Файл: Fsbei he northWestern State Medical University named after I. I. Mechnikov Ministry of Healthcare of the Russian Federation.doc

а) oxidoreductases

b) hydrolase

c) ligase

d) transferase

e) synthetases

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KEYS: " Digestion, carbohydrate absorption, glycogen metabolism "

Тest 1 – а Тest 9 – b

Тest 2 – b Тest 10 – а, e

Тest 3 – b Тest 11 – 1 b, 1 c, 1 d, 2 а, 2 c, 2 e.

Тest 4 – c Тest 12 – b, c.

Тest 5 – b Тest 13 – а, b

Тest 6 – а, b Тest 14 – а, b.

Тest 7 – b, c Тest 8 – а, c. Тest 15 - b

Тopic Dichotomous breakdown of glucose. Gluconeogenesis. Pyruvic acid metabolism.

Venue: Chair of Biochemistry.

Duration - 180 minutes.
2. Objectives: Systematize knowledge on the anaerobic dichotomous pathway of glucose oxidation, gluconeogenesis, and pyruvic acid metabolism.

Master the method for determining pyruvic acid in urine, discuss the value of the method
3. Specific tasks:

3.1.The student must know:

3.1.1. Stages of anaerobic dichotomous breakdown of glucose (glycolysis) and the biological role of each stage.

3.1.2. Reactions of anaerobic dichotomous breakdown of glucose, the role of this process, the energy output of glycolysis.

3.1.3. The process of gluconeogenesis, to be able to write workarounds, the role of gluconeogenesis, hormonal regulation.

3.1.4. The main stages of aerobic oxidation of glucose.

3.1.5. Exchange of pyruvic acid.

3.1.6. The principle of the method for determining glucose in the blood. Norms of blood glucose, the diagnostic value of these indicators.

3.1.7. Diagnostic value of determination of pyruvic acid in urine.

3.2. The student must be able to:

3.2.1. Quantify the content of the determination of pyruvic acid in urine, blood glucose, explain the causes of hypo and hyperglycemia.

3.3. Student must have the skills to use:

3.3.1 algorithm of work on colour density meter; information on changes in biochemical parameters, taking into account the knowledge of the development mechanisms of pathologists and under the influence of harmful environmental factors.

4. Motivation: the knowledge and skills acquired in the lesson are necessary for understanding the mechanisms of development of pathological conditions associated with the processes of impaired energy production. The determination of the concentration of pyroracemic acid PVC in urine and blood is of practical interest to the doctor from the point of view of laboratory diagnosis of acute and chronic intoxications (including alcohol), hypovitaminosis B₁, hypoxic conditions

5. Assignment for self-study:

Study recommended reading list, using questions for self-study.

BasicLiterature

5.1. Лекционный материал по теме: Гликолиз. Глюконеогенез. Обмен пировиноградной кислоты.

2. 
   Березов Т. Т., Коровкин Б. Ф. Биологическая химия. М., Медицина, 1998 г., С. 327-345.
   
3. 
   Биохимия (учебник для ВУЗов под ред. чл.- корр. РАН, проф. Е.С.Северина, М.:, 2003, С. 247-250, 315-316, 333-343, 281-285.
   

2011, С. 245-247, 279-283, 310-312, 328, 339.

5.4. Revise for the laboratory work using the text book «Учебно-методическоe пособие к практическим занятиям по биологической химии, часть 2, СПб., 2013

Additional Literature

5.5 Марри Р., Греннер Д., Мейес П., Родуэлл В. Биохимия человека. М., Мир, 1993, С. 181-188, 196-199, 214-219.

5.6 Study guide for revision for laboratory works

- Медицинские лабораторные технологии и диагностика: Справочник. Т.1, Под редакцией А. И. Карпищенко. – Санкт-Петербург: Интермедика, 1999, С. 83-84;

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- Лабораторные методы исследования в клинике / Под ред. Меньшикова 2002, М:., Медицина, С. 210-212; 240.

5.7. Revision for tests.

6. Questions for self-study:

6.1. What are the breakdown ways for glucose by type of cleavage and oxidation conditions of the molecule.

6.2. What are the main stages of dichotomous breakdown of glucose in anaerobic and aerobic conditions.

6.3. What is glycolysis, what is its biological role?

6.4. Stages of glycolysis (preparatory and oxidoreduction), their biological role.

6.5. What is substrate phosphorylation? At what stages of glycolysis does it occur?

6.6. Glycolysis energy efficiency.

6.7. Under what processes in the body is pyruvic acid formed?

6.8. How does pyruvic acid undergo

1) glycolysis, 2) during aerobic oxidation of glucose, 3) during gluconeogenesis, 4) protein metabolism, 5) lipid metabolism?

6.9. What enzymatic complex is involved in oxidative decarboxylation of pyruvic acid PVA? What are enzymes, co-enzyme- you and the vitamins involved in this process.

6.10. What substances inactivate the pyruvate oxidase system and why?

6.11. Method for determination of PVA in urine, its use for diagnostic and prophylactic purposes.

6.12. Principle of the method for determining pyruvic acid in blood and urine
Question card sample

Choose the number of the correct answer:

1. Glycolysis is an enzymatic process of glucose breakdown:

1) aerobic apotomy

2) anaerobic apotomy

3) aerobic dichotomous

4) anaerobic dichotomous

2. Give an example of a substrate phosphorylation reaction. in the process of glycolysis.

The biological role of pyruvic acid. Pyruvic acid (PVA) is formed in the body during the metabolic transformations of carbohydrates, proteins and lipids. It is formed in tissues during glucose oxidation, glycogen breakdown, glycerol oxidation, during the transamination of amino acids and from lactic acid during gluconeogenesis.

PVA is a key metabolite of anaerobic and aerobic oxidation of glucose. In the process of glycolysis, PVA is reduced to lactic acid, the final product of anaerobic metabolism. Under aerobic conditions, PVA is oxidatively decarboxylated to form acetyl-coA, which undergoes further oxidation in the tricarboxylic acid cycle or is used to synthesize lipids and amino acids. PVA is the main substrate of gluconeogenesis.

The value of determining the concentration of PVA in the blood and urine in sanitary-chemical and clinical studies.

An increase in the concentration of PVA in the blood and urine is observed with hypovitaminosis B₁, when exposed to industrial poisons that block SH-groups of thiol enzymes; hypoxic conditions: pneumonia, lung damage by industrial dust, severe heart failure, acute infectious diseases, lack of oxygen in the environment, hepato-cerebral dystrophy, and cirrhosis, leading to impaired blood circulation, muscular dystrophy and other diseases. The most dramatic increase in PVA in the blood is observed with intense muscular work and hypovitaminosis B₁.

One of the reasons for the accumulation of PVC is the inhibition of the process of its oxidative decarboxylation in the mitochondria of cells.

Vitamin B₁ is part of the coenzyme of thiamine diphosphate, which is the prosthetic group of the first enzyme of the pyruvate dehydrogenase system ­ pyruvate dehydrogenase. With the deficiency of this vitamin, as well as with a violation of its metabolism, a decrease in the intensity of oxidative decarboxylation of PVC is observed. The administration of a vitamin B₁ or thiamine diphosphate drug with the pharmacopoeial name cocarboxylase, on the contrary, stimulates the process of aerobic metabolism of pyruvate and increases the energy supply of cells.

The pyruvate dehydrogenase system includes thiol enzymes ­ dehydrogenases (pyruvate dehydrogenase and dihydrolipoyl dehydrogenase) and coenzymes containing SH-groups (lipoic acid and HS-coA), so the pyruvate dehydrogenase system is blocked by thiol poisons: heavy metal salts, oxidizing salts, and metal oxides.

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The pyruvate dehydrogenase system only works under aerobic conditions, so PVA accumulates in tissues and during hypoxia.
PVA DETERMINATION METHODS

There are several methods for quantifying pyruvic acid PVA in tissues and body fluids.

1. Determination of PVA in the blood by the colorimetric method.

The principle of the method, see the section "Laboratory work".

In the analysis, 0.2 ml of blood from the finger is used.

Normal values are 0.03 - 0.10 mmol / L.

2. Enzymatic method for the determination of PVA in the blood (and in the tissues of experimental animals).

The principle of the method. In the presence of the enzyme lactate dehydrogenase, pyruvate is reduced to lactate in the reaction:

СOO^- COO^-

С=О + NАDН + Н^{+ à} CH-ОН + NAД⁺

СН₃ CH₃

pyruvate lactate

The amount of pyruvate used in the reaction is equivalent to the amount of reduced coenzyme NADN + H ⁺, the loss of which is recorded spectrophotometrically at a wavelength l = 340 nm.

In clinic, 1 ml of venous blood is used for analysis.

Normal values are 0.05-0.114 mmol / L
Laboratory work

Determination of pyruvic acid in urine by colorimetric method

The principle of the method. Pyruvic acid interacts with 2,4-dinitrophenylhydrazine to form hydrazone, which in an alkaline medium acquires a red-brown color, the intensity of which is directly proportional to the concentration of PVA.

Reaction equation.











PVA 2,4-dinitrophenylhydrazine 2,4-dinitrophenyl

phenylhydrazone PVA
Reagents and equipment.

1. 2,4-Dinitrophenylhydrazine (2,4-DNPH), 0.1% solution in 2N HCl.

2. Potassium hydroxide (KOH), 2.5% solution in ethanol.

3. Test tubes with corks, pipettes.

4. Photoelectrocolorimeter.

5. The calibration graph.

The course of work

0.5 ml of a 0.1% solution of 2,4-dinitrophenylhydrazine (2,4-DNPH) is added to 1 ml of diluted 4 times urine and shaken vigorously. Meanwhile, a control sample was prepared containing 1 ml of distilled water instead of urine; all other reagents are added in the same amount as in the experimental sample. After 5 minutes, 2 ml of a 2.5% alcohol solution of potassium hydroxide are added to the control and experimental sample and mixed. After 10 minutes, the samples are photometrically scanned using a green filter (l=560 nm) and cuvettes with a working distance of 10 mm.

Calculations.

A calibration graph of the dependence of the optical density of the colored hydrazone solution on the concentration of PVA in the sample D = f (C) is preliminarily constructed using a standard solution of sodium pyruvate

The content of PVA in the daily volume of urine (C, mg / day) is calculated by the formula

С=Х*4*1500/1000,
where Х ­ the content of PVA in the test sample, determined by the calibration graph, μg / ml; 4 ­ factor for determining the content of PVA in 1 ml of undiluted urine;

1500 ­ average daily urine volume, ml;

1000 ­ coefficient for the conversion of µg to mg.

Compare the results with the norm: 10-25 mg of PVA should be excreted in the urine per day. Indicate the possible causes of increased levels of PVC in urine.

Conclusion

Тest for «Dichotomous breakdown of glucose. Gluconeogenesis.

Pyruvic acid metabolism» Section

Тest 1

choose the correct answer

Glycolysis is an enzymatic process of glucose breakdown :

а) anaerobic apotomy;

b) aerobic dichotomous

c) anaerobic dichotomous

d) aerobic apotomy

Тest 2

CHOOSE THE CORRECT ANSWER

The final product of glycolysis is:

a) lactic acid;

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b) pyruvic acid;

c) two trioses: glyceraldehyde-3-phosphate, dioxiacetone phosphate;

d) acetyl-coA

e) citric acid

Тest 3

CHOOSE THE CORRECT ANSWER

Reaction determining the rate of glycolysis is:

a) hexokinase;

b) aldolase;

c) glyceraldehyde phosphate dehydrogenase;

d) lactate dehydrogenase;

e) phosphofructokinase

Тest 4

CHOOSE ALL CORRECT ANSWERS

Pyruvic acid in cells can:

а) undergo oxidative decarboxylation under aerobic conditions to acetyl-coA

b) recover under anaerobic conditions to lactate

c) turn into alanine in the transamination reaction

d) is a substrate of gluconeogenesis

e) be the end product of gluconeogenesis

Тest 5

CHOOSE ALL CORRECT ANSWERS

Irreversible glycolysis reactions are:

a) lactate dehydrogenase

b) pyruvate kinase

c) aldolase

d) phosphofructokinase

e) hexokinase
Тest 6

CHOOSE ALL CORRECT ANSWERS

Key gluconeogenesis enzymes are:

а) fructose - 1,6 - diphosphatase

b) pyruvate dehydrogenase

c) pyruvate carboxylase

d) glucose - 6 - phosphatase

e) phosphoenolpyruvate carboxykinase

Тest 7

CHOOSE THE CORRECT ANSWER

Substrate phosphorylation is:

а) ATP glucose phosphorylation

b) phosphorylation of fructose-6-phosphate involving ATP

c) the formation of two phosphotriosis in the aldolase reaction

d) ATP synthesis (GTP, etc.) using macroergic energy of substrate bonds

e) ATP synthesis in the respiratory chain
Тest 8

CHOOSE THE CORRECT ANSWER

Glycolysis reaction:	Biological meaning :
conversion of phosphoenolpyruvate into pyruvate	а) the reaction of substrate phosphorylation b) the final reaction of the preparatory stage of glycolysis c) the formation of an active form of glucose d) speed determining reaction

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