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Biochemistry
Biochemistry is the science concerned the chemical reactions occurring in living cells and
organisms. Biochemistry in the medicine is mainly concerned with balance of biochemical
reactions occurring in the body, both in physiological state as in pathology. The program of
teaching biochemistry for medical students consists of lectures, seminars and laboratory
classes. The main object comprises five sections: Structure and Function of Proteins and
Enzymes, Metabolism of Carbohydrates, Lipids of Physiological Significance, Nucleic Acids
and special topics (Nutrition, Digestion, Vitamins, Plasma Proteins, Immunoglobulins,
Haemostasis, Xenobiotics). Total program of teaching in Biochemistry includes: 70 hours
lectures, 68 hours seminars, and 42 hours practical classes. At the end of course students
must take the final examination prepared by Board of Medical Examiners.
Teachers:
Prof. dr hab. med. Józef Kędziora
Dr Jolanta Czuczejko
Dr Karolina Szewczyk-Golec
Dr Mariusz Kozakiewicz
Contact:
[email protected]
Syllabus
I.
Department of Biochemistry
II.
prof. dr hab. med. Józef Kędziora, M.D., Ph.D. Professor
III. Faculty of Medicine, Medical Program, year II
IV. Course coordinator - Józef Kędziora, M.D., Ph.D. Professor
V. Form of classes - lectures, practicals, seminars
VI. Form of crediting - Exam , 16 ECTS points
VII. Number of hours - 60 hours of lectures, 120 hours of practicals and seminars
VIII. Aim of the course:
Acquiring knowledge of the chemical processes occuring in human cells. Studying the chemical
constituents of human cells: structure, properties and function of proteins, saccharydes, lipids,
nucleic acids, vitamins and coenzymes. Studying biochemical aspects of metabolic disorders. Analysis
of the compartmentation, integration and regulation of metabolic pathways. Learning about the
metabolism of the main human organs. Emphasising the relationships between medicine and
biochemistry and the role of biochemical knowledge in medical diagnostics.
IX. Topics:
Biochemistry Lectures
Amino acids, peptides, proteins.
1. Nature of Proteins
- Function: Enzymatic catalysis, transport and storage of small molecules, structural
elements of cytoskeleton, immunity (immune defense system)
2. Amino Acids – fundamental units of proteins
- Composition
- Optical activity
- Amphoteric properties
3. Peptides and polypeptides
- Formation
- Amphoteric properties
4. Purification of proteins
5. Conformation of proteins - primary, secondary, tertiary, quaternary structure
6. Protein Structure – Function Relationship
Oxygen transport proteins
A. Myoglobin and Hemoglobin Structure and Function
B. Hemoglobinopathies (HbS, HbC, HbM, thalassemias)
C. Humoral Immunity - five basic classes of immunoglobulin (structure and function): IgM,
IgD, IgG, IgE, IgA
D. Fibrous proteins - collagen
Enzymes, co-enzymes, vitamins.
1. General characteristics of enzyme
Differences between enzymes and chemical catalysts
Measures of enzyme activity
Enzymes nomenclature
2. Enzyme Kinetics
A.
Quantification of enzyme activity
B.
Quantification of chemical reaction by kinetic order
C
Michaelis – Menten kinetic theory of enzyme action
-significance of the Michaelis constant
-Lineweaver Burk transform
3. Enzyme Inhibition
A.
Competitive
B.
Uncompetitive
C.
Medical relevance of enzyme inhibitor
D.
Regulation of enzymes
4. Enzymes in clinical diagnosis
5. Nomenclature of vitamins - water soluble and fat soluble.
6. Coenzymes:
Nicotinamide-adenine dinukleotide (NAD+)
Nicotinamide – adenine dinukleotide phosphate (NADP+)
Flavin mononucleotide (FMN)
Flavin adenine dinukleotide (FAD)
CoA-SH, ACP
Folic acid
Pyridoxal phosphate (PLP)
Thiamin pyrophosphate (TPP)
Biotin
Cobalamine
Ascorbic acid
7. Cofactors: metal ions of Co, Cu, Mg, Mn, Se, Zn, Fe.
Saccharides, glycolysis pathway, tricarboxylic acid cycle, pentose phosphate pathway.
1. Structures of saccharides
A. Open chain form (asymmetric carbon, isomers, epimers, enantiomers, hemiacetals)
B. Cyclic form (acetals, glycosialles) polysacharydes
2. Carbohydrate derivates
A. Phosphoric acid esters of monosacharides
B. Amino sugars
C. Sugar acids
D. Deoxy sugars
3. Glycoproteins - physiologic functions
A. Glycosaminoglycans (heparin, chondroitin sulfate, dermatan sulfate, heparin sulfate,
keratin sulfate, hyaluronic acid)
4. Glycolysis - anaerobic glycolysis, aerobic glycolisys.
5. The pyruvate dehydrogenase (PDH) enzyme complex, PDH regulation
5. Pentose phosphate pathway
6. Uronic acid pathway (glucuronic acid cycle)
7. Citric acid cycle
Oxidative phosphorylation and biologic oxidation.
Mitochondrial Electron Transport
Localization of electron transport chain
1. The outer membrane
2. The itermembrane space present
3. The inner membrane
4. Organization of the electron transport chain - Complex I, II, III, IV
Lipids
1. Nomenclature of lipids and physiologic significance
2. Phospholipids and glycosphingolipids – structure, function and biosynthesis
3. Fatty acids chain biosynthesis
4. Desaturase & elongase enzyme systems
5. Eicosanoids biosynthesis and their physiologic role
6. Cholesterol biosynthesis (regulation of HMG-CoA reductase activity)
7. Cholesterol as a precursor of steroids (corticosteroids, sex hormones, bile acids, vitamin D)
8. Lipid and cholesterol transport and storage – plasma lipoproteins
9. β-oxidation of saturated, unsaturated and odd number of carbon atoms fatty acids
10. Ketogenesis
11. Lipid peroxidation
12. Interrelationships among carbohydrates and lipids metabolism
*Nucleic acids – structure, function, organization. Molecular genetics.
1. Human genome – definition and structure. Nucleosome and chromatin package. Hetero –
and euchromatin. Structure of nucleic acids. Genetic information and genetic code. Organization
of genes, promoters, microsatellite DNA, pseudogenes.
2. DNA replication – DNA polymerases, start of replication, role of starters in DNA synthesis,
Okazaki fragments. Topology of DNA associated with replication and role of helicases.
3. Transcription of genetic information. RNA synthesis by DNA-dependent RNA polymerase.
Types of RNA and their function. Reverse transcriptase.
4. Translation of genetic information. Components of the translation apparatus. Protein
biosynthesis. Protein maturation and posttranslational modifications. Protein degradation and
turnover.
5. Epigenetics – DNA modifications, DNA methylation. Modulation of genes expression.
Epigenetics and cancer.
6. DNA damage and repair. DNA damage in the way of health & on the way to ageing. Oxidative
DNA damage and repair. Measurement of DNA damage. Biological consequences of oxidative
DNA damage. DNA repair pathways. Mutagenesis. Mutations and polymorphisms in genes
encoding DNA repair enzymes.
7. Mitochondrial genome and its metabolism. Organisation of the human mitochondrial
genome. Maternal inheritance. High mutation rate. Mutations in mtDNA.
*Nutrition, digestion & absorption.
1. Macro- and micronutrients. Digestion and absorption of proteins, carbohydrates and lipids.
Vitamins. Bile acids metabolism.
2. Energy metabolism. Protein-energy intake. Malnutrition. Obesity. Assessment of nutritional
status.
*Blood, hemostasis & thrombosis.
1. The composition of blood. Blood cells and plasma. Oxygenation of blood.
2. Mechanism of blood coagulation.
* Excretory system.
1. Organs of the excretory system. Removal of carbon dioxide excess by lungs. Skin functions.
Break-down of proteins and urea production in liver. Urea cycle.
2. Kidney function. Hormonal control of water and salt
Detoxification processes – liver functions
1. The metabolism of the liver - amino acids, urea cycle, proteins, carbohydrates, lipids
2. Steps of detoxification – cytochrome P450, conjugation (role of reduced glutathione GSH –
biosynthesis of mercapturic acids)
3. Other role of GSH – peroxidase glutathione (GSH-Px), reductase glutathione (GR), transport of
amino acids
Metabolism of amino acids.
Deamination (role of Glu), transamination, decarboxylation, glucogenic and ketogenic
aminoacids, role of aminoacids on biosynthesis, essential and non-essential amino acids
* Hormones and hormonal regulation.
Hormones and the hormonal cascade system. Major polypeptide hormones and their action.
Steroid hormones. Hormone receptors and intracellular hormone signalling.
* lectures conducted by the Department of Clinical Biochemistry
Biochemistry Practicals
Practical 1 Chemical properties of amino acids
The aim of the class: studies on selected properties of amino acids
Theoretical basis: general structure of amino acids, the names (full names and their three-letter
abbrevations) and structures of protein amino acids, characteristics of the chemical
groups attached to amino acid chain (like carboxylic, amino, imino, sulphydryl,
imidazol, guanidine, hydroxyl groups), amphoteric properties of amino acids,
classification of amino acids according to the chemical properties of their side chains
(charged, nonpolar hydrophobic, uncharged polar; aliphatic, cyclic, aromatic; acidic,
basic)
Laboratory tests:
1. Ninhydrin reaction – a characteristic reaction for all amino acids.
2. Characteristic reactions for individual amino acids:
a) xantoprotein reaction – detection of aromatic amino acids
b) Millon reaction – detection of tyrosine
c) Adamkiewicz-Hopkins reaction – detection of tryptophan
d) Pauly reaction – detection of histidine
e) Sakaguchi reaction – detection of arginine
f) cysteine reaction – detection of sulphur amino acids (cysteine, cystine)
Practical 2 Some properties of peptides and proteins
The aim of the class: Some physical and chemical properties of peptides and proteins
Theoretical basis: structure and characteristics of the peptide bond, classification of peptides
according to their structures, the physiologic significance of some peptides in human
body, the characteristics of primary, secondary, tertiary and quaternary structures of
proteins, classification of proteins according to their structures, properties and
functions, the amphoteric properties of proteins (the isoelectric point of proteins)
Laboratory tests:
1. Biuret reaction – detection of peptide bonds.
2. Denaturation of protein:
a) thermal denaturation of protein
b) denaturation of proteins with strong acids
c) denaturation of proteins with strong bases
d) precipitation of proteins with ethanol
e) denaturation of proteins with heavy metal salts
f) denaturation of proteins with alkaloid reagents
3. Amphoteric properties of proteins.
Practical 3 Blood proteins
The aim of the class: Some properties of blood proteins
Theoretical basis: the constituents of the blood, the compositions of blood plasma and blood serum,
characteristics of main blood plasma proteins: albumins, globulins and fibrinogen,
electrophoresis as an important method of plasma (or serum) protein fractionation,
the characteristics of individual protein fractions, the physiologic and pathological
concentrations of blood plasma protein, the diagnostic role of alterations in the
amount of total proteins and in mutual quantitative relationships between individual
fractions, examples of methods for protein concentration determination (biuret
protein assay, Lowry protein assay, Bradford protein assay)
Laboratory tests:
1. Determination of plasma protein concentration by biuret method – preparation of a
calibration curve.
2. Polyacrylamide gel electrophoresis of serum proteins.
3. Fractionation of blood plasma proteins by salting out with ammonium sulphate.
Practical 4 Gel filtration
The aim of the class: molecular filtration for protein separation and desalting of protein
solution
Theoretical basis: The separation of proteins contained in solution by different types of
chromatography, the characteristics of molecular filtration (the types of molecular
sieves and their structure, the principles and applications of gel filtration), different
methods used for measurement of protein molecular weight
Laboratory tests:
1. The separation of mixture of substances with different molecular weight (blue dextran,
hemoglobin, potassium chromate) on chromatographic column filled with Sephadex gel.
SEMINAR / TEST I Amino acids, peptides, proteins
The key problems: The structure of protein amino acids. The classification of amino acids according to
both the polarity and the structural features of their side chains (e. g. polar, nonpolar;
aliphatic, aromatic; sulfur-containing; charged, uncharged; acidic, basic). The amphoteric
properties of amino acids, zwitterions. The structure of some modified amino acids (as
selenocysteine, 4-hydroxyproline, 5-hydroxylysine). The structure of some physiologically
important nonprotein amino acids.
The formation, structure and properties of the peptide bond. Some important peptides in
the human organism (glutathione, peptide hormones). The insulin synthesis.
The classification of proteins according to their structure, properties and functions. The
characteristics of primary, secondary, tertiary and quaternary structures of proteins. The
interactions involved in a protein folding into its final conformation (e. g. the attraction
between positively and negatively charged molecules, the hydrophobic effect, hydrogen
bonding, and van der Waals interactions). Posttranslational modifications of amino acids
in proteins. Structure – function relationships in myoglobin, hemoglobin and
immunoglobulins. The structure and synthesis of collagen. Rybonuclease renaturation as
an example of the importance of primary protein structure. The prions as an example of
medical importance of proper protein folding.
Practical 5 The isolation of enzymes from biological materials
The aim of the class: the isolation and purification of yeast saccharase
Theoretical basis: the structure of enzymes, classification of enzymes, the enzymes names, the
methods of isolation and purification of enzymes from biological materials.
Laboratory tests:
1. The isolation and purification of saccharase (sucrase) from yeast.
2. Samogyi-Nelson method – calibration curve preparation.
Practical 6 The kinetics of the enzymatic reaction
The aim of the class: the determination of the initial velocity and Michaelis constant in
reaction catalysed by saccharase
Theoretical basis: the enzyme-catalyzed reaction, the definitions of initial velocity, maximal velocity
and Michaelis constant, the Michaelis-Menten equation, the standard units of
enzymatic activity (katal, international unit), the influence of some factors on the
enzyme activity (e. g. temperature, pH, the concentration of substrate and enzyme,
competitive and noncompetitive inhibitors).
Laboratory tests:
1. The determination of the initial velocity in reaction catalysed by saccharase.
2. The determination of the Michaelis constant in reaction catalysed by saccharase.
Practical 7 The vitamin C concentration in biological materials
The aim of the class: the determination of the ascorbic acid concentration in different
biological materials
Theoretical basis: classification, structure and functions of water-soluble vitamins, classification,
structure and functions of coenzymes.
Laboratory tests:
1. The determination of the ascorbic acid concentration by Folin method in the blood serum
and some other biological materials (e. g. vegetable and fruit juices)
SEMINAR / TEST II Enzymes and coenzymes
The key problems: The definitions: enzyme, coenzyme, cofactor. The isoenzymes of the
diagnostic importance (lactate dehydrogenase (LDH), creatine phosphokinase (CPK)). The
structure of the active site and models for substrate binding. The specificity of enzymes to
the substrates and the catalysed reaction.
The catalytic mechanisms of the enzymatic reactions. The influence of physical and
chemical factors on the enzyme activity (temperature, pH, the enzyme concentration, the
substrate concentration, the product concentration).
The kinetics of enzymatic
reaction: the initial and maximal velocities, Michaelis constant, the Michaelis-Menten
equation, the Lineweaver-Burk plot.
Regulation of enzyme activity: allosteric enzymes (allosteric activators and inhibitors, the
examples of allosteric enzymes, the sequential and concerted models for an allosteric
enzyme, the kinetics of allosteric enzyme reaction), feedback regulation and its examples
in the human organism, covalent modification of enzymes (phosphorylation), proteolytic
cleavage (proenzymes, zymogens, autocatalysis), reversible inhibition (competitive and
noncompetitive inhibitors, the mechanism of the inhibition, the kinetics of competitive
and noncompetitive inhibitions, medical significance of inhibition: acetylsalicylic acid,
Fluorouracil, methotrexate, penicillin, allopurinol).
The standard units of enzymatic activity (katal, the international unit, the specific activity
of an enzyme). The classes of enzymes (oxidoreductases, transferases, hydrolases, lyases,
isomerases, ligases).
Coenzymes: their structrures and functions in the reactions. Water-soluble and fat-soluble
vitamins: their structures and functions. The trace elements: some enzymatic reactions
that involve the iron, cobalt, zinc, or copper ions.
Practical 8 Some properties of monosaccharides
The aim of the class: some chemical properties of monosaccharides
Theoretical basis: nomenclature of monosaccharides and their isomerism, the structure of
monosaccharides (Fischer projection and cyclic structure), the chemical properties of
monosaccharides, the examples of biologically important monosaccharides.
Laboratory tests:
1. Fuchsine reaction – characteristic reaction for monosacharides in neutral solution
2. Characteristic reactions for monosaccharides in basic solution – the reducing properties of
saccharides:
a) Fehling reaction
b) Benedict reaction
c) Nylander reaction
d) picric acid reaction
3. Characteristic reactions for monosaccharides in concentrated strong acids solutions
a) Molisch reaction – detection of every monosaccharide
b) Seliwanow reaction – detection of ketoses
c) phloroglucinol reaction – detection of pentoses
4. Phenylhydrazine reaction – the formation of osazones.
5. Ethanol fermentation.
Practical 9 Some properties of di- and polysaccharides
The aim of the class: some properties of biologically important disaccharides and
polysaccharides
Theoretical basis: nomenclature of disaccharides and polysaccharides, their structure and properties,
the examples of biologically important di- and polysaccharides, physiologically
significant saccharide derivatives (e. g. heparine) and glycoproteins
Laboratory tests:
1. Molisch reaction – detection of every di- or polysaccharide
2. Seliwanow reaction – detection of ketose presence in saccharose molecule.
3. The reductive test for some dissacharides:
a) Fehling reaction for lactose, maltose and sucrose
b) picric reaction for lactose, maltose and sucrose
4. Barfoed reaction – the characteristic reaction for reducing disaccharides.
5. Iodine test – the detection of starch.
Practical 10 Saccharides of physiological importance
The aim of the class: preparation of glucose tolerance curve, the estimation of sialic
acids concentration in the blood serum
Theoretical basis: the glucose concentration in the blood, the maintenance of blood glucose levels,
regulation of blood glucose level by hormones, the glucose levels in diabetes mellitus,
glucose tolerance test in healthy persons and diabetes mellitus patients, the
physiologic role of sialic acid
Laboratory tests:
1. Determination of the blood glucose levels in the blood samples obtained during oral glucose
tolerance test conducted on the healthy person – an enzymatic reaction with glucose
oxidase.
2. Determination of the blood glucose levels in the blood samples obtained during oral glucose
tolerance test conducted on the diabetes mellitus patient – an enzymatic reaction with
glucose oxidase.
3. The drawing of two glucose tolerance curves – for healthy and diabetes mellitus person.
4. Winzler method – the determination of blood sialic acids concentration.
SEMINAR / TEST III Saccharides
The key problems: Classification of monosaccharides by both the number of contained
carbon atoms (e. g. triose, tetrose etc.) and the type of contained carbonyl group (aldose,
ketose), and their isomerism. Common disaccharides. The structure of important
polysaccharides (starch and glycogen). Physiologically significant saccharide derivatives
(especially amino sugars). Synthesis and functions of sialic acids.
Generation of ATP from glucose: glycolysis (reactions of glycolytic pathway, substratelevel phosphorylation, regulation of glycolysis). Synthesis of 2,3-bis-phosphoglycerate in a
“side reaction” of the glycolytic pathway. Anaerobic glycolysis – (lactate fermentation,
tissues dependent on anaerobic glycolysis, fate of lactate – Cori cycle, lactic acidemia,
ethanol fermentation). Fructose and galactose metabolisms. Synthesis and degradation of
lactose. Formation and degradation of glycogen. Disorders of metabolisms of fructose,
galactose and glycogen. The pentose phosphate pathway. The directions of the pentose
phosphate pathway reactions due to the cellular needs. Hemolysis caused by reactive
oxygen species in the conditions of glucose-6-phosphate dehydrogenase deficiency.
Gluconeogenesis. The maintenance of blood glucose levels by hormones (regulation of
glycolysis and gluconeogenesis, as well as formation and degradation of glycogen by
insulin, glucagon and noradrenaline).
Practical 11 The electron-transport chain.
The aim of the class: studies on selected enzymes of the electron-transport chain and
some enzymes of antioxidative properties.
Theoretical basis: the oxidative-reduction components of the electron-transport chain and their
structures, the electron-transport chain as a major source of the free radicals in the
cell, the antioxidative defense in the human organism, the enzymatic and nonenzymatic components of the antioxidative defense
Laboratory tests:
1.
2.
3.
4.
Demonstration of the cytochrome c oxidase activity in potato juice.
Demonstration of the catalase activity in the hemolysate.
Determination of glutathione peroxidase activity in the blood plasma.
Determination of ceruloplasmin oxidase activity in the blood serum.
SEMINAR / TEST IV # Oxidative phosphorylation and tricarboxylic acid cycle
The key problems: Oxidative fates of pyruvate – oxidation of pyruvate to acetyl CoA by pyruvate
dehydrogenase. The tricarboxylic acid (TCA) cycle (reaction, enzymes, coenzymes,
regulation of this cycle). The energetics of the TCA cycle. Cellular bioenergetics: the
compounds containing high-energy bonds (ATP and the others nucleoside triphosphates,
creatine phosphate, 1,3-bis-phosphoglycerate, acetyl CoA). Oxidative fates of NADH,
produced from glycolysis (glycerol 3-phosphate shuttle and malate-aspartate shuttle).
Transfer of compounds through the inner and outer mitochondrial membranes. The
generation of ATP from glucose (complete aerobic oxidation of glucose, anaerobic
glycolysis).
Oxidative phosphorylation. The electron-transport chain. Chemiosmotic model of ATP
synthesis. The structure of protein complexes of the electron-transport chain. Respiratory
chain inhibitors, chemical uncouplers of oxidative phosphorylation. The generation of
reactive oxygen species (ROS) in the cell (the mitochondrial electron-transport chain and
other sources). ROS-mediated cellular injury. Formation of free radicals during
phagocytosis and inflammation. Cellular defences against oxygen toxicity.
Practical 12 * Some properties of lipids.
The aim of the class: some chemical properties of lipids, lipids of physiologic
significance.
Theoretical basis: categories of lipids, the nomenclature of both saturated and unsaturated fatty
acids, examples of biologically important fatty acids, the structure of glycerolipids, the
structure of sphingolipids, the role of some important lipids in the human organism,
the lipid peroxidation, the structure of cholesterol, the physiological blood
concentrations of both cholesterol and triacylglycerols, the diagnostic significance of
cholesterol, triacylglycerols and lipoproteins levels in the blood.
Laboratory tests:
1.
2.
3.
4.
5.
6.
7.
Acrolein test – detection of glycerol.
Detection of unsaturated fatty acids.
Saponification reaction of triglicerydes.
Emulsifying action of both the soap and the bile acids.
Kreis test – detection of aldehyde products of lipid peroxidation
Determination of the cholesterol concentration in the blood serum – an enzymatic method.
Determination of the triglycerides concentration in the blood serum – an enzymatic method.
SEMINAR / TEST V * Lipids
The key problems: Saturated and unsaturated fatty acids, their nomenclature and structure.
Acylglycerols, phosphoacylglycerols, sphingolipids, steroids, eicosanoids. Structure and
functions of cholesterol. The cholesterol derivatives (vitamin D, steroid hormones, the bile
acids) and their role in the organism. Transport of cholesterol by the blood lipoproteins.
The cholesterol synthesis and its regulation. Medical significance of elevated cholesterol
blood levels. Dyslipoproteinemias.
Activation of long-chain fatty acids and their transport into mitochondria. β-oxidation of
long-chain fatty acids, oxidation of unsaturated fatty acids. Energy yield of β-oxidation.
Odd-chain-lenght fatty acids oxidation. Conversion of propionyl CoA to succinyl CoA.
Metabolism of ketone bodies.
The acetyl CoA transport from mitochondria into cytosol (role of citrate). The sources of
NADPH for fatty acid synthesis. Fatty acid synthesis. Elongation of fatty acids.
Desaturation of fatty acids. Conversion of linoleic acid to arachidonic acid. The synthesis
of eicosanoids and their physiologic significance. Synthesis of triacylglycerols. Synthesis of
glycerophospholipids and sphingolipids. Integration of carbohydrate and lipid metabolism.
Practical 13 * Nucleic acids extraction from yeast
The aim of the class: isolation of nucleic acids from yeast.
Theoretical basis: the structure and nomenclature of nucletides, the structure of nucleic acids: DNA
and RNA, the conditions of nucleic acid separation from yeast
Laboratory tests:
1. The separation and purification of nucleic acids from yeast.
2. Determination of RNA concentration in the sample – orcin method.
3. Determination of DNA concentration in the sample – Feulgen method.
Practical 14 * Some properties of nucleic acids
The aim of the class: studies on the composition and some properties of nucleic acids
Theoretical basis: characteristics of nucleic acids, their structure, metabolism and functions in the
human body
Laboratory tests:
1. The solubility of nucleic acids.
2. Nucleic acid sugar compounds detection:
a) orcin test: detection of ribose
b) diphenylamine test – detection of deoxyribose
3. Nucleic acids hydrolysis
a) detection of purines – by precipitation with Ag+ ions
b) detection of sugar – phloroglucinol reaction
c) detection of phosphate – by reaction with molybdic reagent
4. Absorbsion spectrum of nucleic acids.
SEMINAR / TEST VI * Nucleic acids and their metabolism
The key problems: The nomenclature and structure of purine and pirymidine bases. The structure of
nucleotides. Purine and pyrimidine synthesis and its regulation. The purine nucleotide
salvage pathway. The formation of deoxyribonucleotides. Degradation of purine and
pyrimidine bases. Lesch-Nyhan syndrome, sever combined immunodeficiency disease
(SCID), hyperuricemia.
The structure of the nucleic acids. Synthesis of DNA. Transcription: synthesis of RNA.
Translation: synthesis of proteins. Regulation of gene expression. Use of recombinant DNA
techniques in medicine. The molecular biology of cancer.
Practical 15 Enzymes of digestive tract
The aim of the class: studies on some properties of digestive juices
Theoretical basis: the composition of digestive juices, the physical and chemical properties of
digestive juices, the role of the components of digestive juices in the digestion process.
The characteristics of digestive enzymes.
Laboratory tests:
1. Demonstration of amylase presence in pancreatic juice.
2.
3.
4.
5.
6.
7.
Demonstration of proteolytic enzymes presence in pancreatic juice.
Demonstration of lipase presence in pancreatic juice.
Determination of lipase activity in pancreatic juice.
Detection of some inorganic ions in the saliva.
Measurement of gastric content acidity (free, related and total acidity).
Demonstration of bile acids presence in the bile
a) Hay test
b) Pettenkofer test
Practical 16 * The constituents of the blood
The aim of the class: studies on some properties of some constituents of the blood
Theoretical basis: the constituents of both cellular and noncellular fractions of the blood, major
functions of the blood, the structure of hemoglobin and its role in the respiration
(oxygen and carbon dioxide transport), the oxygen-binding curve of hemoglobin, the
allosteric effectors for oxygen binding to hemoglobin (pH, carbon dioxide, 2,3bisphosphoglycerate), the Bohr effect, some examples of hemoglobinopathies
(hemoglobin M, hemoglobin S, thalassemias), the role of the blood proteins in the
maintenance of homeostasis, the functions of inorganic compounds in the blood
Laboratory tests:
1.
2.
3.
4.
5.
6.
7.
8.
Benzidine reaction – detection of the blood.
Distinction of oxyhemoglobin and carboxyhemoglobin.
Preparation of hematin in acidic and basic conditions.
Demonstration of iron presence in hemoglobine molecule.
Determination of hemoglobin concentration – the cyanmethemoglobin method.
Detection of lipids in the blood plasma.
Van der Bergh reaction - detection of bilirubin in the blood serum.
Detection of bilirubin in the bile.
Practical 17 The blood serum enzymes used in clinical diagnosis
The aim of the class: the analysis of some serum enzymes activities and its role in the
diagnosis of disease processes
Theoretical basis: classification of blood diagnostic enzymes, the major enzymes used in clinical
diagnosis, the role of diagnostic enzymology in diagnosis of myocardial infarction and
some liver diseases, absorption spectra of NAD+ and NADH, the usage of these spectra
for the assays of dehydrogenases activities, the coupled enzyme assays
Laboratory tests:
1. Determination of alanine aminotransferase activity in the blood serum.
2. Determination of aspartate aminotransferase activity in the blood serum.
3. Determination of lactate dehydrogenase activity in the blood serum.
4. Determination of alkaline phosphatase activity in the blood serum.
5. Determination of -amylase activity in the blood serum.
SEMINAR / TEST VII The blood. Nutrition, digestion, and absorption
The key problems: The composition of digestive juices, the enzymes of digestive tract. Digestion and
absorption of carbohydrates, lipids, proteins, vitamins and minerals. The role of
hydrochloric acid and the bile salts in digestion processes. The production of hydrochloric
acid by parietal cell of the stomach. The biosynthesis and degradation of bile salts. The
enterohepatic bile salts circulation.
Synthesis of heme and regulation of this process. Catabolism of heme. The fates of
bilirubin: transport to the liver, conjugation with glukuronic acid, secretion into bile,
reduction to urobilinogen. The enterohepatic urobilinogen cycle. Hyperbilirubinemias,
different causes of jaundice (hemolytic anemia, hepatitis, obstructive jaundice), the
laboratory tests important in helping to distinguish between prehepatic, hepatic and
posthepatic causes of jaundice.
The major functions of the blood, the constituents of the blood. The organic and inorganic
constituents of the blood plasma. The characteristics and functions of plasma proteins.
Structure and functions of red blood cells. Metabolism of the red cell. Reaction of
importance in relation to oxidative stress in blood cells. System for reducing heme Fe 3+
back to the Fe2+ state in the red blood cell. Physiologic roles of hemoglobin and mioglobin,
the oxygen dissociation curves for myoglobin and hemoglobin. The mechanism of binding
O2 to myoglobin and hemoglobin. The cooperative interactions infuencing the binding O2
to hemoglobing, the changes of oxygen-binding curve of hemoglobin (effect of
temperature, pH, carbon dioxide concentration, 2,3-bis-phosphoglycerate concentration).
The Bohr effect. The carbon dioxide transport in the blood. Binding CO to hemoglobin.
Changes of the subunit composition of hemoglobin tetramers during development
(embryonic, fetal and adult subunits). Abnormal hemoglobines. Anemia. The classification
of the causes of anemia.
Practical 18 Urine physiologic parameters
The aim of the class: the analysis of some substances extreted by the kidney from the
body via the urine during physiologic conditions
Theoretical basis: the functions of the kidney (excretion of waste products produced by metabolism,
acid-base homeostasis, osmolality regulation, the blood pressure regulation, hormone
secretion), the production of urine, characteristics and composition of the urine,
glomerular filtration rate (GFR), creatinine clearance as a creatinine-based
approximation of GFR
Laboratory tests:
1. Determination of urea concentration in the urine – Yatzidis method with Ehrlich’s reagent.
2. Harrison’s test – detection of bilirubin in the urine.
3. Detection of creatinine in the urine:
a) Weyl’s test
b) Jaffe method
4. Determination of creatinine concentration in the urine – Jaffe method.
5. Deniges test – detection of indican in the urine.
6. Ehrlich’s aldehyde test – detection of urobilinogen in the urine.
7. Determination of α-amylase activity in the urine – Winslow’s method.
8. Determination of uric acid concentration in the urine and in the blood serum – enzymatic
method.
Practical 19 Diagnostic chemical markers in human urine
The aim of the class: the analysis of some substances excreted by the kidney from the
body via the urine during pathologic conditions
Theoretical basis: the role of the urine analysis in the medical examination, the substances excreted
by the kidney via the urine in selected disease processes, diagnostic urine stripes as a
rapid method of urine analysis
Laboratory tests:
1.
2.
3.
4.
5.
6.
Benzidine method – detection of the blood (or hemoglobin) presence in the urine.
Coagulation urine protein test – detection of the protein presence in the urine.
Benedict’s test – detection of glucose presence in the urine.
Rothera’s test – detection of ketone bodies presence in the urine.
Hay test – detection of the bile salts presence in the urine.
The urine examination using the diagnostic urine stripes.
SEMINAR / TEST VIII Metabolism of amino acids.
The key problems: Fate of amino acid nitrogen. Enzymes important in the process of interconverting
amino acids and in removing nitrogen (dehydratases, transaminases, glutamate
dehydrogenase, glutaminase, deaminases). The convertion of amino acid nitrogen to urea
– the urea cycle.
Degradation of amino acid: fate of amino acid carbon skeletons. Glucogenic and ketogenic
amino acids. The role of pyridoxal phosphate, tetrahydrofolate and tetrahydrobiopterin
coenzymes in amino acid metabolism. Some disorders of amino acid catabolism:
alkaptonuria, phenylketonuria, maple syrup urine disease.
The biosyntheses of biologically important compounds from amino acids (sphingosine,
choline, taurine, creatine, catecholamines, hippuric acid, nitric oxide, glutathione,
glycerophospholipids, purines, pirymidines, carnosine, anserine, bile salts, hem, serotonin,
melatonin, nicotinin acid, coenzyme A). Biosynthetic decarboxylations of amino acids to
amines – the biogenic amines and their biological functions.
The one-carbon carriers in the body: tetrahydrofolate (FH4), vitamin B12, Sadenosylmethionine (SAM). Sources and recipients of one-carbon FH4 pool.The methyltrap hypothesis. The role of SAM in the biosynthesis of the compounds of biological
importance (creatine, phosphatidylcholine, adrenaline, melatonin, methylated
nucleotides, methylated DNA).
Essential and nonessential amino acids. The synthesis of nonessential amino acids in the
human organism.
SEMINAR IX Integrative seminar 1 – Metabolism of tissues and organs.
The key problems: Carbohydrate, lipid and amino acid metabolism of liver, brain, skeletal muscle,
cardiac muscle cells, and kidney. The sources of ATP for skeletal muscle cells (e. g. creatine
phosphate, purine nucleotide cycle). Major functions of the kidney (excretion of waste
products produced by metabolism, acid-base homeostasis, osmolality regulation, the
blood pressure regulation, hormone secretion, γ-glutamyl cycle). The substances excreted
via the urine in normal and pathologic conditions. The metabolism of liver (detoxification
of drugs and metabolites, glutathione S-transferases, metabolism of ethanol). The
functions of glutathione in the organism.
Intertissue relationships in the metabolism of carbohydrates, lipids and amino acids.
SEMINAR X * Integrative seminar 2 – Metabolism regulation – actions of hormones.
The key problems: Classification of hormones according to their structure, a type of hormone
receptor, and a second messenger. The synthesis of thyroid hormones. The hormones
involved in the glucose maintenance in the blood. The major hormones influencing
nutrient metabolism and their actions on muscle, liver, and adipose tissue. The changes in
the fuel metabolism during fasting state and starvation. The metabolism of
carbohydrates, lipids, and amino acids in the diabetes mellitus type I and type II.
Practical 20 End-of-year practical
* practicals and seminars conducted by the Department of Clinical Biochemistry
# practicals and seminars partially conducted by the Department of Clinical Biochemistry
X. Self-study topics XI. Basic booklist
1. Murray RK, Bender DA et al. "Harper`s Illustrated Biochemistry" 28th edition
2. Lieberman M, Marks AD "Marks` Basic Medical Biochemistry a Clinical Approach". 3rd edition
3. Marks DB "Biochemistry" 3rd edition
XII. Detailed list of required practical skills and confirmation of completing
Assessment record
Student name:
Year of the study, group number:
Academic year:
List of required practical skills
Date
of signature
assessment tutor
Gaining
knowledge
of
molecular,
biochemical
physiological mechanisms occuring in human organism
Analysis of laboratory results
and
of comments
Rules and regulations
The main objective of the course is to provide an understanding of biochemical processes
and to gain relevant basic laboratory skills according to the educational requirement defined in the
program of teaching biochemistry for medical students.
The program (180 hrs) consists of lectures (60 hrs), tests and seminars (40 hrs), and practical
classes (80 hrs) and a closing test. All classes are compulsory. Students should check the schedule
carefully and be on time. If students cannot attend class they must have sick note and arrange for an
alternative date to carry out the lab/seminar.
The performance during each laboratory class will be evaluated by the quality of theoretical
preparation, laboratory skills and written protocol from the experiments.
Seminars: Students must be prepared to give a short presentations of topics which will be
given to them by the teacher after each part of material.
Tests: One-choice tests (30 questions, graded 1 point for a correct answer) will be held at the
end of the each part of the material.
Final examination: Students who has earned credit must take the final examination (50
questions, one-choice questions test, graded 1 point for a correct answer).
The final examination and partial tests can be retaken according to the schedule and the
percentage of the points according to the following system:
60-67 % - satisfactory
68-75 % - fairly good
76 - 83 % - good
84 - 90% - better than good
90 - 100 % - very good