due to damage of liver cells. Production of bile is decreased, conjugated bilirubin
partially returns back to blood. Bilirubinemia in this case is caused by increase in
concentration of direct and indirect fractions of bilirubin. Parenchimatous jaundice is
observed in fat degeneration of liver (steatosis), hepatitis (viral, toxic), cirrhosis of liver.
In occlusive jaundice bile is accumulated in bile ducts due to occlusion of bile duct
with bile stones or tumor and bile is returned back to blood. Bilirubinemia in this type
of jaundice is characterized by a marked increase of bilirubin level in blood ( 170-700 
mol/l) mainly due to conjugated fraction of bilirubin.
In neonates at first days of life jaundice is developed due to several causes.
Bilirubin is not transformed to derivatives due to sterility of intestines and is intensively
absorbed to blood from digestive tube. In neonates frequently is observed transient
insufficiency of glucuronyl transferase, which causes the increase of nonconjugated
bilirubin in blood.
Congenital disorders of bilirubin turnover and transport are observed in congenital
jaundices, as such: Crigler-Najjar syndrome ( absence or insufficiency of glucuronyl
transferase), Gilbert disease (insufficiency of glucuronyl transferase and block of
absorption of bilirubin from blood by liver cells – “absorption jaundice”); DubinJohnson syndrome ( disorder of transport of bilirubin diglucuronide from liver cells to
blood - “excretory jaundice”).
Examples of tests „Crock-1”
1. In a 46-year-old woman suffering from cholelithiasis a jaundice.has been developed.
Urine became darkly yellow, feces decolorized, in blood plasma was detected an
increased concentration of conjugated (direct) bilirubin. What type of jaundice can be
suggested?
A. Hemolytic
B. Parenchymal
C. Obturative
D. Jaundice of new-born
E. Gilbert disease
76
2.In preterm born child a yellow color of skin and mucus sheets is observed. A cause of
this state is the temporal lack in activity of the next enzyme.
A. Porphobilinogene synthetase
B. Aminolevulinate synthase
C. Hemoxygenase
D. UDP glucuronyl transferase
E. Biliverdin reductase
3. In a patient with manifested jaundice the next biochemical indexes were detected:: an
increase in concentration of total bilirubin in blood plasma with predominance of
indirect (free) bilirubin, in feces and urine –an increased amount of stercobilin, the
concentration of direct (conjugated) bilirubin in blood plasma – within normal limits.
What type of jaundice can be suggested?
A. Jaundice of new-born
B. Parenchymal
C. Obstructive
D. Hemolytic
E. Gilbert disease
Individual independent student’s work
1.Pathobiochemistry of hereditary jaundices.
Topic № 7. Investigation of biotransformation of xenobiotics and endogenous toxic
compounds. Microsomal oxidation, cytochrome P-450.
Objective: To interpret biochemical mechanisms of detoxofication systems of
liver: reactions of microsomal oxidation and conjugation in biotransformation of
xenobiotics and endogenous toxins. To explain biochemical principles of development
of insufficiency in liver functions due to chemical, biological or radiation damage.
Actuality of the theme. Liver detoxifies endotoxins and xenobiotics, including
drugs, which are foreign for the body and can change or damage the running of
metabolic processes. At the same time drugs and medicinals are used in pathology in
order to normalize metabolism and favor convalescence. Knowledge of kinetics of
absorbtion, transport, distribution and metabolism of drugs in organism is necessary for
development of medicinals and drugs with desired properties.
1.
2.
3.
4.
Theoretical questions
Detoxification function of liver; biotransformation of xenobiotics and endogenous
toxins.
Typer of reactions of biotransformation of foreign substances in liver.
Reactions of microsomal oxidation; inducers and inhibitors of microsomal
monoxygenases.
Conjugation reactions in hepatocytes: biochemical mechanisms, functional
significance.
77
5. Electron transport chains of endoplasmic reticulum. Genetic polymorphismus and
induction of biosynthesis of cytochrome P-450.
6. Development of tolerance to medicinal and drugs and its causes.
Practical part
I. Methods of qualitative detection of phenacetine and its metabolites
Phenacetine – 1-ethoxy-4-acetamidobenzene – possess an anti-inflammatory, pain
relief activity. It is metabolized by dealkylation and hydroxylation and formation of
paracetamol, p-phenetidine, p-aminophenol. Part of phenacetine is excreted with urine
in unchanged form, another part is transformed to glucuronides and sulphates and also
excreted with urine.
Most of reactions for detection of phenacetine in biological material are connected
with detection of p-aminophenol . The last is obtained in a way of extraction of
phenacetine with chloroform in acidic conditions and subsequent hydrolysis of dried
extract with conc. HCl. In hydrolyzate is determined p-aminophenol with several
characteristic reactions.
Experiment 1. Formation of indophenol dye.
To aliquote of tested sample is added droplet of 2% solution of chromic acid
anhydride or 5 % solution of potassium bichromate. Appearance of violet color which
is gradually transformed to cherry red indicates on the presence of p-aminophenol.
Experiment 2. Formation of ammonium salt of indophenol.
To the tested sample is added droplet of 5% phenol solution, several droplets of
freshly prepared solution of calcium hypochlorite , after mixing it appears violetreddish color. After addition of water ammonium solution the color is changed to blue.
Experiment 3. Obtaining of azocompound.
To the tested solution, containing HCl, is added dropwise 1% solution of sodium
nitrite and after several minutes several drops of alkaline solution of 2-naphthol.. In
presence of p-aminophenol it develops a red color or forms red precipitate..
II. Detection of salicylic acid in biological fluids.
Salicylic acid is used in medicine in a form of sodium salts, ointments or chemical
derivatives (aspirin) for treatment of skin diseases, rheumatism and other diseases. It is
excreted from the body partially in unchanged form, as well as conjugates with glycine
or glucuronic acid.. Metabolites of salicylic acid includes 0-hydroxybenzoylglucuronide
and 0-carboxyphenylglucuronide.
Due to hydroxylation are formed also 2,3dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid and 2,3,5- trihydroxybenzoic acid,
which are eliminated with urine.
Experiment 4. Detection of salicylic acid in urine with iron nitrate.
To 0,5 ml of urine specimen are added 4,5 ml of 0,55% solution of iron nitrate in
0,04 n solution of nitric acid. Development of purple red color indicates on the presence
of salicylic acid in tested sample.
78
Side effects of xenobiotics oxidation, significance for pharmacy and
pharmakotherapy. Consumption of some drugs may lead to a state of tolerance, in
which there is a decrease in responsiveness to the drug. This phenomenon is caused by
proliferation of smooth endoplasmic reticulum membrane as well as increase in total
protein and microsomal protein, especially cytochrome P-450 , per gram of liver. Crosstolerance may also occur. Any drug that requires the phenobarbitol-induced enzyme for
its detoxication will also dosage increase to maintain its clinical effectiveness. A
classical example of such an interaction involves the use of the anticoagulant warfarin.
When phenobarbitol and warfarin are administered together, the warfarin soon becomes
ineffective. If phenobarbitol is withdrawn after warfarin dosage has been increased, the
patient quickly becomes susceptible to internal hemorrhaging. In this circumstance a
warfarin overdosage has occurred because without the inducing effect of phenobarbitol,
the induced enzymes quickly revert to their previous levels. Consequently warfarin
accumulates in the body and inhibits blood clotting to a significant extent.
Examples of tests „Crock-1”
1.
During biotransformation of drugs of barbiturates class (hexobarbital,
phenobarbital, pentobarbital) the next type: of reaction take place:
A. Oxidative hydroxylation of alkyl side chains of cyclic compounds
B. Oxidative hydroxylation by the type of benzene hydroxylation
C. Reactions of oxidative dealkylation, in particular N-dealkylation
D. Reduction reactions, in particular reductive dehalogenation
E. Reduction reactions, in particular reduction of nitrogen containing compounds
2. Biotransformation of tetrachloroethane occurs according to the next type:
A. Reduction reactions (without oxygen), in particular reductive alkylation
B. Oxidative dealkylation, in particular N-dealkylation
C. Oxidative hydroxylation by the type of benzene hydroxylation
D. Oxidative dealkylation, in particular O-dealkylation
E. Reduction reactions, in particular reduction of nitrogen containing compounds
3. In clinical practice as an index of protein putrefaction in intestines and functional
ability of liver is investigated the urine excretion of animal indican, which is produced
in liver by the next reaction of conjugation:
A. Sulfation
B. Glucuronation
C. Methylation
D. Acethylation
E. Glycinilation
4. For determination of biotransformation activity of drugs in human body the rate of
the next reaction is measured:
A. Acetylation of sulfonylamide
B. Acetylation of penicillin
C. Acetylation of aspirin
79
D. Methylation of sulfonylamide
E. Methylation of penicillin
Individual independent students work
1. Reactions of microsomal oxidation and conjugation in biotransformation of
xenobiotics and endogenous toxins.
Topic № 8. Investigation of water and mineral metabolism.
Objective: To learn processes of water and minerals turnover in human body. To
learn methods of qualitative and quantitative determination of inorganic substances in
blood serum and to interpret obtained results with diagnostic purpose and in treatment
of some diseases.
Actuality of the theme. Water and minerals are of vital importance for normal
functioning of human body. They are changes in their turnover in different diseases,
thus knowledge and understanding of processes of their turnover as well as their
qualitative and quantitative determination is necessary for evaluation of water-mineral
metabolism.
Theoretical questions
1. Biological role of water and its distribution in human body. Water balance, its
types.
2. Regulation of water and mineral metabolism, its disorders. Dehydratation and
rehydratation, biochemical mechanisms of their development.
3. Mechanism of Na,K-ATPase action and its regulation
4. Biogenic elements, their classification, pathways of their providement.
5. Biological role of macroelements, trace elements and ultramicroelements.
6. Human microelementoses: endogenous and exogenous causes (technogenic,
yatrogenic, etc.). Oligotherapy.
7. Influence of radioactive isotopes, X-rays and other kinds of radiation upon
mineral balance.
8. Methods of investigation of water and mineral metabolism
Practical part
Experiment 1. Colorimetric method of potassium determination in blood serum.
(Lasarev method)
Principle of the method. Potassium ions in presence of Pb=2, Cu+@ and NO2- ions
forms water insoluble compound K 2Pb[Cu(NO 2) 6], which can be dissolved in mixture
of rivanol and conc. acetic acid. Optical density of solution is determined, which is
proportional to the quantity of NO2 ions.
Performance. Into centrifuge tube is added 0,5 ml of sodium acetate, 0,1 ml of
tested blood serum, 0,5 ml of copper acetate, lead acetate and sodium nitrite. The
content of tube is leaved for an hour, therafter centrifugedat 1500 rpm for 10 min.
Supernatant is discarded, to the sediment is added 1 ml of sodium acetate. Tube is
80
centrifuged and sediment wached once again with sodium acetate. To obtained sediment
is added 1 ml of rivanol solution and 2 ml of concentrated acetic acid. Mixture is stirred
in order to dissolve sediment, then solution is adjusted to 25 ml with water and optical
density is measured at 540 nm (green filter). Potassium is estimated according the
formula:
K = A x 36 (mg%), where A is extinction coefficient.
Clinical and diagnostical significance of potassium determination. Investigations
of mineral composition of blood, urine and other biological liquids has an huge value in
diagnostics of violation of water-electrolyte composition, ground of the drinking mode,
diet and therapy. A changes in the row of elements in biological liquids often is specific
for different diseases.
Potassium is cation basic part of which is inside the cell – up to 98%. Insignificant
part of it is contained in intracellular space and does not play a substantial role in
support of osmotic pressure. At the decline of potassium level in blood (less than 3
mM/l) it is come changes in the work of heart, a rhythm and conductivity are violated.
Hyperkaliemia displays in nausea, vomit, bradicardia, violation of cardiac rhythm.
Increase of concentration potassium of plasma higher than 6,5 mM/l – threatening,
higher than 7,5 to 10,5 mM/l – toxic, and higher than 10,5 mM/l – is mortal. Reasons of
hyperkaliemia are: an decreased secretion of potassium with urine at kidney
insufficiency, intravenous injection of potassium-containing solutions, cell necrosis,
insufficiency of adrenal glands etc. Hypokaliemia is accompanied with muscular
hypotonia, by apathy and skin dryness. There is vomit, tachycardia, declined arterial and
growth of venous pressure, arrhythmia, decline of tolerance to cardiac glycosides.
Reasons of hypokaliemia: loss of potassium through a gastro-intestinal tract (vomit,
diarrhoea, reception of diarrhoea pills), during the reception of medications (diuretics,
hypotensive preparations), chronicpyelonephritis and glomerulonephritis etc.
Experiment 2. Determination of calcium in urine (Sulkovitz probe).
Principle of the method. The method is based on formation of insoluble Ca
compounds in its increased quantity in urine. Sulkovitz reagent contains oxalic acid 2,5
g, ammonium oxalate – 2,5 g, conc. acetic acid – 5,0 ml, the volume is adjusted to 150
ml with water.
Performance.To 5 ml of urine are added 2,5 ml of Sulkovitz reagent. In normal
conditions the milky turbidity develops aftyer 30 sec. In increased quantitiy of calcium
the turbidity is more intensive, in decrased calcium content – urine is transparent.
Clinical and diagnostical significance. A calcium almost does not take part in
the support of osmotic pressure, because his maintenance in a intracellular sector is
small and considerable part of ion is binded with proteins. In the regulation calcium
metabolism take part parathormon, derevatives of vitamin D3 and calcitonin. An
increase or diminishing of maintenance of calcium ions in blood plasma can result in
the different pathological states. Medicinal preparations which contain salts of calcium
find in this connection wide application in different industries of medicine. At
hypercalcemia general maintenance of calcium in the blood whey makes more than 2,9
mM/l, and the ionized calcium – more than 1,38 mM/l. Hypercalcemia, that exceeds
3,75 mM/l (15 мg%), can cause the sudden stop of heart. Growth of the concentration of
81
ionized calcium conduces to the pathological states, which display polyuria, vomit,
depression, violation of cardiac rhythm. Hypocalcemia is observed when the level of
total calcium in the blood whey is less than 2,12 mM/l (8,5 мg%) and ionized calcium –
less 1,12 мM/l (4,5 mg%). Hypocalcemia in clinical practice observed more frequent
and flows more difficultly, than hypercalcemia. «Sharp» Hypocalcemia results in
development of tetania, «chronic» calcium insufficiency can be accompanied with
violations in function of skeletal and smooth musculature, sheart and vessels system,
violation of blood coagulation, development of osteoporosis. Reasons of hypocalcemia:
insufficiency of parathyroid glands, violation of absorbtion or increased taking out of
calcium at disorders of digestion and absorbtion, deficit of vitamin D or resistance to
him, at a rachitis etc.
Examples of tests „Crock-1”
1.
A patient which consumed great quantity of fish derived foodstaffs, containinig
excess of vitamin D) was admitted to a hospital due to complaints for increased blood
pressure. A roentgenologic investigation revealed presence of stones in urinary ducts. In
a patient was recognized:
A. Hypercalcemia
B. Hypocalcemia
C. Hyperuricemia
D. Hypouricemia
E. Hyperkaliemia
2.
A tourist in a hot day was deprived of drinking-water for a long time. Finally he
met a spring and satisfied thirst. It resulted in:
A. Increase in osmolality of extracellular fluid
B. Decrease in osmolality of extracellular fluid
C. Increase in osmolality of intracellular fluid
D. Decrease in osmolality of intracellular fluid
E. Increase in intracranial pressure
3. A patient who during long time accepted medication with spironolactone (antagonist
of aldosteron) addressed to the physicianr with complaints on tachicardia and
hypotension. Thecause of such state can be:
A. Hyponatremia
B. Hepernatremia
C. Hypercalcemia
D. Hypocalcemia
E. Hyperkalemia
Individual independent work for students
1. Human microelementosis.
2. Diagnostics of water-electrolyte composition.
82
References
7. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers
International Boston-London. - 1992, 980 p.
8. Biochemistry / Trudy McKee, James R. McKee. - 1996, 654 p.
9. Biochemistry / Trudy McKee, James R. McKee. - 1999, 288 p.
10.Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.
Mayes, Victor W. Rodwell. - 2003, 927 p.
11.Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s
Illustrated Reviews. - 1994, 443 p.
12.Biological chemistry. Methodical instructions for practical exercises. Lviv-2004,
P.III, p. 67-68.
Topic № 9. Functional role of kidneys in urinogenesis. Normal and pathological
constituents of urine.
Objective: To know physico-chemical properties of urine; principal normal and
pathological constituents of urine and pathways of their appearance. To be able to
conduct biochemical analysis of urine and to interpret obtained results.
Actuality of the theme: A biochemical analysis of urine is obviously performed
in ambulatory and clinical conditions for diagnostics of several diseases.
During biochemical investigation both normal and pathological constituents of
urine are determined. The biochemical analysis of urine provides information about the
functional state of kidneys, about the metabolism in different organs and in the body as
a whole, helps to reveal causes, character and prognosis of pathological process, permits
to evaluate efficiency of treatment. Besides, investigation of urine for presence of drugs
or their metabolites permits to evaluate pharmacological effect of drugs and predict
therapeutical effect.
In clinical practice recently are widely used automatic biochemical analyzers for
investigation of urine which enable in relatively short time and in the small volume of
biological material to estimate several important biochemical parameters.
For express diagnostics of diseases different firms produce a test strips, which
contain dry reagents (enzymes etc.), which produce the change in color of indicatory
area as a result of interaction with distinct metabolites in samples of biological material,
for example, urine.
Concrete tasks:
 To interpret the biochemical mechanisms of of water-salt exchange regulation and
role of kidneys in the formation of urine.
 To analyse the biochemical composition of urine in a norm and at during
pathological processes; to estimate the functional value of the final products of
nitrous exchange (urea, urinary acid, creatinine) and products of detoxication
(animal indican, hippuric acid), changes in their dayly secretion.
 To analyse the man state of health on the basis of biochemical parameters of changes
of intermediate and final products of metabolism in blood and urine.
83
Theoretical questions
1. Role of kidneys in regulation of volume, composition of electrolytes and acid-base
equilibrium of biological fluids. Biochemical mechanisms of urine production
(filtration, reabsorption, secretion and excretion). Characterization of renal
clearance and renal threshold, their diagnodtic significance.
2. Humoral mechanisms of regulation of water and mineral metabolism and kidney
function; antidiuretic hormone; aldosteron.
3. Renin-angiotensine system. Natriuretic factors of heart atrium and other tissues.
Biochemical mechanisms of development of renal hypertonia.
4. Physico-chemical properties of urine: volume, color, odor, transparency, acidity
(pH), its dependence from diet. Role of kidneys and lungs in regulation of acidbase equilibrium. Ammoniogenesis.
5. Pathobiochemistry of kidneys. Biochemical changes in glomerulonephritis,
amyloidosis, pyelonephritis.
6. Pathobiochemistry of kidneys. Biochemical changes in acute renal insufficiency.
Diagnostics of chronic renal insufficiency.
7. Conditions of renal stones formation, their chemical nature and preventive
measures.
8. Pathological constituents of urine – blood, hemoglobin, cretain. Causes and
pathways of their appearance in urine.
9. Glucosuria, galactosuria and pentosuria, causes of their development. Clinical
significance of their detection.
10. Clinical significance of detection and determination of indicane, phenylpyruvic and
homogentisic acids in urine.
11. Clinical significance of detection and determination of ketone bodies in urine.
12. Differentiation of jaundices on the appearance of bile acids and bile pigments in
urine.
Practical part
For the laboratory researches is used a morning urine. Urine selection must be
conducted in sterile conditions, in order to avoid the bacteria and mushrooms
contamination. The urine selection, especially dayly, requires the conservating with
such compounds as thymol, toluene, formaldehyde, chloroform. Urine for the enzymes
researches should not contain preservatives; it needs to be cooled or freezed. For our
researches we make collection middle portion of urine during the first morning
urination.
The urine analysis is conducted, beginning from the estimation of physical and
chemical properties: amount, color, smell, transparency, reaction (pH) and density of
urine.
Physical and chemical properties of urine.
A.Urine volume. The quantity of urine excreted in 24 hours is defined as
diurhesis. The volume is measured with graduated cylinder.
Normally healthy person excretes daily in average 1100-1600 ml of urine. Increase
in volume is called polyuria, decrease - oliguria, complete absence of urine - anuria.
84
B. Color. Normally urine has straw-yellow color due to the presence of specific
pigments - urochrome, urobilin, uroerythrin. In pathological conditions color may be
changed due to occurence of bile pigments (bilirubinuria) or hemoglobin
(hemoglobinuria, hematuria). Color of urine depends as well from different pigments in
foodstuffs.
C Odor. Normally urine has specific odor due to the presence of traces of volatile
compounds. Ammonia odor appears in bladder inflammations, purulent - in gangrenous
processes, fruit odor - in diabetes mellitus. Odor of urine also depends from the
presence of specific substances in foodstuffs.
D. Transparency. Normally fresh urine is transparent. By standing it may appear
some sediment due to formation of mucin clots. Turbidity may be caused by sediments
of phosphates (in alkaline medium), urates (in acidic medium), mucin, cellular
elements, microorganisms.
E. Differentiation of organized and non organized sediments (experiment 1).
Into 2 tubes 5 ml of urine are taken and 1 ml of 10 % acetic acid is added to the
first tube and 1 ml of 5 % NaOH - to the second tube. Non organized sediments (
phosphates, carbonates, oxalates) dissolve in acid, urates dissolve after heating or after
addition of alkali. In cases when turbidity does not disappear even after addition of 30%
NaOH , then sediment is classified as organized, i.e. composed from cellular elements
or bacteria.
F. The acidity of urine (pH) is determined with pH sensitive indicator papers or
with pH-meter.
Normally urea has pH in ranges 4,5 - 8,0 and depends from character of diet or
metabolic disorders. Alkaline urine may be in hyperaciditas of gastric juice, in loss of
acid equivalents in massive vomiting, in cystitis or pyelitis . Acidic reaction of urine is
characteristic in diabetes mellitus, starvation (due to the presence of ketone bodies), in
renal insufficiency. Highly acidic urine is in gout and fever .
G. Urine density is measured with urometer ( sort of areometer) with subsequent
scale.
Experiment 2. An aliquote of urine is taken into small cylinder and then urometer
is introduced. The value of density is taken due to the lower meniscus.
In normal conditions urine density is in the range 1,014-1,025 kg/l. Urine density
characterizes the concentrating ability of kidneys, as it is proportional to the quantity of
solids in urine. Decrease in density is called hypostenuria and is characteristic to kidney
diseases (renal insufficiency). Constant excretion of urine with low density is called
isostenuria. Increase in urine density can be observed in water deprivation or organism
dessication, in fever states. In advanced diabetes mellitus urine has higher density due to
the presence of sugar.
Experiment 3. Determination of ammonium in urine (express method).
Principle of the method. Formaldehyde while interacting with ammonium cloride
produce hexamethylene-tetraamine (urotropin) and free HCl. The quantity of HCl is
proportional to the initial content of ammonium ion. HCl is measured by titration with
0,1 n NaOH.
85
Performancy. Into 50 ml flask 10 ml of urine sample are placed, 2-3 drops of
phenol-phtalein solution are added and urine is titrated with 0,1 n NaOH to the
appearance of faint pink color. The 5 ml of neutral formaldehyde solution is added and
the flask is left for 5 min. Thereafter the content of the flask is titrated witn 0,1 n NaOH
to the appearance of pink color, stabile for 30 sec.
Calculation. The quantity of ammonia is calculated according to formula:
CNH3 = 0,10 x a x D / 10, where:
a - volume of 0,1 n NaOH, expended for titration,
D - diurnal diurhesis
10 - urine sample volume.
In normal conditions daily excretion of ammonia is equal to 43-95 mmole/24 hrs.
It is equivalent to 0,6-1,3 g/24 hrs.
The increase in ammonia excretion take place in metabolic or respiratory acidosis,
cystitis and cystopyelitis, fever, hyperaldosteronemia .
Pathological constituents of urine
Experiment 4. Detection of protein in urine.
Principle. The detection of protein in urine is based on the precipitation of
protein with sulfosalicylic acid.
Performance. To 2 ml of urine 5 droplets of 20 % solution of sulfosalicylic acid
are added. In presence of protein white precipitate or turbidity appears. Sometimes
boiling is necessary.
Experiment 5.Quantitative determination of protein in urine after Roberts Stolnikoff method.
Principle of the method. Strong nitric acid induce precipitation of protein at
minimal concentration of the last in urine 0,033 g/1.
Performance. Into 6 tubes two ml of distilled water are placed. To the first tube 2
ml of tested urine are added and after mixing 2 ml are transferred to the second tube.
The procedure is repeated , 2 ml from the last tube are discarded. In such a way
progressive dilution of urine is achieved (2, 4, 8, 16, 32, 64 fold dilutions). Into each
tube 1 ml of 20% solution of nitric acid is carefully underlayered. The appearance of
white precipitate in a form of ring in the interphase between two liquid layers is
registered. The tube is noticed, in which the ring forms at 2-3 min of standing. The
concentration of protein in this tube corresponds to 0,033 g/1. After multiplication of
this concentration on the degree of dilution protein content in tested urine is obtained.
Experiment 6. Detection of sugar in urine.
Principle of the method. All mono- and disaccharides with a free hemiacetal
hydroxyl possess reducing properties and reduce metal ions (silver, copper, bismuth)
with degradation of carbon chain and polymerization. Reactions are performed after
elimination of protein, as it is an interfering substance. To 10-15 ml of urine 10
droplets of 10% acetic acid are added and boiled. The sediment if appeared is
separated by filtration. In the filtrate detection of sugar is performed. By Trommer'
reaction. To 1 -2 ml of test sample 1 ml of 5% NaOH and 2-3 drops of CuS04 solution
are added. A precipitate of Cu(OH )2 is formed. Instead of this reagents 1 -2 ml of
86
Fehling' liquor may be added. After mixing the tube is boiled carefully. Yellow
colored precipitate of cuprous hydroxide is formed. In course of boiling the color of
the solution changes to red as a result of cuprous oxide formation.
Barfoed' reaction. Principle of the method is as described above, but this
reaction is conducted in acidic medium. Barfoed' reaction is positive only with
monosaccharides, which are more strong reducers as di- or tri saccharides.
Performance. Tol ml of tested solution 1 ml of Barfoed' reagent is added and
heated in boiling water bath during 5 min. Red colored precipitate forms in the tube
when glucose is present.
Experiment 7. Quantitative determination of glucose in urine (express method
with glucotest strips).
A strip of glucotest is immersed in the urine specimen for few sec and thereafter
placed on a white surface for 2 min. The color, developed by test band is compared with
a scale, corresponding to 0,1, 0,2 , 0,5, 1,0 % glucose concentration. The concentration
of glucose in tested urine is determined.
Experiment 8. Detection of blood (benzidine probe). To 3 ml of urine 2-3
droplets of 3% H2O2 and 2-3 droplets of benzidine in acetic acid are added. In presence
of blood color changes to green-blue.
Experiment 9. Detection of bile acids (Pettenkofer's probe).
Principle of the method. Bile acids form purple colored product with
hydroxymethyl furfurol, which is formed after an action of cone. sulfuric acid upon
sucrose.
Performance.To 2-3 ml of urine 1-2 droplets of 10 % sucrose solution are added
and 1-2 ml of cone. sulfuric acid are carefully underlayered. In the presence of bile
acids a purple color in the interphase of two layers appears.
Experiment 10. Determination of ketone bodies in urine.
Principle of the method is based on a property of acetone to give violet color
layer after an overlay of ammonia upon the mixture of test solution, sodium
nitroprusside and acetic acid. The rate of the colored layer formation in a form of ring
depends from acetone concentration. It is assumed, that appearance of the ring at 3-4
min corresponds to the acetone content 8.5 mg/1 (8.5 ug/ml).
Performance. 8 tubes are taken for the experiment. Into each tube, except the first
one, 1 ml of distilled water is added. Into the first and the second tubes 1 ml of urine of
the patient with diabetes mellitus are added. Thus, in the first tube urine is undiluted, in
the second one the urine is two times diluted. After the mixing 1 ml of the mixture from
the second tube is taken off and transferred to the third tube, after mixing 1 ml from the
third tube - into the following one and so on. Into each tube 8 drops of 50% ammonium
sulphate solution are added for increasing the density of the solutions, thereafter 8 drops
of concentrated acetic acid and sodium nitroprusside are added. The content of the tubes
is well mixed and into each tube 1 ml of concentrated solution of ammonia is overlaid
carefully beginning from the last tube (be careful with concentrated ammonia!). An
87
appearance of red or violet ring in the tubes is registered. The last tube in which a
colored ring appears between the 3-d and the 4 -th min is registered and the dilution of
urine sample is noted.
Calculation. X=0.85xAx 15, were X - content of acetone in urine (mg/day);
0.85 - empirical index;
A- the dilution of the urine sample;
15 - coefficient for calculation on the daily volume of the urine (1500 ml).
Examples of tests „Crock-1”
1. During laboratory investigation in 18-years-old patient the presence of glucose in
urine at and its normal concentration in blood plasma were detected. The most probable
cause of this state may be disorder of:
A. Insulin secretion
B. Glomerular filtration
C. Glucocorticoid secretion
D. Secretion in renal tubuli
E. Reabsorbtion in renal tubuli
2. A 65-years-old man, which is suffering from gout, complains on pains in the kidneys
area. USI (ultrasound inspection) showed the presence of kidney stones. Increase in
concentration of what substance in urine is the most probable cause of stones formation
in this case?
A. Bilirubin
B. Uric acid
C. Urea
D. Cystine
E. Cholesterol
3. A patient complains on polyuria (5 l per day) and thirst. Biochemical indexes: a
glucose concentration in blood - 5,1 mMoles/l, specific density of urine 1,010. Glucose
and ketone bodies are absent. What is possible cause of such state?
A. Myxoedema
B. Vasopressin insufficiency
C. Diabetes mellitus type 1
D. Thyrotoxicosis
E. Diabetes mellitus type 2
4. During laboratory investigation of the patient the presence of glucose in urine was
revealed at the level of 0,9 %. Clinical investigation revealed no pathology. The content
of glucose in blood is 4,2 mM/l. What may be possible reason of glucose excretion with
urine?
A. Kidneys glucosuria
B. Diabetes mellitus type 1
88
C. Diabetes mellitus type 2
D. Alimentary hypoglycemia
E. Hypoglycemia
5. In urine of new-born child was found citrulline and increased concentration of
ammonia. Biosynthesis of what compound is altered the most brobably?
A. Ammonia
B. Uric acid
C. Urea
D. Citrate
E. Creatine
6. The urine of 2-year-old boy with alcaptonuria develops black color while standing
in a vessel.. Other signs of disease are not observed. This disease is congenital disorder
of the next metabolic pathway:
A. Alanine metabolism
B. Urea synthesis
C. Uric acid synthesis
D. Tyrosine metabolism
E. Cysteine metabolism
Individual independent work of students
1. Pathobiochemistry of kidneys. Clinical and biochemical changes at
glomerulonephritis, amyloidosis, pyelonephritis, sharp and chronic kidney
insufficiency.
Тopic № 10. Investigation of biochemical constituents of connective tissue.
Objective: To learn composition and metabolic peculiarities of connective tissue,
its functioning in normal conditions and in pathology. To conduct a quantitative
determination of hydroxyproline in urine for diagnostics of some diseases.
Actuality of theme: An important problem of modern medicine is investigation
of metabolic disorders in connective tissue in acquired and congenital diseases. In this
connection it is necessary to learn and to know some biochemical tests used for in time
diagnostics of these diseases and correct treatment.
Concrete tasks:
 To give morphological and biochemical description to the components of
connecting tissue (fibrillar structures, basic intercellular substance).
 To explain a biochemical changes in the connecting tissue at a senescence and some
pathological states (mucopolysaccharidosis, collagenosis).
 To assimilate the method of quantitative determination of hydroxyproline and
qualitative reaction of exposure of glycosaminoglycans (mucopolysaccharides) in
urine, to give an estimation of got results.
89
1.
2.
3.
4.
5.
Theoretical questions
General characterization of morphology and biochemical composition of
connective tissue. Biochemical composition of intercellular matrix of loose
connective tissue: fibers (collagen, elastin, reticulin); amorphous material.
Proteins of connective tissue fibers: collagen, elastin, glycoproteins and
proteoglycans. Biosynthesis of collagen and formation of fibrilar structures.
Complex carbohydrates of amorphous matrix of connective tissue –
glycosaminoglycans. Mechanisms of formation of intercellular matrix by molecules
of glycosaminoglycans – (hyaluronic acid, chondroitin-, dermatan-, keratansulphates). Distribution of glycosaminoglycans in different human organs and
tissues.
Pathobiochemistry of connective tissue. Biochemical mechanisms of development
of mucopolysaccharidoses and collagenoses, their biochemical diagnostics.
Rheumatism – its clinical and biochemical diagnostics.
Experimental part
Experiment 1. Quantitative determination of hydroxyproline in urine.
Principle of the method. Hydroxyproline is oxidized to the substance of pyrrole
nature, which after condensation with Ehrlich reagent (p-dimethylaminobenzaldehyde) gives a product of pink color. The intensity of color is
proportional to a concentration of hydroxyproline.
Performance. Into one tube is added 1 ml of tested urine, into another tube – 1 ml
of water. To both tubes are added 1 ml of 0,01 M solution of copper sulphate, 1 ml of
2,5 n NaOH, 1 ml of 6% solution of hydrogen peroxide. Probes are stired for 5 min,
thereafter tubes are placed into boiling water bath for 3 min. After cooling to tubes are
added 4 ml of 3 n solution of sulfuric acid and 1 ml of Ehrlich reagent. Tubes are
heated on boiling water bath for 1 min and after cooling optical density is measured in
a colorimeter at 500-560 nm (green filter) in 10 mm cuvette.
After a calibrate diagram determine maintenance of hydroxyproline in an
experimental sample and calculate the amount of hydroxyproline, secreted with urine
during day using a formula:
а х Vday
Х=
Vexp
,
where a - is an amount of hydroxyproline, founded using a calibrate diagram; V day
- is a day's volume of urine, ml; Vexp - a volume of urine, taken for an analysis, ml.
47
А
0,5
0,4
0,3
0,2
0,1
0,5
1
1,5
2
2,5 С, mg
Curve of dependence of optical density of hydroxyproline solution from its
concentration
Explain the got result. To write a conclusion
Clinical diagnostic significance. The quantity of hydroxyproline excreted with
urine in in healthy adults in 24 hours is about 15-20 mg. In 10-20 years old persons are
excreted round 200 mg of hydroxyproline. Increase in excretion of this amino acid up
to 1 g/day is observed in collagenoses (rheumatism, rheumatoid arthritis, sclerodermia,
dermatomyositis), in hyperparathyroidism, Paget’s
disease (generalized
osteosclerosis). A substantial loss of hydroxyproline is observed in congenital
hydroxyprolinemia, caused by deficiency of hydroxyproline oxidase.
Experiment 2. Qualitative reaction on glycosaminoglycans (Barry-Spinager
test).
Principle of the method. Interaction of glycosaminoglycans with toluidine blue
causes change of color from blue to purple (methachromasia).
Performance. On strip of filter paper are applied spots of 5, 10 , 25 ul of urine.
After drying the paper is immersed into 0,04 % solution of toluidine blue for 1 min.
After destaining in 10% solution of acetic acid the color of spots is registered. If the
quantity of glycosaminoglycans in urine sample is more then 0,10 mg/ml , then spot
shows change color to purple red.
Clinical diagnostic significance. In healthy adults with urine are excreted 2,7-7,5
mg per day of glycosaminoglycans. ( mainly chondroitin sulphates A and C). In
hurgoidism and Hurler’s syndrome the excretion of glycosaminoglycans is increased to
30-80 mg per day.
Examples of tests „Crock-1”
1. In scurvy the disorder in hydroxylation of proline and lysine occurs, which alter the
collagen structure. Inhibition of what biochemical process causes this disorder?
A. Oxidative phosphorylation
B. Microsomal oxidation
C. Lipid peroxidation
D. Peroxide oxidation
E. Tissue respiration
48
2. With ages human cartilage tissue restore proteoglycans more slowly proceeding,
which leads to decrease in hydratation and loss of elasticity of the tissue.that results in
diminishing of degree of their gradation and loss of resiliency of this tissue. What
lyzosomal enzymes activity does increase in this case?
A. Cathepsins, glycosidase
B. Isomerase, dehydrogenase
C. Deaminase, decarboxylase
D. Decarboxylase, lipase
E. Oxidase, transaminase
3. In a patient with Slay syndrome an excretion of heparan sulfate and chondroitin
sulfate with urine is observed. What enzyme deficiency may cause this state?
A. Cathepsin D
B. -amylase
C. Lactate dehydrogenase
D. Arylsulfatase
E. -glucuronidase
4. Increased fragility of vessels, destruction of enamel and dentine in scurvy is caused
mostly by alteration collagen maturation. What stage of collagen post translational
modification is damaged in this avitaminosis?
A. Glycosylation of hydroxylysine residues
B. Cleavage of C-terminal peptide from procollagen
C. Formation of polypeptide chains
D. Cleavage of N-terminal peptide
E. Proline hydroxylation
Topic № 11. Questions for summary lesson “Biochemistry of tissues and
physiological functions”
1.
2.
3.
4.
5.
6.
General characteristic of human nutritional components: macrocomponents
(carbohydrates, lipids, proteins), microcomponents (vitamins, inorganic and
trace elements.
Requirements of human organism in nutrients – carbohydrates, lipids, proteins.
Biological value of some nutrients. Rational nutrition.
Trace elements in human nutrition. Biological function of iodine, bromine, fluor,
copper, mangane, , zinc, cobalt, selenium; manifestations of trace elements
insufficiency.
General characteristic of digestion and absorption of nutrients. Enzymes,
biochemistry of digestion of proteins, carbohydrates, lipids in stomach, small
and large intestines.
Disorders of digestion of distinct nutrients in stomach and intestines; hereditary
enzymopathias.
Changes in biochemical indexes in gastric diseases and their diagnostic
significance.
49
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Disorders of secretory function of pancreas in acute and chronic pancreatitis,
biochemical characterization.
Steatorrhea and its variants: pancreatic (deficiency of lipase), hepatogenic
(deficiency of bile), enterogenic (inhibition of lipolytic enzymes and
triacylglycerides resynthesis in intestines).
Hereditary enzymopathias of intestinal disaccharidases. Biochemical diagnostics
of lactose intolerance.
Vitamins as essential nutritional components. History of vitamins discovery and
development of vitaminology.
Causes of exo- and endogenous hypo- and avitaminoses.
Vitamin B1 and B2, their structure, biological function, sources of supplement,
daily requirement. Symptoms of hypovitaminosis.
Structure and properties of vitamin H and pantothenic acid. Their significance in
metabolism, daily requirement.
Antianemic vitamins (B12, folic acid), their structure, biological function, daily
requirement. Symptoms of hypovitaminosis.
Vitamins B6 and PP, their structure, biological function, nutritional sources,
daily requirement. Symptoms of hypovitaminosis.
Vitamin C and P , their structure, biological function, daily requirement,.
manifestations of insufficiency in human organism.
Vitamins of D group, their structure, biological function, nutritional sources,
daily requirement. Symptoms of hypo- and hypervitaminosis, avitaminosis.
Vitamin A, its structure, biological function, nutritional sources, daily
requirement. Symptoms of hypo and hyper- vitaminosis.
Vitamins E, F, their structure, biological role, mechanism of action, daily
requirement. Symptoms of insufficiency.
Antihemorrhagic vitamins (K group), their water soluble forms, structure,
biological function, nutritional sources, mechanism of action, daily requirement,
symptoms of insufficiency, application in medicine.
Provitamins, antivitamins, mechanism of action and employment in practical
medicine.
Vitaminoids, their structure and biological activity.
Modern vitamin drugs, their application in treatment and prevention of diseases.
Biologically active supplements.
Blood as internal medium of the body. Role of blood in homeostasis.
The composition of whole blood, blood plasma and serum. Physico-chemical
and biological properties of blood.
Blood volume and its pH. Cellular elements of blood: red blood cells, white
blood cells, blood platelets.
Hemoglobin, its structure, properties and molecular forms.
Pathology of hemoglobin: hemoglobinopathias and thalassemias.
Respiratory function of red blood cells in lung capillaries and in peripheral
tissues.
The function of hemoglobin oxygenation from partial pressure of oxygen,
dissotiation curve of oxyhemoglobin, Bohr effect.
50
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
Acid-base equilibrium of blood. Regulation of pH in biological fluids, disorders
of acid-base equilibrium: metabolic and respiratory acidosis, metabolic and
respiratory alkalosis, mechanisms of their development.
Role of kidneys and lungs in support of acid-base equilibrium.
Buffer systems of blood, their types, role of different buffer systems in
providement of constant pH of blood.
Principal types of hypoxias, mechanisms of their development, methods of
diagnostics.
Hypoxia, caused by lowering of oxygen pressure in air.
Hypoxia caused by pathological process: pulmonary type, cardiovascular type,
tissue and exhaustive types.
Hormones as regulators of osmotic pressure in blood. Role of renin-angiotensine
system in regulation of osmotic pressure.
Humoral mechanisms of regulation of acid-base status.
Dependence of biochemical indexes of blood from metabolic processes in
organism.
Principal groups of blood proteins, their composition and content in normal
conditions and in pathology.
Albumins and globulins. Resolution of blood plasma proteins by method of
protein electrophoresis.
Role of glutathion in protection from peroxide oxidation of lipids, antioxidant
vitamins.
Glycoproteins, their structure, biological role, changes in composition in
diseases.
Proteins of acute phase of inflammation: C-reactive protein (CRP),
ceruloplasmin, haptoglobin, cryoglobulin, alpha-1 antitrypsin, alpha-2
macroglobulin, interferon, fibronectin, their diagnostic validity.
Enzymes of blood plasma: genuine (secretory), excretory, indicatory (tissue)
enzymes. Kallicrein-kinine and renin-angiotensine systems, their biological
significance.
Diagnostic value of investigation of enzyme and isoenzyme activity in blood
plasma: creatine kinase (CK), LDH, AsAT, AlAT, amylase, lipase,
cholinesterase.
Definition of total and residual nitrogen in blood. Nonprotein nitrogen
containing compounds of blood, their diagnostic significance.
Nitrogen free organic and inorganic compounds of blood, their metabolic origin.
Middle size molecules, their metabolic origin and diagnostic significance.
Nitrogenemia, its kinds and causes of development, differentiation in clinical
conditions.
Quantitative changes in residual nitrogen in parentheral nutrition, transfusions of
blood, blood plasma, serum or blood substituents.
Biochemical aspects of application of some medical preparations in treatment of
nitrogenemia.
Functional and biochemical characteristics of intrinsic and extrinsic blood
coagulation pathways.
51
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
Blood coagulation system; characteristics of coagulation factors.; intrinsic and
extrinsic blood coagulation pathways. Role of vitamin K in reactions of
hemocoagulation (carboxylation of glutamic acid residues, its role in Ca
binding).
Hereditary disorders of hemocoagulation.
Anticoagulation system of blood, functional characteristics of its components –
heparin, antithrombin III, citric acid, prostacycline. Role of vascular
endothelium.
Fibrinolytic system of blood: stages and factors of fibrinolysis. Medicinal
influencing fibrinolytic process. Activators and inhibitors of plasmin.
Syndrome of disseminated intravascular blood coagulation. Formation of
thrombs and fibrinolysis in atherosclerosis and hypertonic disease.
General characteristic of immune system; cellular and humoral immunity.
Immunoglobulins:
structure,
biological
function,
mechanisms
of
immunoglobulin synthesis. Characteristics of distinct immunoglobulin classes of
human blood.
Mediators and hormones of immune system; (interleukins, interferons, protein
and peptide factors of cell growth and proliferation).
Factors of complement system. Classical
and alternative pathways of
complement activation.
Biochemical mechanisms of immunodeficiencies: primary (hereditary) and
secondary immunodeficiencies; acquired immunodeficiency syndrome (AIDS).
Role of liver in regulation of glycemia (glycogenesis and glycogen breakdown,
gluconeogenesis), lipid metabolism, turnover of bile acids and bile pigments.
Biochemical composition of bile.
Biochemical characterization of liver function insufficiency in chemical,
biological or radiation damage of liver tissue; biochemistry of development of
liver encephalopathia.
Changes in biochemical indexes in acute hepatitis, caused by viruses or alcohol
intoxication, their diagnostic evaluation.
Changes in biochemical indexes in chronic hepatitis, cirrhosis, cholelythiasis,
dyskinesias and cholecystitis. Dependence of excretory function of liver and
disorders of digestion, their diagnostics.
Role of liver in turnover of bile pigments. Hemoglobin catabolism: production
of biliverdin, its transformation to bilirubin, synthesis of bilirubin diglucuronide
and excretion with bile.
Pathobiochemistry of jaundices; hemolytic (prehepatic), parenchimatous
(hepatic), occlusive (posthepatic). Enzymatic congenital jaundices.
Crigler-Najjar syndrome as a consequence of insufficient activity of UDPglucuronyl- transferase.
Gilbert disease – pathology caused by combined disorder of synthesis of
bilirubin diglucuronide and absorption of bilirubin from blood by liver cells
(“absorption jaundice).
Dubin-Johnson syndrome – jaundice caused by disorder of transport of bilirubin
diglucuronide from liver cells to bile (“excretory jaundice”). Enzymatic
jaundices of neonates and methods of their prevention.
52
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
Detoxification function of liver; biotransformation of xenobiotics and
endogenous toxins.
Types of reactions of biotransformation of foreign substances in liver.
Reactions of microsomal oxidation; inducers and inhibitors of microsomal
monoxygenases.
Conjugation reactions in hepatocytes: biochemical mechanisms, functional
significance.
Electron transport chains of endoplasmic reticulum. Genetic polymorphismus
and induction of biosynthesis of cytochrome P-450.
Development of tolerance to medicinal and drugs and its causes.
Biological role of water and its distribution in human body. Water balance, its
types.
Regulation of water and mineral metabolism, its disorders. Dehydratation and
rehydratation.
Mechanism of Na,K-ATPase action and its regulation.
Biogenic elements, their classification, pathways of their providement.
Biological role of macroelements, trace elements and ultramicroelements.
Human microelementoses: endogenous and exogenous causes (technogenic,
yatrogenic, etc.).
Influence of radioactive isotopes, X-rays and other kinds of radiation upon
mineral balance.
Methods of investigation of water and mineral metabolism
Role of kidneys in regulation of volume, composition of electrolytes and acidbase equilibrium of biological fluids. Biochemical mechanisms of urine
production (filtration, reabsorption, secretion and excretion). Characterization of
renal clearance and renal threshold, their diagnodtic significance.
Humoral mechanisms of regulation of water and mineral metabolism and kidney
function; antidiuretic hormone; aldosteron.
Renin-angiotensine system. Natriuretic factors of heart atrium and other tissues.
Biochemical mechanisms of development of renal hypertonia.
Physico-chemical properties of urine: volume, color, odor, transparency, acidity
(pH), its dependence from diet. Role of kidneys and lungs in regulation of acidbase equilibrium. Ammoniogenesis.
Pathobiochemistry of kidneys. Biochemical changes in glomerulonephritis,
amyloidosis, pyelonephritis.
Pathobiochemistry of kidneys. Biochemical changes in acute renal insufficiency.
Diagnostics of chronic renal insufficiency.
Conditions of renal stones formation, their chemical nature and preventive
measures.
Pathological constituents of urine – blood, hemoglobin, creatin. Causes and
pathways of their appearance in urine.
Glucosuria, galactosuria and pentosuria, causes of their development. Clinical
significance of their detection.
Clinical significance of detection and determination of indicane, phenylpyruvic
and homogentisic acids in urine.
Clinical significance of detection and determination of ketone bodies in urine.
53
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
Differentiation of jaundices on the appearance of bile acids and bile pigments in
urine.
General characterization of morphology, biochemical composition of
intercellular matrix of loose connective tissue: fibers (collagen, elastin,
reticulin); amorphous material.
Proteins of connective tissue fibers: collagen, elastin, glycoproteins and
proteoglycans. Biosynthesis of collagen and formation of fibrilar structures.
Complex carbohydrates of amorphous matrix of connective tissue –
glycosaminoglycans. Mechanisms of formation of intercellular matrix by
molecules of glycosaminoglycans – (hyaluronic acid, chondroitin-, dermatan-,
keratan- sulphates). Distribution of glycosaminoglycans in different human
organs and tissues.
Pathobiochemistry of connective tissue. Biochemical mechanisms of
development of mucopolysaccharidoses and collagenoses, their biochemical
diagnostics.
Rheumatism – its clinical and biochemical diagnostics.
Determination of acidity of gastric juice: total acidity, free and bound
hydrochloric acid.
Detection of lactic acid in gastric juice. In what pathological states lactic acid
appears in gastric juice?
Determination of blood glucose level by glucose oxidase method. To write
reactions, which take place in this method.
Determination of bilirubin and its fractions in blood serum. Interpretation of
obtained results.
Determination of alanyl and aspartate aminotransferases activity in blood serum,
diagnostic significance of these tests.
Determination of acidity of urine, interpretation of results.
Determination of enzymatic activity in urine on example of amylase. Diagnostic
application, interpretation of results.
54
CONTENTS
MODUL 4. Molecular biology. biochemistry of intercellular communications…3
Topic № 1. Investigation of biochemical composition and biosynthesis of purine and
pyrimidine nucleotides. Biochemical function of nucleotides and nucleic acids…….. 5
Topic № 2. Catabolism of purine nucleotides, determination of end products of their
metabolism. Hereditary disorders of nucleotide metabolism……………………….. 10
Topic № 3. Replication of DNA and transcription of RNA. Analysis of mutation
mechanisms and DNA reparations. Principles of recombinant DNA technology,
transgenic proteins…………………………………………………………………….14
Topic № 4. Biosynthesis of proteins in ribosomes, initiation, elongation and
termination in synthesis of polypeptide chain. Inhibiting effect of antibiotics. To learn
principles of gene engineering and gene cloning, their application in modern
medicine……………………………………………………..………………………. 20
Topic № 5. The investigation of molecular mechanisms of the effect of hormones of
protein and amino acid nature on target cells. Humoral regulation of calcium
homeostasis……………………………………………………………………………27
Topic № 6. Investigation of molecular and cellular mechanisms of steroid and thyroid
hormones action upon target cells…………………………………………………….33
Topic № 7. Investigation of nerve tissue. Pathochemistry of psychotic disorders…...39
Topic № 8. Biochemistry of muscle tissue. Investigation of mechanism of muscle
contraction…………………………………………………………………………….43
Topic № 9. Summary leson, control work. Module 4……………………………….
MODUL 5. Biochemistry of tissues and physiological functions………………..50
Topic № 1. Investigation of digestion and assimilation of nutritional substances
(proteins, carbohydrates, lipids) in digestive tube…………………………..
Topic № 2. Investigation of functional role of water soluble and fat soluble vitamins in
metabolism and providement of cell functions…………………………………
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Topic № 3. Investigation of acid-base equilibrium in blood and respiratory function of
red blood cells. Pathological forms of hemoglobin………………….
Topic № 4. Investigation of blood plasma proteins: proteins of acute phase, genuine
and indicatory enzymes. Investigation of nonprotein nitrogen containing and nitrogen
free components of blood……………………………………
Topic № 5. Investigation of coagulation, anticoagulation and fibrinolytic systems of
blood. Investigation of principal pathways of immune response. Immunodeficiencies…
Topic № 6. Investigation of end products of heme catabolism. Pathobiochemistry of
jaudices………………………………………………………………………
Topic № 7. Investigation of biotransformation of xenobiotics and endogenous toxic
compounds. Microsomal oxidation, cytochrome P-450…………………………
Topic № 8. Investigation of water and mineral metabolism……………………
Topic № 9. Functional role of kidneys in urinogenesis. Normal and pathological
constituents of urine………………………………………………………………….
Тopic № 10. Investigation of biochemical constituents of connective tissue…….
Topic № 11. Questions for summary lesson “Biochemistry of tissues and
physiological functions”………………………………………………………………
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