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:

  1. Prof. dr hab. med. Józef Kędziora
  2. Dr Jolanta Czuczejko
  3. Dr Karolina Szewczyk-Golec

Contact: Dr Jolanta Czuczejko

Syllabus

I. Department of Biochemistry

II. Head of the Unit: dr hab. n. med.Beata Augustyńska

III. Faculty of Medicine, Medical Program, year II

IV. Course coordinator – dr Jolanta Czuczejko

V. Form of classes - lectures, practicals, seminars

VI. Form of crediting/assessment - 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)

  1. Amino Acids – fundamental units of proteins

-Composition

-Optical activity

-Amphoteric properties

  1. Peptides and polypeptides

-Formation

-Amphoteric properties

  1. Purification of proteins
  2. Conformation of proteins - primary, secondary, tertiary, quaternary structure
  3. Protein Structure – Function Relationship

Oxygen transport proteins

  1. Myoglobin and Hemoglobin Structure and Function
  2. Hemoglobinopathies (HbS, HbC, HbM, thalassemias)
  3. Humoral Immunity - five basic classes of immunoglobulin (structure and function): IgM, IgD, IgG, IgE, IgA
  4. 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

CMichaelis – 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.
  1. 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.
  1. Transcription of genetic information. RNA synthesis by DNA-dependent RNA polymerase. Types of RNA and their function. Reverse transcriptase.
  1. Translation of genetic information. Components of the translation apparatus. Protein biosynthesis. Protein maturation and posttranslational modifications. Protein degradation and turnover.
  1. Epigenetics – DNA modifications, DNA methylation. Modulation of genes expression. Epigenetics and cancer.
  1. 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.
  1. 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.
  1. 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

  1. 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

  1. 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

  1. Phenylhydrazine reaction – the formation of osazones.
  2. 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

  1. Barfoed reaction – the characteristic reaction for reducing disaccharides.
  2. 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, substrate-level 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).