Download part the second - Астраханский Государственный Медицинский

Astrakhan State Medical University
Астраханский государственный медицинский
университет
T.B.Vorobyeva, D.M.Nikulina
Т.Б.Воробьева, Д.М.Никулина
THE MANUAL
ON BIOCHEMISTRY
РУКОВОДСТВО К ЗАНЯТИЯМ
ПО БИОХИМИИ
Part 2
Часть 2
ASTRAKHAN - 2017
АСТРАХАНЬ – 2017
UDC 577.1(076)
LBC 28/902
V75
T.B.Vorobyeva, D.M.Nikulina. The manual on biochemistry. Part 2.
– Astrakhan, Astrakhan State Medical University, 2017, 88с.
English editor S.S.Uzhakina, senior lecturer of Department of Foreign languages, ASU
The training guide contains information which is necessary for individual and group work of students in the classroom and at home.
Reviewers: V.R. Gorst, Doctor of Medicine, Professor of Physiology
Department, ASMU
P.V.Loginov, Candidate of Biology Sciences, Associated
Professor of Chemistry Department, ASMU
T.S. Kirillova, Doctor of Philology, Professor, Chief of
Foreign Languages Department, ASMU
ISBN 978-5-4424-0140-0 (основной)
ISBN 978-5-4424-0193-6 (часть 2)
 T.B.Vorobyeva, D.M.Nikulina.
 Astrakhan State Medical University, 2017
2
FUNCTIONS AND METABOLISM
OF BASIC SUBSTANCES IN THE ORGANISM
SECTION VII. METABOLISM AND FUNCTION OF CARBOHYDRATES
Class 19
DIGESTION AND ABSORPTION OF CARBOHYDRATES. METABOLISM OF GLYCOGEN. QUANTITATIVE AND QUALITATIVE DETERMINATION OF CARBOHYDRATES IN BIOLOGICAL LIQUIDS
The aim of the lesson:
1. To revise physical and chemical properties and to know biological role of carbohydrates.
2. To know processes of digestion and absorption of carbohydrates.
3. To know metabolism of glycogen.
4. To master methods of qualitative and quantitative determination of glucose in urine.
Initial level of knowledge:
- structure of monosaccharides;
- structure and properties of disaccharides;
- structure and properties of homopolysaccharides (starch, glycogen, cellulose);
- classification and the biological functions of carbohydrates;
- classification and the nomenclature of enzymes.
Main topics.
I.2. Stereoisomerism of monosaccharides.
Cyclic forms of monosaccharides.
Reactions with the participation of carboxylic group.
Physical and chemical properties and biological value of maltose,
saccharose and lactose.
Features of structure and the biological functions of homo- and heteropolysaccharides.
3
Digestion and absorption of carbohydrates.
Glycogenesis.
Glycogenolysis.
II.1. Work 1. TEST FOR SUGAR IN URINE
Qualitative Trommer's, Fehling’s, Nilander’s reactions based
on oxidation of the free aldehyde group of carbohydrates (for example, glucose to gluconic acid) and restoration of metals (copper,
bismuth, etc.) from their oxides are applied for the detection of sugar
in urine,. These reactions go fast while heating in the alkaline environment. The restored form of metals is painted in specific color (for
example, cuprous oxide - in red, bismuth - in black)
H
O
C=O + 2CuSO4 + 5NaOН  C–O–Na + 2CuOH + 2Na2SO4 + 2H2O
R
R
Trommer’s reaction
Test with Fehling’s solution is based on the same principle, as
Trommer's reaction. Difference and advantage of this reaction is that
Fehling offered to add Seignette salt (potassium sodium tartrate) for
the linkage of surplus of cupric hydroxide, because the latter at heating turns into cupric oxide (СuO) - the deposit of black color which
is blacking out the reaction when the glucose in the researched sample is in small amount.
COONa
|
(CHOH) 2 + Cu (OH) 2 →
|
COOK
COONa
|
(CHO) 2Cu + 2H2O
|
COOK
Practical procedure.
а) Trommer's reaction.
Add 5 drops of 10% caustic sodium solution and 5 drops of
1% copper sulfate solution to 5 drops of urine and heat up.
RESULT:
4
б) Reaction with Fehling’s solution.
Add 10-20 drops of Fehling’s solution to 20 drops of urine and
heat up the top layer of a liquid to boiling.
RESULT:
CONCLUSION:
WORK 2. EXPRESS ASSAY OF SUGAR ESTIMATION IN URINE.
Practical procedure.
Put the small amount of a powder (structure: 1 part of copper
sulfate + 10 parts of waterless sodium carbonate) into the test tube,
add some drops of urine, heat up.
RESULT:
REFERENCE INTERVAL
Color of a solution
Amount of sugar
Dark blue
No
Flavovirent
0.5% (5 g/L)
Brown - red
1-2% (10-20 g/L)
Red
Over 2% (20 g/L)
RESULT and CONCLUSION:
Clinical-diagnostic value.
In norm sugar in urine is excreted in insignificantly small
amounts and is not determined by usual methods.
Occurrence of sugar in urine in amounts which is possible to
determine by usual clinical methods is called glucosuria. Urine contains, basically, glucose and, less often, fructose and galactose. Glucosuria can be functional (food, emotional) and pathological (at disease of kidneys, at diabetes) when daily urine can contain up to 200300 g of glucose. Sometimes glucosuria is observed at pregnancy.
III.2. Control questions.
What is the monomer of starch and cellulose?
5
What kinds of amylases do you know; where do α- and γ-amylase
function; how do they hydrolize starch (glycogen)?
What is glucosuria?
When is physiological glucosuria marked?
What diseases are accompanied with glucosuria?
III.3. Home assignment.
1. Complete the table.
nos Enzymes needed to
digest the sugars
1 Salivary α-amilase
2
3
4
5
6
7
Place of
synthesis
Specificity
2. Match the name of enzyme (A-D) and their functions (1-4)
A. salivary α-amilase
1. has pH optimum near 7.0
B. amylo α-1,6 glucosidase 2. the end product of reaction is sucrose
C. both
3. is synthesized in pancreas
D. none
4. is hydrolase
3. Match the name of substrate (A-D) and their characteristics (1-4)
A. glucose
1. is the product of sucrose digestion
B. fructose
2. is the product of lactose digestion
C. both
3. is absorbed by simple diffusion
D. none
4. moves across membrane by active transport
(tansporter proteins)
Choose the correct answer.
4. Glycogen is stored in the cells
A. in endoplasmic reticulum membranes
B. as substance in cytosol solution
6
C. in granules
D. in lysosomes
5. The greatest amount of glycogen is in
A. skeletal muscles
B. liver
C. myocard
D. kidney
E. brain
6. Glycogen concentration is higher in
A. skeletal muscle
B. liver
C. myocard
D. kidney
E. brain
7. Enzyme used both in glycogenosis and glycogenolysis is
A. glucokinase
B. hexokinase
C. phosphoglucomutase
D. glycogen synthase
E. glycogen phosphorylase
8. How long is glycogen synthesized after the carbohydrate meal?
A. 10 min
B. 30 min
C. 1-2 hours
D. 3-4 hours
E. to 12 hours
9. Glycogen synthesis in liver is activated by
A. cathecholamines
B. dephosphorylation of phosphorylase b
C. phosphorylation of glycogen synthase
D. increasing of blood glucose
7
E. phosphodiesterase
Material for self-study. I. 1.pp.165-170, 251, 271-276, 539-540
2.pp.102-110; 1454-150, 474-475, 542; II.
Class 20
GLYCOLYSIS, GLYCONEOGENESIS. OXIDATIVE DECARBOXYLATION OF PYRUVATE. DETERMINATION OF GLYCOLYSIS PRODUCTS IN BIOLOGICAL LIQUIDS
The aim of the lesson:
1. To know an anaerobic and aerobic glycolysis.
2. To master methods of qualitative determinaition of glycolysis
products and of vitamin В1.
Initial level of knowledge:
- structure and physical and chemical properties of the basic carbohydrates;
- absorption of carbohydrates in the digestive tract;
- glycogen metabolism;
- tricarboxylic acid cycle;
- classification and the nomenclature of enzymes;
- vitamins В1, В2, В3, В5, lipoic acid.
Main topics.
I.2. Glycolysis: localization in a cell, reactions using formulas, enzymes, coenzymes, biological role, and energy yield.
Similarity and differences between glycolysis and glycogenolysis,
and also between glycolysis and alcoholic fermentation.
Glyconeogenesis: reactions using formulas, enzymes, the biological
function.
Dichotomous way of glucose oxidation: stages, localization in a cell,
reactions using formulas, enzymes, coenzymes, energy yield.
II.1. Work 1. QUALITATIVE DETERMINATION OF SUBSTANCES FORMED IN GLYCOLYSIS
8
а) Qualitative reaction for triosephosphates.
Principle of method.
Various intermediate phosphorylated products (phosphoric
ethers of hexoses, trioses etc.) are formed in glycolysis. The principle
of the method of determination of phosphorus is the formation of
complex phosphomolybdic acid which then is restored by hydroquinone in phosphomolybdate (blue).
Practical procedure.
Reagents (ml)
Protein-free filtrate
2N solution NaОН
Distilled water
Sample
Control
0.5
0.5
0.5
0.5
Mix and leave at room temperature for
15 minutes.
Molybdic reagent
0.5
0.5
2N solution HCl
0.5
0.5
1% solution hydroquinone
0.5
0.5
Mix and leave at room temperature
for 5 minutes.
20% solution NaHSO3
0.2
0.2
RESULT:
б) Qualitative reaction for fructose bisphosphate (Selivanov’s
test)
Principle and chemism of the method.
Hydroxymethyl furfural is formed when fructose is heated with
strong hydrochloric acid. The latter gives the product of condensation of cerise color with resorcin.
HOCH2 O
CH2OH
|
HO |
|
| OH
OH
-3H2O
O
HOH2C-
9
-C=O
H
Practical procedure.`
Pour 0.5 ml of the Selivanov’s reagent into a test tube, mix
with 0.5 ml of protein-free filtrate; warm up slightly on the electric
stove. The colouring depends on the concentration of fructose
bisphosphate.
RESULT:
в) Test for lactic acid.
Principle of method and chemism of the reaction.
The lactic acid at the presence of phenolate of iron (Uffelmann’s reagent) coloured in violet color, forms iron lactate of flavovirent color.
3
-OH + FeCl3
(C6H5O) 3Fe + 3HCl
Ferric phenolate
COOH
|
(C6H5O) 3Fe + 3HCOH
|
CH3
COO Fe
|
HCOH
+ 3 C6Н5OH
|
CH3 3
Ferric lactate
Practical procedure.
Pour 1 ml prepared ex tempore Uffelmann’s reagent (mix 20
dr. of 1% solution of phenol and 2 dr. of 1% solution of ferric chloride) into the test tube and add 0.5 ml protein-free filtrate of muscles.
RESULT:
CONCLUSIONS:
Work 2. QUALITATIVE REACTION FOR VITAMIN В1
Vitamin В1 also is called thiamine since in its structure contains sulfur and nitrogen. Thiamine diphosphate participates in the
10
oxidative decarboxilation of α-ketoacids and in the formation and
degradation of α-ketols by transketolase, being the part of enzymes.
Lack of vitamin В1 in food causes the defeat of peripheral
nervous system known as "beri-beri". It is caused by the collection
of pyruvic acid and other α-ketoacids.
Principle of the method.
In alkaline environment thiamine with diazoreagent forms the
complex compound of orange color (diazo test).
Practical procedure.
Add 1-2 drops of 5% solution thiamine to diazoreagent, consisting of 5 dr. of 1% solution sulfanilic acid and 5 drops of 5% solution sodium nitrate, then on a wall of a test tube cautiously layer 5-7
dr. of 10% solution sodium bicarbonate. Note and write down the
occurrence of the color ring on the border of liquids.
RESULT:
III.2. Control questions.
What reactions of glycolysis are endergonic?
What reactions of glycolysis are exergonic?
What is the energy yield of anaerobic glycolysis (estimated in ATP)?
What is efficiency of glycolysis?
What triosephosphates are formed in glycolysis?
What is the principle of the test for lactate?
III.3. Home assignment.
Choose the correct answer.
1. Anaerobic glycolysis gives … moles of ATP
A. 1
B. 2
C. 6
D. 8
E. 12
2. The energy yield of 2-d stage of complete oxidation of glucose is
(in ATP)
11
A. 2
B. 3
C. 6
D. 8
E. 12
3. … does not take part in oxydative decarboxylaion of pyruvate.
A. pyridoxal phosphate
B. thiamine diphosphate
C. NADH
D. FAD
E. pantothenic acid
4. Choose the wrong answer.The convertation of pyruvate to acetyl
CoA
A. occurs in the cytosol
B. involves the participation of the biotin
C. depends on CoA
D. is catalysed by multimolecular complex
E. donates electrons to ETS
5. Match the parts of the sentences
A. hexokinase
1. catalyzes irreversible reaction
B. pyruvate kinase
2. catalyzes the formation of end product
C. both
3. requies ATP
D. none
4. phosphorylates ADP
6. Match the name of the process and its charecteristic
A. anaerobic glycolysis 1. triosephosphate mutase takes part
B. aerobic glycolyisis
2. pyruvate is the end product
C. both
3. requies GTP
D. none
4. pyruvate is an acceptor of electrons from NADH
7. Put the listed stages of the process in the true order
A. TPP forms the covalent bond with α-atom of carbon of pyruvate
B. the electrons are transferred to NADH+
12
C. the acetyl group is transferred from lipoate to CoA
D. CO2 is released
E. the electrons are transferred from lipoic acid to lipoamide dehydrogynase
Material for self-study. I. 1.pp.251-260, 266-270
2.pp.136-143; 153-155; II.
Class 21
WAYS OF AEROBIC OXIDATION OF CARBOHYDRATES.
REGULATION AND INFRINGEMENTS OF THE CARBOHYDRATE METABOLISM. QUANTITATIVE DETERMINATION
OF GLUCOSE IN BLOOD
The aim of the lesson:
1. To know aerobic ways of oxidation of glucose.
2. To know the regulation and the pathology of carbohydrate metabolism.
3. To master method of quantitative determination of glucose in
blood.
Initial level of knowledge:
- chemistry of monosaccharides;
- anaerobic oxidation of glucose in an organism;
- regulation of activity of enzymes;
- pyridine dependent oxidoreductases.
Main topics.
I.2. The tricarboxylic acid cycle.
Pentose phosphate pathway of oxidation of glucose: localization in a
cell, chemism of the oxidative branch, enzymes, energy yield, and
biological value.
Interrelation of dichotomizing and apotomizing pathways of oxidation of glucose.
Regulation of carbohydrate metabolism: the role of CNS, endocrine
system, c-AMP.
13
Pathology of the carbohydrate metabolism: the reasons, semiology,
examples of enzymopathies.
"Sugar" curves, diagnostic value.
II.1. Laboratory work. QUANTITATIVE ASSAY OF GLUCOSE IN
BLOOD BY ENZYMATIC METHOD
Principle of method and chemism.
Glucoxidase cleaves glucose and releases gluconate and hydrogen peroxide. Produced H2O2 is detected by oxidative condensation. A reaction is catalysed by peroxidase. As a result coloured
product is formed. Concentration of coloured product is measured by
photoelectric colorimeter.
At heating with orthotoluidine in solution of acetic acid glucose forms the complex of blue-green color, its intensity is directly
proportional to concentration of glucose. Concentration of coloured
product is measured by photoelectric colorimeter.
-NH2 + O=C-H
|
|
CH3
R
H2O +
-N=C-H
|
|
CH3 R
Influence of insulin on level of glucose in blood.
Insulin is produced by pancreas and lowers glucose in blood.
The preparation of insulin is applied at treatment of diabetes. CNS is
especially sensitive to the decrease of sugar in blood since glucose is
the basic energy source for it. The decrease of blood glucose level
lower 2.77 mmol/L (50 mg %) causes spasmes and can lead to death.
Under influence of insulin, synthesis of glycogen in a liver and
muscles is stimulated, disintegration of glycogen is broken, glucose
turns in lipids, permeability of glucose through cellular membranes
in the muscular and fatty tissuers increases, activity of hexokinase
(enzyme of glycolysis) is stimulated, Kreb’s cycle is activated.
Influence of adrenaline on the level of glucose in blood.
Adrenaline is produced by adrenal medulla and in high concentration it is strong poison. At introducing of adrenaline the contents of glucose in blood increases, hyperglycemia develops and, if
14
the level of it reaches 8.88 mmol/L (160 mg %), glucosuria appears.
The increase of glucose in blood under the action of adrenaline is the
result, first of all, of disintegration of glycogen in liver up to glucose.
Practical procedure.
Having determined the level of glucose in several samples of
blood, it is necessary to draw a conclusion which is received from an
intact animal, after injection of insulin or adrenaline to the rabbit.
Use centrifugal test tubes.
Reagents (ml)
reagent 3
Standard solution of glucose
Sample 1
Sample 2
Sample 3
Distillated water
Glucose reagent
Supernatant liquid from a corresponding test
tube
Standard
0.5
0.05
Test
tube 1
0.5
-
Test
tube 2
0.5
-
Test
tube 3
0.5
-
Control
tube
0.5
-
0.05
0.05
0.05
0.05
Mix, incubate 10 minutes at room temperature,
centrifuge 10 minutes at 900 rpm, transer into a
dry test tube
StandTest
Test
Test
Control
ard
tube 1a tube 2a tube 3a tube a
2.0
2.0
2.0
2.0
2.0
0.1
0.1
0.1
0.1
0.1
Mix, incubate 30 min at room temperature
Place precisely for 8 min in a boiling water bath. Then cool up to the
room temperature.
Measure optical density against the control in cuvette 10 mm at wave
length 500 nm (490-540 nm).
15
CALCULATION.
Et • 10
Сt =
mmol/L , where
Est
Сt - concentration of glucose in the blood sample;
10 - concentration of glucose in the standard in mmol/L;
Еt - optical density of a sample;
Est - optical density of the glucose standard.
The normal content of glucose in a sample of blood of a man,
determined by the given method, changes within the limits of 3.505.70 mmol/L.
RESULTS:
CONCLUSION:
III.2. Control questions.
What hormones and why can stimulate hyperglycemia?
What hormones and why cause hypoglycemia?
At what concentration of glucose in blood does glucosuria appear?
What character of a "sugar" curve do a healthy man and a patient
with diabetes have?
III.3. Home assignment.
1. Which of the inactivated phosphorilases (liver or muscle) does not
cause low blood glucose level? Why?
Choose the correct answer.
2. Hereditary glucose 6 phosphatase deficiency can lead to
A. glycogen synthesis after a meal
B. glycogen is degraded and glucose enter the blood quickly
C. glycogen has few branches
16
D. glycogen is highly branched
3. The rate of gluconeogenesis in liver is increased by
A. phosphorylation of pyruvate kinase
B. allosteric effects of fructose 1,6-bisphosphate on pyruvate kinase
C. activation of phosphofructokinase 1 by fructose 2,6-bisphosphate
D. allosteric effects of AMP on fructose 1,6-bisphosphatase
4. Pyruvate dehydrogenase activity is regulated by:
A. acceptor control
B. NADH
C. covalent modification
D. all of the above
5. Transfer the glucose from blood into a cell is stimulated by:
A. adrenaline
B. glucagon
C. insulin
D. c-AMP
E. all of the above
6. The enzyme catalyzing an anaplerotic reaction in the TCA cycle is:
A. citrate synthetase
B. isocitrate dehydrogenase
C. pyruvate dehydrogenase
D. pyruvate carboxylase
E. fumarase
7. Choose all correct variants. Pentose cycle gives:
A. 38 ATP
B. availability of coenzymes
C. ribose 5 phosphate
D. NADH
E. NADPH
F. 36 ATP
17
Material for self-study. I. 1.pp.261-265, 277-290, 587-602
2.pp.47, 54-55, 150-152, 155-172, 231; II.
Class 22
COLLOQUIUM ON SECTION
"METABOLISM AND FUNCTIONS OF CARBOHYDRATES"
Questions to a colloquium.
1. The basic carbohydrates of food. General character and classification of carbohydrates. Biological functions in an organism.
2. Digestion and absorption of carbohydrates in digestive tract. Simple diffusion, transport facility, active transport as penetration mechanisms of glucose in cells.
3. Glucose - the major metabolite of a carbohydrate metabolism. The
general circuit of sources and ways of the utilization of glucose in an
organism.
4. Structure, properties, distribution of glycogen in tissues as reserve
polysaccharide. Physiological value of glycogen mobilization, the
chemism.
5. Biosynthesis of glycogen and its physiological value. Inherited
disorders of glycogen metabolism.
6. The role of hormones (adrenaline, glucagon, insulin), c-AMP and
proteinkinases in biosynthesis of glycogen and glycogenolysis.
7. Structure and functions of glycosaminoglycans. The role of hyaluronic acid and chondroitin sulfate in normal functioning of intercellular substance and development of pathological conditions. Value of
heparin in medical practice.
8. Anaerobic glycolysis and glycogenolysis. The further transformation of lactate.
9. Physiological value of the glycolysis and its energy yield. The role
of an anaerobic disintegration of glucose in muscles.
10. The oxidative decarboxylation of pyruvate: biological value and
sequence of reactions.
11. Participation of vitamins at formation of active acetate.
18
12. Stages of the basic aerobic (dichotomizing) way of glucose oxidation, its distribution in the organism of a man and physiological
value.
13. Tricarboxylic acid cycle: sequence of reactions, characteristic of
enzymes.
14. Tricarboxylic acid cycle: physiological role, allosteric mechanism of regulation.
15. Power balance of the dichotomizing way of glucose oxidation.
16. Pentose phosphate (apotomizing) pathway: biological value.
Steps of oxidative stage and power balance.
17. Interrelation of the dichotomizing and apotomizing pathways of
glucose oxidation. A ratio of anaerobic and aerobic pathways of glucose disintegration at muscular work and their role in an organism.
18. Glyconeogenesis: sequence of reactions, the characteristic of enzymes and the biological role.
19. Allosteric regulation of glycolysis and glyconeogenesis (ATP,
ADP, AMP, NAD+, NADH as allosteric effectors). Pasteur effect.
20. Interrelation of glycolysis and glyconeogenesis.
21. Regulation of carbohydrate metabolism in an organism. The concentration of glucose in blood as homeostasis parameter of internal
environment of an organism.
22. Hereditary infringements of a carbohydrate metabolism (monosaccharides and disaccharides).
23. The role of insulin in maintenance of the constant level of sugar in blood. Diabetes mellitus. Sugar curves, their diagnostic value.
III.3. Material for self-study. I. chapter “Metabolism of carbohydrates”; II.
SECTION VIII. METABOLISM AND FUNCTIONS OF LIPIDS
Class 23
DIGESTION AND ABSORPTION OF LIPIDS. PARTICIPATION
OF BILE ACIDS AT THESE PROCESSES
19
The aim of the lesson: 1. To revise physical and chemical properties
and biological role of lipids.
2. To know processes of digestion and absorption of lipids, functions of bile acids.
3. To master methods of qualitative determination of bile acids
and lipase activity.
Initial level of knowledge:
- structure and properties of the saturated (C4-C18) and unsaturated
(C18-C20) fatty acids;
- structure and properties of triacylglycerols;
- structure and properties of complex lipids;
- biological role of lipids;
- classification and the nomenclature of enzymes.
Main topics.
I.2. Structure and properties of fatty acids on the example of palmitic, linoleic and other acids.
Classification of lipids.
Physical and chemical properties and biological value of triacylglycerols.
Features of structure and a biological role of phosphoacylglycerols.
Features of structure and a biological role of sphingolipids and
ceramides.
Features of structure and biological role of cholesterol and cholesterol esters.
Digestion and absorption of lipids.
Physiological value of bile acids.
II.1. Work № 1. INFLUENCE OF BILE ON LIPASE ACTIVITY
Lipase is unspecific enzyme which affects different fats at
рН=9.0, splitting ester bond in α-position (i.e. in external position).
Principle of the method.
Speed of lipase action in separate portions of milk can be
found by the amount of the fatty acids formed at hydrolysis of fat
within a certain time interval. The amount of fatty acids is determined by the titration with alkali at presence of phenolphtalein. If
bile is added to the test, lipase is activated and hydrolysis of milk fats
20
proceeds with the greater speed. Results of determination are expressed in milliliters of the titrating alkali solution. The graphs are
built where amount of 0.05N alkali solution in milliliters used on
neutralization of fatty acids, is put on the axis of ordinates, and the
time in minutes is put on an axis of abscess.
Practical procedure.
Reagents (ml)
Milk
5% pancreatin solution
Distilled water
Bile
Flask № 1
10.0
1.0
1.0
-
Flask № 2
10.0
1.0
1.0
Mix quickly and place in the thermostat at 380С
Every 15 min transfer 1.0 ml of the mix from both flasks into
glasses and immediately titrate with 0.05N NaОН solution up to pale
pink colouring which does no disappear for 30 sec. Carry out 4 tests
in total. According to the received data construct 2 graphs in one system of coordinates.
RESULTS:
ml
0.05N NaOH
solution
0
CONCLUSION:
15
30
45
time in minutes
Work № 2. ACTION OF PANCREATIC PHOSPHOLIPASES
There are some phospholipases in pancreas and its juice.
They accelerate hydrolysis of phosphoacylglycerols, and lecithin in
particular. Lecithin under action of phospholipases А1, А2, C and D
disintegrates to glycerol, fatty acids, phosphoric acid and choline.
Points of action of phospholipases to lecithin (phosphatidylcholine):
21
phospholipase А1
phospholipase А2
СH2-O-CO-R
|
R-CO-O-CH
|
+
CH2-O-PO-O-CH2-СH2-N (CH3) 3
|
OH
phospholipase C
phospholipase D
Principle of the method.
Pancreatic phospholipase activity is determined by free phosphoric acid, capable to form the yellow deposit at heating with ammonium molybdate.
Practical procedure.
Pour 5 dr. of egg yolk suspension into two test tubes. In the
first test tube add 2 dr. of pancreatin, and in the second (control) - 2
dr. of water. Place both test tubes in the thermostat at 380C for 30
minutes. After incubation into both test tubes pour 5 dr. of molybdic
reagent, heat them up and cool with water under the crane.
RESULTS:
CONCLUSION:
Work № 3. QUALITATIVE REACTION FOR BILE ACIDS.
Digestion and absorption of lipids and fat-soluble vitamins is
broken at the inflammation of liver, gall-bladder and at cholelythic
disease.
Principle of the method.
Bile acids can be revealed by Pettenkoffer reaction. At interaction of bile acid with hydroxymethylfurfural formed from reed sugar
under the action of concentrated sulfuric acid, violet colouring appears.
Practical procedure.
22
Put 2 dr of bile, 2 dr of 20% sucrose solution on dry Petri dish,
under which the sheet of the white paper for the best visualization of
results is laid, and carefully mix with the glass stick, then add 7 dr of
concentrated H2SO4 and again mix. After 2-3 min mark and write
down the occurrence of colouring.
RESULT:
III.2. Control questions.
What conditions are necessary for the digestion of lipids?
Where in the intestinal tract does the basic disintegration of lipids occur and why?
What enzymes cause the basic disintegration of lipids in the
intestinal tract?
What phosphoacylglycerols do you know?
What intermediate and end-products of digestion of triacylglycerols, phosphoacylglycerols do you know?
How and where in gastroenteric tract do the lipid digestion
products absorb?
What is the lysolecithine?
What are the pair bile acids?
III.3. Home assignment.
Why fats are suitable for the storage of energy in the body?
Draw the structural formula of the fat molecule (triglyceride) made
of oleic acid, palmitic acid, and glycerol.
Which animal fat has the highest percentage of unsaturated fatty acids?
Which type of lipid molecules is most likely to be present in membranes?
1. Complete the table.
nos Enzymes needed
to digest the fats
1 Lipase
2
Place of synthesis
23
What linkage they
break?
3
4
5
6
Choose the correct answer.
2. Lecithin contains nitrogenous base named
A. ethanolamine
B. choline
C. inositol
D. serine
3. Lecithin contains unsaturated fatty acid at position
A. 1.
B. 2
С. 3
D. 1 or 2
E. any
4. Phospholipase A1 breaks the ester bond in position
A. 1
B. 2
C. 3
D. 4
E. any
5. In the human bile the concentration of glycocholate salt is more
than of taurocholate one as
A. 2:1
B. 3:1
C. 4:1
D. 5:1
6. Choose the wrong answer. Bile acids
A. activate pancreatic lipase
B. emulsify fats increasing area surface
24
C. form micelles
D. stimulate intestinal peristalsis
E. are the bile pigments
Material for self-study. I. 1.pp.175-181, 293-296
2.pp.111-121, 475; II.
Class 24
OXIDATION AND SYNTHESIS OF FATTY ACIDS. METABOLISM OF KETONE BODIES. QUANTITATIVE DETERMINATION OF TRIACYLGLYCEROLS IN BLOOD
The aim of the lesson: 1. To know the sequence of reactions and value of β-oxidation of the fatty acids in tissues.
2. To know the process of fatty acid synthesis in tissues.
3. To know metabolism of ketone bodies.
4. To master methods of qualitative determination of ketone bodies and quantitative determination of triacylglycerols in blood
serum.
Initial level of knowledge:
- structure of fatty acids - components of human lipids;
- structure of neutral fats;
- classification and the nomenclature of enzymes;
- structure and functions of vitamins В2, В3, B5, Н.
Main topics.
I.2. Oxidation of fatty acids in tissues: the localization in a cell, steps
and energy yield (in АТP molecules).
Oxidation of glycerol in tissues.
Power balance of triacylglycerol oxidation.
Synthesis of fatty acids: the localization in a cell, sequence of reactions.
Participation of vitamins at oxidation and synthesis of fatty acids.
Modern representations about synthesis of ketone bodies (localization in an organism, sequence of reactions, biological value).
25
II.1. Work № 1. QUALITATIVE REACTIONS FOR ACETONE
AND ACETOACETATE.
Acetone, β-hydroxybutyrate and acetoacetate are refered to ketone bodies. In blood of healthy people they are in very small amount
– 13.4-185.2 mcmol/L (0.14-1.9 mg%), and in urine they aren’t detected by usual tests.
а) Legal’s test for acetone.
Principle of the method and the sequence of reactions.
Acetone and acetoacetate with sodium nitroprusside in the alkaline environment form a product of orange-red color.
СН3-СО-CH3 + Na2 [Fe (CN) 5NO] + 2NaOH →
Acetone
sodium nitroprusside
→ Na4 [Fe (CN) 5NO=CH-CO-CH3] + 2H2O
complex compound
After adding of ice acetic acid the cherry color compound is
formed.
Na4 [Fe (CN) 5NO=CH-CO-CH3] + CH3COOH →
→ Na3 [Fe (CN) 5NO-CH2-CO-CH3] + CH3COONa
Practical procedure.
Pour 1 dr. of urine, 1 dr. of 10% solution NaOH and 1 dr. of sodium
nitroprusside into a test tube. Note and write down colouring. Add 3
dr. of ice acetic acid.
RESULT:
б) Gerhard's test on acetoacetic acid.
Principle of the method and the sequence of reactions.
Enol form of acetoacetic acid enters into the reaction with ferric chloride with the formation of complex compound of cerise color.
At standing colouring turns pale owing to spontaneous decarboxylation of acetoacetate.
Practical procedure.
Add by drops 5% solution of ferric chloride to 5 dr. of urine.
Fix the occurrence at first a deposit (FePO4), and then the colouring.
N.B. Process proceeds very quickly at boiling.
26
Similar colour is possible after taking of salicylic acid, antipyrine, and other medical products, but colouring does not disappears at standing and boiling.
RESULT:
Clinical-diagnostic value.
Hyperketonemia and ketonuria is observed at diabetes, intake
of ketogenic food (deficiency of carbohydrates), starvation, hyperproduction of hormones (antagonists of insulin), corticosteroids,
von Gierke’s disease.
Hypoketonemia doesn’t have any diagnostic value.
At diabetes when the liver lacks glicogen, there is an amplified
disintegration of fats as the energy source. As the result of fatty acid
oxidation acetyl-CоА is accumulated that promotes its condensation
with formation of ketone bodies.
At early children's age long gastroenteric diseases (the dysentery, toxicoses) can cause ketonemia as the result of starvation and
exhaustion.
Work № 2. QUANTITATIVE DETERMINATION OF TRIACYLGLYCEROLS IN BLOOD SERUM BY ENZYMATIC METHOD
Principle of the method.
Lipase cleaves triacylglycerols to glycerol and fatty acids.
Glycerol is revealed by the reactions, controlled by glycerol kinase
and glycerol phosphate oxidase. Produced hydrogen peroxide is detected by oxidative condensation, a reaction catalysed by peroxidase.
As a result coloured product is formed. Its concentration is measured
by photoelectric colorimeter.
Triacylglycerol
→
glycerol
+ fatty acids
Lipase
Glycerol + ATP →
3 phosphoglycerol + ADP
glycerol kinase
27
3 phosphoglycerol + O2 → dioxiacetone phosphate + H2O2
glycerol phosphate oxidase
2H2O2 + 4-AAP + phenol →
hinoinimin +
peroxidase coloured product
4H2O
Practical procedure.
Reagents (ml)
Work reagent
Distilled water
Reference solution
Blood serum
Control solution
Standard
Sample
2.0
2.0
2.0
0.02
0.02
0.02
Carefully mix contents of test tubes, incubate at room temperature for 15 minutes
Measure optical density of test and the standard against the
control solution in a cuvette 1 sm at wave length 500 nm (490-540
nm).
CALCULATION.
Et • 2.29
Сt =
mmol/L , where
Est
Сt - concentration of triacylglycerols in blood sample;
2.29 - concentration of triacylglycerols in standard in mmol/L;
Еt - optical density of the sample;
Est - optical density of the standard.
In norm the contents of triacylglycerols in blood serum is 0.151.71 mmol/L.
RESULT:
28
CONCLUSION:
III.2. Control questions.
What ketone bodies do you know?
What is the level of ketone bodies in blood in norm?
Where ketone bodies are synthesized?
How is increased elimination of ketone bodies with urine
called?
What are the reasons of ketonuria?
What is the role of triacylglycerols in an organism?
How power value of triacylglycerol oxidation is calculated?
How many triacylglycerols are in blood in norm?
III.3. Home assignment.
Choose the correct answer.
1. Fatty acids for β-oxidation may be taken from
A. meal
B. glucose
C. adipose triacylglycerols
D. all of the above
2. The enzymes of β-oxidation are located in
A. cytoplasm
B. mitochondrial matrix
C. mitochondrial inner membrane
D. ribosome
E. any compartment of cell
3. Synthesis of fatty acids is located in (choose incorrect variant)
A. liver
B. lactating mammary glands
C. kidneys
D.adipose tissue
4. In β-oxidation of fatty acids take part vitamins, except
29
A. niacin
B. riboflavin
C. pantothenic acid
D. biotin
E. carnitine
5. β-oxidation of stearic acid gives …. molecules ATP
A. 36
B. 110
C. 130
D. 147
E. 164
6. Acetyl CoA carboxylase is regulated by
A. citrate
B. long-chain fatty acyl CoA
C. insulin
D. high-carbohydrate diet
E. all of the above
7. Ketone bodies are used as fuel by such tissues as
A. liver
B. brain
C. red blood cells
D. muscles
E. all of the above
Material for self-study. I. 1.pp.296-312
2.pp.173-189; II.
Class 25
BIOSYNTHESIS OF LIPIDS. REGULATION AND INFRINGEMENTS OF LIPID METABOLISM. METHODS OF CHOLESTEROL DETERMINATION
30
The aim of the lesson: 1. To know metabolism of cholesterol in human organism.
2. To know synthesis of triacylglycerols and phosphoacylglycerols.
3. To know regulation and pathology of lipid metabolism.
4. To master the method of estimation of cholesterol in blood.
Initial level of knowledge:
- structure and classification of lipids;
- regulation of enzyme activity;
- structure of c-AMP, ATP, CTP;
- fat-soluble vitamins.
Main topics.
I.2. Synthesis and resynthesis of triacylglycerols.
Synthesis and resynthesis of phosphoacylglycerols.
Synthesis of cholesterol: localization in an organism, stages, steps of
1-st stage.
Biological value and structure of steroids - derivatives of cholesterol.
Elimination of cholesterol from an organism.
Lipoproteins: kinds, structure.
Regulation of lipid metabolism: role of CNS, endocrine system, cAMP.
Pathology of lipid metabolism: the reasons, semiology, examples of
enzymopathies.
II.1. Work № 1. QUALITATIVE REACTION FOR CHOLESTEROL. (LIEBERMANN - BURCHARD TEST)
Cholesterol and its ethers are in different tissues and liquids of
animals: brain, skin fat, blood, bile. In brain in norm cholesterol is
present almost exclusively in the free form, but not as ethers. The
white substance of brain and spinal marrow contains 4-5% of cholesterol of damp weight.
Principle of the method.
The solution of cholesterol in chloroform gives the red colour
changing then in dark blue and green with acetic anhydride and
concentrated sulfuric acid. Under the action of the concentrated sulfuric acid, dehydratation of cholesterol occurs with the subsequent
31
condensation of the unsaturated hydrocarbons combined with sulfuric acid, and formation of the coloured products.
Practical procedure.
Add 10 dr. of acetic anhydride and 2 dr. of concentrated
H2SO4 to 1 ml of the chloroformic extract from a brain, and mix
well. Note and write down the change of colour.
RESULT:
Work № 2. ESTIMATION OF TOTAL CHOLESTEROL IN
BLOOD SERUM BY ENZYMATIC METHOD.
Cholesterol is transported in blood as a component of LDL and
VLDL, 70% of it is in the form of cholesterol esters and 30% - free
cholesterol. Total blood serum cholesterol contains both fractions.
Principle of the method.
Cholesterol is the product of hydrolysis of cholesterol esters
by cholesterol esterase. It is oxidized by air oxygen. Produced hydrogen peroxide is detected by oxidative condensation with appearance of coloured compounds. The reaction is catalysed by peroxidase. Intensity of colour is proportional to the cholesterol level in
blood.
Cholesterol esters + H2O →
cholesterol + fatty acids
cholesterol esterase
Cholesterol + O2
4-cholesten-3-on + H2O2
→
cholesterol oxidase
2H2O2 + phenol
→
coloured product + 4H2O
peroxidase
Practical procedure.
Reagents (ml)
Control solution
32
Standard Sample
Blood serum
Standard sol. (a reagent 1)
Distilled water
Reagent 2 рабочий
0.02
0.02
0.02
2.0
2.0
2.0
Carefully mix, incubate at room temperature not less 15 minutes.
Measure optical density of the sample and the standard against
the control solution in a cuvette 1 sm at wave length 500 nm (490540 nm).
CALCULATION.
Et • 5.17
Сt =
mmol/L, where
Est
Сt - concentration of cholesterol in the sample;
5.17 - concentration of cholesterol in the standard in mmol/L;
Еt - optical density of the sample;
Est - optical density of the standard.
RESULT:
CONCLUSION:
Clinical -diagnostic value.
At infringement of lipid metabolism cholesterol can collect in
blood. The increase of cholesterol in blood (hypercholesterolemia) is
observed at atherosclerosis, diabetes, mechanical jaundice, nephritis,
nephrosis (especially at lipoid nephrosis), hypothyrosis.
Lowering of cholesterol in blood (hypocholesterinemia) is observed at anemias, starvation, tuberculosis, hyperthyrosis, cancerous
cachexia, hepatogenous jaundice, affection of the central nervous
system, feverish conditions, at injection of insulin.
III.2. Control questions.
What forms is cholesterol contained in blood and tissues in?
What is physiological value of cholesterol?
What is normal concentration of cholesterol in blood?
33
What pathology is hypercholesterolemia observed at?
When is hypocholesterinemia observed?
Name initial substances for synthesis of cholesterol.
What biologically active conpounds are synthesized from cholesterol?
III.3. Home assignment.
Choose the correct answer.
1. TAG stored in adipose tissue represents the major reserve of substrate providing energy during the prolonged fast. During such a fast
A. the stored fatty acids are released from adipose tissue into the
plasma as components of the serum lipoprotein particle, VLDL
B. free fatty acids are produced at a high rate in the plasma by the
action of lipoprotein lipase to chylomicrons
C. glycerol produced by the degradation of triacylglycerol is an important direct source of energy for adipocytes and fibroblasts
D. hormone-sensitive lipase is phosphorylated by a c-AMP-activated
protein kinase
2. The highest contents of cholesterol is found in
A. chylomicrons
B. VLDL
C. LDL
D. HDL
3. Regulatory ensyme in synthesis of cholesterol is
A. thiolase
B. HMG-CoA synthase
C. HMG-CoA reductase
D. all of the above
4. Main activator of synthesis of fatty acids is
A. acetyl CoA
B. malonyl CoA
C. citrate
D. biotin
34
E.fatty acids
5. Main inhibitor of degradation of fatty acids is
A. acetyl CoA
B. malonyl CoA
C. citrate
D. biotin
E. long chain fatty acyl CoA
6. A teenager, concerned about his weight, attempts to maintain a
fat-free diet for a period of several weeks. If his ability to synthesize various lipids was examined he would be found to be most
deficient in his ability to synthesize
A. phospholipids
B. glycolipids
C. triglycerids
D. cholesterol
E. prostaglandins
Material for self-study. I. 1.pp.312-339
2.pp.197-229; II.
Class 26
COLLOQUIUM ON SECTION:
"METABOLISM AND FUNCTIONS OF LIPIDS"
Questions to a colloquium.
1. A general characteristic and classification of lipids. Biological role in an organism. Food lipids, norm of daily consumption.
2. Digestion and absorption of triacylglycerols in digestive
tract.
3. Digestion and absorption of phosphoacylglycerols in digestive tract.
4. Bile acids and their role during lipid digestion.
35
5. Resynthesis of triacylglycerols in an intestinal wall and their
synthesis in tissues. Physiological value.
6. Resynthesis of phosphoacylglycerols in an intestinal wall
and their synthesis in tissues. Physiological value.
7. Transport of fats to tissues. Blood lipoproteins: kinds, structure and function.
8. Role of chylomicrons in lipid metabolism. Deposition of lipids in the fatty tissue. Level of lipids in blood.
9. Value of methionine and choline in lipid metabolism.
10. Endocellular lipolysis. The cascade mechanism of lipase
activation.
11. Transport and recycling of the fatty acids formed at lipolysis. Speed change of the use of fatty acids depending on a rhythm of
a meal and muscular activity.
12. β-oxidation of fatty acids. Sequence of reactions, connection with the respiratory chain. Physiological value.
13. Oxidation of glycerol in tissues, its energy yield.
14. Energetic yield of triacylglycerol degradation.
15. Biosynthesis of fatty acids: sequence of reactions, a place
of synthesis, and physiological value.
16. Participation of vitamins in degradation and synthesis of
fatty acids. Role of pentose phosphate pathway of glucose oxidation
for synthesis of fatty acids. Dependence of biosynthesis speed on the
frequency of meal and structure of food.
17. Ketone bodies: a place and regulation of synthesis, sequence of reactions. Recycling of ketone bodies. Its physiological
level in blood, change of this parameter at diabetes and starvation.
18. Biosynthesis of cholesterol: steps of the I-st stage, regulation of the process. Elimination of cholesterol from an organism.
Physiological concentration of cholesterol in blood.
19. Cholesterol as the predecessor of steroid hormones, bile
acids, vitamin D.
20. A role of lipoproteins in cholesterol metabolism. Hypercholesterolemia: its reasons and the consequences. Modern biochemical bases of atherosclerosis.
36
21. Regulation of lipid metabolism. Influence of adrenaline,
glucagon, insulin to mobilization and deposition of fat.
22. Pathology of lipid metabolism, caused by infringement of
fat digestion and absorption. The mechanism of cholelythic disease.
23. Pathology of lipid metabolism. Congenital infringements:
Gauscher’s disease, Niemann-Pick disease, the Tay-Sachs disease,
xanthomatosis etc.
24. The infringements connected to insufficient intake in an
organism of fat-soluble vitamins.
25. Vitamin D, its influence on metabolism of calcium. Infringements of mineral metabolism at rickets.
III.3. Material for self-study. I. chapter “Metabolism of lipids”; II.
SECTION IX. METABOLISM OF AMINO ACIDS AND SIMPLE PROTEINS
Class 27
NITROGENOUS BALANCE. BIOLOGICAL VALUE, DIGESTION AND ABSORPTION OF THE PROTEINS. PUTREFACTION OF THE PROTEINS IN INTESTINES. TEST OF GASTRIC
JUICE
The aim of the lesson: 1. To know processes of digestion and absorption of proteins.
2. To know the processes of rotting of amino acids in intestines
and neutralization of rotting products.
3. To master methods of the quantitative and qualitative test of
gastric juice.
Initial level of knowledge:
- structure and physical and chemical properties of amino acids;
- chemistry and biological role of proteins;
- essential and nonessential amino acids;
- specificity of enzyme action.
37
Main topics.
I.2. Criteria of biological value of proteins.
Nitrogenous balance: types, examples.
Physiological minimum and optimum of protein.
Digestion and absorption of proteins: conditions, departments of the
digestive tract.
Proteolitic enzymes: the name, the place and the mechanism of activation, substrate specificity.
Putrefaction of amino acids in intestine, neutralization of rotting
products.
II.1. Work № 1. PATHOLOGICAL COMPONENTS OF GASTRIC
JUICE AND THEIR DETERMINATION.
Alongside with normal components other substances (lactic
acid, blood, and bilious pigments) can appear in gastric juice at pathology. In clinic these substances are frequently determined using
special reactions.
At achlorhydria alongside with lactic acid other organic acids
(acetic, oil) appear, since under influence of the microorganisms in
the stomach processes of fermentation are developed. Blood pigments can get to gastric juice at ulceration of stomach wall. Bilious
pigments appear in stomach from a duodenal gut owing to antiperistalsis.
а) Detection of lactate.
Principle of the method and sequence of the reactions see lesson №20, (page 10).
Practical procedure.
Add 5 dr. of gastric juice containing lactate to 1 ml of Uffelmann’s reagent.
RESULT:
б) Benzidine test for blood.
Principle of the method and the sequence of reactions.
Reaction is caused by ability of hemoglobin to catalyse the
benzidine oxidation with hydrogen peroxide. Benzidine thus is oxidized to paraquinone diimine and the liquid gets dark blue colour,
38
and at standing - red. This reaction is very sensitive and serves for
detection of the minimal impurity of blood in biological liquids.
+H2O2
H2N–
-NH2 → HN=
═
=NH + 2H2O
Hb
Practical procedure.
Put 5 dr. of 3% solution hydrogen peroxide and 5 dr. of gastric juice
containing blood to 5 dr. of benzidine. Mark and write down colouring.
RESULT:
Work № 2. QUANTITATIVE DETERMINATION OF ACIDITY
OF GASTRIC JUICE.
At determination of gastric juice acidity, the total acidity, the
total hydrochloric acid, a free hydrochloric acid and the connected
hydrochloric acid are distinguished. The sum of all acid-reacted substances understand as the total acidity of gastric juice.
Principle of the method.
The total acidity is measured in ml of 0.1N NaOH solution
spent on neutralization of 1000 ml of gastric juice at the presence of
the indicator phenolphthalein (a zone of transition is 8.3-10.0; when
it is below 8.3 - colorless, when it is higher 10.0 - red). The content
of free HCl is measured in ml of 0.1N NaOH solution, spent on
neutralization of 1000 ml of gastric juice at the presence of the indicator dimethylaminoazobenzene (zone of transition is рН 2.9-4.0;
when it is below - rose-red, when it is higher - yellow).
The hydrochloric acid named "connected" is the form of salt
with proteins and products of their digestion.
Total hydrochloric acid is the sum of the free and connected
hydrochloric acid. It is determined by calculation at titration of gastric juice at the presence of two indicators (dimethylaminoazobenzene and phenolphthalein).
In norm an adult the parameters of acidity vary in the following limits:
Total acidity
- 40-60 mmol/L
Free HCl
- 20-40 mmol/L
39
Connected HCl - 10-20 mmol/L
Total HCl
- 30-60 mmol/L
Practical procedure.
Measure by pipette in flask 5 ml of gastric juice, add 1 dr. of
dimethylaminoazobenzene and 2 dr. of phenolphthalein. At presence
in gastric juice of free HCl it is coloured in red color with a pink
shade, at absence of that immediately there is yellow painting.
Titrate the free HCl by 0.1N alkali solution from microburette
up to the occurrence of yellow-red (orange) colour, write down the
result (n1).
Continue titration without adding alkali in dropping tube till
the occurrence of citreous colour, write down the result (n2).
Continue titration till the occurrence of pink colour (n3).
NB! n3> n2> n1.
Calculation:
n · 1000 · 0.1
X = ——————, where
5
X - acidity of gastric juice;
n - amount of alkali, used on titration of gastric juice.
NB! n=n1
- for determination of free hydrochloric acid;
n=n3 - for determination of the total acidity;
n = (n2+n3):2 - for determination of total hydrochloric acid.
1000
- amount of gastric juice for calculation in ml;
0.1
- amount of mg - equivalent of alkali in 1 ml of 0.1N
solution in mmol;
5
- amount of the gastric juice taken for titration in ml.
RESULTS:
CONCLUSION:
Clinical -diagnostic value.
Acidity can change at diseases of a stomach. At stomach ulcer
or hyperacid gastritis the contents of free HCl and the total acidity
increases (hyperchlorhydria). At hypoacid gastritis or at cancer of a
40
stomach lowering of free HCl amount and the total acidity (hypochlorhydria) is observed. At cancer of a stomach, chronic gastritis
absence of total HCl and significant decrease of the total acidity
(achlorhydria) is marked. At malignant anemia, at cancer of a stomach full absence of HCl and pepsin (achylia) is observed.
III.2. Control questions.
What is the gastric juice?
What components does it have in norm?
What is the role of HCl of gastric juice in digestion?
What pathological components can be found in gastric juice?
What reaction reveals the lactic acid?
What test is used for detection of blood?
What kinds of acidity do you know?
III.3. Home assignment.
1. Complete the table.
nos Enzymes needed
to digest the proteins
1 Pepsin
2
3
4
5
6
7
8
9
10
Place of synthesis
What linkages do they
break?
Choose the wrong answer.
2. Biological significance of digestion of proteins is:
A. source of amino acids, necessary for synthesis of biologically active compounds
B. formation of products easily absorpted in intestine
41
C. formation of products without antigenic specificity
D. source of essential amino acids
E. source of amino acids for synthesis of their own proteins
3. Hydrochloric acid of gastric juice
A. denatures diet proteins
B. makes pH-optimum for pepsin
C. activates pepsin by allosteric way
D. participates in absorbtion of proteins
E. courses partial proteolysis of pepsin
4. Activation of proenzymes can be perfomed by
A. change of primary structure of enzyme
B. change of secondary structure of enzyme
C. change of tertiary structure of enzyme
D. formation of new bonds in molecule of enzyme
E. splitting of peptide from N-terminus
5. Match the name of enzyme and its characteristic
A. trypsin
1. endopeptidase
B. pepsin
2. exopeptidase
C. chymotrypsin
D. carboxypeptidase
E. aminopeptidase
6. Match the parts of sentences. Proteolytic enzyme … is synthesised
in …
A. elastase
1. active form
B. aminopeptidase
2. stomach
C. None
3. pancreas
7. Choose the correct variants. Detoxication of products of rotting of
amino acids
A. is located in liver
B. is the formation of pare compound with UDPGA
C. forms such molecule as animal indican
42
D. forms pare compound with PAPS
E. leeds to synthesis of any amino acids
Material for self-study. I. 1.pp. 50, 170-175, 343-345, 542-545
2.pp. 237, 477-480; II.
Class 28
DEAMINATION, TRANSAMINATION, DECARBOXYLATION
OF AMINO ACIDS. USING OF CARBON SKELETON OF AMINO ACIDS. DETERMINATION OF PRODUCTS OF PROTEIN
METABOLISM IN BIOLOGICAL LIQUIDS
The aim of the lesson: 1. To know the common pathways of metabolism of amino acids (deamination, transamination, decarboxylation).
2. To master methods of qualitative and quantitative test of proteins in urine.
Initial level of knowledge:
- chemistry of amino acids;
- reactions of deamination, decarboxylation;
- classification and the nomenclature of enzymes;
- structure of vitamins В2, nyacin, В6 and coenzymes containing
them.
Main topics.
I.2. The common pathways of metabolism of amino acids.
The mechanism of transport of amino acids in a cell.
Deamination of amino acids and its types.
Transamination of amino acids: the mechanism, enzymes, the function of vitamin В6.
Decarboxylation of amino acids and its types.
Biogenic amines: a structure, a biological role, inactivation.
II.1. Work № 1. QUALITATIVE REACTION FOR PROTEIN IN
URINE.
43
In norm protein in urine is in trace amounts and can not revealed by usual reactions used in clinical laboratory. At some diseases the appreciable amount of protein, from parts of gramm up to 25 g
in day is excreted with urine. Occurrence of protein in urine is
named proteinuria or аlbuminuria as urine contains, basically, serum
albumin.
Proteinuria can be true and false. At true (or renal) proteinuria
the protein of blood serum gets in urine through kidneys. Casual (or
false) proteinuria is observed when slime, blood, pus from urinative
tract gets into urine, but not from kidneys.
Principle of the method.
To determine the protein in urine reactions of sedimentation
with nitric or sulfosalicylic acids are applied. The latter is the most
sensitive.
Practical procedure.
Put 3 dr of 20% solution sulfosalicylic acid to 1 ml of urine.
RESULT:
Work № 2. QUANTITATIVE DETERMINATION OF PROTEIN
IN URINE.
Principle of the method.
The method is based on Geller’s reaction with concentrated
nitric acid. It is experimentally established, that the solutions contained 0.033% of protein (i.e. 0.033 g/L), give a muddy white ringlet
at the end of 2-nd - the beginning of 3-rd minute after layering of
solution of protein on nitric acid. Thickness and speed of occurrence
of a ring depends on amount of protein.
Practical procedure.
Pour 1 ml of 50% nitric acid solution in a test tube, and then cautiously on the wall add 1 ml of urine from a pipette. Due to relatively
small density urine stratifies on the acid. If the ring has appeared at
once, repeat reaction with the urine dissolved in 10, 20, 30 and more
times for reception of negative result.
CALCULATION. Amount of protein (X) is determined according
to the formula:
X = n · 0.033 g/L, where n - dilution of urine.
44
RESULT:
III.2. Control questions.
How can you determine protein in urine?
What is the principle of quantitative determination of protein
in urine?
What kinds of proteinuria do you know?
III.3. Home assignment.
Choose the correct answer.
1. Oxidative deamination of amino acids occurs in:
A. intestine
B. liver and kidneys
C. pancreas
D. adipose tissue E. any tissue
2. The transaminase has ... as coenzyme
A. FAD
B. FMN
C. NADP
D. P-phosphate
E. HS-CoA
3. The most of amino acids are substrates for transamination except
A. alanine
B. glutamate
C. proline
D. valine
E. glycine
4. Match the amino acid and its derivative
A. glutamate
1. cadaverine
B. lysine
2. GABA
C. tryptophan
3. serotonin
D. phenylalanine
4. Norepinephrine
E. tyrosine
5. Indicate the followings as “true” or “false”.
A. histamine is the mediator of pain
B. spermin forms complexes with DNA
C. GABA is activator of CNS
D. insufficiency of dopamine is the cause of parkinsonism
E. serotonin is used for treatment of hypertonia
45
6. Choose correct versions. Inactivation of biogenic amines gives:
A. ammonia
B. amino acids
C. H2O2
D. aldehydes E. uric acid
Material for self-study. I. 1.pp.345-350, 361, 365, 372-374, 386, 391395
2.pp.244, 249-250; II.
Class 29
NEUTRALIZATION OF AMMONIA IN THE ORGANISM. SYNTHESIS OF UREA. DETERMINATION OF PRODUCTS OF
PROTEIN METABOLISM IN BIOLOGICAL LIQUIDS
The aim of the lesson: 1. To know ways of formation and neutralization of ammonia.
2. To master method of quantitative determination of urea in
blood serum.
Initial level of knowledge:
- chemistry of amino acids;
- reactions of deamination, transamination of amino acids;
- classification and the nomenclature of enzymes;
- structure of vitamins В2, В3, and coenzymes containing them.
Main topics.
I.2. Formation of ammonia in an organism.
The ways of utilization of ammonia.
Synthesis of urea: reactions, enzymes, regulation, infringements.
II.1. Work № 1. ESTIMATION OF UREA IN BLOOD SERUM
Principle of the method.
Urea forms the red complex measured by photometry with diacetylmonooxime at the presence of thiosemicarbazide and Fe3+ ions
in a strongly acid medium.
Practical procedure.
46
Prepare the working solution, having mixed one part of the reagent solution with one part of sulfuric acid solution.
Deposit the serum proteins: in centrifugal test tube pour 1 ml
of 5% trichloroacetic acid solution, add 0.1 ml of serum and centrifuge for 10 min at 1500 rot/min Use the supernatant for the test.
Also dilute the reference solution: add 0.1 ml of a reference
solution of urea to 1ml of 5% trichloroacetic acid solution.
Practical procedure.
Reagents (ml)
Working solution
Supernatant
Reference solution
Distilled water
Sample
2.0
0.1
-
Standard
2.0
0.1
-
Control
2.0
0.1
Close the test tubes with the cover made of foil and heat up
exactly for 10 min in the boiling water bath, cool (2-3 min) under the
stream of cold water and not later, than in 15 min measure optical
density of the sample and the standard against the control solution in
cuvette 1 sm at wave length 490-540 nm (an optical filter №5,
green).
CALCULATION.
A
X = ——— · 16.65 mmol/L, where
B
X - concentration of urea in the blood sample;
A - optical density of the test;
B - optical density of the standard.
It is possible to make recalculation of urea in nitrogen of urea by
multiplication to factor 0.466.
The physiological level of urea in blood serum is 2.50-8.32
mmol/L; in urine it is 333-583 mmol/day.
The top border of the urea level depends on intake of proteins with
food. At intake of proteins more than 2.5 g/kg of weight per day the
level of urea in blood can be up to 10 mmol/L.
47
RESULT:
CONCLUSION:
Besides the proteins in blood there are compounds containing
nitrogen, which don’t get precipitated with the protein precipitating
agents (picric acid etc.). After removal of the blood proteins by sedimentation non-protein filtrate only contains the nitrogen named
"non-protein nitrogen" and composed of nitrogen of urea, uric acid,
creatine, creatinine, indican, amino acids and other substances.
III.2. Control questions.
What is normal level of urea in blood? In urine?
In what cases does level of urea in blood raise?
When does blood urea level decrease?
What is the "non-protein nitrogen"?
What is azotemia and when is it observed?
III.3. Home assignment.
Choose the wrong answer.
1. Glutation:
A. is the tripeptide
B. exists in reduced or oxidized states
C. serves as a coenzyme
D. prevents the oxidation of sulfhydryl groups to disulfide groups
E. performs specialized functions in erythrocytes
F. is involved in the transport of amino acids in the intestine and kidney through membrane
G. is involved in the detoxication process
H. is the part of the selenium containing enzyme
Choose the correct answer.
2. The following is the non-protein amino acid
A. ornithine
48
B. homocysteine
C. histamine
D. all of the above
3. The major mechanism of detoxication of ammonia in the brain is
A. synthesis of urea
B. formation of glutamine
C. formation of asparagine
D. synthesis of creatine
E. formation of uric acid
F. all of the above
4. Mainly enzyme glutaminase is present in
A. brain
B. liver
C. muscles
D. kidneys
E. all of the above
5. At insufficiency of liver hyperammoniemia develops. Which one
of the following would be elevated in blood of patient?
A. urea
B. uric acid
C. glutamine
D. asparagine
E. valine
6. Which one of the following statements about the urea cycle is correct?
A. Urea is produced directly by the hydrolysis of citrulline.
B. ATP is required for formation of arginine.
C. All reactions of urea cycle occur in mitochondrion.
D. The two nitrogen atoms of urea enter the cycle as ammonia and
glutamine.
E. Increasing of urea in urine is due to a diet rich in protein.
49
Material for self-study. I. 1.pp.350-354
2.pp.241-248; II.
Class 30
THE GENERAL SCHEME OF SOURCES AND PATHWAYS OF
RECYCLING OF AMINO ACIDS IN TISSUES. CATHEPSINS.
SPECIFIC WAYS OF METABOLISM OF SOME AMINO ACIDS.
THE PATHOLOGY OF THE NITROGENOUS METABOLISM.
ESTIMATION OF THE PRODUCTS OF NITROGENOUS METABOLISM IN BIOLOGICAL LIQUIDS
The purpose of lesson: 1. To know specific ways of metabolism of
some amino acids.
2. To know physiological value of metabolites - derivates of amino acids.
3. To know pathology of nitrogenous metabolism.
4. To master methods of determinaition of products of nitrogenous metabolism in blood and urine.
Initial level of knowledge:
- chemistry of the amino acids;
- essential and nonessential amino acids;
- universal ways of catabolism of amino acids;
- vitamins В3, ВС: a structure, biological role.
Main topics.
I.2. Metabolism of glycine and serine: interconversions, role of tetrahydrofolic acid.
Metabolism of sulphur-containing amino acids: interconversions,
features of deamination, synthesis of creatinine, glutathione.
Metabolism of phenylalanine and tyrosine: disintegration up to fumaric and acetylacetic acids, synthesis of epinephrines, thyronins,
melanins.
Metabolism of tryptophan: serotonin and kynurenine ways.
The scheme of recycling of dicarboxylic amino acids.
50
Congenital and secondary infringements of amino acid metabolism.
II.1. Work № 1. THE DETECTION OF CREATININE IN URINE.
Creatinine is formed from creatine or phosphocreatine by
splitting off phosphoric acid and it is excreted with urine in the
amount of about 2 g/day.
Principle of the method.
The most typical reactions for creatinine are the reaction of
formation of unstable nitrosocreatinine (red) and the reaction of
formation of well soluble creatinine picrate salt (orange-red).
Practical procedure.
а) Nitroprusside test.
Add 0.5 ml of 10% solution caustic soda and 0.5 ml nitroprusside sodium to 1 ml of urine. Mark colouring. After addition of acetic
acid colouring quickly disappears.
RESULT:
б) Reaction with picric acid.
Add 10 dr of 10% picric acid solution and the same amounty
of 10% solution of caustic soda to 1 ml of urine.
RESULT:
Work № 2. ESTIMATION OF BLOOD SERUM CREATININE.
Principle of the method.
In alkaline medium picric acid interacts with creatinine forming the coloured products (orange-red). Colouring is measured by
photometry. The test of blood is carried out after removal of proteins
and in urine it is carried out after cultivation with water.
Practical procedure.
Reagents (ml)
Blood serum
Distilled water
Solution 3
Reagent 3 (TCA)
- Be cautious!
Sample
0.5
1.0
0,5
Control
1.5
0,5
Standard
1.0
0.5
05
Mix, centrifuge sample at 1500 rot/min 10 min.
51
Supernatant
Reagent 4
(picric acid)
Reagent 5 (NaOH)
1.0
0.5
1.0
0.5
1.0
0.5
0.5
0.5
0.5
Mix and exactly in 20 min measure optical density of the sample (А1) and of the standard (А2) against the control solution in cuvette 1 sm at wave length 500-510 nm.
CALCULATION: Amount of creatinine (X) is calculated according to the formula:
A1
X = ——— ∙ 177 · mcmol/L
А2
NB. Determination is not absolutely specific, since some substances with active methylene group and some restoring molecules
(for example, glucose) interfere.
Physiological values.
In norm the parameter for men is
61-115 mcmol/L,
for women is 58 - 97 mcmol/L.
RESULT:
CONCLUSION:
Work № 3. DETERMINATION OF AMMONIUM SALTS IN
URINE
Principle of the method.
After addition the solution of weak alkali into a test tube with
urine the liquid gets a smell of ammonia owing to decomposition of
ammonium salts presenting in urine.
2NH4Cl + Ca(OH)2 → 2NH4OH + CaCl2
Weak alkalis as calcium hydroxyde do not decompose nitrogen-containing organic substances (urea, creatinine, etc.), they destroy only ammonium salts.
Practical procedure.
Pour into a test tube 1-2 ml of urine and the same amount of
solution Ca(OH)2. Put into the liquid a damp red litmus paper. In 1-2
52
min mark and write down the change of colouring of the paper. The
test tube can be closed with cotton wool.
RESULT:
CONCLUSION:
III.2. Control questions.
What products of nitrogenous metabolism are determined in
urine of healthy people?
How much creatinine is excreted with urine daily?
What is the normal level of creatinine in blood?
What color reactions are used for determination of creatinine?
Is BIOTEST on creatinine specific? Why?
III.3. Home assignment.
Choose the correct answer.
1. The following substance is ketogenic
A. fatty acids
B. leucine C. lysine
D. all of them
2. In the synthesis of cysteine, the carbon skeleton is provided by
A. serine
B. methionine C. glutamate
D. alanine
3. The amino acids are said to be ketogenic when the carbon skeleton
is finally degraded to
A. succinyl CoA B. fumarate C. acetyl CoA D. pyruvate
4. Catabolism of phenylalanine begins from reaction of
A. transamination
B. decarboxilation
C. transmethylation
D. hydroxylation
E. dehydration
F. dehydrogenation
5. Match the parts of the sentences
A. 5-hydroxytriptamine 1. is involved in inactivation of niacin.
B. Methionine
2. is synthesized from glycine, methionine
and arginine.
C. Creatin
3. stimulates the central nervous system.
D. Taurine
4. forms pair bile acids
53
6. Which one of the following statements is correct?
A. All essential amino acids are glycogenic.
B. Ornithine and citrulline are found in tissue proteins.
C. In the presence of adequate dietary sources of tyrosine, phenylalanine isn’t an essential amino acid.
D. At lack of methionine in diet cysteine is an essential amino acid.
E. Increasing of urea in urine is due to a diet rich in protein.
Material for self-study. I. 1.pp.50, 54, 69, 355-364
2.pp.238-240, 250-268, 479; II.
Class 31
COLLOQUIUM ON SECTION:
"METABOLISM AND FUNCTIONS OF AMINO ACIDS"
Questions to a colloquium.
1. Concept of biological value of proteins. Essential and nonessential amino acids. Norms of daily intake. Nitrogenous balance.
2. Digestion of proteins and absorption of amino acids in digestive tract. The significance of pancreatic proteinases in development of pancreatitis.
3. Putrefaction of proteins (amino acids) in intestines and neutralization of putrefaction products.
4. General scheme of sources and pathways of recycling of
amino acids in tissues. Protein turnover in an organism.
5. Transamination of amino acids: sequence of reactions, a
role in metabolism. Participation of vitamin В6 in the construction of
enzyme systems of transamination.
6. Amino acids participated in transamination, a special role of
glutamic acid. Specificity of transaminases. Determination of transaminases in blood at some diseases.
7. Disintegration of proteins in tissues. Cathepsins. Sources of
nitrogen for synthesis of amino acids.
54
8. Oxidative deamination of amino acids. Indirect deamination: sequence of reactions, enzymes, biological value.
9. Features of deamination of sulphur-conteining and hydroxyamino acids. Using of carbon skeletons of amino acids in an organism.
10. Ways of formation and neutralization of ammonia in an
organism. Role of kidney glutaminase at acidosis.
11. Biosynthesis of urea. Sequence of reactions, enzymes,
regulation.
12. Total equation of urea cycle. Normal level of urea in
blood. Infringements of synthesis and elimination of urea. Primary
and secondary hyperammoniemia.
13. Decarboxylation of amino acids: the sequence of reactions
on the example of some amino acids.
14. Biogenic amines (histamine, serotonin, GABA), their formation and participation in metabolism and development of pathological conditions. Oxidative deamination of biogenic amines. Aminooxidases.
15. Biosynthesis of serine and glycine. Their participation in
metabolism.
16. Role of serine and glycine in formation of mono-carbon
groups. Participation in this process of H4folate. Insufficiency of folic acid. The mechanism of bacteriostatic action of sulfanilamides.
17. Synthesis of cystein, taurine, glutathione. Their participation in metabolism.
18. Role of methionine in the process of transmethylation. Examples of methylation of compounds.
19. Biosynthesis of choline, creatine, catecholamines.
20. Metabolism of phenylalanine and tyrosine: the sequence of
reactions (disintegration up to fumaric and acetylacetic acids). Physiological value of intermediate metabolites as sources of biologically
active derivates.
21. Biosynthesis of thyroxin and melanin from predecessors.
Their influence on metabolism.
22. Metabolism of tryptophan. Biosynthesis of NAD+ from
predecessors.
55
23. Glucogenic and ketogenic amino acids.
24. Biochemical bases of congenital infringements of metabolisn of some amino acids (phenylketonuria, alkaptonuria, albinism,
Hartnupp’s disease).
25. Physiological role of blood plasma proteins. Protein fractions, value of individual proteins.
26. Hyperproteinemia and hypoproteinemia. Disproteinemia,
paraproteinemia, congenital defectoproteinemia.
27. Antigenes. Specific features of antigenic structure of an
organism as a basis of protein incompatibility. Antibodies. The
mechanism of interaction of antibodies with antigene. Canceroembrionic antigenes.
28. Proteins and the cascade of reactions of blood clotting. The
mechanism of formation of fibrin gel and its stabilization. Role of
vitamin K in blood clotting.
29. Anticoagulative system, antithrombin and heparin. Fibrinolisis
III.3. Material for self-study. I. chapter “Metabolism of amino acids”; II.
SECTION X. METABOLISM OF CONJUGATED PROTEINS
Class 32
METABOLISM OF NUCLEOPROTEINS. BIOSYNTHESIS
AND DISINTEGRATION OF PURINE AND PYRIMIDINE NUCLEOTIDES
The aim of the lesson: 1. To know the scheme of synthesis of purine
and pyrimidine nucleotides.
2. To know catabolism of purines and pyrimidines.
Initial level of knowledge:
- chemistry and properties of proteins;
- chemistry of nucleic acids;
56
- classification and the nomenclature of enzymes;
- metabolism of simple proteins.
Main topics.
I.2. Sources of purine ring.
Synthesis of purine nucleotides from inosinic acid.
Synthesis of pyrimidine nucleotides.
Disintegration of purines up to uric acid.
Disintegration of pyrimidine nucleotides.
Disintegration of nucleic acids in gastrointestinal tract and tissues.
Regulation of metabolism of nucleotides.
Pathology of metabolism of nucleotides.
II.1. Work № 1. QUANTITATIVE ASSAY OF URIC ACID IN
URINE.
Uric acid is an end-product of metabolism of the purine bases
included in the conjugated proteins – nucleoproteins in a human organism.
Principle of the method.
Uric acid is capable to restore phosphotungistic reagent to
phosphotungistic dark blue. Intensity of its colouring is proportional
to the content of uric acid. At titration with red blood salt К3 [Fe
(CN) 6] phosphotungistic dark blue is oxidized and dark blue colour
disappears.
Practical procedure.
Add 1 ml of 20% of solution sodium carbonate and 1 ml of
phosphotungistic reagent to 1.5 ml of urine, mix and titrate with
0.01N solution of red blood salt till the disappearance of colouring.
CALCULATION: content of uric acid (in mg) in daily urine is calculated according to the formula:
0.8 · A · B
X=
, where
1.5
0.8 - amount of uric acid in mg, corresponding to 1 ml of solution
К3[Fe(CN)6];
A - amount of the used red blood salt (in ml);
B - daily diuresis (in ml).
57
Physiological value.
In norm 1.6-3.4 mmol/day (270-600 mg/day) of uric acid is
excreted with urine.
The factor of recalculation to IS (mmol/day) is 0.0059.
RESULT:
CONCLUSION:
Clinical -diagnostic value.
Hypouricuria is marked at gout, nephrytis, and renal insufficiency.
Hyperuricuria – is at alimentary (food) leukaemia, strengthened disintegration of nucleoproteins.
Excretion of uric acid depends on the content of purines in
food, the intensity of metabolism of nucleoproteins, and the age (for
children it is relatively more, than for adults
What is normal daily excretion of uric acid with urine at the
person?
What diseases are characterized by hyperuricuria? hypouricuria?
What factors influence the concentration of uric acid in urine?
What is gout?
III.3. Home assignment.
Choose the correct answer.
1. Name the enzyme associated with hyperuricemia
A. phosphoribosil-pyrophosphate (PRPP) synthetase
B. hypoxantine-guanine phosphoribosyltransferase (HGPRT)
C.glucose 6-phosphatase
D.all of them
2. An enzyme of purine metabolism associated with immunodeficiency disease is
A. adenosine deaminase
B. xanthine oxidase
58
C. PRPP synthetase
D. HGPRT
3. The end product of purine metabolism in human is
A. xanthine
B. uric acid
C. urea
D. allantoin
4. The nitrogen atoms in the purine ring are obtained from
A. glycine
B. glutamine
C. aspartate
D. all of them
5. Administration of which of the following compounds is most likely for patients with orotic aciduria?
A. adenine
B. guanosine
C. hypoxanhtine
D. uridine
E. thymidine
6. The rate of DNA synthesis in experiment could be most accurately
determined by measuring the incorporation of radioactive form
A. adenine
B. guanine
C. cytosine
D. uridine
E. thymidine
E. phosphate
Material for self-study. I. 1.pp.181, 405-418
2.pp.270-284, 478; II.
Class 33
CHEMISTRY AND METABOLISM OF CHROMOPROTEINS.
THE ROLE OF LIVER IN PIGMENTARY, CARBOHYDRATE,
LIPID AND PROTEIN METABOLISM. DETOXICATE FUNCTION OF THE LIVER
59
The aim of the lesson: 1. To revise structure of hemoglobin.
2. To know the synthesis of heme.
3. To know the formation of bilious pigments and removing of
them from an organism.
4. To know the characteristic of the basic types of jaundice.
Initial level of knowledge:
- chemistry of conjugated proteins;
- classification of enzymes;
- metabolism of fats, proteins, carbohydrates;
- neutralization of poisonous products of rotting of amino acids in
intestine.
Main topics.
I.2. Structure of hemoglobin; structural differences of various types
of hemoglobin in globin, in heme.
Synthesis of heme: place of synthesis, chemism before the formation
of porfirinogen.
Disintegration of hemoglobin in tissues: localization, chemism of
transformations before bilirubin is formed, the further transformations and removing of bilious pigments from an organism.
Role of liver in protein, carbohydrate, lipid and pigmentary metabolism.
Neutralizing function of liver.
The characteristic of the basic types of jaundices.
Metabolism of iron in an organism, role of transferrin, ferritin, haptoglobin, hemosiderin.
II.1. Work № 1. ESTIMATION OF BILIRUBIN IN BLOOD.
Bilirubin (free or indirect) is formed during disintegration of
hemoglobin in tissues of reticular endotelial system, bone marrow,
liver, spleen. Then it is transported with albumin in liver where connects with glucuronic acid at participation of enzyme glucuronil
transferase, turning to the connected (direct) bilirubin.
60
Free bilirubin is insoluble in water, but it well dissolves in the
organic solvents, it is toxic, it does not pass through renal filter and it
is not excreted with bile.
Direct bilirubin is less toxic; it well dissolves in water, passes
through renal filter, and secrets into bile. In intestine bilirubin consistently turns to mesobilirubin, mesobilinogen (urobilinogen), stercobilinogen, stercobilin.
Principle of the method.
Diazoreagent gives with direct bilirubin pink colouring whose
intensity is proportional to concentration of bilirubin and can be determined by photometry.
Indirect bilirubin can be transferred in the soluble condition by
additing caffeine reagent to serum and can be determined with diazo
test. Total concentration of both forms of bilirubin in blood serum
makes the total bilirubin. The level of free bilirubin is determined
according to the difference between amount of total and direct bilirubin.
Practical procedure.
Reagents (ml)
Blood serum
Isotonic solution
NaCl
Diazoreagent
Caffeine reagent
1 test tube (direct bilirubin)
0.5
2.0
2 test tube (total
bilirubin)
0.5
-
Control
0.3
-
0.3
2.0
2.0
0.5
0.3
NB! At first add reagents to serum into the 2nd test tube, and in 8
minutes into the 1st and into the control one (then the exposition will
ends simultaneously).
Leave test tubes on the table for 10 minutes. Measure optical
density of the samples against the control in cuvette 10 mm at a
green optical filter (630 nm). Determine concentration of direct and
total bilirubin on calibrating graph, calculate amount of indirect bilirubin.
Clinical -diagnostic value.
61
In norm the content is (in mcmol/L):
The total bilirubin 1.7-20.5
indirect 1.7-17.1
direct 0.86-4.3.
The newborns have the total bilirubin up to 23.17 mcmol/L.
At physiological jaundice the total bilirubin is increased, basically,
due to indirect one owing to functional insufficiency of liver.
Determination of bilirubin fractions has great importance for
differential diagnostics of various types of jaundices.
The factor of recalculation in IS is 17.104.
RESULT:
CONCLUSION:
Work № 2. QUALITATIVE ASSAY OF BILIOUS PIGMENTS
IN URINE.
Bilious pigments appear in urine as alkaline salts at jaundice.
The urine containing bilious pigments has yellowish-brown or green
colouring. The majority of chemical tests on bilious pigments are
based on the ability of bilirubin and biliverdin easily to oxidize with
formation of variously coloured products: biliverdin (green), bilicyanin (dark blue), holetheelin (yellow), etc.
Practical procedure
1. Cautiously layer urine to 20 dr. of concentrated HNO3 so
that liquids do not mix up. Note and write down the occurrence of
variously coloured rings on the border of the unit of liquids. (First
the green ring should appear)
RESULT:
2. Modification of 1st test.
Filter about 2 ml of urine through the small filter. Then put 1
dr. of nitric acid on the filter and observe the occurrence of the coloured rings, as in the 1st test.
RESULT:
62
III.2. Control questions.
What fractions of bilirubin do you know?
What is the normal concentration of the total bilirubin and its
fractions in blood of an adult (a newborn)?
How does direct bilirubin differ from indirect one?
What and how much bilious pigment is excreted with urine in
norm?
What is urobilinogen?
III.3. Home assignment.
Choose the correct answer.
1. δ-aminolevulinic acid forms at condencation of
A. glycine and α-ketoglutarate B. glycine and oxaloacetate
C. glycine and succinyl CoA
D. alanine and succinyl CoA
E. glycine and succinate
2. The catabolism of hemoglobin
A. occurs in eryrthrocytes
B. results in the liberation of carbon dioxide
C. results in the formation of protoporphyrin
D. involves the oxidative cleavage of porphyrin ring
E. is the only source of bilirubin glycine and succinate
3. The most content of ferritin is in
A. liver
B. spleen
D. bone marrow
E. skin
C. muscle
4. Bilirubin is not excreted in urine in
A. obstructive jaundice B. hepatic jaundice
C. hemolitic jaundice
D. all of the above E. none of the above
5. Match the name of substance and its characteristic
A. direct bilirubin
1. forms complex with albumin
B. indirect bilirubin 2. forms in intestine
C. both
3. soluble in water
D. none
4. toxic
63
5. is the product of condensation with glucuronic acid
6. yellow coloured
6. Match the following desease and process
A. obstructive jaundice 1. urobilinogen absent in urine and one
trace to absent in feces
B. hepatic jaundice
2. urobilinogen increased and bilirubin absent in urine and urobilinogen increased
in feces
C. hemolitic jaundice
3. urobilinogen increased and bilirubin present in urine and urobilinogen decreased
in feces
Material for self-study. I. 1.pp.199-224, 506
2.pp.40-47, 293-301; II.
SECTION XI. REGULATION OF METABOLISM.
HIERARCHY OF REGULATORY SYSTEM
Class 34
THE BASIC MECHANISMS OF REGULATION OF METABOLISM. WAYS OF HORMONAL REGULATION ON CELLULAR
AND INTERORGAN LEVEL
The aim of the lesson: 1. To know classification of hormones.
2. To know the role of different hormones at the regulation of metabolism.
3. To master methods of qualitative determination of some hormones.
Initial level of knowledge:
- the general concepts about hormones, their biological value;
- the characteristic of increts and secrets;
- symptoms of the some endocrine diseases.
Main topics.
64
I.1. Reference work.
SUMMARY QUESTIONS FOR A TEST
"HORMONES"
1. Concepts: "hormones", "endocrine glands", "endocrinology". Difference between endocrine and exocrine glands. Increts and
secrets. Similarity and distinction between hormones, vitamins and
enzymes. Mechanism of action of hormones. Examples.
2. Classification and the nomenclature of hormones. Examples.
3. Hormones influencing the metabolism of proteins, lipids,
carbohydrates (to list).
4. Role of hypothalamus in the regulation of endocrine functions. Structure and function of liberins (releasing factors) and
statins.
5. Hormones of anterior pituitary (GH, TSH, FSH, LH,
ACTH, LPH, PRL): the chemical nature, biological role. The principle of a feedback by the example of regulation of secretion TSH and
ACTH. Syndromes of hypo-and hyper-production of GH in adults
and children.
6. Hormones of posterior pituitary. Place of their synthesis,
structure, physiological effects, and hyposecretion of ADH.
7. Hormones, supervising melanogenesis. Structure and biological role of MSH. The chemical nature and biological role of
hormones of epiphysis. Regulation of daily biorhythms of an organism.
8. The basic stages of synthesis of iodinated hormones of thyroid gland: thyroxine (Т4), triiodthyronine (Т3), diiodthyronine (Т2).
Physiological and biochemical effects of thyroid hormones. Mechanism of influence of thyroxine on the speed of biological oxidation.
The diseases caused by infringement of function of thyroid gland (in
adults and children).
9. Hormones regulating calcium balance. Place of synthesis,
chemical nature and biological role of thyreocalcitonin, parathyrin,
calcitriol. Basic symptoms of hypo- and hyper-parathyreoidism.
Treatment of parathyreodic tetany.
65
10. Hormones of pancreas. Basic biochemical effects of insulin. Chemical nature and biological action of glucagon. Diabetes
mellitus.
11. Biosynthesis of catecholamins. The basic biochemical effects of adrenaline. The mechanism of action of catecholamines on
the metabolism of carbohydrates. Inactivation of catecholamins.
12. Structure and biological role of glucocorticoids. The
mechanism of action to organic metabolism. Inactivation of hormones.
13. The structure and mechanism of the action of mineralocorticoids to the water and sodium balance. Excretion of 17-ketosteroid
metabolites.
14. Structure of androgens, their physiological action and the
place of synthesis.
15. Structure of estrogens and progestins, their physiological
action and the place of synthesis. The change of ratio between them
in different phases of menstrual cycle.
16. Structure of the major tissue hormones (hormonoids):
acethyl choline, serotonin, histamine, GABA, prostaglandins, 3', 5'cAMP. Their biological role.
II.1. Work № 1. COLOR REACTIONS FOR INSULIN.
а) Biuret test.
Principle and practical procedure see p.8-9 (Part 1).
RESULT:
б) Ninhydrin reaction.
It is characteristic for the amino groups which are in αposition. Proteins at boling with a water solution of ninhydrin give
the coloured form which has received the name "Ruamann’s blueviolet complex".
Practical procedure.
Add 5 dr. of 0.5% water solution of ninhydrin to 5 dr. of insulin and boil 1-2 minutes. Note and write down the colouring.
RESULT:
66
в) Xanthoprotein reaction (Mulder’s test).
It proves the presence of aromatic amino acids (trp, phe, tyr) in
protein. While processing of the protein with concentrated nitric acid, yellow colouring (nitration) appears. After the addition the alkali
it changes into orange.
Practical procedure.
Add 3 dr. of concentrated HNO3 to 5 dr. of insulin and boil
cautiously. After cooling add 10-15 dr. of 10% solution NaOH. Note
and write down the colouring.
RESULT:
Work № 2. COLOUR REACTION FOR ADRENALINE.
Principle of the method.
Adrenaline has alkalescent reaction, oxidizes easily on air
with formation of adrenochrome owing to it the solution is coloured
in the red color. At interaction with ferric chloride green color is observed which changes into cerise in the alkaline environment. Reaction with ferric chloride is a characteristic for a pyrocatechol ring
which is included in the molecule of adrenaline and noradrenaline.
Practical procedure.
Put 1 dr. of solution FeCl3 to 10 dr. of solution of adrenaline,
note colouring. Then add 3 dr. of 10% solution NaOH.
RESULT:
Work № 3. COLOURE REACTION FOR THYROXIN.
Principle of the method.
At destruction of thyroidin potassium iodide is formed, from
which iodine is easily superseded by potassium iodate. Replacement
of iodine from iodide is oxidation-reduction reaction. Formed iodine
is found with the starch in the sour environment (dark blue colouring).
5KI + KIO3 + 3H2SO4
3I2 + 3K2SO4 + 3H2O
Practical procedure.
Add 3 dr. of 1% solution of starch and 1 dr. phenolphthalein to
24 dr.of hydrolyzate of thyroidin, then add 4 dr. of potassium iodate
67
and 10-15 dr. of 10% solution H2SO4. Note and write down the colouring.
RESULT:
Work № 4. QUALITATIVE DETERMINATION of 17KETOSTEROIDS IN URINE (CIMMERMAN’ reaction).
Principle of the method.
It is based on reaction of 17- ketoctoroids with mdinitrobenzene in the alkaline environment with formation of products of condensation of violet-red color. Intensity of colouring is
proportional to amount of 17- ketoctoroids in urine.
Practical procedure.
Into a dry (!) test tube measure 20 dr. of urine, add 5 dr. of solution
m-dinitrobenzene, shake up and add 6 dr. of 30% solution NaOH.
Observe development of colouring for 2 minutes.
RESULT:
Work № 5. COLOURE REACTION FOR FOLLICULIN.
Reaction for folliculin (estrone) is caused by the formation of
ester compound with sulfuric acid of straw-yellow color with green
fluorescence.
Practical procedure.
Add 30 dr. of concentrated H2SО4 to 2 dr. of oil solution of
folliculin. Note and write down the colouring.
RESULT:
III.2. Control questions.
What is the chemical nature of insulin?
What is qualitative reaction for adrenaline based on?
What is qualitative reaction for thyroxin based on?
What are 17- ketoctoroids?
What is clinical value of determination of 17- ketoctoroids in
urine?
III.3. Home assignment.
+
68
Choose the correct answer.
1. Which of the following substances is involved in the regulation of
plasma calcium level?
A. calcitriol
B. parathyroid hormone
C. calcitonin
D. all of them
2. Which of the following hormones is an amino acid derivate?
A. epinephrine B. norepinephrine C. thyroxine .D all of them
3. The most active mineralocorticoid hormone is
A. cortisol
B. aldosterone
C. deoxycorticosterone
D. corticosterone
4. Name the hormone, predominantly produced in response to fight,
fright and flight
A. thyroxine
B. aldosterone
C. epinephrine
D. ADH
5. The hormone essentially required for the implantation of fertilized
ovum and maintenance of pregnancy is …
A. progesterone
B. estrogen
C. cortisol
D. prolactin
Material for self-study. I. 1.pp.469-503, 451-452, 557
2.pp.434-473; II.
Class 35
COLLOQUIUM ON SECTIONS "METABOLISM OF COMPLEX PROTEINS","INTERRELATION AND REGULATION
OF METABOLISM"
Questions to a colloquium
1. The disintegration of nucleic acids. Nucleases of digestive
path and tissues.
2. Catabolism of purine nucleotides: the sequence of reactions,
end-products. Level of uric acid in blood. Gout.
69
3. Splitting of pyrimidine bases: the sequence of reactions.
Orotataciduria.
4. Biosynthesis of purine nucleotides (an origin of parts of purine ring). The regulation of biosynthesis of purine nucleotides.
5. Biosynthesis of pyrimidine nucleotides. The regulation of
biosynthesis of pyrimidine nucleotides.
6. The disintegration of the heme. Bilious pigments, their formation, transformations and ways of removing from an organism.
7. The infringement of metabolism of bilirubin at jaundices:
hemolytic, obstructive and hepatocellular.
8. The diagnostic value of determination of bilious pigments in
blood and in urine.
9. Synthesis of heme and hemoglobin. The regulation of these
processes.
10. Metabolism of iron: features of transport through an intestinal wall, deposition, daily requirement. Participation of ferritin,
transferrin, haptoglobin in these processes.
11. The uniform circuit scheme of interrelation of metabolism of
the basic substances: proteins, lipids and carbohydrates.
12. The key role of acetyl-CоА. The change of metabolism at
starvation and adiposity.
13. Diabetes - an example of interrelation of metabolism of different substances.
14. Biosynthesis of nonessential amino acids from carbohydrates.
15. Synthesis of glucose from amino acids and glycerol. Glucogenic and ketogenic amino acids. Physiological value of regulation
of gluconeogenesis from amino acids and glycerol.
16. Interrelation of metabolism of fats and carbohydrates: the
scheme of transformations and dependence of speed of transformation on a frequency of a meal and structure of food.
17. The basic mechanisms of regulation of metabolism. The hierarchy of regulatory systems. Hormonal regulation as a mechanism
of intercellular coordination of metabolism. Cells - targets and cellular receptors of hormones. The mechanism of transfer of the signal in
a cell.
70
18. The classification of hormones based on the chemical structure and biological functions. The central regulation of endocrine
system: role of tropic hormones, liberins, statins. Replaceable therapy at hypofunction of hormones.
19. Regulation of metabolism of carbohydrates, the chemical
nature of hormones. Change of the hormonal status and of metabolism at starvation.
20. Regulation of metabolism of lipids, the chemical nature of
these hormones. Change of the hormonal status and of metabolism at
starvation.
21. Regulation of metabolism of amino acids, the chemical nature of these hormones. Change of the hormonal status and of metabolism at starvation.
22. Regulation of concentration of glucose in blood. The reasons of the occurrence of hypo- and hyperglycemia. The major
changes of the hormonal status and of metabolism at diabetes. Biochemical mechanisms of the development of diabetic coma.
23. Regulation of balance of calcium and phosphates. Role of
parathyrin and thyreocalcitonin. Vitamin Д3: structure and metabolism. Hypo- and hypercalciemia: the reasons of the occurrence and
the consequences.
24. Structure, synthesis and metabolism of iodthyronines. Influence on metabolism. Hypo- and hyperthyroidism: the mechanism of
the occurrence and the consequences. Replaceable therapy at hypoproduction of hormones.
25. Role of liver in metabolism of poteins, lipids and carbohydrates.
26. The major mechanisms of neutralization of substances in
liver: microsomal oxidation, reactions of conjugation. Neutralization
of alien (phenol), medicines, inactivaion of hormones.
III.3. Material for self-study. I. chapters “Metabolism of complex
proteins","Regulation of metabolism"; II.
Class 36
71
URGENT BIOCHEMICAL ANALYSIS OF BLOOD AND URINE
The aim of the lesson: 1. To master methods of urgent test express diagnostics of individual components of blood serum and urine.
2. To master complex research of biotests.
Initial level of knowledge:
- chemical compound of blood;
- chemical compound of urine.
Main topics.
I.2. Protein-free nitrogenous components of blood.
Nitrogenous-free organic components of blood.
Organic and mineral components of urine.
Pathological components of urine.
II.1. Work № 1. TEST OF URINE WITH “penta-PHAN".
Each diagnostic strips "penta-PHAN" have five zones of the
indication, stuck on a polymeric base. The reactions of zones are
based on the following principles:
- ketones (white, with a cream shade square) – the zone contains alkaline nitroprusside, giving pink, up to dark-violet colour in
the presence of acetoacetic acid and acetone;
- restoring substances (light yellow square) – the zone contains the acid buffer and phosphomolybdic acid, the latter turns into
molybdenum dark blue under action of strong reducers (mainly,
ascorbic and gentisic acid);
- glucose (bright yellow square) - the zone contains glucosidase, peroxidase, and also chromogenic system which is oxidized
forming the green and even blue products in the presence of glucose;
- protein (light grey-green square) - the zone contains the acid
buffer and the special indicator changing the colouring from yellow
through green up to dark-blue in the presence of proteins;
- рН (orange-red square) - the zone contains the mixed acidbasic indicator which changes the colour from red through yellow
and green to dark blue in an interval of рН 5-9.
72
Practical procedure.
Put a strip into researched urine and take it out.

Immediately write down рН, having compared zones of indication for рН with the corresponding color scale.

Approximately in 30 s. estimate the test on restoring substances.

If the latter are in urine in insignificant amount (the color of
square as "1" on the standard), estimate the test for glucose within
30-60 s.
If the content of restoring substances is in interval "1" - "2",
determine the quantity of glucose for 1-2 min after putting.
If the content of restoring substances is more than "2" it is impossible to estimate the test on glucose. In this case it is necessary to
repeat test not earlier than in 10 hour after intake of ascorbic acid.

Tests on protein and ketons are to be estimated 1 min later
after the putting of a strip into the urine.
Quantitative estimation:
Parameters
Ketones
(mmol/L)
Restoring
substances (mg/L)
Glucose
(mmol/L)
Protein (g/L)
рН
0
<0.3
1
1.5
2
2.9
3
7.3
4
> 14.7
<30
100
200
400
> 600
<1.3
2.78
5.55
16.7
83.3
<0.1
5
0.3
6
1.0
7
3.0
8
> 10.0
9
RESULTS:
CONCLUSION:
Work № 2. DETERMINATION OF UREA IN BLOOD WITH
"UREATEST".
73
Reagent paper "Ureatest" is used for rapid test of the contents
of urea in blood serum. This is strip of chromatographic paper size
120х10 mm impregnated with the solutions of enzyme and indicator.
Zones of their drawing are divided by a red paraffin strip. During the
determination the strip should be held by the free end.
- Nonimpregnated end of a paper
- A paraffin strip
- A red paraffin strip
- A zone of enzyme (a place of drawing of blood
serum
Principle of the method.
The method is based on the specific action of urease to split
urea with the formation of ammonia. Formed ammonia colours the
indicator in blue color. The height of the coloured zone in mm is
proportional to the concentration of urea in mg %, which is determined on the graph. Limits of determination are from 20 up to 250
mg %.
Mm
50 40 30 20 10 |
|
|
|
|
|
|
|
30 60 90 120 150 180 210 240 mg %
Practical procedure.
Dissolve blood serum with distilled water 1:1. On the end of
paper impregnated with enzyme, on distance of 3 mm from red paraffin strip put 0.03 sm3 of the dissolved blood with pipette. Quickly
bring the strip into a clean dry test tube, close tightly with a fuse and
leave in the thermostat for 20 min at 370С or for 30 min at 200С.
Measure the height of the indicator zone coloured in blue color. On
the graph find the content of urea in mg %, corresponding to results
of measurement in mm (see above).
74
In the case blood or undiluted blood serum are used the result
should be divided on 2.
RESULT:
CONCLUSION:
Work № 3. DETERMINATION OF GLUCOSE IN URINE WITH
"GLUCOTEST".
Practical procedure.
Moisten the whole yellow strip of a reagent paper in urine.
Place the piece of the paper on a plastic plate for 2 min (if the chips
were produced 6 months before, the time of exposition should be
increased by 5-10 min). Compare changed colouring of yellow strip
with the color scale.
Notes. For research use freshly gathered urine taken before
meal.
Presence in urine of ascorbic acid influences negatively the
accuracy of the analysis.
Don’t touch yellow strip of a reagent paper. Protect from sun
rays and moisture!
RESULT:
CONCLUSION:
Work № 4. ESTIMATION OF HEMOGLOBIN DISSOLVED IN
BLOOD PLASMA.
Principle of the method and chemism see on page 39.
Practical procedure.
Reagents (ml)
Acetate buffer рН 4.6
0.3% solution hydrogen peroxide
0.1% solution benzidine
Blood plasma
Physiological solution
75
Sample
4.0
2.0
2.0
0.04
-
Control
4.0
2.0
2,0
0.04
In 3 min pour solutions in ditches of 10 mm and measure optical density at red optical filter (680 nm).
In presence of hemoglobin (i.e. at hemolysis) the intensity of
blue colouring increases within 4-5 min. Therefore the optical density is measured twice and maximal indication is registered. 5-6 min
later colouring gets a lilac-brownish shade and optical density decreases.
The amount of dissolved hemoglobin (in mg %) determines at
calibrating curve constructed on the basis of optical density numbers,
received at measurement of solutions with precisely known contents
of hemoglobin.
RESULT:
CONCLUSION:
Clinical -diagnostic value.
In norm plasma of blood contains traces of dissolved hemoglobin (0.2-2.5 mg %).
The increased amount of dissolved in plasma hemoglobin is
the sign of intravascular hemolysis of erythrosytes. At influence of
some industrial toxic substances on the organism it is possible to expect an occurrence of intravascular hemolysis. So, for example, at
acute intoxication with aniline the amount of dissolved in blood
plasma hemoglobin increases up to 5.7 mg.
III.2. Control questions.
How much hemoglobin is contained in blood plasma in norm?
What factors influence the level of hemoglobin dissolved in
plasma?
How much urea is contained in blood serum in norm?
When does glucosuria appear?
When is proteinuria marked?
When is hematuria observed?
What are the reasons of acetonuria?
76
PRACTICAL TASKS
1. The daily diet of the man working at northern area on timber cuttings, containsd 100 g of fat 20%of which is vegetable oil. Is the diet
made correctly?
2. Calculate the requirement of lipids (in grammas) if 60% of power
input of an organism are provided by carbohydrates and 18% by proteins. The total level of power input is equal to 3500 kcal (14665 kJ).
3. How many animal and vegetable lipids should the diet of the person include if his power input is equal to 3500 kcal (14665 kJ)?
Note, that lipids, give 22% of all necessary energy.
4. The patient is diagnosed with the fatty dystrophy of liver (superfluous adjournment of fat). What diet do you recommend to him?
5. What diet and what mode should be recommended to the patient
with propensity to adiposity at a satisfactory condition of cardiovascular system?
6. Patients with atherosclerosis and healthy people as the preventive
measures are recommended to have vegetable oil. Explane why.
7. Parents are concerned with the excessive weight of the child.
Without consulting with the doctor, they have sharply limited the
amount of sugar in food, having increased the contents of proteins,
but they have not reduced the amount of fat. In some days the child
health has worsened, vomitting has brgun. What infringements of
metabolism is it connected with? What biochemical mechanism will
confirm the infringement of this kind of metabolism?
8. When the patient with atherosclerosis was being discharged from
the hospital the doctor recommend him the diet stimulating the outflow of bile and activation of peristalsis of intestines. Explane why.
9. The weight of a man of 55 started to increase due to obesity. Name
the main reasons of it.
10. Patient with a chronic pathology of liver and intestine has the
broken absorption of fats. What accompanying hypovitaminoses
complicate the condition of a patient?
77
11. At discharging from sanatorium of a patient with adiposity, the
doctor - dietician suggested him having enough of cottage cheese
with food. Why?
12. A patient has a plenty of fat in feces (steatorrhea). Name the
main causes of infringement of digestion and absorption of fats.
13. For the regulation of secretion of digestive juices the doctor can
use various products. What products can be recommended for the
stimulation and inhibition of gastric juice production, for intensification of bile secretion?
14. The level of glucose in blood of a sportsman before start increased up to 6.5 mmol/L as one of FFA up to 1.2 mmol/L (norm
0.4-0.9 mmol/L). Explain.
15. The level of triacylglycerols and phospholipids in a cardiac muscle is 1.6-2 times higher, than in a skeletal muscle. What biochemical
sense does this distinction have? Is high sensitivity of myocardium to
oxygen insufficiency not connected with it?
16. Carbohydrate metabolism In tissues of a sportsman running long
distances changes to lipid one. How many times will output of the
ATP at oxidation of 1 mol of tripalmitoglycerol in comparison with 1
mol of glucose increase?
17. The patient for some days did not have food due to medical starvation. Will the level of glucose and free FA in blood change?
18. A patient excretes ketone bodies with urine. How is this phenomenon named? What are its main reasons? What are the main ways of
formation of ketone bodies?
19. How do biochemical parameters of lipid metabolism change at
diabetes?
20. The elderly patient with diabetes suddenly has lost consciousness
(diabetic coma). Could a doctor diagnose the type of coma without
laboratory analysis?
21. Carbohydrates in a diet of the student give 6704 kJ (1600 kcal).
Does he receive enough carbohydrates?
22. Dishes from a potato, prepared without salt, seem tasteless. Does
addition of salt have physiological sense or only taste?
23. The patient is injected with a solution of adrenaline. How does
the concentration of glucose in blood change? Explain mechanism.
78
24. The patient has asked the doctor, whether it is possible to receive
all daily diet of carbohydrates as glucose. What is your opinion?
25. It is necessary for 7-year child to test glucose in blood to reveal
diabetes. The child before procedure in laboratory is very worried,
cried. It is established, that the level of glucose in blood is higher
than the norm. Is it possible to assert that the child is ill?
26. A patient is diagnosed with high hyperglycemia, glucosuria, acetonuria, and decrease in alkaline reserves. Is it necessary to introduce
hormone? Does the injection of glucose simultaneously with hormone cause a negative effect?
27. The sportsman has run a hundred-meter distance. Will the level
of lactic acid in blood change? Why?
28. One sportsman has run at competitions 100m, another – 5000m.
Which sportsman has a higher level of lactate in blood?
29. How does the ratio of pentose phosphate pathway and glycolysis
in the case of a patient after bleeding change? What enzymes can test
to confirm the assumption?
30. 9.6 mmol/L glucose is revealed in blood of the patient. What are
the probable reasons of it? What analyses are to be carried out to
specify its character?
31. The patient has drunk some milliliters of insulin solution. How
does the level of glucose in blood change?
32. The level of glucose in blood of the student before an exam was
equal to 7.2 mmol/L. Is it deviation from a norm? Explain the mechanism.
33. What carbohydrate - glucose or fructose is more useful to the
patient with diabetes?
34. Why do doctor recommend to diabetics to change glucose to sorbitol?
35. A child has vomiting and diarrhea after having of confectionery
products, sweets. He feels badly after having sweet tea whereas milk
does not cause negative reactions. What is the reason? How can we
check up the assumption?
36. In hospital the child was admitted with complaints on vomiting
after having milk. What is the reason?
79
37. On the basis of the data on the level of glucose in blood
(mmol/L) draw a sugar curve, determine probable infringements of a
carbohydrate metabolism. Blood was investigated at the interval of
30 minutes.
Up to loading
after loading
The patient A
5.9
7.0 9.2 9.0 8.0 7.0
The patient B
4.5
7.0 7.5 5.5 5.4 5.0
What additional researches are necessary to check the assumption?
38. The laboratory tests of a patient at laboratory research have
showed:
Blood - total bilirubin 13 mkmol/L,
Urine – urobilinogen - traces
bilirubin is absent
Feces - stercobilinogen +++
Draw the conclusion.
39. A woman suffering with cholelithic illness, has pains in the field
of a liver, icteric coloring of the sclera and skin has quickly developed, feces have become colorless, urine has got color of strong tea.
What are the reasons of infringement of pigmental metabolism?
40. The doctor has found that the patient has weight lose, the increase of irritability, subfebrile in the evening, exophtalmos, the increase of the general metabolism, the increase in absorption of oxygen, hyperglycemia, hyperazotemia. What pathology of endocrine
gland is?
41. A patient has bright yellowness of skin, sclera and mucous, urine
has color of beer. The laboratory test has showed:
Blood - direct bilirubin has increased
Résistance of erythrocytes has decreased
Urine - stercobilinogen +, bilirubin is present
Feces - stercobilinogen ++.
What type of jaundice is it?
42. A patient has an increased level of indole in blood and urine, the
amount of indican has decreased. What function of liver is broken
according to the results of laboratory test?
80
43. A patient with disease of liver has been admitted to hospital. The
level of urea in blood has been tested. Is this test afficient for the estimation of severity of liver disease?
44. After the examination, the blood test of a patient showed the level of uric acid is equal to 0,55 mmol/L, the test of urine showed 2,2
g/day. A doctor has prescribed hypoxanthine, and has recommended
to have less meat. What disease does the patient have? Explain the
recommendations of the doctor.
45. Why is the patient with acute and chronic intoxication prescribed
potassium orotate?
46. In case of hereditary orotataciduria the retardation in physical
and mental development is observed, process of hemopoiesis is broken. The introduction to the patient of uridine or cytidine reduces
pathological symptoms. Why?
47. A patient has chronic hepatitis. Does it illness influences character of the person?
48. Patient С., 62, has complained in clinic to dryness in oral cavity,
thirst, plentiful and often urination, weakness, sleeplessness, loss of
weight. What diseases are these symptoms characteristic for? What
laboratory tests are necessary to confirm the diagnosis?
49. What external signs are the symptoms of hypofunction of anterior pituitary or the thyroid gland at the early age?
50. At diabetes there are complaints of increased urination (polyuria), thirst (polydipsia), the raised appetite (polyfagia). Explain, why
a patient feels hunger though he eats a lot of food and the level of
glucose in blood is increased.
51. A doctor examines two patients suffering diabetes. An elderly
one has adiposity; while the young one has the weight thay is lower
than the norm for his age. What factors influence on lipid metabolism? What is the reason of distinction in lipid metabolism in case
with these patients?
52. What disease can we diagnoze if a patient excretes up to 5 l
urine daily, the density of urine is 1,002, the level of glucose in blood
equals to 4,2 mmol/L?
81
53. At some kinds of the pituitary tumor the synthesis of GH increases. How does the person look?
54. In case of some diseases (for example, pituitary nanism) patients
are treated with Growth hormone. How does the overdose of the
hormone influence the metabolism of carbohydrates and lipids?
55. The patient complains to unquenchable thirst, significant urination (up to 6 l/day). Laboratory examination has revealed: the level
of glucose in blood is 5,2 mmol/L; urine is colorless with density
1,002, sugar is absent. Name the probable reasons of polyuria.
56. The patient suffering from chronic allergic disease takes prednizolon (analogue of cortizole) for a long time. After the cancellation
of the medicine the symptoms of hypocorticism have appeared
(weakness, hypotonia, hypoglycemia), the level of 17-ketocteroids in
urine has decreased. Explain the reason of changes. Will introduction
of corticotropin help the patient?
57. The patient with signs of hyperthyroidism has strongly grown
thin, the hypodermic fatty layer is not expressed. Why?
58. The level of the total calcium in blood serum of the child is 1,8
mmol/L. Is it a deviation from the norm? What factors influence the
level of calcium in blood?
59. 5-month child has the marked symptoms of a rickets. Disturbances of digestion are not present. The child spend enough time in
the open air. The cource of vitamin D-therapy appeared to be inefficient. Come out with the assumption of the reason of changes.
60. The patients with hyperparathyreoidism have the muscular
weakness, osteoporosis, deformation of the skeleton, the formation
of renal stones. What processes have become more active?
82
LIST
of laboratory and clinical - laboratory skills,
received by students at biochemistry department
1. Skills of laboratory and experimental work:
- with aggressive substances;
- separation of substances by the methods of sedimentation, dialysis;
- with measured glasswares, micropipettes;
- at photocolorimeter;
- quantitative determination using indicators;
- at centrifuge;
- chromatographic studing of researched samples;
- separation of protein mixes into fractions;
- acquaintance to the principles of experimental scientific work;
- getting up the report of the test;
- statistical processing of digital material by results of research;
- with scientific literature.
2. Clinical-laboratory skills:
- quantitative and qualitative determination of carbohydrates used in
clinical-biochemical researches;
- determination of amylase activity in urine;
- quantitative determination of parameters of lipid metabolism in
blood (cholesterol, triacylglycerols);
- quantitative determination of catalase in blood;
- determination of protein in biological liquid;
- quantitative determination of total protein in the samples;
- determination of vitamins and hormones in solutions;
- quantitative determination of the common acidity, the free and connected hydrochloric acid in gastric juice;
- determination of lactate and blood in the samples;
- determination of homogentisinic and phenylpyruvic acid in urine;
- determination of creatinin, uric acid and urea;
- quantitative determination of bilious pigments and hemoglobin;
- acquaintance to the urgent biochemical analyses of some parameters of blood and urine.
83
LITERATURE
1.
2.
3.
4.
5.
Биохимический практикум. /Под ред. Д.М.Никулиной. Издво АГМА, Астрахань, 2010, 146с.
Practical and laboratory studies on biochemistry. Part 2 /Edited
by prof. O.V.Ostrovskiy.-Volgograd, 2004-120p.
U. Satyanarayana //Biocnemistry. Books and allied (p) Ltd. 8/1
Chintamoni Das Lane, Kolkata, 700009 (India), 2004.
Robert.K.Murray, D.K.Granner, P.A.Mayes, V.W.Rodwell
//Harper’s Illustrated Biochemistry.26-th edition. McGraw-Hill
Companies, Inc., USA, 2003
A. Lehninger, D. Nelson, M. Boyle Cox //Principles of biochemistry (4th ed.), 2005
84
CONTENTS
Class 19. Digestion and absorption of carbohydrates. Metabolism of glycogen. Quantitative and qualitative determination
of carbohydrates in biological liquids ……...
Class 20. Glycolysis, glyconeogenesis. Oxidative decarboxylation of pyruvate. Determination of glycolysis products in
biological liquids ………………………………..
Class 21. Ways of aerobic oxidation of carbohydrates. Regulation and infringements of the carbohydrate metabolism.
Quantitative determination of glucose in blood ……................
Class 22. Colloquium on section "Metabolism and functions
of carbohydrates"……….……………………………………..
Class 23. Digestion and absorption of lipids. Participation of
bile acids during these processes ……………………………...
Class 24. Oxidation and synthesis of fatty acids. Metabolism
of ketone bodies. Quantitative determination of triacylglycerols in blood ……...……………...………………..
Class 25. Biosynthesis of lipids. Regulation and infringements
of lipid metabolism. Methods of cholesterol determination ….
Class 26. Colloquium on section: "Metabolism and functions
of lipids"……………………………………………………….
Class 27. Nitrogenous balance. Biological value, digestion
and absorption of the proteins. Rotting of the proteins in intestines. Test of gastric juice ………………………………..
Class 28. Deamination, transamination, decarboxylation of
amino acids. Using of carbon skeleton of amino acids. Determination of products of protein metabolism in biological liquids …………………………………………………………
Class 29. Neutralization of ammonia in the organism. Synthesis of urea. Determination of products of protein metabolism
in biological liquids ……………………………...
Class 30. The general scheme of sources and pathways of recycling of amino acids in tissues. Cathepsins. Specific ways of
metabolism of some amino acids. The pathology of the ni-
85
3
8
13
18
20
25
31
36
37
43
46
trogenous metabolism. Estimation of the products of nitrogenous metabolism in biological liquids…………...……
Class 31. Colloquium on sections: "Metabolism and functions
of amino acids"………………………………………………...
Class 32. Metabolism of nucleoproteins. Biosynthesis and
disintegration of purine and pyrimidine nucleotides ………….
Class 33. Chemistry and metabolism of chromoproteins. The
role of liver in pigmentary, carbohydrate, lipid and protein
metabolism. Detoxicate function of the liver …………………
Class 34. The basic mechanisms of regulation of metabolism.
Ways of hormonal regulation on cellular and interorgan level .
Class 35. Colloquium on sections "Metabolism of complex
proteins", "Interrelation and regulation of metabolism"………
Class 36. Urgent biochemical analysis of blood and urine …...
Practical tasks……………..…………………………………...
List of laboratory and clinical - laboratory skills, received by
students at biochemistry department………………………….
Literature……………………………………………………...
.
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Воробьева Татьяна Борисовна
Никулина Дина Максимовна
THE MANUAL ON BIOCHEMISTRY. Part 2
РУКОВОДСТВО К ЗАНЯТИЯМ
ПО БИОХИМИИ. Часть 2
Учебное пособие
Компьютерный набор - авторский
Технический редактор – Нигдыров В.Б.
ISBN 978-5-4424-0140-0 (основной)
ISBN 978-5-4424-0193-6 (часть 2)
Подписано к печати 17.06.2016
Гарнитура Times New Roman. Формат 60 80 1/16
Напечатано на ризографе
Усл. печ. лист – 5,0
Заказ №
Тираж 100 экз.
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Издательство Астраханский государственный медицинский
университет 414000, г. Астрахань, ул. Бакинская, 121
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