51
DANYLO HALYTSKYI LVIV NATIONAL medical UNIVERSITY DEPARTMENT OF BIOLOGICAL CHEMISTRY BIOLOGICAL CHEMISTRY METHODICAL INSTRUCTION FOR PRACTICAL EXERCISES for students of dentistry faculty Part І Lviv – 2011

Methodical Guide 2011

Embed Size (px)

Citation preview

Page 1: Methodical Guide 2011

DANYLO HALYTSKYILVIV NATIONAL medical UNIVERSITY

DEPARTMENT OF BIOLOGICAL CHEMISTRY

BIOLOGICAL CHEMISTRY

METHODICAL INSTRUCTION FOR PRACTICAL

EXERCISES

for students of dentistry faculty

Part І

Lviv – 2011

Page 2: Methodical Guide 2011

Methodical instructions prepared by:

Prof. Sklayrov A.Ya., M.D., PhD.

Prof. Lutsik M.D., PhD.

Fomenko I.S., PhD.

Klymyshin D., PhD.

Editor: prof. Sklayrov A.Ya., M.D., Ph.D.

Page 3: Methodical Guide 2011

MODULE 2. GENERAL PRINCIPLES OF METABOLISM

Sense module № 5. An introduction to biochemistry. Biochemical constituents of the cell.

Topic №1. An object and assignments of biochemistry, its principal trends and parts. Objectives and methods of biochemical investigation;their clinical and diagnostic significance.

Objective: Introduction to the assignments and methods of biochemical investigation; to learn specific methods of investigation of biologically active substances as well to make an acquaintance with instruments and devices used in biochemistry.

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.

Theoretical questions:1. The objectives and assignments of biochemistry and its principal trends and

parts.2. A short history of biochemistry and its main periods.3. Contribution of scientists of the Dept. of Biochemistry of Lviv National Medical

University to development of biochemistry.4. The significance of biochemistry in the development of medical science and

practical health care.5. Chemical constitution of living organisms, the characteristic features of living

systems: the turnover of matter and energy and their relations with the external environment.

Page 4: Methodical Guide 2011

6. The structural components of the prokaryotic and eukaryotic cells, their resolution with ultracentrifugation methods.

7. Methods in biochemical investigations:• fractionation of material using differences in solubility• optical methods in biochemical investigations (photoelectrocolorimetry,

spectrometry, spectrophotometry, fluorescent analysis)• electrophoresis (zonal electrophoresis, electrophoresis in gels, isoelectrofocusing,

immunoelectrophoresis).• chromatography (ion-exchange chromatography, affinity chromatography, thin

layer chromatography - TLC, gel filtration)• polarographic methods• manometric and radioisotopic methods• enzyme immunoassays

8. Objective of biochemical investigations: the whole organism and its organs, tissues, cells, homogenates, subcellular organelles, extracts and molecular biocomplexes.

9. Clinical and diagnostic significance of biochemical investigation.10. Biological material used in biochemical investigations.11. Errors in biochemical investigations.

Essay of the history of biochemistry unitUnit 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-

Page 5: Methodical Guide 2011

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 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

Page 6: Methodical Guide 2011

concentration Experiment 1. Measurement of optical density (D) 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 color intensity of molybdene blue complex.

Method. Add the following solutions to five tubes (as indicated in the table):

№ ReagentsTube number

1 2 3 4 51 Standard phosphate solution (10

mg/ml), ml- 0.5 1.0 2.0 3.0

2 Distilled water, ml 5.0 4.5 4.0 3.0 2.03 Molybden reagent, ml 5.0 5.0 5.0 5.0 5.04 Phosphorus concentration, mg% 0 50 100 200 3005 Results of optical density

measurementAfter mixing a color develops, which is measured on a photoelectrocolorimeter

with a red light filter. Optical density must be measured at distinct time after the addition of the last reagent, usually after 5 min. According to obtained results the plot is constructed (a calibration curve).

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 molecolarn 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

Page 7: Methodical Guide 2011

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 tube, add 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. Denaturated proteins are readily digested by proteolitic enzymes. Gastric hydrochloric acid causes the denaturation of proteins of food stuffs. As a result they are more readily hydrolysed 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, sulfosalicylic acid.

Methods of quantitative determination of proteinsProtein 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.

Experiment 3. Biuret method of protein quantitation.Principle. Proteins in alkaline medium interact with copper sulfate with

formation of compound with violet color.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. To the first tube 0,1 ml of saline is added (control), to the second tube 0,1 ml of standard protein solution is introduced, into the third and fourth tubes 0,1 ml of tested protein solutions are added. To all tubes 5 ml of biuret reagent are added, mixed and after 10-30 min the extinction (optical density) is measured on a photocolorimeter using green light filter against control solution.

Calculation. The concentration of protein in test solution is calculated according to formula

X= (AxB)/C, whereX –protein concentration in test solution, (g/l),A – protein concentration in standard solution (g/l),B – extinction of tested protein solution,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

Page 8: Methodical Guide 2011

loss, 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.

Hyper p r o t e i nem i a i s obser ved i n p l asm ocy t om a, Wal denst r om macroglobulinemia, rheumatoid arthritis, collagenoses, liver cirrhos as well as in diseases accompanied with dehydration, that is during diarrhea, vomiting, 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 tests1 Proteins have a role in the following biochemical processes:A. Protein synthesis;B. Digestion of proteins in the digestive tract;C. Replication of DNA;D. Muscle contraction;E. Transport of glucose across membranes.

2. Which of the following techniques are used for proteins: purification?A. Affinity chromatography;B. Electrophoresis on polyacrylamide gel;C. Fractionation by precipitation with ammonium sulphate ; D.Selective denaturation of other proteins in the mixture; E. Ion exchange chromatography.

3. Which of the following techniques depend on the functional properties of proteins:A. Affinity chromatography;B. Electrophoresis on polyacrylamide gel;C. Fractionation by precipitation with ammonium sulphate;D. Selective denaturation of other proteins in the mixture;E. Ion exchange chromatography.

4. Biuret test for detection of proteins in a sample is developed due to the next type of chemical bonds in protein molecule:A.Hydrogen bondsB.Peptide bondsC.Disulphide bondsD.Hydrophobic and resonance interactionE.Ionic bonds between amino acid side chains

Page 9: Methodical Guide 2011

Individual independent students work1. 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 structure and fuctional significance of proteins and nucleic acids.

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International

Boston-London, 1992.- 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003.- 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994.- 443 p.6. Mardashko O.O., yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The

Odessa State Medical University, 2003.-416p.

Sens module № 6. Enzymes and coenzymes. Regulation of metabolism.

Topic № 2. Investigation of structure and physico-chemical properties of enzymes. The determination of enzymatic activity, investigation of mechanisms and kinetics of enzymatic reaction.

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 objectives:¬ To interprete biochemical principles of structure and functioning of different

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.

Theoretical questions:1. The concept of enzymes, difference between enzymes and inorganic catalysts.2. Physico-chemical properties of enzymes (chemical properties, solubility,

denaturation etc).3. Simple and conjugated enzymes; prosthetic groups of enzymes (cofactors,

Page 10: Methodical Guide 2011

coenzymes).4. Structure of enzymes: active centers and allosteric sites.5. Levels of structural organization of enzymes. Multi-enzyme complexes, ensembles,

polyfunctional enzymes and their advantages.6. International classification and nomenclature of enzymes (E.C.); the principal

classes of enzymes. 7. Modern concepts of enzyme kinetics (the dependence of enzymatic reaction from

the concentration of the substrate and ezyme; temperature, pH of medium; Michaelis-Menten equation;

8. Specificity of enzymes.9. The localization of enzymes in cells and organs; the peculiarities of enzyme

functioning in biomembranes.10. Units of enzymatic activity.11. Enzymes of salivary glands12. Isoenzymes.

Practical partExperiment 1. Investigation of the termolability of salivary amylase. Principle. Salivary amylase is a protein and consequently is termosensitive.

Boiling inactivates enzyme due to denaturation, low temperature (near 0 0C) substantially lowers the rate of reaction. The hydrolysis of starch is monitored by probes with iodine and appearance of reductive properties due to production of maltose (see above).

Method. Add 2 ml of diluted saliva to a tube and boil it for two min on a flame. Into three other tubes add 5 ml of starch solution. To the first tube add 2 ml of boiled saliva, to the second and the third tubes add 2 ml of nonboiled diluted saliva. The second tube is placed in the water bath at 37o C for 10 min, the third tube is placed for 10 min in a cold water (near 4-6 oC). Thereafter the content of each tube is divided into two equal portions. To the first portion of each test solution are added 3 drops of iodine and changes in colour are registered. The second portion of the probes is used for performance of Trommer reaction (see above).

Explain the results and draw a conclusion.

Experimen 2. The investigation of the influence of pH of medium on the amylase activity.

Principle. Starch with iodine gives a blue coloured complex. Dextrins become red-brown or they do not change colour at all. Maltose also does not form a coloured complex with iodine, but it can be detected with the Trommer reaction.

Method. Add 2 ml of starch solution to each of the three tubes. Add to the first tube 2 ml of phosphate buffer, pH 5.0, to the second 2 ml of phosphate buffer, pH 7.0, to the third 2 ml of phosphate buffer, pH 9.0. Add into each tube 1 ml of diluted saliva; incubate tubes at 37 o C. After the finish of incubation the content of tubes is divided into two parts and with one a probe with iodine is performed. With another part the Trommer reaction is conducted as described above.

Explain the results and draw a conclusion.

Page 11: Methodical Guide 2011

Clinical and diagnostic significance. Amylase (E.C. 3.2.1.1) hydrolizes 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 1 of plasma in 1 minute.

An increase of amylase activity in serum is observed in pancreatitis, peritonitis, mesentheric 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 of n.facialis. A decrease in amylase activity is noted in some cases of psychoses, accompanied by depression or excitation and in gastric secretion disorders (anaciditas).

Experiment 3 Quantitative determination of amylase activity in urine according to Wohlgemuth.

Principle. A minimal quantity of an enzyme, which cleaves a distinct quantity of substrate, is determined. Arbitrary unit of amylase activity in urine corresponds to the quantity of 0,1 % solution of starch, which can be cleaved by 1 ml of urine at 45 °0 in 15 min.

Method: Into 9 ennumerated tubes is added 1 ml of saline. To the first tube 1 ml of urine is added, mixed and transfere 1 ml of the first tube content to the second tube. With the same manner 1 ml of the mixture after mixing is transferred to the third tube and so on. A series of dilution of urine is obtained as a geometric progression with a coefficient 2.

Into each tube 1 ml of saline is added, thereafter 2 ml of 0,1 % of starch solution. Content of tubes is substancially mixed and tubes are placed on a water bath at 45°C for 15 min.

After incubation tubes are taken out of the bath, cooled by tap water and to each tube 2 drops of Lugol solution (iodine in potasium iodide solution) are added. A colour reaction in tubes is registered. Results are registered in a table and the activity of amylase is calculated.Tube N 1 2 3 4 5 6 7 8 9Urine dilution

1:2 1:4 1:8 1:16 1:52 1:64 1:128 1:256 1:512

Colour with iodine

yel. yel. yel. yel. blue blue blue blue blue

Starch cleavage

+ + + + - - - - -

Example. 1/16 ml of urine cleaved 2 ml of 0,1 % starch solution (yellow colour in tubes NN 1-4). In the and 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.

Page 12: Methodical Guide 2011

Normally amylase activity in blood serum and in urine consists around 16-64 units (Wohlgemuth units). Increase of amylase activity to more than 256 units indicates to acute inflamation of pancreatic gland. In renal insufficiency amylase is absent in urine.

Examples of Krock-1 tests1. The following statements describe coenzymes and prosthetic groups:A. Some enzymes contain metal ions such as zinc or copper that are essential for

their activity;B. Some enzymes contain organic molecules that are derived from vitamins and

that are essential for activity. These molecules are called prosthetic groups.C. Coenzymes are small polypeptides that are involved in transfer reactions

catalysed by enzymes;D. Coenzymes are required in some enzyme catalysed reactions as carriers of acyl

groups or phosphate groups;E. Coenzymes are required in some enzyme catalysed oxidation-reduction reactions

involving hydrogen transfer.

2. Allosteric enzymes have the following features:A. Allosteric enzymes have ligand-binding sites other than the active centers to

which metabolites or other signal molecules can bind and switch the enzyme on or off;

B. Allosteric enzymes always have subunits and multiple active centers;C. Allosteric enzymes do not obey Michaelis kinetics;D. Allosteric control is irreversible; E. Allosteric enzymes are often involved in negative feedback control mechanisms.

3. Which one of the following statements is not characteristic of allosteric enzymes?A. They frequently catalyze a committed step early in a metabolic pathway;B. They are often composed of subunits; C. They frequently show cooperativity for substrate binding.D. They follow Michaelis-MenIen kinetics; E. The binding of a positive allosteric effector results in an increase in enzymic

activity. Individual independent students work

1. Multi-enzyme complexes and their advantages.2. The employment of enzymes in biochemical investigations.

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers

International Boston-London, 1992.- 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003.- 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Page 13: Methodical Guide 2011

Illustrated Reviews, 1994.- 443 p.6. Mardashko O.O., yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The

Odessa State Medical University, 2003.-416p.

Topic № 3. Investigation of regulation of enzymatic activity and mechanisms of enzymopathias. The role of cofactors, vitamins and their coenzyme forms in enzyme catalysis. Medical enzymology.

Objectives: 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. To learn the structure, principles of classification and funtion of coenzymatic vitamins. To master the methods of qualitative and quantitative determination of vitamins.

Actuality of the theme: Enzymes are biocatalysts with changeble activity, submitted to regulatory influences. Estimation of enzymatic activity is routinely used in laboratory investigations with diagnostic purposes. Enzymes are also employed as medicinals and drugs in practical medicine. Water soluble vitamins are participants of metabolism as coenzymes and activators in many enzymatic reactions.

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 medicinals

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.

¬ To interprete the role of vitamins and their biologically active derivatives in mechanism of catalysis by enzymes of different classes.

¬ 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. Activation and inhibition of enzymes

• activators;• enzyme inhibition (reversible, irreversible, competitive, noncompetitive).

2. Regulation by change in catalytic activity of enzymes.• allosteric enzymes;• covalent modification of enzymes;• activation of enzymes by limited proteolysis;• the effect of regulatory proteins;• cyclic nucleotides in regulation of enzymatic processes.

3. Cofactors, coenzymes as prosthetic groups of enzymes. Classification of

Page 14: Methodical Guide 2011

coenzymes due to their chemical nature and type of catalytic reaction 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 • metalloporphyrins5. 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

7. Isoenzymes (isozymes), multiple forms of enzymes. Isoenzymes in enzymodiagnostics.

8. Changes in enzymatic activity of blood plasma and serum as diagnostic indexes (markers) of pathological processes in distinct organs - myocardial infarction, liver disease, pathology of muscle tissue.

9. Inborn (hereditary) and acquired metabolic defects, their clinical and laboratory diagnostics.

10. Enzymotherapy – the application of enzymes as drugs and medicinals.

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. Saliva is diluted twofold . Then three tubes are taken. Into tube #1 is poured 1 ml of water, tube # 2 – 0,8 ml of water and 0,2 ml of 1 % NaCl, #3 – 0,8 ml of water and 0,2 ml of 1% solution of CuSO4. Into each tube is added 1 ml of diluted saliva , mixed and then 2 ml of 1% starch solution. Tubes are incubated at 37C during 15 min and then reaction with iodine is performed. (0,1% solution of iodine in 0,2% sodium iodide). Changes in color development are observed and registered in a table (see below).

Page 15: Methodical Guide 2011

Tube content№ of tube

1 2 3Water (ml) 1 0,8 0,8NaCl, 1 %-solution (ml) - 0,2 -CuSO4, 5 %-solution (ml) - - 0,2

Saliva 2 fold diluted (ml) 1 1 1Starch, 1 %-solution (ml) 2 2 2Final 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. A copper probe for 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.

Examples of Krock-1 tests

Pharmaceuticals, containing mercury, arsen or other heavy metals, inhibit enzymes, posessing sulfhydril groups. What amino acid is used for reactivation of these enzymes?

HistidineIsoleucineCysteineAspartic acidGlycine

2. 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?

AllostericIrreversibleUncompetitiveCompetitiveNoncompetitive

Page 16: Methodical Guide 2011

3. After laboratory investigation in blood of patient an increase of LDH activity was detected, which is characteristic symptom of heart, liver or kidney diseases. What additional biochemical investigation must be performed in differential diagnostics?

Estimation of blood glucose level Ketone bodies level in bloodDetermination of LDH isozymesDetermination of blood cholesterol levelAmylase activity in blood

4. In a patient with complaints on pain in cardiac area a myocardial infarction was recognized after estimation of enzymes activity in blood. Indicate, what enzyme activities were determined?

Amylase, lipase, phosphataseLDH, creatine kinase, aminotransferasePeptidase, arginase, glucokinaseTrypsin, lysozyme, citrate synthaseAldolase, succinate dehydrogenase, hexokinase

5. In a patient with symptoms of acidosis (pH lowering in blood .) in urine was detected significant quantity of methylmalonic acid. This is caused by insuficiency of the next vitamin:B2B5B12 CD

Individual independent students work1. Regulators of enzymatic activity and their employment in clinical practice.2. Enzymodiagnostics. The use of enzymes in enzymodiagnostics.

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers

International Boston-London, 1992.- 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003.- 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994.- 443 p.6. Mardashko O.O., yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The

Odessa State Medical University, 2003.-416p.

Sense module № 7. Principles of metabolism. Tricarboxylic acid cycle.

Page 17: Methodical Guide 2011

Topic № 4. 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 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.

Theoretical questions:1. Conception of turnover of material and energy (metabolism). Characterization of

catabolic, anabolic and amphibolic reactions and their significance.2. Exergonic and endergonic biochemical reactions, role of ATP and other

macroergic phosphate containing compounds in their coupling. 3. Intracellular location of metabolic pathways, compartmentalization of metabolic

reactions in the cell. Methods of investigation of metabolism.4. Catabolic transformation of biomolecules: proteins, carbohydrates, lipids, its

characterization.5. The most important metabolites of amphibolic pathways in turnover of proteins,

carbohydrates, lipids, their significance for integration of metabolism in the cell.6. 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.

7. Anaplerotic and amphibolic reactions of TCA cycle.

Practical part

Experiment 1. Isolation of mitochondria from liver by method of differential

Page 18: Methodical Guide 2011

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 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. Three tubes – a control one, experimental N1 and N 2 – are filled with reagents as indicated in the table.

Content of tubes TubesControl Exp. N 1 Exp. N 2

Phosphate buffer, pH 7,4, ml 2,0 2,0 2,0Sodium pyruvate solution, ml 0,5 0,5 0,5

Page 19: Methodical Guide 2011

Malonic acid, ml 0,5Saline, ml 0,5 0,5Ca(OH)2 solution, ml 0,5 0,5 0,5

Suspension of mitochondria 0,5 0,5Boiled suspension of mitochondria 0,5

Incubation in thermostate 15 min at 37 oC0,1 M solution of sulphuric acid 1,0 1,0 1,0Result: production of CO2 bubbles

All tubes are placed in a thermostate for 15 min at 37 oC. Thereafter into each

tube is added 1,0 ml of 0,1 M solution of sulphuric acid and appearance of CO2 bubbles is observed and registered.

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. Three tubes (control, experiment 1 and experiment 2) are prepared as indicated in a table.

Reagents added TubesControl Exp.N1 Exp.N2

Phosphate buffer, pH 7,4, ml 2,0 2,0 2,0Solution of sodium pyruvate, ml 0,5 0,5 0,5Solution of malonate, ml - - 0,5Saline 0,5 0,5 -Suspension of mitochondria, ml - 0,5 0,5Suspension of boiled mitochondria, ml

0,5 - -

Solution of methylene blue, ml 0,5 0,5 0,5

Incubation at 37 oC in thermostateResults: time of medium decoloration

.Explain the results and draw a conclusion.

Experiment 4. Determination of FAD-dependent succinate dehydrogenase activity, an enzyme of respiratory chain .

Principle. Succinate dehydrogenase catalyses the elimination of two hydrogens from succinic acid transforming it into fumaric acid. As coenzyme is used FAD, which

Page 20: Methodical Guide 2011

is reduced in course of reaction to FAD H2 . The last reduces methylene blue which is changed to colorless substance..

Method. Two tubes are prepared as indicated in a table.

Reagents added TubesControl Experiment

Phosphate buffer, pH 7,4, ml 1,0 1,0Solution of succinic acid, ml 1,0 1,0Suspension of mitochondria - 0,5Suspension of boiled mitochondria, ml 0,5 -Solution of methylene blue, ml 0,5 0,5

Incubation at 37 oC in thermostateResults: 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 tests1. Enzymes of tricarboxylic acids cycle oxidize acetyl-CoA and produce 3 molecules of reduced NAD and one molecule of reduced FAD. Where are localized these enzymes? A. On plasma membraneB. In cell cytoplasmC. On external mitochondrial membraneD. In mitochondrial matrixE. On inner mitochondrial membrane

2. 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. Fatty acids biosynthesisB. Oxidative decarboxylation of α-ketoglutarateC. Neutralization of superoxide anionsD. Coupling of oxidation and phopsphorylationE. Microsomal oxidation

3. Substrate phosphorylation is the process of direct transfer of phosphate residue from substrates, which contain macroergic bond, upon ADP and production of ATP. What

Page 21: Methodical Guide 2011

enzyme of TCA cycle catalyzes the reaction of substrate phosphorylation?A. Citrate synthaseB. Succinate dehydrogenaseC. FumaraseD. Succinyl thiokinaseE.Complex of α-ketoglutarate dehydrogenase

4. What substance is the main fuel material for TCA cycle?A. Acetyl-CoA .B. GlucoseC. Amino acids D. Fatty acids E. Succinyl-CoA

Individual independent students work1. 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.

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers

International Boston-London, 1992.- 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003.- 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994.- 443 p.6. Mardashko O.O., yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The

Odessa State Medical University, 2003.-416p.

Sense module № 7. Molecular basis of bioenergetics.

Topic № 5. Molecular basis of bioenergetics.

Objective: to learn general principles of enzymatic respiratory chain organization in mitochondria, distinct oxido-reductases and their functional significance in tissue respiration. To master the methods of investigation of the next oxido-reductases: phenol oxidase, aldehyde dehydrogenase, peroxidase, cytochrome c. To learn the mechanisms of tissue respiration and oxidative phosphorylation.

Actuality of the theme: oxido-reductases 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, oxidative phosphorylation and its changes in different functional status of the body. Chemiosmotic theory of oxidative

Page 22: Methodical Guide 2011

phosphorylation explains molecular mechanisms of ATP production in course of biological oxidation.

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 NAD-H2 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.¬ To explain mechanisms of coupling of biological oxidation and oxidative

phosphorylation – biosynthesis of ATP.¬ To explain the main principles of chemiosmotic theory of oxidative

phosphorylation. ¬ To analyse the action of inhibitors and uncouplers of oxidative phosphorylation

of natural and artificial origin and their physiologicasl significance.

Theoretical questions:1. Reactions of biological oxidation and their functional significance:

• dehydrogenases• oxidases• oxygenases (mono- and dioxygenases)

2. Pyridine dependent dehydrogenases, structure of NAD and NADP, their role in reactions of oxidation and reduction.

3. Flavine dependent dehydrogenases. Structure of FAD and FMN, their role in reactions of oxidation and reduction.

4. Cytochromes and their role in tissue respiration. Structure of their prosthetic group.5. Molecular organization of electron transport chain of mitochondria

• constituents of respiratory chain in mitochondria• the sequence of electron transporters in respiratory chain • the significance of redox potentials in transport of electrons and protons

6. Points of introduction of reducing equivalents (electrons and protons) into respiratory chain of mitochondria.

7. Chemiosmotic thery of oxidative phosphorylation – molecular mechanism of ATP production in course of biological oxidation.

8. 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.

9. Inhibitors of electron transport in a respiratory chain of mitochondria.10. Uncouplers of electron transport and oxidative phosphorylation in a respiratory

chain of mitochondria.11. Microsomal oxidation.

Page 23: Methodical Guide 2011

Practical part

Experiment 1. Study of phenoloxidase activity. Principle. Method is based on the phenomenon of pyrocatechol oxidation by

oxygen in the presence of phenoloxidase. Method. The source of enzyme is fresh potato juice. 1 - 2 g of potato are minced

in a mortar, 25 ml of distilled water are added, mixed and press the juice into tube. (Iron instruments must not be used as ions of iron induce the darkening of juice).

Into 4 ennumerated tubes 0,5 ml of potato juice are added. To the first tube add 0,5 ml of pyrocatechol solution, to the second – 1 - 2 drops of inhibitor Na2S and 0,5 ml of pyrocatechol. The juice in the third tube is boiled and then O,5 ml of pyrocatechol is added. To the fourth tube O,5 ml of water are added. Tubes are placed on a water bath at 37o C for 30 min. The content is periodically shaken for better aeration. In the first tube brown colour appears due to oxidation of pyrocatechol . The color of other tubes does not 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 decoloration of methylen blue stain during its

reduction by aldehyde in presence of an enzyme aldehyde dehydrogenaseMethod. Into three ennumerated tubes the following solutions are added: 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. Tubes are placed for 30 min on a water bath at 37 C.

Result. Content of the first tube is decolorized due to the enzyme activity. Colour in the rest tubes does not change , as in the 2 tube substrate is absent, in the 3 - enzyme is heat denaturated.

Explain the results and draw a conclusion.

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. Into 3 ennumerated tubes the following reagents are added: (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. In the first tube a blue colour is developed due to oxidation of benzidine by H2O2 under catalysis of peroxidase. In the 2 and 3 tubes colour is not developed.

Explain the results and draw a conclusion.

Page 24: Methodical Guide 2011

Examples of Krock-1 tests1. NAD is: a participant of biological oxidation and can be classified as:A. Cofactor of dehydrogenasesB. MononucleotideC. HormoneD. LipidE. Vitamin

2. The component of respiratory chain enzymes cytochrome oxidase (cytochrome a+a3), operates as a transporter of:A. ProtonsB. ElectronsC. HydrogenD. Phosphate residuesE. Hydroxyl groups

3. Dehydrogenases are enzymes which catalyze transfer of the next chemical elements of the molecule:A. Phosphate groupB. Methyl groupC. HydrogenD. Amino groupE. Oxygen anion

4. The next process occurs in suspension of mitochondria with ruptured inner membrane and provided with malate and oxygen A. Transport of electrons along enzymes of respiratory chain B. Phosphorylation of ADPC. Decrease of pH in the external medium D. Increase of pH in mitochondrial matrixE. Oxydative phosphorylation will take place

Individual education of students.1. Modern data about the organization and functioning of respiratory chain as a

single polyenzyme complex.2. Uncouplers of oxidative phosphorylation and regulation of thermogenesis

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers

International Boston-London, 1992.- 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996.-654 p.3. Biochemistry / Trudy McKee, James R. McKee , 1999.- 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003.- 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Page 25: Methodical Guide 2011

Illustrated Reviews, 1994.- 443 p.6. Mardashko O.O., yasinenko N.Y. Biochemistry. Texts of lectures.-Odessa. The

Odessa State Medical University, 2003.-416p.

Topic № 7. Investigation of breakdown and biosynthesis of glycogen. Regulation of glycogen metabolism, biosynthesis of glucose – gluconeogenesis.

Objective. To learn reactions of synthesis and breakdown of glycogen; 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 a principal form of carbohydrate reserve in the body. It is deposited as as granular particles mainly in liver and muscle cells. The synthesis (glycogenesis) and decomposition (glycogenolysis) of glycogen are under strict regulation and control depending from nutrition, silent or active state of the body, etc. The principal regulatory role play the key enzymes of glycogen metabolism – glycogen phosphorylase and glycogen synthetase. Their activity is regulated by reversible phosphorylation and partially by mechanism of allosteric regulation.

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.

Theoretical questions:1. Glycogenesis: enzymatic reactions, biological significance. Regulation of glycogensynthetase activity.2. Splitting of glycogen in muscles and liver. Regulation of glycogen phosphorylase activity.3. Mechanism of regulation of gluconeogenesis and glycogenesis by the process of cascade ATP-dependent phosphorylation of enzymes. 4. Role of adrenalin, glucagone and insuline in hormonal regulation of glycogen metabolism in liver and muscles5. Hereditary disorders in enzymes of glycogen synthesis and breakdown. Glycogenoses, aglycogenoses, their characterization and causes.6. Guconeogenesis: reactions, mechanisms of regulation, biological significance of the process.7. Relations between glycolysis and gluconeogenesis (Cori cycle). Glucose-lactate and glucose-alanine cycles.

Practical part:Experiment 1. Reaction on polysaccharides.Principle. Iodine in presence of starch produce complex compounds with blue

Page 26: Methodical Guide 2011

color.Reagents: 1% solution of starch, Lugol solution (iodine dissolved in potassium

iodide), tubes, pipettes.Method. Into the tube is introduced 0,5 ml of starch solution and 1-2 drops of

Lugol solution are added. The appearance of blue color is observed. Changes in color during heating and cooling are observed. Note the results, draw the conclusions.

Make a conclusion. Experiment 2. Detection of glycogen in the liver.Principle. Glycogen in presence of iodine produces complexes with a red-violet

color (starch gives clear blue color).Reagents and materials. Fresh or frozen liver tissue, Lugol solution (iodine

dissolved in potassium iodide), 1% solution of acetic acid, porcelain mortar, water bath, paper filters, pipettes.

Method. 0,5 g of liver tissue is minced with scissors, mixed with 4 ml of boiling distilled water , transferred to the tube and boiled 2-3 min in order to inactivate enzymes, then transferred to the mortar and grinded to uniform mass. This homogenate is transferred to the tube, mortar washed with water and combined with the homogenate and boiled on a water bath for 20 min. It is recommended to add 5-10 droplets of acetic acid solution.

Precipitated proteins are eliminated by filtration through paper filter. To the filtrate are added 2-3 droplets of Lugol solution. In presence of glycogen a red-violet color develops.

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/. Increased concentration of glycogen in blood is observed in some infection diseases, which are accompanied with leukocytosis, diabetes mellitus, malignancies.

An important clinical significance has cytochemical detection of glycogen in blood cells, bone marrow cells and liver cells.

Control of laboratory work fulfillment1. What color compound is formed after adding of iodine solution to the homogenate, containing glycogen?А. Blue color compoundВ. Red color compoundС. Green color compoundD. Colorless compoundЕ. Violet color compound

2. Patient has cramps in muscles during strenuous physical work. Muscle tissue biopsy revealed significant excess of glycogen. The concentration of glucose in the blood is below normal. Deficiency of what enzyme is observed in this case?

3. It is known that glycogen delays as a reserve in liver and muscles, but does not create a reserve in the brain, which use glucose in large quantities. Explain why glycogen does not store in brain?

1. Examples of Krock-1 tests

Page 27: Methodical Guide 2011

1. 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 synthaseB. PhosphorylaseC. AldolaseD. Fructose bis-phosphataseE. Pyruvate carboxylase

2. Phosphorolysis of carbohydrates plays a key role in mobilization of polysaccharides. Under the action of phosphorylase from glycogen is produced the next substance:

A. Glucose -1-phosphateB. Glucose 1,6-bis-phosphateC. Glucose 6-phosphateD. GlucoseE. Fructose 6-phosphate

3. In an infant with point mutations in genes the absence of glucose 6-phosphatase, hypoglycemia and hepatomegaly were revealed. What disease is characterized by these symptoms?

A. Edison diseaseB. Gierke diseaseC. Parkinson diseaseD. Cori diseaseE. Mac Ardle 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. PhosphofructokinaseB. Glucose 6-phosphate dehydrogenaseC. Glycogen phosphorylaseD. Glycogen synthaseE. Glucose 6-phosphatase

5. Glycogen synthesis takes place under the action of several enzymes. Indicate, what enzyme provides the formation of 1,6-glycosidic bonds in glycogen molecule?

A. Glycogen synthaseB. HexokinaseC. GlucokinaseD. Glycosyl 4,6-transglycosidaseE. Glucose 1-phosphate uridil transferase

Individual independent students work1. Principles of regulation of glycogen biosynthesis and breakdown.2. Hereditary disorders of synthesis and breakdown of glycogen and

glycoconjugates.

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers

International Boston-London, 1992. – 980 p.

Page 28: Methodical Guide 2011

2. Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.3. Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003. – 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994. – 443 p.

Topic № 8. Alternative pathways of carbohydrate metabolism. Investigation of mechanisms of metabolic and hormonal regulation of carbohydrate metabolism. Diabetes mellitus.

Objectives: To understand the sequence of enzymatic reactions of pentose phosphate pathway. 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 during diabetes mellitus.

Actuality of the theme: The concentration of glucose in blood depends from the equilibrium between its secretion into blood and utilization by tissues. The regulatory system involves hormones insulin, glucagon, epinephrine, glucocorticoids, as well as interaction between distinct organs, i.e. liver, muscles, brain, etc. 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 interpret biochemical patterns of alternative pathways of monosaccharide’s metabolism.¬ 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. Pentosophosphate pathway of glucose oxidation; enzymatic reactions and biological significance.2. Metabolic pathways of fructose and galactose metabolism; enzymatic disorders of their metabolism.3. Biochemical processes which provides the constant blood glucose level. Role of different pathways of carbohydrate metabolism in regulation of blood glucose level.4. Hormonal regulation of carbohydrate metabolism (insulin, its structure, mechanism of action, role in carbohydrate metabolism; adrenalin and glucagon, mechanism of their regulatory effects on carbohydrate metabolism; Glucocorticoids, their effect on carbohydrate metabolism).5. Characterization of hypo- and hyperglycemia, glucosuria.6. Insulin dependent and noninsulin dependent forms of diabetes mellitus.7. Characterization of metabolic disorders in diabetes mellitus.8. Biochemical tests for evaluation of conditions of patients with diabetes mellitus.9. Disorders of carbohydrate metabolism during starvation

Page 29: Methodical Guide 2011

Practical partExperiment 1. Quantitative determination of glucose concentration in blood by

orto-toluidine method.

Principle. Heating of glucose with orto-toluidine in acetic acid gives compound of blue-green color, the intensity of color is proportional to glucose concentration.

Reagents and materials. Blood sample, 3% solution of trichloroacetic acid (TCA), orto-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.Procedure. Into each of two centrifuge tubes 0,9 ml of trichloroacetic acid are added. Into test tube 0,1 ml of blood specimen is introduced, into control tube – 0,1 ml standard glucose solution. Tubes are centrifuged at 3000 rpm for 10 min. From each tube 0,5 ml of supernatant is transferred to the clean tubes and 4,5 ml of orto-toluidine reagent is added. Tubes are placed on a boiling water bath for 8 min. Thereafter tubes are cooled and the intensity of color is measured in a colorimeter at wavelength 630 nm (red filter) in 1 cm pathway cuvette.

Calculation. The concentration of glucose is calculated using the formula: Ctest = Cstand × Atest/Astand

where: Ctest – concentration of glucose in blood , mmoles/L;Cstand – concentration of glucose in standard solutionAtest – optical density of test probeAtest – optical density of standard glucose probe.

Compare the obtained result with normal value, draw the conclusion.Experiment 2. Determination of blood glucose concentration by glucose oxidase

method.Principle. Glucose is oxidized by an enzyme glucose oxidase with production of

hydrogen peroxide. The last is used for oxidation of phenol and 4-aminoantipyrine in presence of peroxidase with formation of colored compound. The intensity of color is proportional to glucose concentration.

Reagents and materials. Working solution of enzymes, which contains 150 mg/l of glucose oxidase, 1,1 mg/l of horse radish peroxidase, 1,0 g/l of phenol, 0,15 g/l of 4-aminoantipyrine in 0,1 M phosphate buffer, pH 7,0. Standard glucose solution 5,55 mM/l, sample of blood serum or plasma.

Method. In tubes are mixed solutions as indicated in a table.Reagents Sample tube Glucose standard Control tubeBlood serum or plasma

0,02 ml – –

Glucose standard – 0,02ml –Working solution of enzymes

2,0 ml 2,0 ml 2,0 ml

Page 30: Methodical Guide 2011

After standing in room temperature for 30-60 min solutions are measured in colorimeter with a light filter 490-540 nm (blue-green light).The concentration of glucose is calculated according to a formula:

Ctest= Cstand × Atest /Astandwhere:Ctest – concentration of glucose in tested sampleCstand – concentration of glucose in standard sampleAtest – optical density of tested sampleAstad – optical density of standard sample.Compare the obtained result with a normal value and draw the conclusion.

Clinical and diagnostic significance. Physiological hyperglycemia is observed in emotional states, eating lots of carbohydrates with food. Pathological hyperglycemia is associated with diseases of the endocrine system, in diabetes, tumors of the adrenal and pituitary glands, liver function disorders, thyroid hyperfunction, organic lesions of the nervous system.

Hypoglycemia can be caused by starvation, heavy physical work, after overdose of insulin treatment, kidney diseases, accompanied by decrease in renal threshold for glucose.

Control of laboratory work fulfillment1. The concentration of glucose in the blood of a healthy individual varies within the following limits:А. 2 – 4 mmol/lВ. 10 – 25 mmol/l С. 3,5 – 5,5 mmol/lD. 6 – 9,5 mmol/lЕ. 1 – 2 mmol/l

3. In some people after a load of sugar a content of glucose in the blood may decrease below the initial level. Explain why?4. Patient transported to hospital ambulance. His status is heavy, consciousness marred, and such symptoms as adynamia, tachycardia, acetone smell from mouth are observed. What is the evidence of this pathology? What biochemical tests should be performed?

Examples of Krock-1 tests1. Increase in blood glucose concentration under the action of glucagone is caused by activation of the following enzyme:

A. HexokinaseB. GlucokinaseC. AldolaseD. Glycogen phosphorylaseE. Glycogen synthase

2. During the investigation in some person was revealed blood glucose concentration

Page 31: Methodical Guide 2011

on a level 4,5 mMoles/l. It may be the next interpretation of obtained result:A. A person is healthyB. A person has diabetes mellitusC. A person possess increased tolerance toward glucoseD. A person has diabetes insipidusE. A person has steroid diabetes

3. A patient was admitted to a hospital in comatose 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. HyperglycemicB. HypoglycemicC. AcidoticD. Hypothyroid E. Hepatic

4. A woman 46 years old complains on the feeling of dryness in mouth, thirst, frequent urination. In biochemical investigation of blood hyperglycemia was detected. In urine are present glucose, ketone bodies. In a patient can be suggested:

A. Alimentary hyperglycemiaB. Diabetes mellitusC. Acute pancreatitisD. Diabetes insipidusE. Cardiac ischemia

5. 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 diabetesB. Insulin dependent diabetes mellitusC. Addison diseaseD. Glycogenosis of the 1 typeE. Myxoedema

References:1. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International

Boston-London, 1992. – 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.3. Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003. – 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994. – 443 p.

Topic № 9. Investigation of catabolism and biosynthesis of triacylglycerols and phospholipids. Intracellular lipolysis and molecular mechanisms of its regulation.

Objective. To learn the processes of biosynthesis of phospholipids and

Page 32: Methodical Guide 2011

triacylglycerols and the main pathways of intracellular metabolism of lipids. To perform the methods of determination of phospholipids and activity of lipase and interpret the obtained results.

Actuality of the theme: The knowledge of principal pathways of intracellular metabolism of lipids in normal conditions and in pathology are necessary to 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.¬ 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. Biological functions of simple and complex lipids in human body (reserve,

energetic, thermoregulatory, production of biologically active substances)2. Involvement of lipids in formation of structure and function of biological

membranes.Fluid mosaic model of biomembranes. Liposomes. Application of liposomes in medical practice.

3. Circulatory transport and deposition of lipids in adipose tissue. Endothelial lipoproteinase.

4. 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.

5. Biosynthesis of triacylglycerols and phospholipids, the significance of phosphatidic acid as a precursor.

6. Metabolism of sphingolipids. Genetic anomalies of sphingolipid metabolism – sphingolipidoses. Lysosomal diseases.

7. Humoral regulation of lipolysis involving adrenaline, noradrenalin, glucagon and insulin

Practical partExperiment 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. Into the centrifuge tube 0,2 ml of blood serum and 2 ml of water are

Page 33: Methodical Guide 2011

added. 3 ml of 10 % solution of trichloroacetic acid is added and after 2–3 min the mixture is centrifuged 5 min at 3000 rpm. Supernatant is carefully discarded. To sediment, which contains lipoproteins, 1 ml of 56 % HClO4 is added and heated on a boiling water bath for 30 min. The final solution must be colorless.

Thereafter to the tube are added 5 ml of water, 1 ml of ammonium molybdate and 1 ml of 1% solution of ascorbic acid. Simultaneously a standard solution of phosphorus is prepared for comparison by mixing 1 ml of standard phosphorus solution (0,05 mg/ml), 5 ml of water, 1 ml of ammonium molybdate and 1 ml of ascorbic acid solution. After 15-20 min the extinction of probes is measured in a colorimeter.

Calculation: conducted according to formula: Total phospholipids in serum = (Eexp × 0,05 / Est × 0,2) x 25 mg/ml or g/lwhere Eexp – extinction of a sample probe,Est – extinction of phosphorus standard0,05 – concentration . of phosphorus in standard (mg/ml)0,2 – volume of analyzed serum25 – 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. Increasing of phospholipids level in blood serum (hyperphospholipidemia) is observed in heavy form of diabetes mellitus, nephrosis, obturative jaundice. Decreasing of phospholipids level (hypophospholipidemia) is observed in atherosclerosis, anemia, fever, alimentary distrophias, liver diseases.

Experiment 2. The influence of bile on the lipase activity.Principle. The lipase activity is estimated according to the production of fatty

acids during hydrolysis of fat. The quantity of fatty acids is determined by titration with alkali in the presence of phenolphthalein. Bile activates lipase thus accelerating the cleavage of lipids.

Method. Into two tubes or flasks 10 ml of milk and 0.5 ml pancreatine solution are added. Into the first tube 1 ml of water is added, into the second one - 1 ml of bile. After the mixing 1 ml of the mixture from each tubes is taken off and transferred to the other tubes, into which 1-2 drops of 0.5% phenolphthalein solution are added. The mixture is titrated with 0.05 N NaOH up to the pink color, which doesn’t disappear during 30 seconds. The volume of alkali is registered. The first two tubes with digestive mixture are placed at 38°C. Every 15 min 1 ml of the mixture from each tube is taken off and transferred to the clean tubes and the titration is conducted. 4-5 subsequent determinations are conducted. The results are registered as ml of alkali solution, expended for titration. Obtained results are noted in the table.

Time, min 0 15 30 45 60

Quantity of NaOH expended for titration, ml

Page 34: Methodical Guide 2011

Lipase activity in presence of bile

Lipase without bileLipase activity in absence of bile

Lipase without bile

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 emulsifies 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 emulsification, digestion and absorption of lipids and fat soluble vitamins as well may take place in diseases of liver, gall bladder or biliary ducts (cholelythiasis).

Control of laboratory work fulfillment1. What is the principle of the method of phospholipids determination in blood serum?

A. A. Precepitation of phospholipids by trichloroethanoic acid and determining the content of phosphorus B. Formation of complex compound in the presence of acetate anhydride and solution of acetate and sulphate acidsC. Formation of colored products with oxymethylfurfurolD. Formation of a blue color with 1,2-dihydrophenol and reduction by ascorbic acid

2. Indicate the concentration of total phospholipids in blood serum of healthy adult:A. 0,25 – 1,25 g/lB. 1,52 – 3,62 g/lC. 3,75 – 5,40 g/lD. 10,15 – 12,30 g/l

Page 35: Methodical Guide 2011

E. 9,80 – 15,72 g/l

3. How can we estimate the activity of lipase?A. For the ability of enzyme to hydrolyze peptide bondsB. By the concentration of pyruvic acid formation C. With the ability to decolorize methylene blue during its reduction by aldehydeD. By formation of fatty acids after hydrolysis and their determination with titration at the presence of phenolphthaleinE. For their ability to inhibit reduction of nitrotetrazolium blue at the presence of NADH2

4. A person was limited in the use of products containing phospholipids for a long period of time. To what metabolic disorders it can lead?5. The analysis of human serum revealed 5 mmol/l of total cholesterol, 3,2 mmol/l – of phospholipids, 1,5 mmol/l of triglycerides. How can you explain these results?6. The concentration of phospholipids in blood is 0,7 g/liter. What is the main reason of this state and what metabolic disturbances are also observed in this case?

Examples of Krock-1 tests1. Arachidonic acid as essential nutrient is needed for normal growth and development of animal and man. It is precursor of biologically active substances. Indicate what compounds are synthesized from arachidonic acidA. CholineB. NoradrenalinC. EthanolamineD. TriiodothyronineE. Prostaglandin E1

2. After consumption of fat meal in a patient appears nausea and heartburn, steatorrhea also develops. A cause of such symptoms may be:Amylase insufficiencyHypersecretion of lipaseDisorder in phospholipase synthesisDisorder in trypsin synthesisBile acids insufficiency

3. A victim of a snake’s beat almost dyed due to very intensive hemolysis. Blood analysis detected unusually high content of lysolecitine. The toxic effect of snake’s venom was caused by the presence of:Phospholipase A1Phospholipase A2Phospholipase CPhospholipase DNeuraminidase

Page 36: Methodical Guide 2011

4. The form in which most dietary lipids are packaged and exported from the intestinal mucosa cells is as follows:Free fatty acidsMixed micellesFree triacylglycerol2-monoacylglycerol E. Chylomicrons

5. Which of the following substances is involved in the synthesis of both glycerol-containing phospholipids and triacylglycerol?A. Acetoacetyl CoAB. CholineC. Phosphatidic acid D. CDP-EthanolamineE. 3-Hydroxyburyrate

References:1. Medical biochemistry / Bhagavan N.V. Jones and Bartlett Publishers International Boston-London, 1992. – 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.3. Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell, 2003. – 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s Illustrated Reviews, 1994. – 443 p.

Topic № 10. β–Oxidation and biosynthesis of fatty acids. Investigation of metabolism of ketone bodies.

Objective. To learn reactions of biosynthesis and oxidation of fatty acids. To know metabolic pathways of ketone bodies formation in normal conditions and in pathology states, to determine their concentrations in urine.

Actuality of the theme: Oxidation of lipids, as well as ketone bodies metabolism is an important constituent of energetic metabolism in sense of providing tissues and cells with ATP. Determination of ketone bodies concentration in blood and in urine has an important significance in diagnostics of several pathological processes.

Specific aims:¬ To interpret biochemical patterns of 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

Page 37: Methodical Guide 2011

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; reactions and 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 NADP H2 for biosynthesis of long chain fatty acids; the sequence of enzymatic reactions in biosynthesis of long chain fatty acids regulation of biosynthetic process on level of acetyl-CoA-carboxylase and fatty acid synthetase; elongation of carbon chain of fatty acids; synthesis of unsaturated 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; the notions of ketoacidosis, ketonemia, ketonuria.

Practical partExperiment 1. Qualitative reaction on acetone and acetoacetic acid (Lange

probe).Principle. It is based on a property of acetone to produce violet color compound

after an overlaying of concentrated ammonia solution over the mixture of test sample, containing sodium nitroprussid and acetic acid.

CH3 – CO – CH3 + Na2 [Fe (CN)5 NO] + 2 NaOH → Na4 [Fe (CN)5 NO = CHCOCH3] + 2 H2O

Acetoacetic acid can also form cherry-red colored complex compound of iron chloride (III).

Na4 [Fe (CN)5 NO =CHCOCH3] + CH3COOH →Na3 [Fe (CN)5 NOCH3COCH3] + CH3COONa

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).

Reagents and materials. Urine of patient with diabetes mellitus and urine of healthy person, 10% sodium nitroprussid solution, concentrated acetic acid, 10% solution of NaOH solution, ammonium sulfate, concentrated ammonia solution, 10% solution of iron chloride (III), pipettes, test tubes, funnel, filter paper

Method. Into two tubes 0.5 ml of tested urine is added: to the first tube - from healthy person (control), to the second one – from patient with diabetes mellitus (test sample). To both tubes 2 drops of concentrated acetic acid, 5 drops of fresh 10% sodium nitroprussid solution are added; thereafter the content of the tubes is mixed. An equal volume of concentrated ammonia is overlaid carefully omitting the mixing of two liquids. It may be observed an appearance of red-violet or violet colored layer in a form of ring at the border of two layers in the second tube with a test sample,

Page 38: Methodical Guide 2011

containing ketone bodies. Explain the results, draw a conclusion.Acetoacetic acid can also produce a complex compound of cherry red color after

interaction with ferric chloride. This is used in Gerhardt probe for detection of acetoacetic acid in urine.

Method. Into the tube 2 ml of tested urine are added and by droplets a solution of FeCl3 is introduced up to complete precipitation of iron phosphates. The precipitate is eliminated by filtration and several additional droplets of FeCl3 solution are added. In presence of acetoacetic acid a cherry red color appears.

Explain the results.

Experiment 2. Quantitative determination of ketone bodies in urine.Principle. (See previous experiment).Method. 8 tubes are taken for the experiment. Into each tube, except the first one,

1 ml of distilled water is added. Into the first and the second tubes 1 ml of urine of the patient with diabetes mellitus are added. Thus, in the first tube urine is undiluted; in the second one the urine is two times diluted. After the mixing 1 ml of the mixture from the second tube is taken off and transferred to the third tube, after mixing 1 ml from the third tube – into the following one and so on. Into each tube 8 drops of 50% ammonium sulfate solution are added for increasing the density of the solutions, thereafter 8 drops of concentrated acetic acid and sodium nitroprussid are added. The content of the tubes is mixed and into each tube 1 ml of concentrated solution of ammonia is overlaid carefully beginning from the last tube (be careful with concentrated ammonia!). An appearance of red or violet ring in the tubes is registered. The last tube in which a colored ring appears between the 3-d and the 4-th min is registered and the dilution of urine sample is noted.

Calculation: X = 0.85 × A × 15, wereX – content 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).Explain the results.

Clinical and diagnostic significance. Ketone bodies include the following substances – acetoacetic acid, -hydroxybutyric acid and 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 persons 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 in 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

Page 39: Methodical Guide 2011

value. In early childhood prolong disorders of digestion (toxicosis, dysenteria) may lead to ketonemia due to the permanent starvation and exaggeration.

Control of laboratory work fulfillment1. What is the principle of the method of determination of ketone bodies by Lange’s test?

A. A property of acetone to produce violet color compound after overlaying of concentrated ammonia solution over the mixture of test sampleB. Formation of colored condensation products with oxymethylfurfurolC. Formation of fine-grained emulsionD. Formation of green colored compound with acetic anhydride and solution of acetic and sulfuric acids2. Indicate the content of ketone bodies in blood in healthy person:2,5 – 8 mmol/lA. 0 – 2,5 mmol/lB. 180 – 250 mmol/lC. 13 – 185 mmol/lD. 205 – 317 mmol/l3.

3. What test should be used for detection of acetoacetate in urine?Gerhardt probe A. Barfood probeB. Bohomolov probeC. Pettenkofer's ProbeD. Hmelin’s probe

4. What is the principle for the detection of ketone bodies by Gerhardt probe?A. Formation of a red colored solution with oksymetylfurfurolB. Appearance of red colored complex compound of iron chloride (III)C. Reduction of benzidine by oxygenD.Formation of yellow colored iron lactate E. The ability to form a red color with diazo reagent

6. In patient’s blood was detected a high concentration of free fatty acids. What is the main reason for this?

Examples of Krock-1 tests1. Lipids are obvious energetic material for the body. What is the main pathway of fatty acids metabolism in mitochondria?A. ReductionB. DecarboxylationC. β-OxidationD. α -OxidationE. -Oxidation

2. In diabetes mellitus and starvation there is an increase of ketone bodies content in blood, which are utilized as energetic material by tissues. Note the substance which is used in ketone bodies synthesis.A. CitrateB. Acetyl-CoAC. Succinyl-CoAD. α –Ketoglutarate

Page 40: Methodical Guide 2011

E. Malate

3. During the prolong starvation in blood of the person an increase in ketone bodies content occurs. It is caused by the next factors:A. Decrease of free fatty acid level in blood plasmaB. Mobilization of high density lipoproteins C. Production of acetyl-CoA.D. Enhance of fatty acids oxidation in liverE. Decrease of triacylglycerols in adipose tissue

4. In a patient suffering from diabetes mellitus in blood was detected acetone. Note the process of its production in the body.A. In course of б-oxidation of fatty acidsB. In course of в-oxidation of fatty acidsC. By condensation of two molecules of acetyl-CoAD. In course of г-oxidation of fatty acidsE. In tricarboxylic acid cycle.

5. Which one of the following statements about the absorption of lipids from the intestine is correct?A. Dietary triacylglycerol is partially hydrolyzed and absorbed as free fatty acids and monoacylg.ycerol;B. Dietary triacylglycerol must be completely hydrotyzed to tree fatty acids and glycerol before absorption; C. Release of fatty acids from triacylglycerol in the intestine is inhibited by bile salts; D. Fatty acids that contain ten carbons or less are absorbed and enter the circulation primarily via the lymphatic system; E. Formation of chylomicrons does not require protein synthesis in the inteslinal mucosa.

References:1. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International

Boston-London, 1992. – 980 p.2. Biochemistry / Trudy McKee, James R. McKee, 1996. – 654 p.3. Biochemistry / Trudy McKee, James R. McKee, 1999. – 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A.

Mayes, Victor W. Rodwell, 2003. – 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s

Illustrated Reviews, 1994. – 443 p.

Topic № 11. Investigation of biosynthesis and biotransformation of cholesterol. Pathologies of lipid metabolism.

Objective. To learn general pathways of cholesterol metabolism and principal disorders of lipid metabolism.

Actuality of the theme: Disorders in cholesterol biotransformation processes cause a variety of diseases, such as atherosclerosis, obesity et al. In this connection the

Page 41: Methodical Guide 2011

investigation of lipid metabolism indexes is obvious for diagnostics and treatment of different diseases.

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 and excretion of cholesterol from the body.

¬ To explain biochemical bases of genetic disorders of lipid metabolism. Atherosclerosis, diabetes mellitus, obesity, steatorrhea.

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.2. Pathways of cholesterol biotransformation (esterification, production of bile acids and steroid hormones, synthesis of vitamin D3, excretion from the body).3. Atherosclerosis, mechanism of its development, role of genetic factors, hypercholesterolemia, WHO classification of hyperlipidemias.4. Disorders of lipid metabolism in diabetes mellitus.5. Pathological processes which leads to the development of obesity6. Lipoproteins of blood plasma, structure and physiological significance.

Practical partExperiment 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.

Reagents and materials. Bile, 20% sucrose solution, concentrated H2SO4, tubes.Method. 10-15 droplets of bile are introduced into tube, then one droplet of fresh

20% sucrose solution. This mixture is overlaid carefully onto 1 ml of conc. sulfuric acid in another tube without mixing two fluids. In the borderline between two fluids the precipitation of bile acids is observed and appearance of violet color ring.

Clinical and diagnostic significance. 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).

Experiment 2. Determination of low-density lipoprotein (LDL).Principle. The method is based on precipitation of LDL in the presence of СаCl2

and heparin, causing turbidity of the solution, which is proportional to the

Page 42: Methodical Guide 2011

concentration of LDL. Reagents and materials. Blood serum, 0.025 M calcium chloride solution,

pipettes, micropipettes.Method. In the test tube are added 2 ml of 0.025 M calcium chloride, 0,2 ml of

blood serum. The content is mixed thoroughly and the optical density is measured (E1) at a wavelength of 630 – 690 nm (red light filters) against distilled water. The solution is poured from into a tube, filled up with 0.04 ml 1% solution of heparin and carefully mixed. In 4 min. the optical density of solution (E2) is determined under similar conditions.

The difference (E1 - E2) indicates the optical density, due to the presence of LDL.Calculations should be performed according to the formula:

Х = (Е1 – Е2) × 100The content of LDL is expressed in units of optical density, multiplied on the

dilution (100). Normal values of LDL is 35 – 55 cuExplain the result. Make a conclusion.Clinical and diagnostic significance. LDL are synthesized in the liver and

represent about 5% of blood plasma proteins. In the lipid part of LDL predominate sterols: cholesterol and its esters – 45% phospholipids – 25%, triglycerides – 10%. High content of cholesterol and small sizes of molecules facilitate their infiltration into aorta. This leads to atherosclerosis. In this regard, this fraction of lipoprotein is called atherogenic lipoproteins.

In normal conditions the content of LDL and HDL is 3.6 and 2.6 g /l, respectively. The ratio between LDL and HDL in the organism is essential. For example increasing of concentration of LDL is observed in atherosclerosis, jaundice, hepatitis, diabetes, obesity etc.

Control of laboratory work fulfillmentWhat qualitative reaction is used for detection of bile acids in biological fluids?

A. Lange probeB. Biuret testC. Petenkoffer reactionD. Benzidine probeE. Foll reaction

What is the significance of bile acids in digestion and assimilation of lipids?A. Hydrolysis of lipidsB. Accelerating of digestion and absorption of lipidsC. Transfer of monosaccharidesD. Prevents proteolysisE. Excretion of lipids from the organism

The concentration of cholesterol in blood is 12 mg/liter. Give clinical interpretation of these results, indicate the possible causes and consequences for the organism

4. A high level of cholesterol in the LDL fraction was revealed during the blood test. What are the causes and possible consequences for the patient’s organism.

Page 43: Methodical Guide 2011

Examples of Krock-1 tests1. A man 67 years old suffers from brain vessels atherosclerosis. After investigation hyperlipidemia was detected. What class of lipoproteins in blood plasma will be increased most of all in biochemical investigation?HDLNon esterified fatty acids in complex with albuminChilomicronsLDLVLDL

2. A child 5 years old suffers from transient abdominal pains.On a skin – xanthoma plaques are found.. After investigation a hepatosplenomegala is detected.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?HDLIDLChylomicronsLDLVLDL

3. In a patient suffering from diabetes mellitus an increase in concentration of VLDL and triacylglycerols was detected. Cholesterol and HDL content in normal values.What type of lipid metabolism disorder can be classified such changes of indicated data?Hyperlipoproteinemia type IIHyperlipoproteinemia type YHypelipoproteinemia type IIIHyperlipoproteinemia type IYHyperlipoproteinemia type II b4. Bile acids are derivatives of:A. HemeB. CholesterolC. SphingomyelineD. Phosphatidyl cholineE. Long chain fatty acids

5. In course of investigation of the patient with symptoms of atherosclerosis there was detected substantial decrease of HDL, increase of LDL, cholesyterol concentration 11 mM/L. Decrease of activity of what enzyme is the most possible cause of changes observed?Blood lipoprotein lipaseTissue lipasesPancreatic phospholipasesCholesterol esteraseLecitin cholesterol acyl transferase

References:

Page 44: Methodical Guide 2011

1. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International Boston-London. – 1992, 980 p.2. Biochemistry / Trudy McKee, James R. McKee. – 1996, 654 p.3. Biochemistry / Trudy McKee, James R. McKee. – 1999, 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell. – 2003, 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s Illustrated Reviews. – 1994, 443 p.

Topic № 12. Studies on turnover of amino acids. Biogenic amines. Investigation of synthesis and detoxification of ammonia. Urea biosynthesis.

Objective. To learn general pathways of turnover of amino acids and pathways of production, transport and neutralization of ammonia in human body. To learn the methods of determination of urea, phenylpyruvic acid in biological fluids and interpretation of obtained results.

Actuality of the theme. Protein metabolism plays the most important role in general whole body metabolism. The competence and understanding of general pathways of amino acids transformations, their metabolic intermediates, determination of activity of enzymes, participating in these processes are criteria for evaluation of protein metabolism. In course of amino acid metabolism are produced metabolites, which can be detected and quantified in blood and urine and may be used in diagnostics and treatment monitoring.

Specific aims.¬ To explain biochemical patterns of intracellular metabolism of amino acids: processes of dezamination, transamination, decarboxylation, explain the biological mechanism of action of biological amines: serotonin, histamine, gamma-amino butyric acid, etc.¬ To interpret metabolic pathways of production and neutralization of ammonia, circulatory transport of ammonia, urea biosynthesis.¬ To analyze changes in processes of transport and neutralization of ammonia in hereditary anomalies of enzymes of ammonia turnover.¬ To identify alaninaminotransferase activity in blood serum and to interpret the obtained result¬ Quantitative determination of urea in biological fluids. Interpretation of obtained results and conclusions

Theoretical questions:1. Pathways of formation and maintainance of free amino acid pool in human body. General pathways of free amino acid turnover.2. Types of reactions of amino acid deamination their final products. Mechanism of oxidative deamination, oxidases of D- and L- amino acids, their enzymatic activity and specificity. 3. Glutamate dehydrogenase structure of the enzyme, reactions, biological significance4. Transamination of amino acids, substrates for transamination reaction. Mechanism of transamination. Reaction. Aminotransferases, their localization in tissues and organs.

Page 45: Methodical Guide 2011

Clinical diagnostic significance of determination of aminotransferases activity.5. Decarboxylation of amino acids, decarboxylases. Production of biogenic amines (GABA, histamine, setrotonin, dopamine). Decarboxylation of amino acids in putrifaction of proteins in intestines. Oxidation of biogenic amines. 6. Pathways of ammonia production. Toxicity of ammonia and mechanisms of its detoxification. Circulatory transport of ammonia (glutamine, alanine).7. Biosynthesis of urea: enzymatic reactions, ammonia sources; energetics.8. Hereditary defects of enzymes involved in urea synthesis.

Practical part Experiment 1. Determination of activity of alanine aminotransferase.Principle. Under the influence of aminotransferase amino group from alanine is

transferred to alpha-ketoglutarate resulting the formation of pyruvate and glutamate. Pyruvate forms a colored hydrazone after reaction with dinitrophenylhydrazine. In alkaline conditions pyruvate hydrazone gives a product of red-brown color, which intensity is proportional to concentration of pyruvate. According to the quantity of formed pyruvate the activity of subsequent amino transferase can be evaluated quantitatively.

Method. Into control and test tubes 0.5 ml of substrate mixture are added and tubes are preincubated at 37°C during 5 min. Thereafter to the test tube 0.1 ml of serum is added, into control tube – 0.1 ml of water. Both tubes are incubated 30 min at 37°C. After finishing of incubation time into each tube 0.5 ml of 0.1% solution of dinitrophenylhydrazine are added and tubes are left at room temperature for 20 min. Thereafter to each tube 5 ml of 0.4 M solution of NaOH are added and left at room temperature for 10 min more. Then optical density (extinction) is measured in colorimeter at 500-560 nm wavelength. The quantity of pyruvate (in μg) is determined according to calibration curve or according to formula:

X = D / 0.09 , where X – quantity of pyruvate in μg,D – Optical density.

Calculation of transaminase activity. 1 unit of alanine aminotransferase is such quantity of enzyme, which produce 1 μg of pyruvate in described conditions. The calculation of enzyme activity to micromoles of pyruvate, formed by 1 ml of serum in one hour is provided according to formula:A = (X´2´10) / 88 ,where X - quantity of pyruvate in g,

2 – coefficient for calculation for an hour of incubation,10 – coefficient for calculation for 1 ml of serum,88 – molecular weight of pyruvate.

Clinical diagnostic significance. In human body the process of transamination occurs in the liver, hear, skeletal muscles, kidneys and other organs and tissues. In blood plasma transaminase activity is very low during normal conditions. When a cell membrane is damaged and the integrity of cell is breached and aminotranferase migrates into the blood. Thus the estimation of aminotranferase activity in blood serum is important in diagnoses, especially in myocardial infarction, viral hepatitis and liver

Page 46: Methodical Guide 2011

cirrhosis.Considerable increase in ALT activity (10 – 100 times normal values) is observed

in cases of viral and toxic hepatitis, blood circulation insufficiency during shock and hypoxia.

Moderate increase of ALT activity occurs during liver cirrhosis, chronic hepatitis during the acute phase, obstructive jaundice, liver swelling during cardiac insufficiency and degenerative processes of the kidneys, lungs, muscles and pancreatic gland.

Considerable increase in AST activity is observed during myocardial infarction, viral hepatitis, toxic liver destruction and blood circulation insufficiency during shock and hypoxia.

Moderate increase of AST activity is detected during liver cirrhosis, obstructive jaundice, liver malignancies, skeletal muscules destruction, pancreatitis, pneumonia and hemolytic anemia.

Normal values in blood serum: ALT – 0.1 – 0.7 moles/hr´ml,AST – 0.1 – 0.45 moles/hr´ml.

Explain the results, draw the conclusion.Experiment 1. Determination of urea in urine.Principle. The method is based on the property of urea to form with

paradimethylaminobenzaldehyde in acidic medium a complex compound of yellow colour. The intensity of colour is proportional to the concentration of urea in urine and may be determine colorimetrically.

Reagents and materials. Urine, 2% paradimethylaminobenzaldehyde solution, standard solution of urea (2,5%), pipettes, micropipettes, dry test tubes

Method. Into tube 0.2 ml of urine is added and well mixed with 1.2 ml of 2% paradimethylaminobenzaldehyde solution. After 15 min extinction of the probe is measured in a colorimeter with blue lightfilter in cuvette with 20 mm pathway. The concentration of urea is calculated according to calibration curve, prepared for distinct urea concentrations.

Explain the results, draw the conclusion.

Control of laboratory work fulfillment1. What substrate mixture is used for determination of the activity of ALTА. α- ketoglutarate and alanineВ. α- ketoglutarate and glycineС. α- ketoglutarate and aspartateD. Oxaloacetate and alanineЕ. Oxaloacetate and glutamate

2. Hydrazone of pyruvic acid in alkaline medium forms a reddish-brown color, the intensity of which is proportional to the concentration of… А. PyruvateВ. OxaloacetateС. α- ketoglutarateD. AlanineЕ. Aspartate

3. Patient, complaining of pain in the left half of the chest, shortness of breath during

Page 47: Methodical Guide 2011

the rest was admitted to the hospital. Diagnose myocardial infarction. Laboratory tests: creatine kinase 12 mmol/min.l (normal 06), AST 60 mmol/min.l (normal 220), LDH 15 mmol/(h.ml) (normal 4,08,8). Is this diagnosis confirmed the results of laboratory testing?

4. What is the content of urea in blood and urine of healthy person? What method is used for its determination?

Examples of Krock-1 tests1. In a child, consuming meal of plant origin for a long period of time growth retardation, anemia, liver and kidney impairment were observed. The cause of such state is deficiency in diet of the next nutrients:Lipids Essential amino acidsCarbohydrates Mineral macroelements Carotene2. Which of the following amino acids can undergo deamination by dehydration?A. GlutamineB. LeucineC. Threonine D. Valine E. Lysine

3. According to clinical indications a patient was administered pyridoxal phosphate. What processes is this medication intended to correct? ATransamination and decarboxylation of aminoacids B Oxidative decarboxylation of ketonic acids C Desamination of purine nucleotide D Synthesis of purine and pyrimidine bases E Protein synthesis

4. The greater amount of nitrogen is excreted from the organism in form of urea. Inhibition of urea synthesis and accumulation of ammonia in blood and tissues are induced by the decreased activity of the following liver enzyme: ACarbamoyl phosphate synthetase B Aspartate aminotransferase C Urease D Amylase E Pepsin

Individual independent students work1. Features of urea cycle in normal and pathological conditions

References:1. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International Boston-London. 1992, 980 p.

Page 48: Methodical Guide 2011

2. Biochemistry / Trudy McKee, James R. McKee. 1996, 654 p.3. Biochemistry / Trudy McKee, James R. McKee. 1999, 288 p.4. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell. 2003, 927 p.5. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s Illustrated Reviews. 1994, 443 p.

Topic № 13. Investigation of turnover of specialized pathways of amino acids in tissues.

Objective. To know specialized pathways of amino acids turnover in humans. To understand methods of determination of phenylpyruvic acid in biological fluids and creatinine in blood serum. To interpret the obtained results.

Actuality of the theme. Determination of distinct metabolic intermediates of amino acids metabolism in blood and urine is widely used in medical practice.

Specific aims.• To explain general metabolic pathways of nitrogen free residues of amino acids and peculiarities in transformation of aromatic and heterocyclic amino acids.• To explain biochemical basis in development and manifestation of genetic anomalies in metabolism of aromatic and heterocyclic amino acids, accumulation of distinct metabolic intermediates in phenylketonuria, alkaptonuria, albinism.¬ To identify phenylpyruvic acid in biological fluids and creatinine in blood serum. ¬ Interpretation of obtained results and conclusions

Theoretical questions:1. General metabolic pathways of nitrogen free residues of amino acids in human body. Glucogenic and ketogenic amino acids.2. Special pathways of noncyclic amino acids metabolism. Metabolism of glycine and serine, role of tetrahydrofolate in transfer of one carbone fragments. Inhibitors of dihydrofolate reductase as antitumor agents.3. Metabolism of sulfur containing amino acids, reactions of methylation.4. Peculiarities of metabolism of branched chain amino acids, role of vitamin B12 in metabolism of amino acids.5. Metabolism of arginine. Biological significance of nitric oxide, NO-synthase.6. Specific pathways of metabolism of aromatic amino acids phenylalanine and tyrosine, sequence of enzymatic reactions. 7. Hereditary enzymopathias of phenylalanine and tyrosine metabolism – phenylketonuria, alkaptonuria, albinism.8. Metabolism of tryptophane: kinurenic and serotonin pathways. Hereditary enzymopatias. 9. Glutathion, structure and its role in metabolism of organic peroxides.10. Production of creatine and creatinine, clinical and diagnostic significance of disorders in their metabolism.

Practical partExperiment 1. Qualitative reaction on phenylpyruvic acid. (Fehling test).

Page 49: Methodical Guide 2011

Principle. Phenylpyruvic acid forms a complex compound with Fe+3 ions, which has a blue-green color.

Materials and reagents. 10% solution of FeCl3, filters, pipettesMethod. To 2 ml of prefiltered urine are introduced 8–10 droplets of 10%

solution of FeCl3 In the presence of phenylpyruvic acid a blue-green color appears after 30–60 seconds, which gradually disappear during 5–30 min of standing in dependence from the content of phenylpyruvic acid in urine

Clinical and diagnostic significance. Accumulation of phenylpyruvic acid in blood and tissues leads to general intoxication of organism. As a result a violation of normal brain development and severe neurological disorders are observed. Diagnostic criteria for this hereditary disease are the high content of phenylalanine in the blood and the presence phenylpyruvic acid in urine.

Normally, children’s blood contains the following concentrations of phenylalanine: before 1 month – 0.133 mmol/l, from 1 month – 1 year – 0.095 mmol/l, from 1 year – 14 years – 0.115 mmol / liter.

Experiment 2. Method of creatinine determination according to Jaffe color reaction. (Popper et al. ).

Principle. After interaction of creatinine and picric acid in alkaline medium a colored compound is formed, the intensity of color is proportional to the concentration of creatinine.

Materials and reagents. Solution of saturated picric acid (about 2%); standard solution of creatinine: 100 mg of creatinine iare dissolved in 100 ml of 0,1 n HCl; 10% solution of NaOH; 0,1 n solution of HCl; centrifuge, water bath, photocolorimeter.

Method. 2 ml of blood serum are mixed with 6 ml of picric acid solution. After 5 min the tube is placed into boiling water bath for 20-30 sec and then formed sediment is eliminated by centrifugation or filtration. To 4 ml of supernatant fluid are added 0,2 ml of 10% NaOH , mixed and adjusted the volume up to 10 ml. Standard probe is prepared using 2 ml of standard solution of creatinine instead of serum. Control probe is also prepared by taking 2 ml of water instead of test samples. After standing for 10 min probes are measured in a colorimeter at green light beam (530 nm) in a cuvete with an optical pathway 20 mm. Concentration of creatinine is calculated according the formula:

X = (Eexp/Est )×0,088 mMoles/l, where:Eexp – extinction of tested sampleEST – extinction of standard solution 0,088 mMoles/l – concentration of creatinine in standard solution.

Clinical and diagnostic significance. Creatinine is an end product of creatine metabolism. Concentration of creatinine in blood plasma is relatively constant and reflects the amount of muscle mass and does not depend from diet or other factors. Creatinine is not reabsorbed in renal tubules and its clearance value is used for estimation of glomerular filtration in kidneys.

In normal conditions concentration of creatinine in women – is 0,044 – 0,097 mMoles/l, in men – 0,044 – 0,115 mMoles/l. Increase in creatinine concentration is

Page 50: Methodical Guide 2011

observed in acute and chronic kidney diseases, a decrease – in the 1-2 trimesters of pregnancy, in lowering of muscle mass due to age or myodystrophic changes.

Control of laboratory work fulfillment1. After interaction of creatinine and picric acid in alkaline medium a colored compound is formed, the intensity of color is proportional to the concentration of … А. CreatinineВ. CreatineС. α- ketoglutarateD. AlanineЕ. Aspartate

2. Normative values of creatinine in blood serum for women (mmol / l) is: А. 0,044 – 0,097 В. 0,440 – 0,970 С. 4,400 – 9,700 D. 44,000 – 97,000 Е. 440,000 – 970,000

3. Normative values of creatinine in blood serum for men (mmol / l) is:A. 0,053 – 0,115B. 0,53 – 1,15C. 5,3 – 11,5D. 53 – 115E. 530 – 1150

4. What reaction is used for detection of phenylketonuria?

Examples of Krock-1 tests1. Laboratory examination of a child revealed increased concentration of leucine, valine isoleucine and their ketoderivatives in blood and urine. Urine smelt of maple syrup. This disease is characterized by the deficit of the following enzyme: A. Dehydrogenase of branched amino acids B. Aminotransferase C. Glucose-6-phosphatase D. Phosphofructokinase E. Phosphofructomutase

2. A 13-year-old boy complains of general weakness, dizziness, tiredness. He is mentally retarded. Increased level of valine, isoleucine, leucine is in the blood and urine. Urine has specific smell. What is the diagnosis?A. Maple syrup urine diseaseB. Addison's diseaseC. TyrosinosisD. HistidinemiaE. Graves' disease

3. Albinos can't stand sun impact - they don't aquire sun-tan but get sunburns. Disturbed metabolism of what aminoacid underlies this phenomenon?

Page 51: Methodical Guide 2011

A. Phenilalanine B. Methionine C. Tryptophan D. Glutamic acid E. Histidine

4. Nappies of a newborn have dark spots that witness of formation of homogentisic acid. Metabolic imbalance of which substance is it connected with? A. Thyrosine B. Galactose C. Methionine D. Cholesterine E. Tryptophane

5. A 1,5-year-old child presents with both mental and physical lag, decolorizing of skin and hair, decrease in catecholamine concentration in blood. When a few drops of 5\% solution of trichloroacetic iron had been added to the child’s urine it turned olive green. Such alterations are typical for the following pathology of the amino acid metabolism: A. Phenylketonuria B. Alkaptonuria C. Tyrosinosis D. Albinism E. Xanthinuria

Individual independent students work1. Biological role of nitrogen oxide, types and significance of NO-synthase.

References:6. Medical biochemistry/ Bhagavan N.V. Jones and Bartlett Publishers International Boston-London. 1992, 980 p.7. Biochemistry / Trudy McKee, James R. McKee. 1996, 654 p.8. Biochemistry / Trudy McKee, James R. McKee. 1999, 288 p.9. Harper′s Biochemistry. 26th edition / R. K. Murray, Daryl K. Granner, Peter A. Mayes, Victor W. Rodwell. 2003, 927 p.10. Biochemistry. 2nd edition / Pamela C. Champe, Richard A. Harvey. Lippincott′s Illustrated Reviews. 1994, 443 p.