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FSBEI HE

North-Western State Medical University named after I.I.Mechnikov

Ministry of Healthcare of the Russian Federation

EDUCATIONAL AND METHODICAL GUIDE FOR STUDENTS OF THE GENERAL MEDICINE FACULTY FOR PRACTICAL LESSONS IN

BIOLOGICAL CHEMISTRY

(with tests )


Edited by prof. V.A. Dadali,

Ass.Prof. Zh.V. Antonova, R.N. Pavlova

Volume 2

Chemistry and carbohydrate exchange.

Regulation of carbohydrate exchange.

Hormones.

Saint-Petersburg

2020


УДК 577.1(076.5)
Chemistry and carbohydrate exchange. The reaction of carbohydrate excahnge. Hormones: a teaching aid for practical classes in Biological Chemistry for medical students; ed. 3rd, rev. and add. / ed. prof. V.A. Dadali, Ass. Prof. Zh.V. Antonova, R.N. Pavlova; English text by E.Yu.Lavrova.­ SPb.: North-Western State Medical University named after I.I. Mechnikov Publishing House, 2020 .­ 88 p.


The Study guide is intended for students in the specialty "General Medicine".

The present book formulates the purpose and motivation of each topic, questions for self-study, the main and additional literature for each topic, the methodology of practical work. After each topic, educational tests with answers are given, part of the classes are offered in a form of situational problems, for the solution of which the students are to use the additional literature offered in the lesson.

The present study guide was prepared by the team of the Chair of Biological and General Chemistry of the North-Western State Medical University named after I.I. Mechnikov: Antonova Zh.V., Beishebaeva Ch.R., Ivanova L.V., Kuleba V.A., Makarova M.N.., Pavlova R.N., Smertina M.N., Sokolova E.A. edited by prof. V.A. Dadali, Ass. Prof. Zh.V. Antonova, R.N. Pavlova.

Approved

as a Study guide by the

Methodical Council of

NWSMU n.a.I.I.Mechnikov,
Composite authors, 2020


Topic: Digestion of carbohydrates in the gastrointestinal tract. Glycogen exchange. Practical work ­ quantification of amylase in urine

1. Venue – Chair of Biochemistry.

Duration - 180 minutes.

2. Objectives: To systematize students' knowledge on the process of digestion of carbohydrates in the gastrointestinal tract and the reasons for the violation of this process; glycogen metabolism (synthesis and breakdown); causes of glycogenosis. To introduce students to the use of exercise tests in the diagnosis of carbohydrate metabolism disorders. Discuss the diagnostic value of amylase studies in various biological substrates.

3. Specific tasks:

3.1. The student must know:

- classification and structure of carbohydrates;

- the sequence of digestion of carbohydrates in the gastrointestinal tract (intermediate and final products of digestion of carbohydrates, the participation of enzymes), assessment of the processes of digestion of carbohydrates and absorption of carbohydrates,

- the stages of the synthesis and breakdown of glycogen, the regulation of these processes and their violation, types of glycogenoses. Diagnostic value of the study of amylase activity.

3.2.The student must be able to:

- write the formulas of mono-, di- and polysaccharides and indicate the enzymes that digest them, use the knowledge about stress tests used to detect disorders of the digestion of carbohydrates in the gastrointestinal tract;

- write down the stages of glycogen synthesis and breakdown and explain their regulation;

- master the method for determining the activity of amylase in the urine, evaluate the results of determining the activity of amylase in the blood and urine.

3.2. The student must have the skills to:

deal with the information on changes in biochemical parameters, taking into account the knowledge of the mechanisms of development of pathologies of pathology with a lack or excess of the main components of food and violation of their digestion.

4. Motivation:

The knowledge and skills acquired in the class are necessary for understanding the mechanisms of the development of pathological conditions that develop when there is a violation of the digestion of carbohydrates with enzyme deficiency, with hereditary diseases ­ glycogenoses, with diseases of the pancreas and salivary glands.

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Assignment for self-study:

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

Basic Reading

5.1. Revise the lecture on the topic: Digestion of carbohydrates. Glycogen exchange (synthesis, breakdown).

2. 
   Биохимия (учебник для ВУЗов под ред. чл.- .корр. РАН
   

проф. Е.С.Северина,М.: - 2003, С. 298-333., 2011, С. - 293-328

2. 
   Использовать при подготовке учебник: Березов Т.Т., Коровкин Б.Ф. «Биологическая химия».М., Медицина, 200, С. 169-186, 319-327, 361 – 362.
   

5.3. Подготовиться к лабораторной работе по методическому пособию: «Учебно-методическое пособие к практическим занятиям по биологи ческой химии, часть 2, Санкт-Петербург., 2013

FILL IN THE GAPSльная литература

5. 
   Изучить раздел в учебнике Марри Р.
   

«Биохимия человека», М,Мир, 1993., С. 108-112.

5. 
   Лабораторные методы исследования в
   

клинике. Справочник под ред. проф. В.В.Меньшикова, М.: Мед. 2002 - С. 191-192.

5. 
   Медведев В.В., Волчек Ю.З. Клиническая лабора-
   

торная диагностика. Справочник для врачей. СПб.:

Гиппократ.1995. С. 66-67, 108, 132, 138-139

5.8. Долгов В.В., Аметов А.С., Щетникович К.А.,

Ройтман А.П., Днмидова Т.Ю.Лабораторная диагностика нарушений обмена углеводов, сахарный диабет. Учебное пособие.М.: Лахема. 2000,63 с.

6. Questions for self-study.

6.1. Why is there a hydrolytic breakdown of carbohydrates in the gastrointestinal tract prior to the absorption (absorption) of carbohydrates from the intestine?

6.2. Enzymes that hydrolyze glycosidic bonds of sugars, their localization in the juices of the gastrointestinal tract, and their bonds are cleaved. The participation of specific enzymes in the hydrolysis of specific poly and disaccharide molecules. The causes for the lack of hydrolysis of fiber. The sequence of action of individual enzymes.

6.3. How is the absorption of carbohydrates in the intestine performed?

6.4. Stages of synthesis and breakdown of glycogen in tissues (write formulas). What are the features of muscle glycogen metabolism compared to the liver.

Equipment used at the class:

Thermostat, photoelectric colorimeter, automatic and glass pipettes, test tubes.

Question card sample:

1. Write a fragment of the structure of amylopectin

2. Stages of glycogen breakdown
Laboratory work

Determination of urine amylase activity

by the Smith-Ro method in the Ugolev modification.

Method principle

The method is based on the determination of the amount of starch cleaved by amylase, which is determined by the change in the intensity of the blue starch in the presence of iodine. Definition Progress

Measure 1 ml of phosphate buffer (pH = 7.2) and 1 ml of a 0.15% starch solution, mix with shaking and add 0.5 ml of urine diluted in "R" times. At the same time, a control sample containing 0.5 ml of water instead of urine is examined (the remaining reagents are the same).

Experimental and control samples are incubated in a thermostat at a temperature of 380 for 20 minutes. After removal from the thermostat, 1 ml of 1N hydrochloric acid, 4 ml of distilled water and 0.25 ml of a 0.25% iodine solution are quickly added to the test and control samples to stop the reaction. The tubes are shaken, the resulting blue color is measured against water with a red filter (640 nm).

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Calculations

The optical density of the control sample containing undigested starch is taken as 100%.

1. Calculate the percentage of undegraded starch in the experimental sample according to the formula

Х= D₂ * 100

D₁

where D₁ – optical density of the control sample,

D₂ – optical density of the experimental sample.

2. 
   Find the percentage of split starch
   

Y=100% - Х

3. 
   Amylase activity is calculated, which is expressed in arbitrary units by the formula:
   

А (unit.) = Y*Р

t

where У – the amount of split starch in %

Р – urine dilution rate (e.g. 40, i.e. 1:40 dilution)

t - time – incubation time in minutes (20 min).

Note: with high amylase activity, urine is diluted 100, 200 or more times. Norm 15-50 conventional units

Conclusion.
Clinical and diagnostic value of the method for determining the activity of amylase in different biological substrates

a - amylase / CF 3.2.1.1./ is synthesized by the salivary glands and pancreatic gland. A small activity of the enzyme synthesized in lysosomes is also noted in other organs.

Separation of blood amylase by gel filtration on DEAE - Sephadex revealed 2 isoenzymes a - amylases: S - type - salivary and P - type - pancreatic. The content of isoenzymes in blood serum: 70% - type S and 30% - P-type.

Urine contains 70% P type, because the molecular weight of P - type a - amylase (48,000 - is quite small and it is filtered by the kidneys much easier ).

I. Saliva. Identification of pathologies accompanied by lesions of the salivary glands.

II. Blood serum or plasma. You can determine the total activity and the ratio of S - and P - types. Hyperamylasemia can be observed in acute pancreatitis - a significant increase in plasma activity of the enzyme; viral hepatitis, pancreatic cancer, damage to the salivary glands, renal failure, cholecystitis, peritonitis, burns, acute appendicitis, the use of certain pharmacological drugs: corticosteroids, salicylates, tetracycline, furosemide, histamine.

III. Pancreatic and intestinal juice

Diagnosis of poly and disaccharidase deficiency is based primarily on clinical manifestations, as well as on the determination of the activity of disaccharidase enzymes in biopsy specimens of the small intestine mucosa, determination of lactose, sasarose in feces, lowering fecal pH, and performing stress tests.

IV. Urine. Mostly P-type a - amylases are determined.

Hyperamilazuria occurs in acute pancreatitis, viral hepatitis, pancreatic cancer, cholelithiasis, pancreatic necrosis. In acute pancreatic pathology, the growth of amylazuria is "delayed" compared with amylasemia by about 6 hours. There is evidence that in severe pancreatitis in some patients, urine amylase activity may remain normal, while blood amylase activity increases.

Loading tests.

1. Diagnosis of disaccharide digestion disorders

On an empty stomach, orally (previously dissolved in water), one of the disaccharides is prescribed sequentially (based on 1 g per kg of weight): sucrose, maltose, lactose. In this case, the concentration of glucose in the blood is recorded in dynamics - before taking the disaccharide and 1 and 2 hours after taking the disaccharide. In healthy people, 1 hour after the introduction of carbohydrates, an increase in serum glucose levels of 1-1.5 mmol / l is observed. A test is considered positive if there is no increase. In some diseases, the load with carbohydrates causes diarrhea, abdominal pain, flatulence. Undigested lactose (or another disaccharide) enters the colon, where it is broken down by bacteria to organic acids and carbon dioxide. An increase in the concentration of lactose and organic acids increases the osmolarity in the intestinal lumen, increases fluid secretion, the volume of chyme, intestinal motility increases, and diarrhea develops. Diagnosis of disaccharidase deficiency is also carried out by determining the activity of disaccharidase enzymes in biopsies of the small intestine mucosa, determining lactose, sucrose in the feces, lowering the pH of feces. At the same time, hydrogen ions generated by the breakdown of organic acids are able to enter the bloodstream. The lungs are included in the removal of excess hydrogen ions, the condition is manifested by an increase in the concentration of hydrogen in the condensates of exhaled air (this test is widespread in the USA)

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To diagnose the absorption of monosaccharides, glucose, galactose, and fructose are administered orally according to the same scheme. In case of malabsorption, the level of these monosaccharides in the blood does not increase compared to the initial one.

2. Diagnosis of glycogenosis.

а) Glucogon test. On an empty stomach, i/v or i/m glucagon (up to 2.8 mg) or adrenaline (up to 1.0 mg) is administeredt, and the glucose content in the blood serum is recorded in dynamics (before and after administration). Healthy people have an increase. Otherwisents, no increase occurs (positive test).

b) Ischemic test for the diagnosis of muscle glycogenosis

A physical exercise with ischemia is performed on an empty stomach as follows: two cuffs from the manometer are applied to the middle third of the shoulder and to the lower third of the forearm. Air is pumped up to 200 mm Hg. Blood from a cubital vein is taken for the first time before work and before air is injected into the cuff located on the shoulder. 2 minutes after work (45 sec. of squeezing the pear from the pressure gauge with a hand), the cuff located on the shoulder is removed and blood is taken from the cubital vein for 10-15 minutes.

In both blood samples, the concentration of glucose and lactate is measured. In healthy people, against the background of a halving of the glucose concentration, an increase in lactate by 3.3–5.5 mmol / l is observed. In patients with a slight decrease in glucose concentration, an increase in lactate occurs only by 0.55 - 1.1 mmol / l (positive test).

All loading tests are prescribed after a preliminary diagnosis in the presence of an appropriate clinical and morphological picture to clarify the diagnosis.

In case of contraindications, tests are not used. In some cases, the final diagnosis is made only after determining the activity of the corresponding enzymes in the biopsy


Tests for «Digestion, carbohydrate absorption, glycogen metabolism» Section

Тest 1

Choose correct answer

A study of changes in amylase activity in the blood and urine is necessary for:

а) diagnosis of pancreatic diseases

b) assessment of the digestion of carbohydrates

c) assessment of energy metabolism in tissues

d) assessment of carbohydrate absorption processes

Тest 2

Choose correct answer

Carbohydrate digestion processes are evaluated by:

а) studies of the activity of the amylase enzyme in the blood and urine

b) studies of the activity of enzymes that break down di- and polysaccharides in the intestinal and pancreatic juices

c) studies of the activity of tissue enzymes

d) determination of glucose in the urine

Тest 3

Choose correct answer

The biological significance of the digestion of complex polysaccharide and disaccharide molecules is to:

а) create a large concentration of monosaccharides in the intestinal lumen

b) turn complex polysaccharide molecules into simple monosaccharides used by tissues

c) remove the species specificity of biopolymers

d) evaluate the work of the endocrine system

Тest 4

Choose correct answer

What digestive juice does not contain enzymes that digest carbohydrates:

а) saliva

b) pancreatic juice

c) gastric juice

d) intestinal juice

Тest 5

Choose correct answer

The action of the hormone on carbohydrate metabolism is carried out through enzymes:

Hormone	Enzymes
adrenaline	а) hexokinase b) phosphorylase c) phosphoglucomutase d) glucose-6-phosphatase e) lactate dehydrogenase

Тest 6

Choose all correct answers

Glucose absorption requires:

а) carriers

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b) the work of ATP

c) changes in osmotic pressure

d) the formation of micelles

Тest 7

Choose all correct answers

Features of muscle glycogen metabolism:

а) glycogen stores serve as a source of glucose for the whole body

b) glycogen stores are used for energy costs only by the tissue itself

c) regulates the breakdown of glycogen adrenaline

d) regulates the breakdown of glycogen adrenaline and glucagon

Тest 8

Choose all correct answers

Features of glycogen metabolism in the liver:

а) the ability to synthesize glycogen with an excess current of glucose in the blood

b) the ability to quickly convert glycogen to glucose-1-phosphate

c) glycogen stores serve as a source of glucose for the whole organism

d) glycogen reserves are used for energy costs only by the tissue itself

e) the ability to synthesize glycogen, regardless of the amount of glucose entering the tissue

Тest 9

Choose correct answer

The action of the hormone on carbohydrate metabolism is carried out through enzymes:

Hormone	Enzymes
glucagon	а) hexokinase b) phosphorylase c) phosphoglucomutase d) glucose-6-phosphatase e) lactate dehydrogenase

Тest 10

Choose all correct answers

The action of the hormone on carbohydrate metabolism is carried out through enzymes:

Hormone	Enzymes
insulin	a) hexokinase b) phosphorylase c) phosphoglucomutase d) glucose-6-phosphatase e) lactate dehydrogenase f) glycogen synthetase

Тest 11

Match the correspondence

(the correct answer can be used once or twice)

Processes	Enzymes
1) glycogen synthesis 2) glycogen breakdown	а) phosphorylase b) hexokinase c) phosphoglucomutase d) glycogen synthetase e) glucose-6-phosphatase

Тest 12

Choose all correct answers

Amylase breaks down:

а) lactose b) starch c) glycogen d) maltose e) sucrose

Тest 13

Choose all correct answers

Glycogenosis is a disease caused by:

а) hereditary diorder of glycogen synthesis;

b) a hereditary violation of the breakdown of glycogen;

c) disorders of hormonal regulation of glycogen metabolism

d) violation of the glycolysis process

e) impaired gluconeogenesis

Тest 14

Choose all correct answers

The mechanisms of glucose uptake into cells

а) facilitated diffusion using special carrier proteins GLUT-1, GLUT-2, GLUT-3, GLUT-4, GLUT-5

b) glucose concentration gradient transport

c) transport against glucose concentration gradient

d) transport through membranes with the expense of ATP.

Тest 15

Choose correct answer

Carbohydrate digestion enzymes belong to the class of

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