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DANYLO HALYTSKYI LVIV NATIONAL MEDICAL UNIVERSITY DEPARTMENT OF BIOLOGICAL CHEMISTRY LABORATORY MANUAL ON BIOLOGICAL CHEMISTRY for students of pharmaceutical faculty PART І LVIV – 2016 1 Methodical instructions prepared by: Prof. Sklayrov A.Ya., M.D., Ph.D. Fomenko I.S., Ph.D. Klymyshin D.O, PhD. Nasaduk Ch.M., M.D., PhD Editor: prof. Sklayrov A.Ya., M.D., Ph.D. Reviewed by: prof. Pinyazhko O.R., M.D., PhD 2 PREFACE Biological chemistry is a fundamental medical discipline. A competence in biochemical processes, which take place on different levels of organization – cellular, organ, tissues and in a whole body – is necessary to medical students for understanding of metabolic processes and turnover of substances in tissues, energy production, anabolic and catabolic reactions, transfer of genetic information, processes providing elementary physiological functions as well for interpretation of biochemical data and their diagnostic and prognostic value. In each practical lesson students would be learning topics, which are determined by a program, will perform investigations of different substances and compounds, and interpret their significance. In each sense modul the themes for independent studies are proposed A considerable attention is paid to clinical aspects of biological chemistry – inborn and acquired disorders of metabolism, enzymopathias, to employment of enzymes as medicinals, as well as to a pathochemical processes, which take place in development and in course of diabetes mellitus, atherosclerosis, rheumatism, myocardial infarction, diseases of digestive system, etc. In order to improve the learning of educational material students are recommended to use modern textbooks and instruction materials, prepared by leading ukrainian specialists as well by coworkers of the Department of Biochemistry of Lviv national medical university. The final aims in learning of the subject “Biological and bioorganic chemistry“ are determined with a thesis, that student in his future professional activity ought to be able: To analyze a correspondence of bioorganic compounds structure to physiological function, which fulfill these compounds in the body. To interpret peculiarities of physiological status of organism and development of pathological processes on basis of laboratory investigations data. To analyze reaction ability of carbohydrates, lipids and amino acids which provides their functional properties and metabolic transformations in the body. To interpret peculiarities of structure and transformations in tissues of bioorganic compounds as a background of their pharmacological activity as medicinals and drugs. To interpret biochemical mechanisms of pathogenesis of different disorders in human body and principles of their correction. To explain the main mechanisms of biochemical effect and directed action of different classes of pharmacological agents. To explain biochemical and molecular basis of physiological functioning of cells, organs and systems of human body. To analyze enzymatic reactions which occur in cell membranes and organelles for explanation an integration of metabolic processes To classify the results of biochemical investigation and changes of biochemical and enzymatic data, which are used in diagnostics of the most spread human diseases To interpret the significance of biochemical processes and their regulation in providement of normal function of organs, systems and whole body 3 LABORATORY SAFETY Avoid loose or synthetic clothing for lab work; remove loose jewelry; secure hair and clothing away from flames, equipment, and chemical contamination. A laboratory is a workplace. The list of things not permitted in chemistry labs is long – begin with eating, drinking, cooking, applying makeup, smoking and anything else that might increase the chance of ingesting lab chemicals. Careful workers do not touch hands to their faces while working in lab. Professional and serious behavior is expected at all times; rowdy or boisterous play or pranks of any kind will be deemed cause for expulsion from lab. Housekeeping. Store backpacks and other extra materials away from work areas and off floors to protect them and to keep walkways clear. Keep work areas clear; store extra glassware and materials as soon as you finish with them, keeping only essential materials on the workbench. Use cotton towels to dry wet hands & clean surfaces. Use paper toweling for absorbing hazardous materials. Dispose of dirty waste paper in trash receptacles. Clean your work area: Check to be sure all reagents and waste containers are securely closed; Clean lab surfaces with sponges; Paper with absorbed hazardous chemicals should be placed in solid hazardous waste containers, not in the general trash. Broken glass contaminated with hazardous or smelly materials can be rinsed with appropriate solvent before placing shards in the broken glass container. Handle hazardous materials with correct techniques. Never touch hazardous chemicals with bare hands; use tools such as tongs and scoops. Never remove chemicals from the laboratories. Do not attach samples to lab reports or notebook pages. In addition to causing disposal problems, taking samples from lab creates the potential for an accidental exposure. Anhydrous materials (such as NaOH, CaCl2, MgSO4 or NaSO4) may absorb water from the air and MUST be kept tightly closed between uses. Left in the air, NaOH or KOH pellets will absorb moisture and produce a puddle of concentrated corrosive liquid on the work bench – a serious skin exposure hazard. 4 Thematic plan of practical lessons in Semester 1 N Topics of lessons 1. Introduction to biochemistry. Methods of biochemical investigation. Amino acid composition, structure, physico-chemical properties, classification and functions of simple and conjugated proteins 2. Enzymes: structure, physico-chemical properties, classification and mechanism of action of enzymes. Methods of detection of enzymes in biological material. 3 Kinetics of enzymatic reactions. Regulation of enzymatic activity, determination of enzymatic activity. 4. Regulation of enzymatic processess enzymopathias and mechanisms of their development. Application of enzymes as pharmaceutical preparations. 5. Role of cofacors and coenzymatic vitamins in catalytic activity of enzymes. 6. General principles of turnover of substances and energy. Functioning of citric acid cycle. 7 Biological oxidation, oxidative phosphorylation and ATP synthesis. Inhibitors and uncouplers of tissue respiration and oxidative phosphorylation in respiratory chain of mi9tochondria 8 Glycolisis – anaerobic oxidation of glucose, alternative pathways of carbohydrate metabolism. 9 Aerobic oxidation of glucose. Alternative pathways of carbohydrate metabolism. 10 Catabolism and biosynthesis of glycogen. Regulation of glycogen metabolism Biosynthesis of glucose - gluconeogenesis, 11 Mechanisms of metabolic and humoral regulation of carbohydrate metabolism. Disorders of carbohydrate metabolism. 12 Catabolism and biosynthesis of triacylglycerols and phospholipids. Intracellular ipolysis and molecular mechanisms of its regulation. 13 -Oxidation of fatty acids and their biosynthesis. Investigation of fatty acids ketone bodies metabolism. 14 Biosynthesis and biotransformation of cholesterol/ Regulation of lipid metabolism and its disorders. Hours 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 Thematic plan of lectures in Semester 1 N 1 2 3 4 5 6 7 8 9 10 Theme of the lecture Hours Introduction to biochemistry. Methods of biochemical investigations. 2 Enzymes: structure, properties and classification. Mechanism of action and regulation of enzymatic activity. Kinetics of enzymatic reactions. 2 Role of cofactors in catalytic activity of enzymes. Regulation of enzymatic processes and analysis of enzymopathias 2 appearance. Medical enzymology General principles of turnover of biomolecules and energy. 2 Tricarboxylic acid cycle. Molecular principles of bioenergetics. Biological oxidation. Oxidative 2 phosphorylation and its regulation. Carbohydrates: structure, classification, functional significance. Metabolism of monosaccharides, anaerobic and aerobic oxidation of 2 glucose. Gluconeogenesis. Metabolism of polysaccharides. Gluconeogenesis. Alternative pathways 2 of carbohydrate metabolism. Regulation of carbohydrate metabolism and its disorders. 2 Lipids: structure, classification and functional significance. Transport 2 forms of lipids in blood. Metabolism of simple lipids. Metabolism of complex lipids and its regulation. Correction of disorders 2 of lipid metabolism by pharmaceutical preparations. 6 Topic №1. Introduction to biochemistry. Methods of biochemical investigation. Amino acid composition, structure, physico-chemical properties, classification and functions of simple and conjugated proteins Objective: Introduction to the subject and assignments of biological chemistry, their practical employment; to make an acquaintance with methods of biochemical investigation. To demonstrate some physico-chemical methods of investigation as well to learn about devices used in biochemistry. To learn structure, classification and physico-chemical properties of proteins; to perform qualitative reactions on amino acids and quantitative determination of protein. Actuality of the theme: Biochemistry is a science which investigates chemical composition of living organisms, chemical structure of constituents, their properties, localization, the pathways of their appearance and transformations, as well as chemical processes, which take place in living cell and provide turnover of matter and energy in the cell. Biochemistry is on the way of solution of important problems and questions of natural history and medicine, e.g. problem of protein synthesis, life span prolongation, etc. Modern physical, chemical and mathematical methods are used in biochemical investigations. Biochemical data are used in medical diagnostics, treatment and prevention of diseases Specific aims: To know principal stages and regularities in origin and development of biochemistry as fundamental medical and biological science and educational discipline. To recognize priciples of methods of investigation of functional status in human body in health and disease. To interprete data of biochemical investigations and evaluate the status of selected metabolic pathways To determine optical density of colored solutions at distinct light wavelength using a photocolorimeter, to interpret obtained results properly. 1. 2. 3. 4. 5. Theoretical questions: The objectives and assignments of biochemistry and its principal trends and parts. A short history of biochemistry and its main periods. Methods of biochemichemical analysis: Optical methods in biochemical investigations. Electrophoresis. Chromatography. Radioisotopic methods. Enzyme immunoassays (ELISA). General characterization of amino acids. Classification of amino acids (due to their structural, electrochemical, biological properties). Biologically active peptides, their significance and employment in medicine (glutathione, hormones of hypophysis and hypothalamus, insulin etc.). 7 6. Modern concept of structural levels in organization of protein molecule and types of chemical bonds in protein molecule. Physical and chemical properties of proteins. Isoelectric point, proteins as amphoteric electrolytes. 7. Classification of proteins. Characterization of simple proteins. 8. Conjugated proteins, their characteriristics. Essay of the history of biochemistry unit Unit of Biological Chemistry was founded in 1894 at the Medical department of Lviv University by Prof. V. Niemilovych (1863-1904) who had arrived from Vienna in 1891 upon invitation to work as an associate professor in Pharmacognosy and as a lecturer in chemistry. From 1904 to 1919, the unit was headed by Prof. Bondzinski (1862-1929). He was the founder and the first president of the Academy of the Medical Sciences of Poland. Research of the unit's faculty under Prof. S. Bondzinski was the so-called oxyproteins - the products of protein turnover (metabolism) excreted in small amounts within urine. From 1919 to 1922, the unit was supervised by Prof. W.Morachewsky, who also became rector of the Academy of Veterinary medicine in Lviv. W. Morachewsky was certified to teach medical chemistry at the University of Lviv in 1918 and he began his work as Professor at Lviv Academy of Veterinary medicine in 1921. From 1922 until 1941 the unit was headed by the world-well known scientist Jakub Oscar Parnas. His most significant work at the Lviv schoole was connected with enzymatic products, their formation in association with muscular activity and alcohol fermentation. Some of his investigations have become classic works in biochemistry. They concern the rapid autolytic increase of ammonium content in extravasate blood and "postmortem" or traumatic ammoniogenesis in muscular tissue. Significantly important was his discovery of the higher phosphorylated derivatives of adenylic acid found in muscular fibers and erythrocytes, namely ATP and ADP. As alternatives to adenylic acid they are not subject to enzymatic deamination in cells. J.Parnas and his schoole studied biological and chemical properties of muscular adenylic acid and its derivatives and introduced analytical methods for its quantitative determination. J. Parnas achieved worldwide recognition in his research of anaerobic degradation of carbohydrates. His research is called in biochemical literature as the Embden-Meyerhof-Parnas pathway or "EMP scheme of glycolysis ". J. Parnas was the first who introduced isotopic methods into biochemical investigations. From 1944 to 1973, the unit was headed by Prof. B.A. Sobchuk who began his scientific activity under J. Parnas conduction. B. A. Sobchuk investigated carbohydrate metabolism in the muscular tissue and yeast. He was awarded the degree of Doctor of Medicine (1937) for his research on the significance of pyruvate in glycogenolysis. For his thesis, "Synthesis of xanthopterin and its derivatives and their effect on experimental tumors in animals", B.A .Sobchuk was awarded the degree of Doctor of Biological Sciences in 1959. A year later he attained professorship of biochemistry. The co-workers of department studied the peculiarities of carbohydrate metabolism in tumor cells (the Krebtry's effect), as well as the synthesis and specific effect of xanthopterin and its derivatives iodopterin and porphyropterin on tumors. Another significant direction of the unit's work was the 8 investigation of iodine metabolism, function of the thyroid gland and toxicology of carbon monoxide. From 1974 to 1995, the unit was headed by prof. M.P. Shlemkevych. In close collaboration with the unit of oncology, the department of biochemistry studied the mechanisms of sensitivity and resistancy of tumor cells of stomac cancer to chemotherapeutic agents, namely 5-fluorouracyl. From 1995 to 1998, the head of biochemistry unit was prof. M.F. Tymochko. The main goal of his scientific research was the investigation of the fundamentals of adaptive-compensatory processes under various experimental conditions with emphasize on changes in oxygen-dependent reactions. He studied metabolic backgrounds of oxygen homeostasis in different functional conditions and dealt with solving of a wide range of important problems in medical practice. He also investigated the effect of harmful environmental factors on the functions of digestive system, the role of energy exchange in the pathogenesis of chronic diseases of the liver and cardiovascular system. He determined the degree of risk in surgery in abdominal cavity, endocrine and cardiovascular surgery and endogenous intoxication in oncological diseases. Since 1998, the Biochemistry unit has been headed by O. Ja. Sklyarov, who started his scientific and educational activities at the unit of normal physiology. In 1993, for his thesis, "Mechanisms of combined action of mediators and hormones on the secretory function of gastric glands", he obtained the scientific degree doctor of medical sciences. In 1997 he was nominated as honorary associate professor by the fund of Soros and was noted by the fund "Revival". Prof. O.Ja.Sklyarov is concerned with the study of the mechanisms of regulation of gastric secretion under the influence of the combined action of neurohormonal substances. Alongside his research, prof. O.Ja.Sklyarov pays significant attention to educational work. Currently, the unit's researchers are investigating the systemic influence of malignant tumors and endogenous intoxication in patients and in experimental animals. They are also studying the effects of stress on cytoprotective and ulcerogenic mechanisms of gastric mucosa, the ion-transport and metabolic processes in it and the action of exogenous factors on endoecological conditions. Practical part Determination of optical density of colored solutions according to their concentration The photoelectrocolorimetric method of analysis serves for determination of substances concentrations in coloured solutions, biological liquids or tissue extracts. It may be also used for determination of concentrations of colourless substances if they can be transformed into coloured state with the specific reagents. The method is one of the most widespread in biochemistry and clinical medicine. Experiment 1. Measurement of optical density (A) of solutions which contain different quantities of phosphorus. Principle. Phosphates in presence of sulfuric acid form phosphomolybdate complexes with molybdate, which are then reduced to a compound called molybdene blue. The amount of phosphates is then measured colorimetrically according to a 9 color intensity of molybdene blue complex. Method. Pipette the solutions into five labelled test tubes according to the table: Tube number № 1 2 3 4 5 Reagents Standard phosphate solution (10 mg/ml), ml Distilled water, ml Molybden reagent, ml Phosphorus concentration, mg% Results of optical density measurement 1 - 2 0.5 3 1.0 4 2.0 5 3.0 5.0 5.0 0 4.5 5.0 50 4.0 5.0 100 3.0 5.0 200 2.0 5.0 300 After mixing a color develops. After 5 min measure the optical density on a photoelectrocolorimeter with a red light filter. Then draw a plot (calibration curve) according to obtained results. Experiment 2. Protein precipitation with sulfosalicylic acid. Principle. Proteins dissolve in water with formation of homogenous solutions. There are two main factors of the stabilization of proteins in solutions. The first, the existence of electric charges, and, the second, the formation of hydrate envelopes, which are due to the attachment of water molecular dipoles round charged hydrophilic residues of amino acids. The electric charge of protein molecules depends from the ionization of the functional groups of side chains of amino acids. Thus, the solubility of proteins is due to their amino acid composition, the unique structural organization of the protein molecule and the properties of the solvent. Neutral salts increase solubility of proteins because of the interaction of their ions with the polar groups in proteins. There are reversible and irreversible (denaturation) methods for the precipitation of proteins. The reversible precipitation occurs with high concentration of neutral salts (salting out), alcohol, and acetone (the last two conducted at below zero temperatures). The mechanism of precipitation consists in removing the hydrate envelopes and masking the charges of the protein molecule. During the denaturation process the alteration of spatial structure of protein molecule occurs. This is accompanied by decrease or complete loss of solubility, changes in chemical properties, and loss of biological activities of a protein. The biological activity of proteins such as hormone effect, enzymatic activity, virulence of microorganisms is lost after denaturation (called inactivation). Cations of heavy metals, organic acids (trichloroacetic, sulfosalicylic, tannic acids), concentrated mineral acids, heating, ultraviolet and ionizing radiation exhibit a denaturing effect on proteins. Though denaturation is an irreversible process, it is not connected with destruction of the primary structure of a protein. Method. Add 1.0 ml of protein solution into the tube, and 3-5 drops of 20% sulfosalicylic acid and observe the appearance of a precipitate. Clinical and diagnostic significance. Proteins are important cell constituents. They play an important role in metabolic processes. Denaturation is usually met in natural conditions and in experimental research. Denatured proteins are readily 10 digested by proteolitic enzymes. Gastric hydrochloric acid causes the denaturation of proteins of food. As a result they are more readily hydrolyzed by digestive enzymes. Milk proteins, such as casein, bound heavy metals and are used in treatment of poisoning with heavy metals. In clinical laboratories the proteins of blood and serum are denatured by precipitation with organic acids like trichloroacetic acid and sulfosalicylic acid. Methods of quantitative determination of proteins Protein quantity in the specimen can be estimated by physical and chemical methods. Physical methods include refractometry, spectrophotometry, etc. Chemical methods employ color reactions of proteins and the intensity of color is measured by colorimetry (e.g. biuret test, Lowry method). Chemical methods include also determination of nitrogen by Quieldahl method. 3 Standard tube Serum A 4 5 1- Serum C 2 Serum B 1 Control tube Experiment 3. Biuret method of protein quantitation. Principle. Proteins in alkaline medium interact with copper sulfate forming a compound with a violet colour. Optical density of this compound solution is directly proportional to the concentration of protein. Reagents. Blood serum, standard protein solution (50 g/l ), biuret reagent (0.15 g copper sulfate, 0.6 g sodium, potassium tartrate are dissolved in 60 ml of water, 30 ml of 10% NaOH solution are added, volume adjusted to 100 ml, then 0.1 g KJ is added). Method. Take five clean dry tubes and do the following procedures: 1. Add to the first tube (control) 0.1 ml of saline. 2. Add to the second tube 0.1 ml of standard protein solution. 3. Add to the third, fourth and fifth tubes 0.1 ml of tested blood serum A, B and C respectively. 4. Add 5 ml of biuret reagent to all tubes, mix and incubate for 10-15 min. 5. Measure the extinction (optical density) on a photocolorimeter using a green light filter against control solution. 2- - 0.1 ml of standard solution 3- - 0.1 ml of blood serum A 4- - 0.1 ml of blood serum B 5- - 0.1 ml of blood serum C + 5 ml of biuret reagent - 0.1 ml of saline Incubation for 10-15 min (room temperature) Measurement of the optical density using a green light filter C RESULTS: BA BB BC Calculation. The concentration of protein in the test solution is calculated according to the formula: X= (AB)/C, where X – protein concentration in test solution, (g/l), A – protein concentration in standard solution (50 g/l), B – extinction of tested protein solution, 11 C – extinction of standard protein solution. Clinical and diagnostic significance. In a healthy person the content of protein consists from 60 g/l to 80 g/1. Below 60 g/1 is hypoproteinemia and above 80 g/1 is hyperproteinemia. A decrease in blood protein concentration occurs as a result of a decrease in protein biosynthesis, of water balance disorders, or as a result of an increase in protein breakdown and protein loss. Hypoproteinemia is observed in nephritic syndrome, malabsorption syndrome (enteritis, chronic pancreatitis), exudative enteropathias, skin diseases (combustion, exematous lesions), during massive blood loss, in retention of water and mineral salts (chronic kidney diseases), agammaglobulinemias, hypogamma-globulinemias, and during starvation or improper nutrition. Decrease in blood protein concentration is observed during cardiac failure as a result of water retention causing a swelling of tissues or during kidney diseases when proteins are excreted in the urine. Protein biosynthesis disturbances take place during cancer cachecsia and during chronic inflammatory diseases, when accompanied by degenerative processes. Hyperproteinemia is observed in plasmocytoma, Waldenstrom macroglobulinemia, rheumatoid arthritis, collagenoses, liver cirrhosis as well as in diseases accompanied with dehydration, that is during diarrhea, vomiting, and diabetes insipidus. As a result of heavy mechanic lesions (traumas) the increase in blood protein concentration may be due to the loss of a substantial part of the intravascular fluid. In acute infections the increase in blood protein concentration may due to the increased production of acute phase proteins, in chronic infectious diseases it may be due to enhanced synthesis of immunoglobulins. Examples of Krock-1 tests 1. Choose from listed below methods ONE, which is used for fractionation of protein mixtures and isolation of individual proteins (enzyme, hormone, toxin etc): A. Affinity chromatography D. Proteolysis B. Precipitation with nitric acid E. Radioimmunoassay C. Boiling of extracts 2. Determination of C-reactive protein (CRP) in blood plasma is conducted with the use of antisera, containing specific antibodies against CRP. What type of analytical method is used in this case? A. Immunoprecipitation D. Chromatography B. Spectrophotomenry E. Polarography C. Electrophoresis 3. Proteins are biopolymers of principal significance in cell building, they are composed from amino acids as monomers, which are connected into chain by the next main type of chemical bond: A. Peptide bond C. Ionic bond B. Phosphodiester bond D. Hydrogen bond 12 E. Glycosidic bond 4. Protein preparations from human blood plasma are frequently used in clinical medicine for treatment of many diseases. Fractionation of blood plasma and preparation of distinct protein fractions is achieved by the next method: A. Fractional precipitation with C. Precipitation with salts of heavy ammonium sulphate metals B. Fractional precipitation with ethanol D. Electrophoresis in agarose gel by Cohn YI method E. Ultracentrifugation Individual independent students work 1. History of biochemistry and its main periods. The significance of biochemistry in the development of medical sciences and practical health care. 2. The fundamental discoveries in a branch of structural and functional significances in proteins and nucleic acids. References: 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 2. Enzymes: structure, physico-chemical properties, classification and mechanism of action of enzymes. Methods of detection of enzymes in biological material. Objective: Life in all its diverse manifestations is an extremely complex system of chemical reactions which are catalyzed by enzymes. The last play vital role in nearly all life processes. They are involved in living organisms in a vast multitude of interrelated chemical reactions such as synthesis, degradation and interconversion of a large number of chemical compounds. An understanding of their implications provides a deep insight into the sense and innermost enigmas of the fascinating phenomenon that we call life. Specific aims: To interpret biochemical principles of structure and functioning of different 13 classes of enzymes. On the basis of physical and chemical properties of enzymes as proteins to explain the dependence of enzymatic activity from pH of medium, temperature and other factors. To analyze values of the activity of enzymes in blood plasma in dependence from their localization in the cell, tissue or organ. To analyze methods of determination of enzymatic activity for an optimal application in clinical biochemistry. 1. 2. 3. 4. 5. 6. 7. 8. Theoretical questions: Enzymes: definition, properties of enzymes as biological catalysts, difference between enzymes and inorganic catalysts. Nomenclature and classification of enzymes. Physico-chemical properties of enzymes. Simple and conjugated enzymes. Role of non-protein part of conjugated enzymes. Structure of enzymes: active centres and allosteric sites. Levels of structural organization of enzymes. Multi-enzyme complexes, their advantages. Specificity of enzymes. The localization of enzymes in cells and organs. Practical part Salivary amylase Amylase – is an enzyme that catalyses the hydrolysis of starch into sugars. Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of carbohydrates digestion. Salivary gland makes amylase to hydrolyse α-1,4-glycosidic bonds of starch into disaccharides (maltose) which are converted by other enzymes to glucose to supply the body with energy. As diastase, amylase was the first enzyme to be discovered and isolated (by A. Payen in 1833). Experiment 1. Investigation of the thermolability of salivary amylase. Principle. Salivary amylase is a protein and consequently is thermo-sensitive. Boiling inactivates enzymes due to denaturation, low temperature (near 0 oC) substantially lowers the rate of reaction. The hydrolysis of starch is monitored by probes with iodine and the appearance of the of maltose, which is the final product of starch hydrolysis by salivary amylase. Starch with iodine gives a blue coloured complex. Dextrins (intermediate products of starch hydrolysis) in presence of iodine form red-brown colour or no change in colour at all. Maltose also does not form a coloured complex with iodine, but it can be detected with the Trommer’s reaction. Method. I. Dilution of saliva. To obtain diluted saliva (the source of amylase), rinse one’s mouth with distilled water during 1-2 min and collect that portion of fluid (saliva + water) into separate tube and use it for the analysis. 14 II. Preparation of experimental mixtures and incubation. Take 3 clean tubes and do the following procedures: 1. Add 2 ml of diluted saliva to the first tube and boil it for two min on a flame. 2. Add 2 ml of diluted (nonboiled) saliva to the second and the third tubes. 3. Add 5 ml of starch solution into each tube. 4. Place the first and the second tubes into the termostate at 37 oC for 10 min. 5. Place the third tube in a cold water (near 4-6 oC) for 10 min. 6. Divide the content of each tube into two equal portions. III. Color reactions for the detection of starch and maltose. 1. Add 3 drops of iodine to the first portion of each test solution. Register changes in color. 2. Use the second portion of the probes for the performance of the Trommer’s reaction. Preparation of mixtures and incubation 2 1 - 2 ml of boiled diluted saliva 3 - 2 ml of diluted saliva - 5 ml of starch solution Incubation for 10 min (T=4-60C) Incubation for 10 min (T=370C) Color reactions for the detection of starch and maltose 1a Trommer’s reaction (+CuSO4+NaOH) 1b + I2 2a 2b Trommer’s reaction (+CuSO4+NaOH) + I2 3a Trommer’s reaction (+CuSO4+NaOH) 3b - 2.5 ml of mixture after incubation + I2 Results: – + + – – + Trommer’s reaction. Add 5 ml of 5% NaOH and several drops of CuSO4 solution, mix the content of these tubes and heat it on a flame to boiling. The appearance of yellow-red sediment indicates on the presence of reducing substances, namely, reducing disaccharide maltose. Explain the results and draw a conclusion. Experiment 2. The investigation of the influence of pH of medium on amylase activity. Principle. Each enzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive. Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8). Method. I. Dilute saliva as described above (Experiment 1) 15 II. Preparation of experimental mixtures and incubation. Take 3 clean tubes and do the following procedures: 1. Add 2 ml of starch solution to each of the three tubes. 2. Add to the first tube 2 ml of phosphate buffer, pH 5.0. 3. Add to the second 2 ml of phosphate buffer, pH 7.0. 4. Add to the third 2 ml of phosphate buffer, pH 9.0. 5. Add into each tube 1 ml of diluted saliva. 6. Incubate tubes at 37 oC for 10 minutes. 7. Divide the content of each tube into two equal portions. Stage 1 Preparation of mixtures and incubation 2 1 - 2 ml of of starch solution 3 - 2 ml of phosphate buffer pH 9 pH 7 pH 5 - 1 ml of diluted saliva Incubation for 10 min (T=370C) Stage 2. Color reactions for the detection of starch and maltose 1a Trommer’s reaction (+CuSO4+NaOH) 1b + I2 2a 2b Trommer’s reaction (+CuSO4+NaOH) + I2 3a Trommer’s reaction (+CuSO4+NaOH) 3b - 2.5 ml of mixture after incubation + I2 Results: – + + – – + III. Color reactions for the detection of starch and maltose. 1. Add 3 drops of iodine to the first portion of each test solution. Register changes in color. 2. Use the second portion of the probes for the performance of the Trommer’s reaction (see Experiment 1). Explain the results and draw a conclusion. Experiment 3. Investigation of substrate specificity of salivary amylase and yeast sucrase. Principle. Enzymes exhibit selectivity to substrates, which is called substrate specificity. In many cases this property is the essential characteristic that distinctly differs enzymes from inorganic catalysts. The high specificity of enzymes depends from the conformational complementarity between the molecules of enzyme and substrate due to the unique structure of active centre of the enzyme. 16 Method: I. Specificity of amylase: 1. Add 1 ml of two fold diluted saliva in each of two tubes. 2. Add 2 ml of 1% solution of starch into the first tube. 3. Add 2 ml of 1% solution of sucrose into the second tube. 4. Incubate both tubes at 37oC in thermostat for 15 min. 5. Conduct the Trommer’s reaction (see Experiment 1) with the content of both tubes. II. Specificty of yeasts sucrase: 1. Add 1 ml of sucrase from yeasts solution in each of two tubes. 2. Add 2 ml of 1% solution of sucrose to the first tube 3. Add 2 ml of 1% starch solution to the second tube. 4. Incubate both tubes at 37oC in thermostat for 15 min. 5. Conduct the Trommer’s reaction (see Experiment 1) with the content of both tubes. Part 3. Note the results of the experiment in the table. Tube № Enzyme Substrate Result of Trommer reaction 1 Amylase Starch 2 Amylase Sucrose 3 Sucrase Sucrose 4 Sucrase Starch Explain the results and draw a conclusion. Clinical and diagnostic significance. Amylase (E.C. 3.2.1.1) hydrolyzes starch and dextrins. It is produced predominantly in salivary and pancreatic glands, although amylase activity can be detected in tissues of liver, kidneys, intestines and lungs. During normal conditions amylase activity in blood serum corresponds to 0.42-0.96 g of starch, hydrolyzed by 1 m1 of enzyme in 1 minute. An increase of amylase activity in serum is observed in pancreatitis, peritonitis, mesenteric vessels thrombosis, rupture of oviduct in case of extrauterine pregnancy and after the injection of some drugs, e.g. morphine, caffeine, ACTH and cortisol. The increase of the activity of amylase in saliva is observed in renal insufficiency, stomatitis, neuralgia. A decrease in amylase activity is noted in some cases of psychoses, accompanied by depression or excitation and in gastric secretion disorders (anaciditas). Examples of Krock-1 tests 1. After the addition of an extract of pancreatic gland to the tube with starch solution a blue coloration of the sample with iodine have disappeared, which indicates on starch hydrolysis. What pancreatic enzyme is involved in this reaction? A. Amylase D. Lipase B. Trypsin E. Aldolase C. Chymotrypsin 17 2. In human saliva there is an enzyme able to hydrolyze the α[1→4] glucosidic bonds in the molecule of starch. Name this enzyme: A. α-Amylase D. β-Galactosidase B. Phosphatase E. Lysozyme C. Fructofuranosidase 3. In a patient was detected disorder in digestion of protein in stomach and small intestines. What group of enzymes may cause this disorder? А. Proteinases D. Lyases В. Amylase Е. Aminotransferases С. Lipase 4. The first position in classification of enzymes is occupied by: A. Oxidoreductases D. Hydrolases B. Transferases E. Ligases C. Isomerases Individual independent students work 1. Multi-enzyme complexes and their advantages. 2. The employment of enzymes in biochemical investigations. 1. 2. 3. 4. 5. 6. 7. 8. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 3. Kinetics of enzymatic reactions. Regulation of enzymatic activity, determination of enzymatic activity. Objective: To show the catalytic role of enzymes on the basis of pancreatic proteinases and amylase activity measurements. To estimate clinical and diagnostic significance of determination of amylase activity in urine. Actuality of the theme: Enzymes are biocatalysts with changeable activity, submitted to regulatory influences. The knowledge of kinetics of enzymatic reactions is important for the understanding of metabolic processes in cells, tissues and organs of human body. 18 Specific aims: To explain the mechanism of enzymatic action on basis of the affinity of enzyme to substrate and events which occur in enzyme-substrate complex. To interprete changes in activity of enzymes in biological fluids under conditions of different pathological processes. To explain the employment of inhibitors and activators of enzymes in cases of metabolic disorders or distinct pathology To analyse the mechanism of enzyme action using an example of chymotrypsin and acetylcholinesterase. Theoretical questions: 1. Enzyme kinetics. Factors affecting enzymatic activity: concentration of enzyme concentration of substrate; effect of temperature; effect of pH 2. Michaelis-Menten constant and equation; 3. Lineweaver-Burk double-reciprocal plot; 4. Mechanism of enzyme action: substrate-enzyme complex formation and dissociation; lock and key model or Fisher’s template theory; induced fit theory or Koshland’s model; substrate strain theory. 6. Units of enzymatic activity. 7. Methods of enzymatic activity assays. Practical part Methods of enzymatic activity assays Methods for the determination of enzymatic activity can be divided into: direct, indirect and coupled assays. The direct method is based on measurement of the substrate or product concentration as a function of time. For example, the enzyme cytochrome c oxidase catalyzes the oxidation of the heme-containing protein cytochrome c. In its reduced (ferrous iron) form, cytochrome c displays a strong absorption band at 550 nm, which is significantly diminished in intensity when the heme iron is oxidized (ferric form) by the oxidase. One can thus measure the change in light absorption at 550 nm for a solution of ferrous cytochrome c as a function of time after addition of cytochrome c oxidase; the diminution of absorption at 550 nm that is observed is a direct measure of the loss of substrate (ferrous cytochrome c) concentration. In some cases the substrate and product of an enzymatic reaction do not provide a distinct signal for convenient measurement of their concentrations. Often, however, product generation can be coupled to another, nonenzymatic, reaction that does produce a convenient signal; such a strategy is referred to as an indirect assay. Dihydroorotate dehydrogenase (DHODase) provides an example of the use of an indirect assays. This enzyme catalyzes the conversion of dihydroorotate to orotic acid in the presence of the exogenous cofactor ubiquinone. During enzyme turnover, 19 electrons generated by the conversion of dihydroorotate to orotic acid are transferred by the enzyme to a ubiquinone cofactor to form ubiquinol. It is difficult to measure this reaction directly, but the reduction of ubiquinone can be coupled to other nonenzymatic redox reactions. A third way of following the course of an enzyme-catalyzed reaction is referred to as the coupled assays method. Here the enzymatic reaction of interest is paired with a second enzymatic reaction, which can be conveniently measured. In a typical coupled assay, the product of the enzyme reaction of interest is the substrate for the enzyme reaction to which it is coupled forconvenient measurement. An example of this strategy is the measurement of activity for hexokinase, the enzyme that catalyzes the formation of glucose 6-phosphate and ADP from glucose and ATP. None of these products or substrates provide a particularly convenient means of measuring enzymatic activity. However, the product glucose 6-phosphate is the substrate for the enzyme glucose 6-phosphate dehydrogenase, which, in the presence of NADP+, converts this molecule to 6-phosphogluconolactone. In the course of the second enzymatic reaction, NADP+ is reduced to NADPH, and this cofactor reduction can be monitored easily by light absorption at 340 nm. Experiment 1. Quantitative determination of amylase activity in urine according to Wohlgemuth. Principle. Wohlgemuth’s method is based on the ability of amylase to breakdown (hydrolyze) the starch. A minimal quantity of an enzyme, which is able to hydrolyze 1 ml of 0.1% starch solution at 45 °0 during 15 min, is considered as an enzymatic unit of amylase activity Method: Take 9 clean tubes and do the following procedures: 1. Add 1 ml of saline into each of 9 enumerated tubes. 2. Add 1 ml of urine to the first tube, mix it and transfer 1 ml of the mixture from the first tube to the second tube. With the same manner, transfer 1 ml of the mixture after mixing from the second to the third tube and so on. Then remove 1 ml of mixture from tube 9. 3. Add 1 more ml of saline into each tube. 4. Add 2 ml of 0.1 % of starch solution. 5. Mix substantially the content of tubes and place tubes are into incubator (45°C for 15 min). 6. Cool tubes by tap water. 7. Add 2 drops of Lugol solution (iodine in potassium iodide solution) to each tube. 8. Registered a colour of solution in each tube. Results write in a table and calculate the activity of amylase. Tube N 1 2 3 4 5 6 7 8 9 Urine dilution 1:2 1:4 1:8 1:16 1:52 1:64 1:128 1:256 1:512 Colour with iodine Starch cleavage 20 1 2 3 4 5 6 7 8 9 - 1 ml of saline - 1 ml of urine - 1 ml of mixture from the previous tube Add 1 ml of saline into each tube and add 2 ml of 0.1 % of starch solution 1 2 3 4 5 6 7 8 9 Blue Blue - 2 ml of 0.1 % of starch solution Incubation for 15 min (T=450C) + I2 Results: Yelow Yelow Yelow Yelow Blue Blue Blue Example. 1/16 ml of urine cleaved 2 ml of 0.1 % starch solution (yellow color in tubes NN 1-4). In the end subsequent tubes starch was not cleaved (blue colour). Amylase activity: X = 1×2/ 1/16 = 2×16 / 1 = 32 (un.), where 16 – urine dilution, 2 – quantity of starch added. Explain the results and draw a conclusion. Clinical and diagnostic significance. The determination of amylase activity in biological fluids is widely used in clinical practice for diagnosis of pancreas diseases. Normally amylase activity in blood serum and in urine consists around 16-64 units (Wohlgemuth units). An increase of amylase activity in urine to more than 256 units indicates is called amylasuria. It may be a sign of: acute inflammation of the pancreatic gland (acute pancreatitis), alcohol consumption, cancer of the pancreas, ovaries, or lungs, cholecystitis, Gallbladder disease, intestinal obstruction, pancreatic duct obstruction, pelvic inflammatory disease. Decreased amylase levels may be due to: damage to the pancreas, kidney disease, pancreatic cancer, toxemia of pregnancy Experiment 2. Quantitative determination of pancreatic proteinases activity. Principle. The following method is based on the ability of proteinases (proteases, peptidases) to hydrolyze peptide bonds of proteins. Thus free amino acids become released from proteins. After amino groups of amino acids can be blocked by formaldehyde, then carboxyl groups can be measured by Zorensen’s method of titration with NaOH in presence of phenolphthalein. Method. 21 1. Put into each of two flasks 5 ml of 6% casein solution and 2 ml phosphate buffer, pH 7.6. 2. Add into the first flask a suspension of 0.5 g of pancreatin in small volume of water and mix well. 3. Place both flasks (the first one is the test; the second the control) in a thermostat at 37 C for 30 min. 4. Add into both flasks 2-3 drops of phenolphthalein and then titrate with 0.2 n NaOH solution until a faint pink colour appears (pH - 8.3). At this pH the quantity of amino groups is equal to the quantity of carboxyl. 5. Add into both flasks 5 ml of formalin mixture. Formaldehyde binds with amino groups causing the carboxyl groups to shift the pH to acidic side. 6. Titrated both flasks with 0.02 n NaOH solution till faint pink colour (pH 8.3) and add additionally several drops of 0.02 n NaOH to attain a bright red colour (pH 9.1). Test Control - 5 ml of 6% casein solution - 2 ml phosphate buffer, pH 7.6 - 0.5 g of pancreatin Incubation for 30 min, 37oC Test Control - 2-3 drops of phenolphthalein Titration by 0.2N NaOH to appearance of faint pink colour Test pH - 8.3 Control - 5 ml of formalin mixture Titration by 0.2N NaOH to appearance of pink colour + several drops of 0.02N NaOH to attain a bright red colour Calculation: The volume of 0.02 n NaOH solution, expended for titration of the test sample is determined as the difference between test and control probes. The activity of proteinases is expressed in arbitrary units. 1 arbitrary unit corresponds to 1 ml of 0.02 n NaOH solution expended for titration of test sample Explain the results and draw a conclusion. Clinical and diagnostic significance. Proteinases and peptidases are hydrolytic enzymes, decomposing proteins and peptides to amino acids. In living organism these 22 enzymes are found inside living cells. They are also excreted by specialized exocrine glands into the digestive tract. The highest activity exhibit by proteinases and peptidases are those of the pancreas - trypsin (E.C. 3.4.21.4), chymotrypsin (E.C. 3.4.4.5), and carboxypeptidase (E.C. 3.4.2.1). In blood plasma there exists a potent antiproteolytic system, which binds and inactivates proteinases appearing in blood. This system includes α-1 antitrypsin, α-1 antichymotrypsin, antiplasmin, and α-2 macroglobulin. Proteolytic activity can be detected in blood in some diseases, when the quantity of proteinases exceeds the binding capacity of antiproteolytic system. This situation can take place during acute pancreatitis, gastric ulcers, heavy combustions. There are a group of specialized proteinases, which perform limited proteolysis and generate biologically active peptides and proteins. They include blood coagulation enzymes (such as the Hageman factor, Stewart-Prower factor, thrombin, plasmin), kallikrein-kinine system, and renin. The last generates angiotensin I peptide which is transformed to angiotensin II and increases blood pressure. Limited proteolysis is a process of a great importance for regulation of vital processes in the cell. Examples of Krock-1 tests 1. Michaelis-Menten constants of two enzymes are 1,3x10-5 M/l and 2,3x10-3 M/l subsequently. Indicate true statement about the affinity of these enzymes to substrate. A. The second enzyme has higher to substrate affinity to substrate D. For decision an information on B. Enzymes possess equal affinity to concentration of enzyme is needed substrate E. Data are incomplete and it is C. The first enzyme has higher affinity impossible to draw a conclusion 2. In an enzyme assay the substrate concentration was taken much higher than Km. In this conditions the rate of the reaction will be as follows: A. Shows zero-order kinetics D. Is independent of enzyme B. Approaches 50 % value of Vmax concentration C. Is proportional to substrate E. Is independent of temperature concentration 3. Activity of many enzymes depends from the presence of free thiol groups in active center. What amino acid residue provides presence of these groups in enzyme molecule? A. Cysteine D. Methionine B. Lysine E. Serine C. Tryptophan 4. Michaelis-Menten constant (Km) reflects the next property of enzyme: A. Affinity to substrate D. Affinity to a product of reaction B. Sensitivity to pH of medium E. Sensitivity to competitive inhibitors C. Thermolability 23 1. 2. 3. 4. 5. 6. 7. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. Topic № 4. Regulation of enzymatic processes enzymopathias and mechanisms of their development. Application of enzymes as pharmaceutical preparations Objective: To learn the main principles of regulation of metabolic pathways, the consequences of alteration of enzymatic activity in the cell and employment of enzymes in medicine. Actuality of the theme: Enzymes are biocatalysts with changeable activity, submitted to regulatory influences. Estimation of enzymatic activity is routinely used in laboratory investigations with diagnostic purposes. Enzymes are also employed as medicines and drugs in practical medicine. Specific aims: To analyze pathways and mechanisms of regulation of enzymatic reactions as a background of metabolism in health and disease. To explain the application of activators and inhibitors of enzymes as medicines and pharmaceuticals for correction of metabolic disorders in pathology. To explain changes in metabolic pathways and accumulation of distinct metabolic intermediates in the inborn (hereditary) and acquired disorders of metabolism – enzymopathias. To analyze changes in activity of indicatory enzymes in blood plasma in pathology of distinct organs and tissues. Theoretical questions: 1. Enzyme inhibition (reversible, irreversible, competitive, non-competitive). 2. Regulation of enzyme activity in the living system: allosteric regulation; feedback regulation; covalent modification of enzymes; activation of latent enzymes by limited proteolysis; cyclic nucleotides in regulation of enzymatic processes. 3. Control of enzymes synthesis (constitutive and adaptive enzymes). 4. Application of enzymes: 24 enzymes as therapeutic agents; enzymes as analytic agents; immobilized enzymes. 5. Diagnostical importance of enzymes (plasma specific and non-plasma specific enzymes. 6. Changes in enzymatic activity of blood plasma and serum as diagnostic indexes (markers) of pathological processes in distinct organs – myocardial infarction, acute pancreatitis, liver disease, pathology of muscle tissue. 7. Isoenzymes, their role in enzymodiagnostics. 8. Inborn (hereditary) and acquired metabolic defects, their clinical and laboratory diagnostics. 9. Application of enzyme inhibitors as medicinal and drugs – acetylsalicylic acid, allopurinol, sulfonamides. Practical part Experiment 1. Study of the influence of activators and inhibitors on activity of salivary amylase. Principle. Compounds, which enhance the activity of enzymes – called activators – are a number of metal ions, e.g. Na+, Mg2+, Mn2+, Co2+, as well as organic substances, especially metabolic intermediates. Amylase is activated by sodium chloride (NaCl), inhibited – by copper sulphate (CuSO4). As indicator of the influence of mentioned compounds on the activity of amylase is a degree of starch cleavage under the action of amylase in the presence of NaCl or CuSO4. Method. I. Dilution of saliva. Dilute saliva two fold. II. Preparation of experimental mixtures and incubation. Take 3 clean dry tubes and do the following procedures: 1. Add 1 ml of distilled water into the first tube. 2. Add 0,8 ml of distilled water and 0,2 ml of 1 % NaCl into the second tube. 3. Add 0,8 ml of distilled water and 0,2 ml of 1% solution of CuSO4 into the third tube. 4. Add 1 ml of diluted saliva into each tube 5. Add 2 ml of 1% starch solution into each tube. Mix them well and place tubes to thermostat at 37C during 15 min. III. Color reaction. Add 0,1% solution of iodine in 0,2% sodium iodide). Changes in color development are observed and registered in a table (see below). № of tube Tube content Water (ml) NaCl, 1 %-solution (ml) CuSO4, 5 %-solution (ml) Saliva 2 fold diluted (ml) Starch, 1 %-solution (ml) 1 2 3 1 1 2 0,8 0,2 1 2 0,8 0,2 1 2 25 Final color after addition of iodine Explain the results and draw a conclusion. Clinical and diagnostic significance. Inhibitors of enzymes are widely used in medicine as drugs and medicinals, e.g. acetylosalicylic acid (aspirin) is an inhibitor of cyclooxygenase (prostaglandin syntase) and employed as anti-inflammatory drug. Trasylol (inhibitor of trypsin) and contrical – inhibitor of different proteinases – (one of them are kallikreins) are used in treatment of pancreatitis, allopurinol –inhibitor of xanthine oxidase – is used for treatement of gout, etc. Experiment 2. The influence of phosphacol and calcium ions on activity of cholinesterase. Principle. Organic phosphate compounds are irreversible inhibitors of cholinesterase and acetyl-cholinesterase due to irreversible binding with an active site of enzyme and suppress its activity. Preparations of phosphorganics are strong poisons for insects (pesticides) as well as for higher animals. Mechanism of inhibitory action consists in covalent binding with OH group of serine in active center of enzyme. In conduction of nerve excitation an increase of calcium ions in nerve ending occurs and this is a signal for release of acetylcholine into the synaptic cleft and subsequent interaction with acetylcholine receptors of postsynaptic membrane and hydrolytic cleavage by cholinesterase. Besides, calcium ions are strong acticvators of cholinesterase. Method of quantitative determination of cholinesterase is based on the titration with alkali the acetic acid, which is liberated after acetylcholine hydrolysis. The quantity of alkaline solution, expended for titration, is a measure of enzyme activity. Method. Take three clean dry tubes and add reagents according to the table: Reagents in tubes N of tube 1 2 3 Blood serum (ml) 0,5 0,5 0,5 0,5 % solution of CaCl2,(drops) --5 --0,5 % solution of phosphacol ----5 (drops) Incubation at room temperature 5 min 2 % solution of acetylcholine (ml) 1,5 1,5 1,5 Incubation for 10 min Phenolphthaleine (drops) 2 2 2 Quantity of 0,1 М NaOH expended for titration, ml Explain the results and draw a conclusion. Clinical and diagnostic significance. Cholinesterase catalyses hydrolysis of acetylcholine, a known neuromediator with formation of choline and acetic acid. In human blood there are two forms of cholinesterase. In blood plasma is present nonspecific cholinesterase (EC 3.1.1.8), which cleaves not only acetylcholine, but also other esters of choline. In red blood cells there is a specific, or true, cholinesterase, which cleaves acetylcholine only (EC 3.1.1.7). 26 In normal conditions the activity of cholinesterase is about 44,4-94,4 ucat/l (colorimetric method according to hydrolysis of acetylcholine chloride). Physiologically active substances, which are inhibitors of cholinesterase, have an important pharmacological and toxicological significance, as they cause a significant increase in concentration of neuromediator in some parts of CNS, as well as in body in general. Reversible inhibitors of acetylcholine esterase are employed in medicine for enhancement of cholinergic impulsation, which is alterede in some neurological diseases, e.g. atonia of intestines or bladder. In these cases are used preparations proserine, physostigmine, galantamine. Irreversible inhibitors of acetylcholinesterase are potent neurotoxins, which causes a marked excitation of nerve system manifested as convultions, disorders of cardiovascular, digestive and other systems of the organism. The most wide spreded irreversible inhibitors are organic phosphates. They are employed for killing of harmfull insects (dichlophos, chlorophos, metaphos, etc.). Neuroparalytic toxins which are used as warfare, are zarine, soman, tabun and others. High sensitivity of cholinesterase to this phosphorganic compounds permits to use this enzyme as a marker for detection of traces of phosphorganic compounds in human body. Examples of Krock-1 tests 1. Marked increase of activity of МВ-forms of CPK (creatinephosphokinase) and LDH-1 were revealed on the examination of the patient's blood. What is the most likely pathology? A. Miocardial infarction D. Pancreatitis B. Hepatitis E. Cholecystitis C. Rheumatism 2. 12 hours after an accute attack of retrosternal pain a patient presented an increase of aspartate aminotransferase activity in blood serum. What pathology is this deviation typical for? A. Myocardium infarction D. Diabetes mellitus B. Viral hepatitis E. Diabetes insipidus C. Collagenosis 3. Desulfiram is widely used in medical practice to prevent alcocholism. It inhibits aldehyde dehydrogenase. Increased level of what metabolite causes aversion to alcochol? A. Acetaldehyde D. Propionic aldehyde B. Ethanol E. Methanol C. Malonyl aldehyde 4. In course of tuberculosis treatement a patient was administered isoniazide – a structural analogue of nicotinamide and pyridoxine. What type of inhibition by mechanism of action exhibits isoniazide? A. Competitive D. Allosteric B. Irreversible E. Noncompetitive C. Uncompetitive 27 Individual independent students work 1. Regulators of enzymatic activity and their employment in clinical practice. 2. Enzymodiagnostics. The use of enzymes in enzymodiagnostics. References: 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 5. The role of cofactors, vitamins and their coenzyme forms in enzyme catalysis. Objectives: To learn the structure, principles of classification and funtion of coenzymatic vitamins. To learn the methods of qualitative and quantitative determination of vitamins. Actuality of the theme: Water soluble vitamin take part in metabolism as coenzymes and activators for many enzymatic reactions. Deficiency in vitamin supply of the body or disorders of their metabolism which is caused by alteration of their absorption or transformation into coenzyme forms, substantially decrease the intensity of energetic and plastic metabolism. This is accompanied with functional disorders of brain, heart, liver and other organs, suppression of immune response to infection, loss of ability to accommodate effectively to unfavorable environmental conditions. Specific aims: To interprete the role of vitamins and their biologically active derivatives in mechanism of catalysis by enzymes of different classes. To explain the application of antivitamins as inhibitors of enzymes in contagious diseases and in disorders of homeostasis. To explain the role of metals in mechanisms of enzymatic catalysis. To classify distinct groups of coenzymes according to their chemical nature and type of the reaction, which they catalyze. Theoretical questions: 1. Cofactors, coenzymes and prosthetic groups of enzymes. 2. Role of metal ions in function of enzymes 3. Classification of coenzymes due to their chemical nature and type of catalytic reaction. 28 4. Coenzymes as transporters of hydrogen atoms and electrons (examples of distinct reactions): NАD+, NАDP+ coenzymes – derivatives of vitamin РР; FАD, FМN coenzymes – derivatives of vitamin В2 – riboflavin; Role of vitamin C in oxidative-reductive reactions metalloporphyrins 5. Coenzymes as transporters of chemical groups (examples): pyridoxal phosphate; НS-CоА – coenzyme of acylation; lipoic acid; THF – derivatives of folic acid 6. Coenzymes of isomerisation, synthesis and cleavage of C-C bonds (examples) : thiamine pyrophosphate – coenzyme form of vitamin В1; biocytin – coenzyme form of vitamin Н – biotin; methylcobalamin and deoxyadenosylcobalamin – coenzyme forms of vitamin В12 Practical part In complex enzymes a protein component (apoenzyme) can be found out with the biuretic reaction. An unprotein component, containing a derivative of any vitamin, can be opened with the appropriate qualitative reactions for each vitamin. Experiment 1. A method for the detecton of nicotinic acid. Principle. Heating of a mixture of nicotinic acid and copper acetate gives a blue sediment of copper nicotinate. Method. Dissolve 10 mg of nicotinic acid in 10-20 drops of 10% acetic acid. Heat the tube to boiling and then add an equal volume of 5% solution of copper acetate. The liquid turns blue and after standing blue sediment appears. Explain the results and draw a conclusion. Experiment 2. Probe for pyridoxine with FeCl3.. Principle. In the presence of pyridoxine FeCl3 solution turns red. Method. Add to 5 droplets of pyridoxine solution one drop of 5% solution of FeCl 3. The liquid in tube becomes red due to formation of complex, a compound of iron of the phenolate type. Explain the results and draw a conclusion. Experiment 3. Reduction of K3Fe(CN)6 by ascorbic acid. Principle. Ascorbic acid reduces K3Fe(CN)6 to K4Fe(CN)6. The latter reacts with FeCl3 to produce a bright blue pigment – Berliner blue. Method. 1. Add into two tubes one drop of 5% solution of K3Fe(CN)6 and one drop 1% solution of FeCl3. 2. Add to the first tube 5-10 drops of an extract from canine rose fruits. Color changes to blue (sometimes a blue sediment of Berliner blue pigment appears). 3. Add into the second 5-10 drops of distilled water. No changes occur in the second tube. Explain the results and draw a conclusion. 29 Examples of Krock-1 tests 1. In case of enterobiasis acrihine – the structural analogue of vitamin B2 - is administered. The synthesis disorder of which enzymes does this medicine cause in microorganisms? A.FAD-dependent dehydrogenases D. NAD-dependet dehydrogenases B. Cytochromeoxidases E. Aminotransferases C. Peptidases 2. Hydroxylation of endogenous substrates and xenobiotics requires a donor of protons. Which of the following vitamins can play this role? A. Vitamin C D. Vitamin E B. Vitamin P E. Vitamin A C. Vitamin B6 3. A newborn child has convulsions that have been observed after prescription of vitamin B6. This most probable cause of this effect is that vitamin B6 is a component of the following enzyme: A. Glutamate decarboxylase D. Aminolevulinate synthase B. Pyruvate dehydrostase E. Glycogen phosphorylase C. Netoglubarate dehydromine 1. 2. 3. 4. 5. 6. 7. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. Topic № 6. Metabolic pathways and bioenergetics. Tricarboxylic acid cycle and its regulation. Objectives: To learn the sequence of reactions in tricarboxylic acids (TCA) cycle and biological significance of TCA cycle as the final stage of catabolic pathway in the cell. To make an aquaintance with methods of TCA cycle investigation in mitochondria and to examine the effect of malonic acid upon this process. Actuality of the theme: The peculiarities of TCA cycle functioning have an important significance in evaluation of its role for providement of the cell with energy as well as for understanding of its amphibolic significance. The analysis of 30 TCA cycle function is necessary for estimation of its role in turnover of matter and energy in the cell. Specific aims: To interprete biochemical principles of metabolic pathways: catabolic, anabolic, amphibolic pathways. To explain biochemical mechanisms of regulation of catabolic and anabolic reactions. To interprete biochemical principles of TCA cycle functioning and its anaplerotic reactions and their amphibolic sense. To explain biochemical regulatory mechanisms in TCA cycle and its principal position in turnover of matter and energy. 1. 2. 3. 4. 5. 6. 7. Theoretical questions: Conception of turnover of material and energy (metabolism). Characterization of catabolic, anabolic and amphibolic reactions and their significance. Exergonic and endergonic biochemical reactions, role of ATP and other macroergic phosphate containing compounds in their coupling. Intracellular location of metabolic pathways, compartmentalization of metabolic reactions in the cell. Methods of investigation of metabolism. Catabolic transformation of biomolecules: proteins, carbohydrates, lipids, its characterization. The most important metabolites of amphibolic pathways in turnover of proteins, carbohydrates, lipids, their significance for integration of metabolism in the cell. Tricarboxylic acid (TCA) cycle: Cellular location of TCA cycle enzymes; Sequence of TCA cycle reactions; Characterization of enzymes and coenzymes participating TCA cycle; Reactions of substrate phosphorylation in TCA cycle; The effect of allosteric modulators upon TCA cycle reactions; Energetic effect of TCA cycle. Anaplerotic and amphibolic reactions of TCA cycle. Practical part Experiment 1. Isolation of mitochondria from liver by method of differential centrifugation (a demonstration). Principle. Liver cells contain a great amount of specific subcellular organelles – mitochondria, in which reactions of tricarboxylic acid (TCA) cycle take place as well as processes of tissue respiration and oxidative phosphorylation. Enzymes of TCA cycle are localized in mitochondrial matrix, while components of respiratory chain – on inner mitochondrial membrane. Mitochondria are isolated from the tissue after its breakdown (homogenization) in special devices, called homogenizer. There exists several types of homogenizers, the most frequently is used glass homogenizer with a teflon pestle. Tissue is homogenized in 0.25 M sucrose solution. Thereafter homogenate is submitted to 31 centrifugation at different speeds (revolutions per minute - rpm). At low speeds are sedimented nuclei and cell debris, which are discarded, mitochondria are sedimented at higher speeds (about 7-10 thousands rpm). Method. Liver of experimental animal (albino rat) is washed from the blood, minced with a scissors and homogenized in 10 fold volume of cold solution N1 (0,25 M sucrose in 0,01 M solution of EDTA, pH 7,6). All subsequent procedures are conducted at 1-20 C. Homogenate is centrifuged at 700 rpm during 6 min in order to eliminate nuclei and large cell debris. Supernatant is collected, transferred to another tube and submitted to centrifugation 10 min at 7000 rpm. The sediment of mitochondria is washed with solution N1 by suspending it in original volume of the solution and recentrifugation 10 min at 7000 prm. The obtained sediment is suspended in a small volume of solution N2 (0,25 M sucrose in 0,02 M tris buffer, pH 7,5), protein concentration is determined and obtained suspension is used for investigation of TCA cycle reactions, tissue respiration, oxidative phosphorylation etc. Experiment 2. Investigation of TCA cycle functioning in mitochondria and the effect of malonate upon this process. Principle. The transformations of acetyl-CoA in presence of mitochondrial enzymes is accompanied with production of CO2 . As a source of acetyl-CoA, which is further incorporated into CTA cycle, is used pyruvate. The last under the action of multimeric pyruvate dehydrogenase complex is submitted to oxidative decarboxylation and acetyl-CoA and CO2 are produced. If TCA cycle is inhibited with malonate bubbles of gaze does not occur. Malonate is a classic competitive inhibitor of succinate dehydrogenase – enzyme of TCA cycle. It binds with active center of this enzyme and hinder binding of true substrate – succinic acid (or succinate). For binding of released CO2 into incubation medium is added Ca(OH)2. At the end of incubation a bound CO2 is detected due to a production of gaze bubbles after addition of sulphuric acid solution into incubation medium. Method. Fill three tubes – a control one, experimental 1 and 2 with reagents as indicated in the table: Content of tubes Control Tube 1 2,0 0,5 Tubes Exp. 1 Tube 2 2,0 0,5 Phosphate buffer, pH 7,4, ml Sodium pyruvate solution, ml Malonic acid, ml Saline, ml 0,5 0,5 Ca(OH)2 solution, ml 0,5 0,5 Suspension of mitochondria 0,5 Boiled suspension of mitochondria 0,5 Incubation in a thermostate for 15 min at 37oC 0,1 M solution of sulphuric acid 1,0 1,0 Result: production of CO2 bubbles Exp. 2 Tube 3 2,0 0,5 0,5 0,5 0,5 1,0 32 Place tubes in a thermostat for 15 min at 37 oC. Thereafter add 1,0 ml of 0,1 M solution of sulphuric acid into each tube and observe the appearance of CO2 bubbles. Explain the results and draw a conclusion. Experiment 3. Investigation of TCA cycle activity in mitochondria according to the rate of reductive equivalents production and the influence of malonate upon this process. Principle. During the oxidation of acetyl-S-CoA in TCA cycle NAD+ and FAD are reduced by hydrogen taken from the substrates. Reduced NAD and FAD in turn reduce methylene blue and make it colorless. Time of decoloration of reaction medium, containing methylene blue, corresponds to the intensity of reactions in TCA cycle of mitochondria. Method. Fill three tubes – a control one, experimental 1 and 2 with reagents as indicated in the table: Reagents added Tubes Control Exp.N1 Exp.N2 Tube 1 Tube 1 Tube 1 Phosphate buffer, pH 7,4, ml 2,0 2,0 2,0 Solution of sodium pyruvate, ml 0,5 0,5 0,5 Solution of malonate, ml 0,5 Saline 0,5 0,5 Suspension of mitochondria, ml 0,5 0,5 Suspension of boiled mitochondria, ml 0,5 Solution of methylene blue, ml 0,5 0,5 0,5 o Incubation at 37 C in a thermostate Results: time of medium decoloration . Explain the results and draw a conclusion. Clinical and diagnostic significance. Many substances, including medicinals and drugs, may influence the bioenergetics of the cell by changing the effectiveness of oxidative phosphorylation and ATP production. They can be devided into activators and inhibitors of energetic metabolism. Activators are represented by acid participants of TCA cycle (citric, succinic, malic acids) as well as by other compounds (glucose, amino acids, etc.). They are used in medical practice. Citric acid as sodium salt is additionally used as anticoagulant, as well as a component of some other drugs. Examples of Krock-1 tests 1. A patient was admitted into hospital with a diagnosis diabetes mellitus type I. In metabolic changes the decrease of oxaloacetate synthesis rate is detected. What metabolic passway is damaged as a result? A. Tricarboxylic acid cycle D. Glycogen mobilization B. Glycolysis E. Urea synthesis C. Cholesterol biosynthesis 33 2. Substrate phosphorylation is a process of phosphate residue transfer from macroergic donor substance to ADP or some other nucleoside diphosphate. What enzyme of tricarboxylic acid cycle participates in reaction of substrate phosphorylation? A. Succinyl thiokinase D. Fumarase B. Citrate synthase E. Alpha-ketoglutarate dehydrogenase C. Succinate dehydrogenase complex 3. In a patient are manifested symptoms of intoxication with arsenic compounds. What metabolic process is damaged taking into account that arsen containing substances inactivate lipoic acid? A. Oxidative decarboxylation of αD. Coupling of oxidation and ketoglutarate phopsphorylation B. Fatty acids biosynthesis E. Microsomal oxidation C. Neutralization of superoxide anions 4. Mitochondria are subcellular organelles and are present in a cytoplasm of every cell exept mature red blood cells, bacteria, blue-green algae. What method is used principally for their isolation? A. Differential centrifugation D. Spectrophotometry B. Chromatography E. Gel-filtration C. Electrophoresis Individual independent students work 1. Anaplerotic and amphibolic role of tricarboxylic acid cycle. 2. Role of the most important metabolites (pyruvate, α-ketoglutarate, acetyl-CoA, succinyl-CoA) in the integration of metabolism. 1. 2. 3. 4. 5. 6. 7. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. 34 Topic № 7. Biological oxidation and oxidative phopshorylation. Mechanisms of ATP synthesis. Objective: to learn general principles of enzymatic respiratory chain organization in mitochondria, distinct oxidoreductases and their functional significance in tissue respiration. To master the methods of investigation of the next oxido-reductases: phenol oxidase, aldehyde dehydrogenase and peroxidase. Actuality of the theme: oxidoreductases catalyze reactions connected with transfer of electrons and protons and are in the background of macroergic compounds production. Investigation of their activity is necessary for detailed understanding of the mechanisms of tissue respiration and its changes in different functional status of the body. Specific aims: To explain processes of biological oxidation of different substrates in the cell and reservation of released energy in a form of macroergic bonds of ATP. To analyze reactions of biological oxidation and their role in providement of fundamental biochemical processes in tissues. Malate-aspartate and glycerophosphate shuttle systems of transmembrane transfer of reduced NADH2 into mitochondria and their significance To explain the structural organization of electron transport chain and its macromolecular complexes. To interpret role of biological oxidation, tissue respiration and oxidative phosphorylation in generation of ATP in aerobic conditions. Theoretical questions: 1. Biological oxidation of substrates in cells. 2. Reactions of biological oxidation and their functional significance. 3. Pyridine dependent dehydrogenases, structure of NAD and NADP, their role in reactions of oxidation and reduction. 4. Flavine dependent dehydrogenases. Structure of FAD and FMN, their role in reactions of oxidation and reduction. 5. Cytochromes and their role in tissue respiration. Structure of their prosthetic group. 6. Molecular organization of electron transport chain of mitochondria. 7. Supramolecular complexes of respiratory chain in inner membrane of mitochondria. 8. Oxidative phosphorylation. Sites of oxidative phosphorylation. P/O ratio. 9. The scheme of chemiosmotic mechanism of coupling of electron transport in respiratory chain with ATP synthesis. Molecular structure and principles of functioning of ATP-synthetase. 10.Inhibitors of electron transport in a respiratory chain of mitochondria. 11.Uncouplers of electron transport and oxidative phosphorylation in a respiratory chain of mitochondria. Practical part Oxidoreductases 35 Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule, the reductant, also called the electron donor, to another the oxidant, also called the electron acceptor. Phenol oxidase, aldehyde dehydrogenase, peroxidase and many others belong to oxidoreductases. Polyphenol oxidase is an enzyme that catalyses the hydroxylation of monophenols to o-diphenols. They can also further catalyse the oxidation of odiphenols to produce o-quinones. It is the rapid polymerisation of o-quinones to produce black, brown or red pigments (polyphenols) that is the cause of fruit browning. O OH + Ѕ О2 Phenol oxidase OH Hydroquinon (o-diphenol) + H2O O Quinon Aldehyde dehydrogenases are a group of enzymes that catalyse the oxidation (dehydrogenation) of aldehydes. To date, nineteen ALDH genes have been identified within the human genome. These genes participate in a wide variety of biological processes including the detoxification of exogenously and endogenously generated aldehydes. Peroxidases are a large family of enzymes that typically catalyze a reaction of the form: ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides Experiment 1. Study of phenol oxidase activity. Principle. Method is based on the phenomenon of pyrocatechol oxidation by oxygen in the presence of phenol oxidase. Method. I. The source of enzyme is fresh potato juice. Mince in a mortar 1 - 2 g of potato add 25 ml of distilled water, mix it and press the juice into tube. (Iron instruments must not be used because ions of iron induce the darkening of juice). II. Add 0.5 ml of potato juice into 4 tubes. Add 0.5 ml of pyrocatechol solution to the first tube; 1-2 drops of Na2S (an inhibitor of phenol oxidase) and 0,5 ml of pyrocatechol – to the second; boiled juice and 0.5 ml of pyrocatechol - to the third and 0.5 ml of water to fourth tube. Place tubes into the thermostat at 37o C for 30 min. Periodically shake the tube’s content for better aeration. 36 2 1 3 - 0.5 ml of potato juice 4 - 0.5 ml of boiled potato juice - 0.5 ml of pyrocatechol solution - 1-2 drops of Na2S - 0.5 ml of water Incubation for 30 min (T=370C) Result: 1. brown 2. colorless 3. colorless 4. colorless III. Result. In the first tube brown colour appears due to oxidation of pyrocatechol. The colours of other tubes don’t change: due to the presence of inhibitor (2 tube), denaturation of enzyme (3 tube ) and absence of substrate (4 tube). Explain the results and draw a conclusion. Experiment 2. A study of aldehydedehydrogenase activity. Principle. Method is based on the decolouration of methylene blue during its reduction by aldehyde in presence of an enzyme aldehyde dehydrogenase (ADH). О ОН ADH + Н2О Н С НС ОН Н ОН Formaldehyde НС Hydrated form of formaldehyde ОН ADH ОН + Мb ОН Methylene blue Н С О ОН Formiatic acid Method. Add the following solutions into three ennumerated tubes: (1) – 1 ml of fresh non boiled milk, 1-2 drops of neutralized formaldehyde, 2 drops of methylene blue solution; (2) – 1 ml of fresh milk and 2 drops of methylene blue; (3) – 1 ml of boiled milk, 1-2 drops of formaldehyde and 2 drops of methylene blue. Place tubes to the termostate for 30 min to at 37 °C. 1 2 3 - 1 ml of fresh non boiled milk - 1 ml of boiled milk - 1-2 drops of neutralized formaldehyde - 2 drops of methylene blue solution Incubation for 30 min (T=370C) Result: 1. colorless 2. blue 3. blue 37 Result. Content of the first tube is decolorized due to enzyme activity. Color in the rest of the tubes does not change, as in the second tube – substrate is absent, in the third – enzyme is heat denaturated. Explain the results and draw a conclusion. Clinical and diagnostical importance. Aldehyde dehydrogenase plays a crucial role in maintaining low blood levels of acetaldehyde during alcohol oxidation. In this pathway, the intermediate structures can be toxic, and health problems arise when those intermediates cannot be cleared. When high levels of acetaldehyde occur in the blood, facial flushing, light headedness, palpitations, nausea, and general “hangover” symptoms occur. Experiment 3. A study of peroxidase activity. Principle. Method is based on the oxidation of benzidine by hydrogen peroxide in the presence of peroxidase. Blue product is formed during the oxidation of benzidine. Method. Add the following reagents into 3 tubes: (1) – 3-4 drops of benzidine solution in acetic acid, 3-4 drops of 3 % solution of H2O2, 3-4 drops of horseradish extract. (2) – 3-4 drops of benzidine solution, 3 - 4 drops of H2O2 solution, 3 - 4 drops of a boiled horseradish extract. (3) – 3-4 drops of benzidine solution, peroxidase inhibitor, 3 - 4 drops of horse radish peroxidase and 3-4 drops of H2O2 solution. Place Result. In the first tube a blue color is developed due to oxidation of benzidine by H2O2 under catalysis of peroxidase. In the 2 and 3 tubes color is not developed. 1 2 3 - 3-4 drops of horseradish extract - 3 - 4 drops of a boiled horseradish extract - 3-4 drops of 3 % solution of H2O2 - 3-4 drops of benzidine solution in acetic acid Incubation for 30 min (T=370C) 1. blue - 3-4 drops of peroxidase inhibitor Result: 2. colorless 3. colorless Explain the results and draw a conclusion. Clinical and diagnostical importance. Horseradish peroxidase can be conjugated to a labeled molecule. It produces a coloured, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. Horseradish peroxidase is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to horseradish peroxidase may be used to detect a small amount of a specific protein in a western blot. Here, the antibody provides the specificity to locate the protein of interest, and the horseradish peroxidase enzyme, in the presence of a substrate, produces a detectable signal. Horseradish peroxidase is also commonly used in techniques such as ELISA and 38 Immunohistochemistry due to its monomeric nature and the ease with which it produces coloured products. Peroxidase, a heme-containing oxidoreductase, is a commercially important enzyme which catalyses the reductive cleavage of hydrogen peroxide by an electron donor. Examples of Krock-1 tests 1. Enzymes of respiratory chain perform oxidation of substrates and transfer of reductive equivalents to oxygen with production of water molecules. Where they are located? A. On inner mitochondrial membrane. D. On outer mitochondrial membrane B. On cytoplasmic membrane E. In nucleus C. In cytoplasm 2. Cytochromes are components of respiratory chain in mitochondrias, which transfer electrons from ubiquinon to molecular oxygen. What part of cytochrome molecule take part in oxydative-reductive reactions? A. Iron atom D. Proteinous part B. Vinyl residue E. Methen bridge C. Pyrrole cycle 3. The next process occurs in suspension of mitochondria with ruptured inner membrane and provided with malate and oxygen: A. Transport of electrons along D. Increase of pH in mitochondrial enzymes of respiratory chain matrix B. Phosphorylation of ADP E. Oxydative phosphorylation will C. Decrease of pH in the external take place medium 4. Redox potential (EO volts) of ubiquinone, OX/RED system is: A. +0.04 D. +0.29 B. +0.08 E. + 0.35 C. +0.10 5. The correct sequence of cytochrome carriers in respiratory chain is: A. Cyt b→cyt c1→cyt c→cyt aa3 D. Cyt b→cyt aa3→cyt c→ cyt c B. Cyt aa3→ cyt b→cyt c→cyt c1 E. Cyt aa3→ cyt c1→cyt c→ cyt b C. Cyt b→cyt c→cyt c1→cyt aa3 Individual independent students work 1. Development of knowledge on biological oxidation, works of A. Bakh, V. Palladin, O.Warburg in this field. 2. Modern data about the organization and functioning of respiratory chain as a single polyenzyme complex. References: 39 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 8. Glycolisis – anaerobic oxidation of glucose, alternative pathways of carbohydrate metabolism. The aim of the lesson: To learn fundamental principles of intracellular oxidation of glucose in anaerobic and aerobic conditions and pathways of its regulation. To interpret the role of coenzymes and enzymes in glycolytic pathway. To explain reactions of oxidative phosphorylation and ATP synthesis. Actuality of the theme: The occurrence of uninterrupted glycolysis is very essential in skeletal muscle during strenuous exercise where oxygen supply is very limited. Glycolysis in the erythrocytes leads to lactate production, since mitochondria - the centres for aerobic oxidation - are absent. Brain, retina, skin, renal medulla and gastrointestinal tract derive most of their energy from glycolysis. Concentration of lactate in blood increases after hard muscle exercises and in some diseases. Under anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6 ATP are synthesized-depending on the shuttle pathway that operates. Specific aims: To interpret biochemical pathways of intracellular oxidation of glucose in anaerobic conditions To analyze peculiarities of glycolytic reactions, which occur with involvement of ATP To analyze peculiarities of substrate phosphorylation and production of ATP in this way To interpret role of coenzymes and enzymes in glycolytic reactions To analyze regulatory mechanisms of glucose oxidation in anaerobic conditions Theoretical questions: 1. Glucose as an important metabolite in carbohydrate metabolism: general scheme of sources and turnover of glucose in the organism 2. Anaerobic oxidation of glucose: the sequence of reactions in glycolysis; enzymatic reactions of anaerobic and aerobic glycolysis; 40 characterization of glycolytic reactions, which occur with utilization of energy; characterization of enzymatic reactions of substrate phosphorylation in glycolysis; mechanism of glycolytic oxido-reduction and reactions, which provide this process. 3. The contribution of works of Embden, Meyerhof and Parnas in detection of sequence of enzymatic glycolysis reactions. 4. The role of lactate dehydrogenase (LDH) in glycolysis, mechanism of reaction and its peculiarities. Isoenzymes of LDH and their clinical diagnostic significance. 6. Energetic effect of anaerobic oxidation of glucose. 7. Alcohol fermentation, common and different reactions in glycolysis and fermentation. Practical part Experiment 1. The utilization of inorganic phosphate by yeast cells in fermentation process. A. The preparation of fermentation mixture. 1. Put 2 g of dry glucose into a test tube; afterwards add 5 ml of phosphate buffer (pH 7.2); 2. Incubate the solution at 37° C. 3. Add 4 ml of the yeast suspension and mix the solution. B. The investigation of phosphate binding rate in probes. From the fermentation mixture take 1 ml probes and allow 0, 30, 60 and 90 min for incubation. a) Precipitation of yeast cells. Add to 1 ml of mixture 3 ml of 0.1 n acetic acid. In a weak acidic medium yeast cells form a sediment, which is eliminated by filtration. b) Dilution of phosphates. 1. Add to 1 ml of the filtrate 19 ml of water. 2. From this solution take 1 ml and mix with 4 ml of water; repeat (a) and (b) 3 times after every 30 min. c) Determination of phosphate concentration by colorimetry. 1. To the 5ml of the above obtained solution add 4.5 ml of molybdate reagent and subsequently 0.5 ml of ascorbic acid (0.5%). 2. Mix the content of tubes and after 2 min (exactly) 3. Measure the optical density (A) in a colorimeter at red light. The concentration of phosphate is determined according to calibration curve, which is obtained as indicated below. Explain the results and draw a conclusion. The calibration curve for phosphate: In 5 tubes are mixed the following reagents (see the table): № Reagents 1 2 3 4 1. 2. 3. 4. Standard solution of phosphate Distilled water Molybdate reagent Ascorbic acid solution 0 0.5 1.0 2.0 5 4.5 0. 4.5 4.5 0.5 4.0 4.5 0.5 3.0 4.5 0.5 41 5. Phosphate concentration 0 50 100,0 200,0 (mg%) 6. Optical density 0.01 0.06 0.11 0.21 According to optical density values a calibration curve is constructed, where the concentration of phosphate is on the abscissa and corresponding optical density values is on ordinate axis. Using a calibration curve the concentration of phosphate in the examined probes is determined. From data obtained a graph (the dependence of inorganic phosphate utilization in the course of fermentation) is constructed. Calibration curve (example) Graph of concentration of inorganic phosphate from the incubation time (example) Experiment 2. Quantitative determination of lactic acid in blood serum after Buchner method. The occurrence of uninterrupted glycolysis is very essential in skeletal muscle during strenuous exercise where oxygen supply is very limited. Glycolysis in the erythrocytes leads to lactate production, since mitochondria-the centres for aerobic oxidation-are absent. Brain, retina, skin, renal medulla and gastrointestinatl ract derive most of their energy from glycolysis. Principle. By heating with concentrated sulfuric acid lactic acid is converted to acetic aldehyde, which, when followed by a reaction with hydroquinone, forms a redbrown substance. Preparation of a filtrate. 1. Pour into 2 clean tubes 6 ml of water. 2. Add to the first tube 1 ml of standard solution of lactate (test tube) and to the 42 second one add l ml of blood serum (control tube). 3. For protein precipitation add to each tube l ml of metaphosphoric acid and after several minutes filter the solutions. 4. Add to each filtrate l ml of copper sulfate (10%) and 0.5g of calcium hydroxide. Mix the probes with a glass rod and filter after 5 min. Method. 1. Add 1 ml of each filtrate into 2 clean, dry tubes, 2. Add 0.1 ml of copper sulfate, mix the solutions 3. Add 4 ml of concentrated sulfuric acid to each tube and place them into a boiling water bath for 1.5 min. 4. After cooling add 0.1 ml of alcohol solution of hydroquinone and incubate the tubes in boiling water bath for 15 min. 5. Cool the tubes and measure optical density at blue light filter. Calculations. Lactic acid concentration is calculated according to a formula: Where, C is concentration and A is optical density. Explain the results and draw a conclusion. Clinical diagnostic significance. Venous blood of healthy person contains 0.5 2.2 mmol/1 of lactic acid. An increase of lactic acid content is associated with strenuous muscular effort during short time term intervals when there is a deficiency of oxygen. In this case the oxidative decarboxylation of pyruvate to acetyl-CoA does not occur and the production of the lactate takes place. Lactate can be utilized during a restoration period with a sufficient supply of oxygen. The increase production of lactic acid is also observed during epilepsy, tetanies, convulsions and hypoxia, and it 43 is associated with cardiac and pulmonary failure, malignant tumors, liver diseases and other pathologies. Examples of Krock-1 tests 1. An untrained person who has not been practicing physical exercises for a long time complains of a muscle pain as a result of intensive manual work. What is the probable reason of the pain syndrome? A. Accumulation of lactate in muscles D. Accumulation of creatinine in B. Decreasing of lipids level in muscles muscles E. Increase of ATP level in muscles C. Increased disintegration of muscle proteins 2. The high speed sprint causes a feeling of pain in skeletal muscles of untrained people that occurs due to lactate accumulation. The activation of what biochemical process is it resulting from? A. Glycolysis D. Lipogenesis B. Gluconeogenesis E. Glycogenesis C. Pentose phosphate pathway 3. A 7-year-old girl manifests obvious signs of anemia. Laboratory tests showed the deficiency of pyruvate kinase activity in erythrocytes. The disorder of what biochemical process is a major factor in the development of anemia? A. Anaerobic glycolysis D. Oxidative phosphrylation B. Deamination of amino acids E. Breaking up of peroxides C. Tissue respiration 4. During consumption of cakes or sweets in mixed saliva a transient increase in lactate level takes place. Activation of what biochemical process causes this effect? A. Anaerobic glycolysis D. Gluconeogenesis B. Tissue respiration E. Microsomal oxidation C. Aerobic glycilysis 5. In yeast cells occurs a process which is similar to glycolysis- an alcohol fermentation. In course of this process through several stages from pyruvate is produced: A. Lactate D. Glyceraldehyde B. Ethanol E. Pyruvate C. Acetaldehyde Individual independent students work 1. Disorders of carbohydrate metabolism and its pharmacological correction. 2. Principles of regulation of glucose metabolism. Characterization of regulatory enzymes in glycolysis. 44 References: 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 9. Glucose oxidation under aerobic conditions and alternative metabolic pathways of monosaccharides metabolism. Objective: To learn the role of pyruvate dehydrogenase multienzyme complex in aerobic metabolism of glucose. To interpret metabolic pathways of fructose and galactose in human body. To learn the sequence of enzymatic reactions in Pentose Phosphate Pathway (PPP). Actuality of the theme: Metabolism of carbohydrates includes all complex process of carbohydrates transformation starting from digestion, absorption, transport and utilization in cells up to formation of end products – CO2 and H2O. In aerobic conditions pyruvate, as a product of glycolysis, releases CO2 and is transformed to acetyl-CoA, which is further oxidized in tricarboxylic acid cycle (Crebs cycle) to CO2 and H2O. The rate of reactions in TCA cycle depends from requirements of the cell in ATP. Regulatory reactions in TCA cycle are synthesis of citrate and oxidative decarboxylation of alpha-oxoglutarate, which are regulated by amount of ADP, succinyl-CoA and NADH2. Specific aims: To interpret mechanisms of monosaccharides transformation to final metabolic products and energetic effect in aerobic conditions To analyze structural and functional peculiarities of pyruvate dehydrogenase complex To explain the sequence of reactions in PPP and significance of this process To analyze metabolic pathways of fructose and galactose transformations in human body. 45 1. 2. 3. 4. 5. 6. Theoretical questions: Stages of aerobic oxidation of glucose. Oxidative decarboxylation of pyruvic acid. structure of multienzyme pyruvate dehydrogenase complex. peculiarities of function of pyruvate dehydrogenase complex. mechanism of oxidative decarboxylation of pyruvate. role of vitamins and coenzymes in transformation of pyruvate to acetyl-CoA. Energetic effect of aerobic oxidation of glucose. Pentose phosphate pathway (PPP) of glucose utilization: scheme of reactions in oxidative and nonoxidative stages of PPP; enzymes and coenzymes of PPP reactions; biological significance of PPP; disorders of PPP in red blood cells; enzymopathias of glucose-6-phosphate dehydrogenase. Enzymatic reactions of fructose turnover in human body. Hereditary enzymopathias of fructose metabolism. Enzymatic reactions of galactose metabolismr in human body. Hereditary enzymopathias of galactose metabolism. Practical part Experiment 1. Quantitative determination of pyruvic acid in urine by colorimetric method. Pyruvate is converted to acetyl CoA by oxidative decarboxylafion. This is an irreversible reaction, catalysed by a multienzyme complex, known as pyruvate dehydrogenase complex(PDH), which is found only in the mitochondria. High activities of PDH are found in cardiac muscle and kidney. The enzyme PDH requires five cofactors (coenzymes), namely-TPP, lipoamide, FAD, coenzyme A and NAD+ (lipoamide contains lipoic acid linked to amino group of lysine). Principle. Pyruvic acid and 2.4-dinitrophenyl hydrazine form in alkaline medium a 2.4-dinitrophenylhydrazone pyruvate (brown-red color compound). The intensity of the color is proportional to the pyruvic acid concentration and is evaluated calorimetrically. 46 Materials and reagents. Sample of urine, standard solution of pyruvic acid (625 mg in 100 ml of water), 0.1% solution of 2.4-dinitrophenylhydrazine (in 2 N hydrochloric acid), 12% solution of sodium hydroxide, distilled water, tubes, pipettes, colorimeter. Method. To one tube is added 0.1 ml of tested urine, to another tube – 0.1 ml of standard pyruvate solution. To each tube is added 0.9 ml of distilled water. Thereafter is added 0.5 ml of 2.4-dinitrophenylhydrazine and tubes are leaved for 20 min in a dark place. Then to each tube is added 1 ml of 12% solution of sodium hydroxide and after 10 min the intensity of color is measured in colorimeter with a blue light filter. Calculation: Pyruvic acid concentration (Cexp) is calculated according to the formula: where: Cstand. – concentration of standard pyruvate Cexper. – concentration of pyruvate in a sample of urine Aexper. – optical density of tested urine Astand. – optical density of standard pyruvate V – daily volume of excreted urine (1500 ml) a – 0.1 ml of urine, taken for analysis. 47 Compare the obtained result with normal value. Draw the conclusion. Examples of Krock-1 tests 1. A newborn child with the signs of cataract, growth and mental retardation, who manifested vomiting and diarrhea, was brought to an emergency clinic. A presumptive diagnosis of galactosemia was made. The deficiency of what enzyme occurs in case of this disease? A. Galactose-1-phosphate uridyl C. UDP-galactose-4-epimerase. transferase. D.Hexokinase. B. Glucokinase. E. Glucose-6-phosphate dehydrogenase 2. A cataract and fatty degeneration of the liver develop in the conditions of high galactose and low glucose level in blood. What disease do these symptoms testify to? A. Galactosemia. D. Steroid diabetes. B. Diabetes mellitus. E. Fructosemia. C. Lactosemia. 3. In a patient are manifested symptoms of intoxication with arsenic compounds. What metabolic process is damaged taking into account that arsenic containing substances inactivate lipoic acid? A. Oxidative decarboxylation of D. Coupling of oxidation and pyruvate phopsphorylation B. Fatty acids biosynthesis E. Microsomal oxidation C. Neutralization of superoxide anions 4. A 2-year-old boy has the increase of liver and spleen sizes detected and eye cataract present. The total sugar level in blood is increased, but glucose tolerance is within the normal range. The inherited disturbance of the metabolism of what substance is the cause of the indicated state? A. Galactose. D. Maltose. B. Fructose. E. Saccharose. C. Glucose. Individual independent students work 1. Pathobiochemistry of disorders in pentosophosphate pathway. 2. Pathobiochemistry of enzymopathias in connection with genetic defects in synthesis of enzymes of fructose and galactose metabolism. References: 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 48 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. Topic № 10. Catabolism and biosynthesis of glycogen. Regulation of glycogen metabolism Biosynthesis of glucose – gluconeogenesis Objectives: To learn reactions of synthesis and breakdown of glycogen and mechanisms of humoral regulation of glycogen metabolism in liver and muscles. To interpret reactions of gluconeogenesis, their peculiarities and principles of their regulation. Actuality of the theme: Glycogen is the storage form of glucose in animals, as is starch in plants. It is stored mostly in liver (6-8 %) and muscle (1-2 %). Due to more muscle mass, the quantity of glycogen in muscle (250 g) is about three times higher than that in the liver (75 g). Glycogen is stored as granules in the cytosol, where most of the enzymes of glycogen synthesis and breakdown are present. Specific aims: To explain characteristic features of glycogen breakdown and biosynthesis. To analyze mechanisms of humoral regulation of glycogen metabolism in liver and muscles. To explain hereditary disorders of glycogen metabolism. To analyze specific features of gluconeogenesis reactions and substrates of this process. To explain and interpret regulatory mechanisms of gluconeogenesis. 1. 2. 3. 4. 5. 6. 7. 8. Theoretical questions: Mechanism and peculiarities of enzymatic reactions of glycogenesis. Glycogenolysis, common and different reactions with glycolysis. Cascade mechanisms of ATP-dependent regulation of glycogen phosphorylase and glycogen synthase activities. Peculiarities of hormonal regulation of glycogen metabolism in liver and muscles. Hereditary disorders in enzymes of glycogen synthesis and breakdown. Glycogenoses, aglycogenoses. Metabolic pathways and substrates of gluconeogenesis, mechanisms of regulation, compartmentalization of enzymes, biological significance of the process. Relations between glycolysis and gluconeogenesis (Cori cycle). Irreversible reactions of glycolysis and their shunt pathways. Glucose-lactate and glucosealanine cycles. Regulation of gluconeogenesis in human organism. 49 Practical part Experiment 1. Iodine Test for Starch. Principle. Starch is a polysaccharide consisting of glucose units joined together by glycosidic bonds. The chains formed during the condensation reaction are either linear or highly branched molecules. Linear - both straight and helical - molecules of starch are referred to as Amylose. Whereas branched molecules of starch are called Amylopectin. The Iodine Test for Starch is used to determine the presence of starch in biological materials. The chains formed during the condensation reaction are either linear or highly branched molecules. Iodine on its own (small non-polar molecule) is insoluble in water.Therefore Potassium triiodide solution - Iodine dissolved in potassium iodide solution - is used as a reagent in the test. To be more specific, potassium iodide dissociates, and then the Iodide ion reacts reversibly with the Iodine to yield the the triiodide ion. A further reaction between a triiodide ion and an iodine molecule yields the pentaiodide ion. (C6H10O5)n I2 starch heating (C6H10O5)n I2 complex of starch with Iodine Since molecular iodine is always present in solution, the bench iodine solution appears brown; the iodide and triiodide pentaiodide ions are colourless. The triiodide and pentaiodide ions formed are linear and slip inside the helix of the amylose (form of starch). Reagents: 1% solution of starch, Lugol solution (iodine dissolved in potassium iodide), tubes. Method. 1. Add 1ml of the starch solution to a clean, dry test tube. 2. Add about 5 drops of Lugol solution to the test tube. 50 3. Note any colour changes. Write a conclusion. Experiment 2. Detection of glycogen in the liver. Principle. A positive test for glycogen is a brown-blue color. A negative test is the brown-yellow color of the test reagent. Glycogen, as well as starch, forms a colored compound with iodine (starch forms blue, glycogen - a red-brown compound). It is thought that starch and glycogen form helical coils. Iodine atoms can then fit into the helices to form a starch-iodine or glycogen-iodine complex. Starch in the form of amylose and amylopectin has less branches than glycogen. This means that the helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine complex. Reagents. Fresh or frozen liver tissue, Lugol solution (iodine dissolved in potassium iodide), 1% solution of acetic acid, porcelain mortar, water bath, paper filters. Generation of filtrate 1. Put 0.5 g of liver tissue into a tube, add 4 ml of distilled water and boil it (2-3 min in order to inactivate enzymes). 2. Transfer liver into the mortar and grind it. 3. Transfer the obtained homogenate into the tube, add 1 ml of distilled water and boil the solution on a water bath (20 min). Add 5-10 droplets of acetic acid solution for protein precipitation. 4. Eliminate precipitated proteins by filtration using paper filters. Method. 1. Take a clean, dry tube. Put 1 ml of filtrate. 2. Add 2-3 droplets of Lugol solution. 3. Note any colour changes. (In presence of glycogen a red-violet color is observed). Compare the color with a color obtained in the previous experiment. Explain the results. Clinical diagnostic significance. Glycogen is a polysaccharide, which serves as a main reserve of carbohydrates in the body. It is stored mainly in liver and muscles. Normal blood level – 16.2 – 38.7 mg/l. The prime function of liver glycogen is to maintain the blood glucose levels, particularly between meals. Liver glycogen stores increase in a well-fed state which are depleted during fasting. Muscle glycogen serves as a fuel reserve for the supply of ATP during muscle contraction. The metabolic defects concerned with the glycogen synthesis and degradation are collectively referred to as glycogen storage diseases. These disorders are due to defects in the enzymes which may be either generalized (affecting all tissues) or tissue-specific. The inherited disorders are characterized by deposition of normal or abnormal type of glycogen in one or more tissues. Increase in blood glycogen concentration is observed in some infection diseases, which are accompanied with leukocytosis, diabetes mellitus, and malignancies. Examples of Krock-1 tests 1. In a weak apathic infant an enlarged liver was detected, which in investigation of biopcia pieces showed an excess of glycogen. Blood glucose concentration is under the normal value. What may be the cause of this disease? 51 A. Lowered activity of glycogen phosphorylase in a liver B. Lowered activity of glycogen synthase C. Lowered activity of glucose 6- phosphate isomerase D. Lowered activity of glucokinase E. Deficiency of gene responsible for synthesis of glucose 1-phosphate uridyl transferase 2. During biochemical investigation of blood in a patient was detected hypoglycemia in fasting condition. Investigation of liver bioptates revealed the failure of glycogen synthesis. What enzyme deficiency may cause such status? A. Glycogen synthase D. Fructose bis-phosphatase B. Phosphorylase E. Pyruvate carboxylase C. Aldolase 3. In an infant with point mutations in genes the absence of glucose 6phosphatase, hypoglycemia and hepatomegalia were revealed. What disease is characterized by these symptoms? A. Gierke disease D. Cori disease B. Adison disease E. Mac Ardle disease C. Parkinson disease 4. In a patient a lowering in ability to physical load was revealed, while in skeletal muscles the glycogen content was increased. The decrease in activity of what enzyme may cause this condition? A. Glycogen phosphorylase D. Glycogen synthase B. Phosphofructokinase E. Glucose 6-phosphatase C. Glucose 6-phosphate dehydrogenase Individual independent students work 1. Principles of regulation of glycogen biosynthesis and breakdown. 2. Hereditary disorders of synthesis and breakdown of glycogen glycoconjugates. 1. 2. 3. 4. 5. 6. 7. and References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. 52 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. Topic № 11. Mechanisms of metabolic and humoral regulation of carbohydrate metabolism. Disorders of carbohydrate metabolism Objectives: To interpret the role of hormones in regulation and maintenance of constant blood glucose level. To learn the peculiarities of changes in metabolism of carbohydrates, lipids and proteins in diabetes mellitus. Actuality of the theme: Diabetes mellitus is a clinical condition characterized by increased blood glucose level (hyperglycemia) due to insufficient or inefficient insulin. In other words, insulin is either not produced in sufficient quantity or inefficient in its action on the target tissues. As a consequence, the blood glucose level is elevated which spills over into urine in diabetes mellitus. Determination of blood glucose level in clinical laboratory investigations is of great importance in diagnostics of diabetes mellitus and many other diseases and disorders. Specific aims: To analyze the principal sources and metabolic pathways of utilization of blood glucose To explain the role of hormones in maintenance of constant glucose level in blood To explain disorders in metabolism of carbohydrates in diabetes mellitus. Theoretical questions: 1. Biochemical processes which provides the constant blood glucose level. Role of different pathways of carbohydrate metabolism in regulation of blood glucose level. 2. Hormonal regulation of carbohydrate metabolism: Insulin, its structure, mechanism of action, role in carbohydrate metabolism. Adrenalin and glucagone, mechanism of their regulatory effects on carbohydrate metabolism. Glucocorticoids, their effect on carbohydrate metabolism. 3. Characterization of hypo- and hyperglycemia, glucosuria. 4. Insulin dependent and noninsulin dependent forms of diabetes mellitus. 5. Characterization of metabolic disorders in diabetes mellitus 6. Biochemical tests for evaluation of conditions of patients with diabetes mellitus. Glucose tolerance test and its alteration in diabetes mellitus. Biochemical criteria of diabetes mellitus. Practical part Experiment 1. Estimation of blood glucose by o-toluidine method. Principle. In this method of glucose determination, a primary aromatic amine, otoluidine, reacts in hot glacial acetic acid with the terminal aldehyde group of glucose to produce a blue-green color. The absorbance of this product is measured photometrically and glucose concentration can be calculated. The absorbance in 600 700 nm region is directly proportional to the glucose concentration. 53 Reagents and materials. Blood sample, 3% solution of trichloroacetic acid (TCA), o-toluidine reagent, glucose standard (4 mM/L, i.e. 720 mg of glucose dissolved in 1 L of water), distilled water, tubes, pipettes, micropipette 0.1 ml, centrifuge, centrifuge tubes, electrocolorimeter, water bath. Preparation of supernatant. Add 0.9 ml of trichloroacetic acid into two centrifuge tubes. Into test tube add 0.1 ml of blood specimen, into control tube – 0.1 ml of standard glucose solution. Centrifuge the tubes at 3000 rpm for 10 min. Separate test and control supernatants for further investigations. Method. 1. Take two clean, dry tubes. Into the first tube add 0.5 ml of control supernatant, into second – 0.5 ml of tested supernatant. 2. Add 4.5 ml of o-toluidine reagent to each tube. 3. Place the tubes on a boiling water bath for 8 min. 4. Cool tubes and measure optical density (A) in a colorimeter at wavelength 630 nm (red filter). Calculation. The concentration of glucose is calculated using the formula: where: Ctest – concentration of glucose in blood, mmoles/L; Cstand – concentration of glucose in standard solution Atest – optical density of test probe Atest – optical density of standard glucose probe. Compare the obtained result with normal value, draw the conclusion. Clinical diagnostic significance. The fasting blood glucose level in normal individuals is 3.3-5.5 mmol/l and it is very efficiently maintained at this level. When the blood glucose concentration falls, the symptoms of hypoglycemia appear. The manifestations include headache, anxiety, confusion, sweating, slurred speech, seizures and coma, and, if not corrected, death. All these symptoms are directly and 54 indirectly related to the deprivation of glucose supply to the central nervous system (particularly the brain) due to a fall in blood glucose level. Elevation of blood glucose concentration is the hallmark of uncontrolled diabetes. Hyperglycemia is primarily due to reduced glucose uptake by tissues and its increased production via gluconeogenesis and glycogenolysis. When the blood glucose level goes beyond the renal threshold, glucose is excreted into urine (glycosuria). Examples of Krock-1 tests 1. A 46-year-old woman complains of dryness in the oral cavity, thirst, frequent urination, general weakness. Biochemical research of the patient's blood showed hyperglycemia and hyperketonemia. Sugar and ketone bodies are revealed in the urine. Diffuse changes in myocardium are marked on the electrocardiogram. Make an assumptive diagnosis of the illness. A. Diabetes insipidus. D. Diabetes mellitus. E. Ischemic cardiomyopathy. B. Alimentary hyperglycemia. C. Acute pancreatitis. 2. A patient was admitted to a hospital in comatous state. The accompanying mates explained that the patient loss his consciousness during the training on the last stage of marathon distance. What coma type can be recognized? A. Hypoglycemic D. Hypothyroid B. Hyperglycemic E. Hepatic C. Hypovolemic 3. A patient addressed to physician with complaints on permanent thirst. In laboratory investigation it was revealed hyperglycemia, polyuria and increased content of 17-ketosteroids in urine. What disease is the most probable? A. Steroid diabetes D. Glycogenosis of the 1 type B. Insulin dependent diabetes mellitus E. Myxoedema C. Addison disease 55 4. A 38-year-old man is receiving treatment for schizophrenia in hospital. Fhe initial levels of glucose, ketone bodies and urea in the blood are within the normal range. Shock therapy put into practice by regular insulin injections resulted in the development of the comatose state which improved the clinical status of the patient. What is the most probable cause of insulin coma? A. Hypoglycemia. D. Ketonemia. B. Dehydratation of tissues. E. Hyperglycemia. C. Metabolic acidosis. 1. 2. 3. 4. 5. 6. 7. 8. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd edition” – 2008. – 488 p. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M. – 2012. – 308 p. Topic № 12. Catabolism and biosynthesis of triacylglycerols and phospholipids. Intracellular ipolysis and molecular mechanisms of its regulation Objective: to learn the processes of biosynthesis of phospholipids and triacylglycerols and the main pathways of intracellular metabolism of lipids. To learn the methods of determination of phospholipids concentration and activity of lipase and to interpret the obtained results. Actuality of the theme: The knowledge of main pathways of intracellular metabolism of lipids under normal conditions and in pathology are necessary for medical students in further studies of general pathology, pharmacology and related clinical disciplines for correct interpretation of results of laboratory investigations and recognition of metabolic disorders in distinct cases. Specific aims: To interpret biochemical function of simple and complex lipids in organism: their involvement in formation of structure and function of biological membranes, reserve and energetic significance, the role as precursors in biosynthesis of biologically active compounds of lipid nature. To explain the principal pathways of intracellular lipid metabolism. To explain enzymatic reactions of catabolism and biosynthesis of triacylglycerols. To interpret enzymatic reactions of synthesis of phospholipids and sphingolipids. 56 To analyze the main pathways of lipid metabolism in human body in normal conditions and in pathology. To explain hormonal regulation of lipid metabolism. Theoretical questions: 1. Classification of lipids. Biological functions of simple and complex lipids in human body (reserve, energetic, thermoregulatory, production of biologically active substances). 2. Structure and role of fatty acids (saturated, unsaturated and polyunsaturated). 3. Triacylglycerols (TAG) their structure and properties. 4. Phospholipids: structure of main glycerophospholipids (phosphatidic acid, lecithin, cephalin, phosphatidylinosit, phosphatidylserine, plasmalogen) and sphingolipids (sphlngomyeliln). Their biological role. 5. Structure and occurrence of main steroids. 6. Amphipathic lipids Involvement of lipids in formation of structure and function of biological membranes. Liposomes. Application of liposomes in medical practice. 7. Digestion of lipids. 8. Catabolism of triacylglycerols: characterization of intracellular lipolysis, its biological significance; enzymatic reactions; neurohumoral regulation of lipolysis: role of epinephrine, norepinephrine, glucagone, insulin; energetic balance of triacylglycerol oxidation. 9. Biosynthesis of triacylglycerols and phospholipids, the significance of phosphatidic acid as a precursor. 10.Metabolism of sphingolipids. Genetic anomalies of sphingolipid metabolism – sphingolipidoses. Lysosomal diseases. Practical part Experiment 1. Quantitative determination of phospholipids in blood serum. Principle: Phospholipids are precipitated with trichloroacetic acid together with plasma proteins. After mineralization of sediment the quantity of phosphorus is determined colorimetrically and content of phospholipids is calculated. Method. I. Precipitation of phospholipids with trichloroacetic acid (Attention!!!! This part of experiment was previously done by technitions): 1. Take a clean dry centrifuge tubes. 2. Add 0.2 ml of blood serum, 2 ml of distilled water and 3 ml of 10 % solution of trichloroacetic acid. 3. Mix the content of the tube and after 2-3 min. centrifuge it during 5 min at 3000 rpm. 4. Carefully remove the supernatant. Sediment contains lipoproteins. Add to the sediment 1 ml of 56 % HClO4 put the tube into a bath with a boiling water for 30 min. The final mineralizate must be colorless. II. Color reaction: 1. Take 2 clean dry tubes. 57 2. To the 1st (experimental) tube add 5 ml of mineralizate, 1 ml of ammonium molybdate and 1 ml of 1% solution of Exp St ascorbic acid. 3. To the 2nd tube (standard) add 5 ml of standard phosphorus 1 ml of 1% solution of solution (0,05 g/l) 1 ml of ascorbic acid ammonium molybdate and 1 1 ml of ammonium molybdate ml of 1% solution of ascorbic 5 ml of mineralizate 5 ml of standard acid. 4. Mix the content of both tubes and incubate 15-20 min at room temperature. III. Measurement of optical desity: After 5 min measure the optical density on a photoelectrocolorimeter with a red light filter. IV. Calculation: Use the following formula: Aexp 0,05 [Total phospholipids in serum] = -------------- 25 g/l Аst 0,2 where Aexp – optical density of the experimental probe; Ast – optical density of the standard probe; 0.05 – concentration of phosphorus in standard (mg/ml) 0.2 – volume of analyzed serum 25 – coefficient for calculation of total phospholipids content. Clinical and diagnostic significance. The determination of phospholipid content in blood has an important diagnostic significance. The concentration of total phospholipids in blood serum of healthy adult is 1.5 – 3.6 g/l. The increase of phospholipids level in blood serum (hyperphospholipidemia) is observed in heavy form of diabetes mellitus, nephrosis, obturative jaundice. Decrease of phospholipid level (hypophospholipidemia) may be observed in atherosclerosis, anemias, feaver, alimentary distrophias, liver diseases. Experiment 2. The influence of bile on the lipase activity. Principle. The lipase activity is estimated according 2 1 to the production of fatty acids 1 ml of bile during hydrolysis of fat. The 0.25 g of pancreatine quantity of fatty acids is determined by titration with 10 ml of milk alkali in the presence of Mix well phenolphthalein. Bile activates 1 ml from lipase thus accelerating the tube 1 cleavage of lipids. Method. I. Preparation of mixtures: 1 ml of H2O 1 ml from tube 2 1-2 drops of 0.5% phenolphthalein Titrate with 0.05 N NaOH to appearance the pink color, which doesn’t disappear during 30 seconds. 58 1. Take 2 clean dry tubes. 2. Add 10 ml of milk and 0.25 g of pancreatine into both tubes. 3. Add 1 ml of bile into 1st tube and 1 ml of water into 2nd. Mix them well. II. Titration: 1. Take 1 ml of the mixture from tube 1 (with bile) and transfer it to the flask 1. 2. Take 1 ml of the mixture from tube 2 (without bile) and transfer it to the flask 2. 3. Add 1-2 drops of 0.5% phenolphthalein solution into both flasks. 4. Titrate mixtures of both flasks with 0.05 N NaOH up to the pink color, which doesn’t disappear during 30 seconds. Register the volume (in ml) of used alkali. Time, min 0 min 15 min 30 min 45 min Quantity of NaOH expended for titration, ml Lipase activity in presence of bile Lipase activity in absence of bile 60 min Lipase without bile Lipase without bile III. Incubation. The first two tubes with digestive mixture (see I) place to the termostate at 38C. Every 15 min take 1 ml of the mixture from each tube into flasks and repeat the titration (see II). Conduct 4-5 subsequent determinations. The results are registered as ml of alkali solution, expended for titration. Note the obtained result into the table bellow. Explain the results, draw a conclusion. Clinical and diagnostic significance. Digestion of lipids occurs predominantly in intestines under the action of active pancreatic lipase, which acts only on emulsified lipids, e.g. milk fat. In gastric juice the activity of lipase is negligible. Lipids must be emulsified, which is achieved due to the action of bile acids. As bile emulsify lipids and activates lipase, hydrolysis of lipids proceeds more quickly. As lipase is the main enzyme in lipid digestion, changes in its activity may indicate on disorders in some processes of lipid metabolism. Deficiency of pancreatic lipase causes pancreatogenic steatorrhea, which is observed in chronic pancreatitis, hereditary or acquired deficiency of pancreatic gland, in mucoviscidosis. The amount of bile pigments in feces in such conditions is accompanying with the lowering of non esterified fatty acids content and significant increase in unhydrolized lipids. The alteration in emulcification, digestion and absorption of lipids and fat soluble vitamins as well may take place in diseases of liver, gall bladder or biliary ducts (cholelythiasis). Examples of Krock-1 tests 1. In patients suffering from diabetes mellitus an increase in a content of non esterified fatty acids (NEFA) in blood is observed. It may be caused by: A. Increase in activity of triacylglycerol C. Activation of synthesis of lipase apolipoproteins A1 , A2, A3 B. Stimulation of ketone bodies utilization 59 D. Decrease in activity phosphatidylcholine-cholesterolacyltransferase in blood plasma of E. Accumulation palmitoyl-CoA in cytosol of 2. The essence of lipolysis, that is the mobilization of fatty acids from neutral fats depots, is an enzymatic process of hydrolysis of triacylglycerols to fatty acids and glycerol. Fatty acids that release during this process enter blood circulation and are transported as the components of: A. Serum albumins D. LDL B. Globulins E. Chylomicrons C. HDL 3. Which one of the following statements about the absorption of lipids from the intestine is correct? D. Fatty acids that contain ten carbons A. Dietary triacylglycerol is partially or less are absorbed and enter the hydrolyzed and absorbed as free fatty acids circulation primarily via the lymphatic and monoacyl glycerol system B .Release of fatty acids from triacylglycerol E. Formation of chylomicrons does not in the intestine is inhibited by bile salts require protein synthesis in the C. Dietary triacylglycerol must be intestinal mucosa. completely hydrotyzed to tree fatty acids and glycerol before absorption 4. After consumption of lipids in the body than begins their digestion and absorption in intestines. What products of lipid hydrolysis are absorbed in the intestine? A. Monoacylglycerol, fatty acids D. Monosacharides B. Amino acids E. Lipoproteins C. Polypeptides 5. After the consumption of animal food rich in fats, a patient feels discomfort, and droplets of fats are found during laboratory investigation of his feces. Bile acids are revealed in the urine. The cause of such state is the deficiency of ___ in the digestive tract. A. Bile acids. D. Triacylglycerols. B. Fatty acids. E. Phospholipids. C. Chylomicrons. Individual independent students work 1. Metabolism of sphingolipids under normal conditions and in pathology. Abnormalies of sphingolipid metabolism – sphingolipidoses. References: 1. Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. 2. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. 3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. 60 4. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. 5. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. 6. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 7. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd edition” – 2008. – 488 p. 8. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 13. β –Oxidation and biosynthesis of fatty acids. Studies on metabolism of fatty acids and ketone bodies. The aim of the lesson: To learn reactions of biosynthesis and oxidation of fatty acids. To know metabolic pathways of ketone bodies under normal conditions and in pathology and to determine their amount in urine. Actuality of the theme: Oxidation of lipids, respectively fatty acids, as well as ketone bodies metabolism are important constituents of energetic metabolism in sense of providing tissues and cells with ATP. Determination of ketone bodies concentration in blood and in urine has important significance in diagnostics of several pathological processes. Specific aims: To study reactions β -oxidation of long chain fatty acids. To interpret biosynthesis of long chain fatty acids and regulation of biosynthetic process on the level of acetyl-CoA-carboxylase and fatty acid synthetase. To analyze the metabolism of ketone bodies. To explain the mechanism of excessive accumulation of ketone bodies in diabetes mellitus and in starvation. Theoretical questions: 1. β –Oxidation of long chain fatty acids: localization of the process of β-oxidation of fatty acids; activation of fatty acids, the role of carnitin in transport of fatty acids into mitochondria; the sequence of enzymatic reactions in β –oxidation of fatty acids; energetic balance of β –oxidation of fatty acids; 2. Mechanism of glycerol oxidation, bioenergetics of this process. 3. Biosynthesis of long chain fatty acids: localization of biosynthesis of long chain fatty acids; metabolic sources for biosynthesis of fatty acids; stages in synthesis of saturated fatty acids; characteristic of the synthetase of long chain fatty acids, the significance of acyl transporting protein and biotin; sources of NADPH2 for biosynthesis of long chain fatty acids; the sequence of enzymatic reactions in biosynthesis of long chain fatty acids 61 regulation of biosynthetic process on level of acetyl-CoA-carboxylase and fatty acid synthetase; elongation of carbon chain of fatty acids; 4. Metabolism of ketone bodies. enzymatic reactions of ketone bodies biosynthesis (ketogenesis); reactions of ketone bodies utilization (ketolysis), energetic effect; metabolism of ketone bodies in pathology. Mechanism of excessive accumulation of ketone bodies in diabetes mellitus and in starvation. Practical part Experiment 1. Qualitative reaction on acetone and acetoacetic acid (Lange reaction). Principle. It is based on a property of acetone and acetoacetic acid to produce compounds with sodium nitroprussid of red color in the basic pH of medium. CH3 – CO – CH3 + Na2 [Fe (CN)5 NO] + 2 NaOH → → Na4 [Fe (CN)5 NO = CHCOCH3] + 2 H2O. Under the action of acetic acid it turns to a violet product: Na4 [Fe (CN)5 NO =CHCOCH3] +CH3COOH → Na3 [Fe (CN)5 NOCH3COCH3] + CH3COONa. compound after an overlay of conc. ammonia solution over the mixture of test sample, containing sodium nitroprussid and acetic acid. The rate of color ring formation between two layers of liquids depends from acetone concentration. It is assumed, that appearance of the ring after 3-4 min corresponds to the acetone concentration 0.0085 g/l (8,5 mg/ l). Method. 1. Take two clean dry tubes. 2. Add 0.5 ml of urine of a healthy person to the first tube (blank). 3. Add 0.5 ml of urine of a patient with diabetes mellitus to the second one (test sample). 4. Add 0.5 ml of 10 % NaOH and 5-7 drops of fresh 10% sodium nitroprussid solution into both tubes. Color in the 2nd tube turns red. 5. Add 5-7 drops of acetic acid into both tubes. Color in the 2nd tube turns violet. Explain the results, draw a conclusion. Experiment 2. Quantitative determination of ketone bodies in urine. Principle. (see the previous experiment). Method. 1. Take 5 clean dry tubes. 2. Add 1 ml of distilled water into each tube (except the first one). 3. Add 1 ml of urine of the patient with diabetes mellitus into the first and the second tubes. Thus, in the first tube urine is undiluted, in the second one the urine is two fold diluted. 4. Mix the content of the second tube thereafter transfer 1 ml of this mixture to the third tube and mix it. Now the third tube conatains 4 fold diluted urine. 62 5. Remove 1 ml from the third tube - into the forth and mix it - 8 fold dilution. 6. Remove 1 ml from the fourth tube - into the fith one and mix it - 16 fold dilution. 7. Add 8 drops of 50% ammonium sulfate solution into each tube for increasing the density of the solutions 8. Add 8 drops of concentrated acetic acid into each tube. 9. Add 8 drops of sodium nitroprussid each tube. Mix well the content of the tubes 10. Overlay carefully 1 ml of concentrated solution of ammonia into each tube starting from the last tube (be careful with concentrated ammonia!). 11. Register an appearance of red or violet ring in the tubes. Notice the last tube in which a colored ring appears between the 3-d and the 4-th min. Calculation: X = 0.85 A 15, were X – concentration of acetone in urine (mg/day); 0.85 - empirical coefficient, corresponding to 0,85 mg of acetone in 100 ml; A- the dilution of the urine sample; 15 - coefficient for calculation on the daily volume of urine (1500 ml). Draw the conclusions. Clinical and diagnostic significance. Ketone bodies include the following substances – acetoacetic acid, -hydroxybutyric acid, acetone. Biosynthesis of ketone bodies (ketogenesis) takes place in liver from intermediates of fatty acid oxidation, namely, from acetyl-CoA. Acetoacetic acid, produced in liver, is transported to body tissues (brain, muscles, kidneys, heart etc.), where it serves as an energetic material. In healthy person the content of ketone bodies in blood is in ranges of 13-185 moles/l (1.5-20 mg/l). With urine is excreted 20-40 mg of ketone bodies daily, which are preferentially acetoacetic and hydroxybutyric acids. Acetone appears under pathological conditions. The increase of ketone bodies concentration in blood (ketonemia) and in urine (ketonuria) is observed in diabetes mellitus, deficiency of sugar in nutrition, overproduction of hormones, antagonistic to insulin (corticosteroids, thyroxine, hormones of adenohypophysis). The decrease of ketone bodies content has no clinical value. In early childhood prolong disorders of digestion (toxicosis, dysenteria) may lead to ketonemia due to the permanent starvation and exaggeration. Examples of Krock-1 tests 1. In a patient suffering from diabetes mellitus in blood was detected acetone. Note the process of its production in the body. A. By condensation of two molecules of D. In course of γ-oxidation of fatty acetyl-CoA acids B. In course of α-oxidation of fatty acids E. In tricarboxylic acid cycle. C. In course of β-oxidation of fatty acids 2. Aerobic oxidation of substrates is typical of a cardiac muscle. Which of the following is the major oxidation substrate of a cardiac muscle? A. Fatty acids. D. Glucose. B. Triacylglycerols. E. Amino acids. C. Glycerol. 63 3. Carnitine is recommended to a sportsman as a preparation that increases physical activity and improves achievements. What biochemical process is mostly activated under the action of carnitine? A. Transport of fatty acids into C. Lipids synthesis. mitochondria. D. Tissue respiration. B. Ketone bodies synthesis. E. Steroid hormones synthesis. 4. A 1-year-old child was brought to a clinic with signs of muscle weakness. Through the inspection, the deficiency of carnitine in the muscles was determined. The biochemical mechanism of the development of this pathology consists in the disorder of the process of: A. Transport of fatty acids into C. Substrate level of phosphorylation. mitochondria. D. Utilization of lactate. 2+ B. Regulation of the level of Ca in E. Synthesis of actin and myosin. mitochondria. 5. Lipids are obvious energetic material for the body. What is the main pathway of fatty acids metabolism in mitochondria? A. β-Oxidation D. Reduction B. Decarboxylation E. γ-Oxidation C. α -Oxidation 1. 2. 3. 4. 5. 6. 7. 8. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2nd edition” – 2008. – 488 p. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. Topic № 14. Biosynthesis and biotransformation of cholesterol. Pathology of lipid metabolism: steatorrhea, atherosclerosis, obesity. Transport forms of lipids: lipoproteins of blood plasma. Objective: To learn the pathways of cholesterol metabolism and principal disorders of lipid metabolism. To interpret free radical reactions, mechanisms of lipid peroxidation and its significance in biological processes in normal conditions and in pathology. 64 Actuality of the theme: Disorders in cholesterol biotransformation processes cause several diseases, such as atherosclerosis, obesity et al. In this connection the investigation of lipid metabolism indexes is obvious for diagnostics and treatment of different diseases. Production of free radicals and products of peroxide oxidation of lipids are normal metabolic processes. In normal conditions the level of products of peroxide oxidation of lipids is regulated by antioxidative enzymatic system. Disorders in relations between activity of pro- and anti- oxidative systems is manifested in form of different pathological features. This makes necessary the investigation an evaluation of oxidative stress indexes in pathology. Specific aims: To interpret stages of cholesterol biosynthesis. To explain regulation of cholesterol production in human body. To analyze pathways of cholesterol biotransformation: esterification, synthesis of bile acids, steroid hormones, vitamin D3, excretion of cholesterol from the body. To interpret pathology of lipid metabolism: atherosclerosis, diabetes mellitus, obesity, steatorrhea. To explain processes of lipid peroxidation under normal conditions and in pathology. To interpret regulation of free radical reactions in human body. Theoretical questions: 1. Biosynthesis of cholesterol in human body. localization of the process and its significance; stages of cholesterol biosynthesis; enzymatic reactions of biosynthesis of mevalonic acid; regulation of cholesterol synthesis. 3. Pathways of cholesterol biotransformation (esterification, production of bile acids and steroid hormones, synthesis of vitamin D3, excretion from the body). 4. Atherosclerosis, mechanism of its development, role of genetic factors, hypercholesterolemia. Hypercolesterolemia in diabetes mellitus, myxoedema, obstructive jaundice, nephritic syndrome. Control of hypercholesterolemia 5. Lipoproteins: structure, classification, characteristics of apolipoproteins. 6. Metabolism of lipoproteins – a general view. 7. Disorders of plasma lipoproteins (classification of hyperlipoproteinemias, characteristics of hypolipoproteinemias. 8. Fatty liver (steatosis), lipotropic factors. 9. Pathological processes which leads to the development of obesity. 10. Lipid peroxidation and mechanisms of antioxidant enzymatic system action: Lipid peroxidation under normal conditions and in pathology. Regulation of free radical reactions in human body. Characterization of prooxidants and antioxidants, their significance in peroxide oxidation of lipids. Practical part 65 Experiment 1. Qualitative reaction on bile acids (Petenkoffer reaction) Principle. The reaction is based on formation of colored products after condensation of bile acids with hydroxymethylfurfurol. The last is produced under the action of sulfuric acid upon fructose, which in turn is formed from sucrose due to its hydrolysis with sulfuric acid. Method. 1. Take a clean dry tube. 2. Add 10-15 droplets of bile. 3. Add ~5 droplets of a fresh 20% sucrose solution and mix it. 4. Carefully overlay 1 ml of conc. sulfuric acid without mixing of two fluids. 5. Observe the precipitation of bile acids in the borderline between two fluids (appearance of violet color ring). 6. Mix it well and observe the appearance of red-violet color. Clinical and diagnostic significance. The bile acids and their salts are detergents that emulsify fats in the gut during digestion. They are synthesized from cholesterol in the liver by a series of reactions. The bile acids are produced in liver, about 10-15 g daily. Bile acids include cholic, deoxycholic, chenodeoxycholic, lithocholic et al., which are excreted in bile in free state or conjugated with glycine or taurine. Bile acids emulsify lipids, activate lipase, are involved in absorption of fatty acids, form choleinic complexes, stabilize cholesterol. Deficiency of bile acids in intestines may be caused with liver diseases (occlusive jaundice, hepatitis, cirrhosis), disorders in gallbladder or bile ducts (cholelithiasis, tumors of bile ducts). In coprologic investigations decrease or complete absence of bile pigments in feces are observed, as well as high content of soaps, espetially calcium soaps. Experiment 2. Investigation of antioxidative enzymatic system. Quantitative determination of catalase activity. Principle. Catalase belongs to a class of oxido-reductases and catalyzes the transformation of hydrogen peroxide to water and oxygen: 2H2O2 2 H2O + O2 Biological significance of this enzyme consists in the defense of the body from harmful effect of hydrogen peroxide, which forms in the course of intracellular oxidation of different substances. According to chemical structure, catalase is a chromoprotein, containing heme as a prosthetic group. Molecular weight of catalase is 225 – 240 kDa. It is widely distributed and extremely active enzyme. The determination of catalase is based on the estimation of hydrogen peroxide, which is decomposed by the enzyme in a distinct period of time. Hydrogen peroxide is titrated with permanganate, the reaction occurs according to the equation: 5H2O2 + 2 KMnO4 + 3 H2SO4 → K2SO4 + 2 MnSO4 + 8 Н2О + 5О2 Method. Take two flasks and do the following procedures: 1. Add 1 ml of diluted 1:1000 blood into both flasks then add 7 ml of water. 2. Add to the first flask (the test) 2 ml of 1% solution of hydrogen peroxide 3. and 5 ml of 10 % sulfuric acid to the other flask (the control). Leave both flasks at room temperature for 30 min. Then, add to the test flask 5 ml of 10 % sulfuric acid, and to the control flask 2 ml of 1% solution of H2O2. Titrate the content of flasks by 0.1 N KMnO4 to appearance of pink colour. 66 Test - 1 ml of diluted 1:1000 blood Control - 7 ml of water - 2 ml of 1% solution of H2O2 - 5 ml of 10 % sulfuric acid Incubation for 30 min (room temperature) Test Control A - the volume (ml) of KMnO4 used for titration of the control probe, B - the volume (ml) of KMnO4 used for titration of the test probe. Titration by 0.1 N KMnO4 to appearance of pink colour Calculate catalase activity according to formula: Cf = (A – B) 1.7, where A - the volume of KMnO4 solution, used for titration ofthe control probe, B - the volume of KMnO4 solution used for titration of the test probe. Explain the results and draw a conclusion. Clinical and diagnostic significance. Catalase (E.C. 1.11.1.6. ) is one of the most active and the most wide spread enzymes. It accelerates the breakdown of hydrogen peroxide and its derivatives. Normal value of catalase activity in blood is in ranges of 10 – 15 units/L. The estimation of catalase activity data on red blood cells count in blood is of low diagnostic value as catalase activity is tightly connected with red blood cells. In clinical laboratory the catalase index determination is used, which is equal to catalase activity in units over number of: red blood cells (in ml/mm3). In normal conditions this index is in 2 – 3 10 – 6 ranges. Examples of Krock-1 tests 1. A patient suffers from arterial hypertension due to atherosclerotic injury of blood vessels. The consumption of what dietary lipid needs to be limited? D. Monooleateglycerol. A. Cholesterol. E. Phosphatidylserine. B. Oleic acid. C. Lecithine. 2. Fats of phospholipids is disordered due to fat infiltration of the liver. Indicate which of the presented substances can enhance the process of methylation during phospholipids synthesis? A. Methionine D. Glycerin B . Ascorbic acid E. Citrate C. Glucose 67 3. In a patient after investigation it was detected an increased content of low density lipoproteins in blood serum. What disease can be expected in this patient? A. Atherosclerosis D. Acute pancreatitis B. Pneumonia E. Kidney disease C. Gastritis 4. A child 5 years old suffers from transient abdominal pains. Blood serum is turbid in fasting conditions. Cholesterol content – 4,3 mmoles/l, total lipids – 18 g/l. For precisement of diagnosis electrophoresis of blood lipoproteins is administered. What classes of lipoproteins are expected to be increased? A. Chylomicrons D. LDL B. HDL E. VLDL C. IDL 5. In cases of complete or partial restriction of lipotropic factors in humans develops a fat degeneration of liver. What substances can be considered as lipotropic? A. Choline D. Cholesterol B. Pyridoxine E. Triacylglycerols C. Fatty acids Individual independent students work 1. Lipid peroxidation, its role under normal conditions and in pathology. 1. 2. 3. 4. 5. 6. 7. 8. References: Satyanarayana U., Chakrapani U. “Biochemistry”, Third Edition. – 2006. – 792 p. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003.- 927 p. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p. Lehninger A. Principles of Biochemistry. – New York. – W.H.Freeman and Company. – 2005. – 1010 p. Mardashko O.O., Yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The Odessa State Medical University, 2003.-416p. Devlin T.M., ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. Toy E.C., Seifert W. E., Strobel H.W., Harms K.P. “Case Files in Biochemistry. 2 nd edition” – 2008. – 488 p. MCQs / Prof. Sklyarov A.Ya., Lutsik M.D., Fomenko I.S., Klymyshin D.O., Nasadyuk C.M.. – 2012. – 308 p. 68