MINISTRY OF HEALTH OF UKRAINE

ZAPORIZHZHIA STATE MEDICAL UNIVERSITY

Biological Chemistry Department

Biological chemistry

A manual for independent work at home and in class

preparation for licensing examination “KROK 1”

on semantic modules 8, 9, 10 of module 2

for students of International Faculty

(the second year of study)

Zaporizhzhia, 2016

2

UDC 577.1(075)

BBC 28.902я73

B 60

Reviewers:

Prihodko O. B., Head of Department of Medical Biology, Parasitology and

Genetics. Dr. Hab, assoc. professor

Voskoboynik O. Yu., assoc. professor of Organic and Bioorganic Chemistry

Department, Ph. D.

Authors:

Aleksandrova K.V.

Krisanova N.V.

Ivanchenko D.G.

Rudko N.P.

Levich S.V.

Tikhonovska M.A.

Biological chemistry : a manual for independent work at home and in class

preparation for licensing examination "KROK 1" on semantic modules 8, 9, 10 of

module 2 for students of International Faculty (the second year of study) / K. V.

Aleksandrova, N. V. Krisanova, D. G. Ivanchenko, N. P. Rudko, S. V. Levich, M.

A. Tikhonovska. – Zaporizhzhia : ZSMU, 2016. – 187 p.

This manual is recommended for II year students of International Faculty of specialty

"General medicine" studying biological chemistry, as additional material to prepare for practical

training semantic modules 8, 9, 10 of module 2 and licensing exam "KROK 1: General medical

training".

Біологічна хімія : навч.-метод. посіб. для самостійної роботи при

підготовці до ліцензійного іспиту "КРОК 1" змістових модулів 8, 9, 10

модулю 2 для студентів 2 курсу міжнар. ф-ту / К. В. Александрова, Н. В.

Крісанова, Д. Г. Іванченко, Н. П. Рудько, С. В. Левіч, М. А. Тихоновська. -

Запоріжжя : ЗДМУ, 2016. – 187 с.

UDC 577.1(075)

BBC 28.902я73

©Aleksandrova K.V., Krisanova N.V., Ivanchenko D.G.,

Rudko N.P., Levich S.V., Tikhonovska M.A.2016

©Zaporizhzhia State Medical University, 2016

3

INTRODUCTION

The handbook "Biological chemistry. A manual for independent work at

home and in class preparation for licensing examination ―KROK 1‖ on semantic

modules 8 ―Biochemistry of Vitamins‖, and 9 ―Functional biochemistry of organs

and tissues‖, and 10 ―Biochemical indexes of blood and urine in diagnostics of

metabolic disorders‖ of module 2 ―Molecular Biology. Biochemistry of cell-to-cell

interactions. Of tissues and physiological functions‖ for students of International

Faculty (the second year of study) speciality «General Medicine» contains a

summary of the theory, which facilitates finding the right answer test tasks.

Tests of this manual are similar in content and form to the test tasks,

provided Testing Center of Ministry of Health of Ukraine. Each test task has only

one either correct or more correct answer that must be chosen among the available

ones by a student. As a self-study students are invited to give rationale for the

choice of the correct answer, identify key words for case described in a test task.

The authors hope that this special form of student work with test tasks, with

detailed explanation described in these tasks mostly clinical situations allow

foreign English-speaking students to prepare properly and pass licensing exam

"KROK 1: General medical training".

4

THE ROLE OF WATER-SOLUBLE AND FAT-SOLUBLE VITAMINS IN

THE METABOLISM OF HUMANS.

VITAMIN SIMILAR SUBSTANCES. ANTIVITAMINS

(Rudko N. P.)

INFORMATIONAL MATERIAL

Vitamins are a group of organic nutrients of various nature required in small

quantities for multiple biochemical reactions for the growth, survival and

reproduction of the organism, and which, generally, cannot be synthesized by the

body and must therefore be supplied by the diet. The most prominent function of

the vitamins is to serve as coenzymes (or prosthetic group) for enzymatic reactions.

Vitamins are grouped together according to the following general biological

characteristics:

1. Vitamins are not synthesized by the body and must come from food. An

exception are vitamin B3 (PP), which active form NADH (NADPH) can be

synthesized from tryptophan and vitamin D3 (cholecalciferol), synthesized from 7-

dehydrocholesterol in the skin. Amount of those ones and vitamins partially

synthesized by intestinal microflora (В1, В2, В3, B5, В6, К, and others) is normally

not sufficient to cover the body's need them.

2. Vitamins are not plastic material. Exception is vitamin F.

3. Vitamins are not an energy source. Exception is vitamin F.

4. Vitamins are essential for all vital processes and biologically active

already in small quantities.

5. They influence biochemical processes in all tissues and organs, i.e. they

are not specific to organs.

6. They can be used for medicinal purposes as a non-specific tools in high

doses for: diabetes mellitus - B1, B2, B6; colds and infectious diseases - vitamin C;

bronchial asthma - vitamin PP; gastrointestinal ulcers - vitamin-like substance U

and nicotinic acid; in hypercholesterolemia - nicotinic acid.

5

Since only a few vitamins can be stored (A, D, E, B12), a lack of vitamins

quickly leads to deficiency diseases (hypovitaminosis or avitaminosis). These

often affect the skin, blood cells, and nervous system. The causes of vitamin

deficiencies can be treated by improving nutrition and by administration vitamins

in tablet form. An overdose of vitamins leads to hypervitaminosis state only, with

toxic symptoms, in the case of vitamins A and D. Normally, excess vitamins are

rapidly excreted with the urine.

Lack of vitamins leads to the development of pathological processes in the

form of specific hypo- and avitaminosis. Widespread hidden forms of vitamin

deficiency have not severe external manifestations and symptoms, but have a

negative impact on performance, the overall tone of the body and its resistance to

various adverse factors.

Avitaminosis is a disease that develops in the absence of a particular vitamin.

Currently avitaminosis are not commonly found, but hypovitaminoses are observed

with vitamin deficiency in the body. Numerous examples you can see in the table 1.

Table 1. Vitamin functions and manifestations of hypo- and avitaminoses

Vitamin Functions Hypovitaminosis symptomes

B1 Thiamin Functional part of

coenzyme TPP in pyruvate

and α-ketoglutarate

dehydrogenases,

transketolase; poorly

defined function in nerve

conduction

Peripheral nerve damage (polyneuritis

beriberi) or central nervous system lesions

(Wernicke-Korsakoff syndrome)

Concentration of pyruvate is increased in the

patient's blood, the most of which is excreted

with urine

B2 Riboflavin Functional part of

coenzymes FAD, FMN in

oxidation-reduction

reactions

Epithelial, mucosa, cutaneous, corneal

lesions: lesions of corner of mouth, lips, and

tongue; seborrheic dermatitis

B3

(PP)

Niacin,

nicotinic

acid,

nicotin-

amide

Functional part of

coenzymes NAD+, NADP

+

in oxidation-reduction

reactions

Pellagra: photosensitive dermatitis, glossitis

(tongue inflammation), alopecia (hair loss),

edema (swelling), diarrhea, depressive

psychosis, aggression, ataxia (lack of

coordination), dementia, weakness

B5 Pantothenic

acid

Functional part of

coenzyme CoA (universal

acyl carrier in Krebs cycle,

fatty and other carboxylic

acid metabolism) and

phosphopantetheine (acyl

carrier protein in fatty acid

Numbness in the toes, burning sensation in

the feet, the defeat of mucous membranes of

internal organs, early graying, hair loss,

various disorders of the skin: the

development of small cracks in the corners of

the mouth, the appearance of white patches

on various parts of the body. There may also

6

synthesis) be depressed mood, fatigue.

B6 Pyridoxine,

pyridoxal,

pyridox-

amine

Functional part of

coenzyme PLP in

transamination and

decarboxylation of amino

acids and glycogen

phosphorylase

Dermatitis of the eyes, nose, and mouth.

There is mental confusion, glossitis and

peripheral neuropathy, convulsions (due to

lack of inhibitory neurotransmitter GABA)

B7

(H)

Biotin Coenzyme in carboxylation

reactions in

gluconeogenesis and fatty

acid synthesis

Seborrheic dermatitis, anemia, depression,

hair loss, high blood sugar levels,

inflammation or pallor of the skin and

mucous membranes, insomnia, loss of

appetite, muscle aches, nausea, sore tongue,

dry skin, high blood cholesterol

B9 Folic acid Functional part of

coenzyme THFA in transfer

of one-carbon fragments

Megaloblastic anemia: red tongue, anemia,

lethargy, fatigue, insomnia, anxiety, digestive

disorders, growth retardation, breathing

difficulties, memory problems.

Deficiency during pregnancy is associated

with neural tube defects

B12 Cobalamin Functional part of

coenzymes

adenosylcobalamin

(Methylmalonyl Co A

mutase) and

methylcobalamin

(Methionine synthase) in

transfer of one-carbon

fragments and metabolism

of folic acid

Vitamin B12-deficiency anemia (in other

words pernicious anemia or Addison–

Biermer anemia) is one of many types of

megaloblastic anemias with degeneration of

the spinal cord, anemia, fatigue, depression,

low-grade fevers, diarrhea, weight loss,

neuropathic pain, glossitis (swollen, red and

smooth appearance of the tongue), angular

cheilitis (sores at the corner of the mouth)

Possible manifestations are also hypochromic

anemia, splitting hair and loss of hair,

increased nail bottling and taste alteration

C Ascorbic

acid

It serves as a donor of

protons in hydroxylation

reaction for:

- collagen synthesis (prolyl-

and lysyl residues are

hydroxylated by prolyl

3(4)-hydroxylase and lysyl

5-hydroxylase respectively;

- catecholamines and

steroid hormone synthesis;

It has properties of

antioxidant; enhances

absorption of iron

Scurvy: general weakness, subcutaneous

hemmorhages (frequent hemorrhages from

internals and mucous membranes), gingival

hemmorhages, loss of teeth, formation of

spots on the skin, spongy gums, yellow skin,

fever, neuropathy

Multiple hemorrages in the places of clothes

friction are possible if a person often

experiences acute respiratory infections

A Retinol Functional part of visual

pigments (rhodopsins and

iodopsins) in the retina;

regulation of gene

expression and cell

differentiation; β-carotene

(provitamin A) is an

antioxidant

Vision impairment hemeralopia (night

blindness), xerophthalmia; keratinization of

skin

7

D Calciferol Stimulation of Ca2+

absorption through

intestinal wall, maintenance

of calcium balance and

mobilization of bone

mineral

Rickets = poor mineralization of bone;

osteomalacia = bone demineralization

Osteoectasia of the lower extremities and

delayed mineralization of cranial bones are

onserved in infants

E Tocopherols Antioxidant, especially in

cell membranes

Extremely rare is a serious neurologic

dysfunction

K Phyllo-

quinone,

mena-

quinones

Coenzyme in formation of

γ-carboxyglutamate

residues in structure of:

- factors II (prothrombin),

VII, IX, X, XIV, protein S

(blood coagulation system);

- bone matrix proteins

Impaired blood clotting, hemorrhagic

disease, osteoporosis and coronary heart

disease

Intestinal dysbacteriosis occurs hemorrhagic

syndrome

External causes for hypovitaminosis

1. Lack of the vitamin in the diet or presence of food factors hindering the

absorption of vitamin. For example, use of large amounts of raw eggs (they contain

protein avidin binds vitamin H (biotin)) as a result may develop a state of

hypovitaminosis H.

2. Do not take into account the need for a particular vitamin. For example, in

protein-free diet is increasing demand for vitamin PP (with normal diet it may be

partially synthesized from tryptophan). If a person consumes much protein, it can

increase the need for vitamin B6 and reduce the need for vitamin PP.

3. Social reasons: urbanization, power and extremely high purity of canned

food; antivitamin presence in food. People are not enough exposed to sunlight in

large cities - so it can be hypovitaminosis D. In such cases, the medicine uses

ultraviolet radiation in the form of different physical treatments, which activate the

synthesis of vitamin D3 from 7-dehydrocholesterol in the skin cells.

Internal causes of hypovitaminosis

1. Physiological increased need for vitamins, for example, during pregnancy,

with heavy physical labor.

2. Long-term severe infectious diseases, as well as during the recovery

period.

8

3. Disturbance of vitamin absorption in some diseases of the digestive tract,

for example impaired absorption of fat-soluble vitamins is observed at

cholelithiasis; vitamin B12 is done with atrophy of the gastric mucosa and a

deficiency of Castle intrinsic factor. Another case if a person who hadn’t been

consuming fats but had been getting enough carbohydrates and proteins for long

time revealed dermatitis, poor wound healing, vision impairment. Lack of vitamins

A, D, E, K, F (linoleic, linolenic, arachidonic acids) is probable cause of the

metabolic disorder.

4. Intestinal dysbacteriosis. It has the meaning as some vitamins are

synthesized by the intestinal microflora (these vitamins are B3, B6, B7 (H), B9, B12,

and K).

5. Cirrhosis. The liver is the major depot of many vitamins, particularly fat-

soluble (especially high hepatic reserves of fat soluble vitamins A, D), but also

certain water-soluble, such as B9, B12, etc. In case of vitamin consumption increase

and reducing their dietary intake, which is usually the case, for example, in

alcoholism, megaloblastic anemia is developed in a short time as a characteristic

sign of hypovitaminosis B9. Patients with cirrhosis may experience blurred vision

in the twilight due to malabsorption of vitamin A in the intestine and its reduced

deposit in the liver.

6. Genetic defects of some enzymatic systems. For example, vitamin D-

resistant rickets occurs in children lack the enzymes involved in the formation of

the active form of vitamin D - calcitriol (1, 25-dihydroxycholecalciferol).

Classification of vitamins

1. Fat-soluble vitamins: A (retinol), D (calciferol), E (tocopherol), K

(naphthoquinone), F (polyunsaturated fatty acid: linoleic, linolenic, arachidonic).

2. Water-soluble vitamins:

- Group B: B1 (thiamine), B2 (riboflavin), B3 or PP (nicotinamide, niacin), B5

(pantothenic acid), B6 (pyridoxine), B7 or H (biotin), B9 or Bc (folic acid), B12

(cyanocobalamin) ;

- Vitamin C (ascorbic acid);

9

- Vitamin P (rutin and other bioflavonoids).

3. Also vitamin-like substances are separated:

- fat-soluble: Coenzyme Q (ubiquinone),

- water-soluble vitamins: B4 (choline), B8 (inositol), BT or B11 (carnitine), , B13

(orotic acid), B15 (pangamic acid), U (S-methylmethionine), N (lipoic acid).

Water-soluble vitamins are usually functioning as precursors of coenzymes

and prosthetic groups of enzymes. For example, coenzyme form of

- Vitamin B1 is TPP (thiamine pyrophosphate) (trade name - cocarboxylase);

- Vitamin B2 is FMN (flavin mononucleotide) and FAD (flavin adenine

dinucleotide);

- Vitamin B3 is NAD+ (nicotinamide adenine dinucleotide) or NADP

+

(nicotinamide adenine dinucleotide phosphate);

- Vitamin B5 is Coenzyme A (coenzyme of acylation);

- Vitamin B6 is PLP (pyridoxal phosphate);

- Vitamin B9 is THFA (tetrahydrofolic acid);

- Vitamin B12 is adenosylcobalamin and methylcobalamin.

Holoenzymes containing coenzymes (as its non-protein part) which are often

vitamin derivatives perform multiple functions. For example, the first enzyme in

gluconeogenesis pyruvate carboxylase uses biotin for carboxylation of pyruvate;

but the transformation of the pyruvate to acetyl-CoA by pyruvate dehydrogenase

complex requires five coenzymes: TPP, lipoic acid, CoA, FAD, NAD+. Since TPP

is involved in this conversion first, pyruvate accumulation in cells of the nervous

system (primarily) and then increase in pyruvate content in the blood and urine of

patients in the case of vitamin B1 deficiencies becomes obvious.

Most water-soluble vitamins must be supplied regularly with food, as they

are quickly removed or destroyed in the body. Fat-soluble vitamins can be

deposited in the body. Furthermore, they are poorly excreted, therefore,

hypervitaminosis as diseases associated with high doses of fat-soluble vitamin

intoxication of organism are observed. Such diseases are described for vitamins A

and D.

10

Currently, vitamins and antivitamins widely used to prevent and treat a

variety of disorders of metabolism. For example:

- vitamin K or menadione, or vicasol (both are synthetic water-soluble analogue of

vitamin K) are prescribed to stimulate the synthesis (specifically post-translational

γ-carboxylation of glutamic acid residues) such enzymes of coagulation system as

factors II (prothrombin), VII, IX and X in the liver. They are usually used after

long-term antibiotic treatment (if there is increased bleeding with small injuries,

increase in blood clotting time) and in the preoperative period;

- vitamin K antagonist (antivitamin K) dicumarol reduces the efficiency of

the blood coagulation promoting blood thinning thereby it use for the treatment of

blood clotting diseases, in particular, thrombosis, thrombophlebitis;

- Vitamin A and its derivatives like retinol acetate are used for treating of

vitamin A deficiency. For example, they can be administered a patient in order to

restore his vision if the patient suffers from vision impairment hemeralopia (night

blindness, twilight vision impairment), age-related glaucoma, cataracts etc.

Vitamin A drug is also used for skin conditions including acne, eczema, psoriasis,

cold sores, wounds, burns, sunburn. It is also used for gastrointestinal ulcers, gum

disease, urinary tract infections, diseases of the nervous system;

- drug isoniazid which is antivitamin nicotinic acid and pyridoxine is used In

the treatment of patients with pulmonary tuberculosis;

- the structural analogue of vitamin B2 acriсhine is formerly widely used as

an antimalarial drug but superseded by chloroquine in recent years. It has also been

used as an anthelmintic (in enterobiasis) and in the treatment of giardiasis and

malignant effusions. The mechanism action of the drug is based on preventing of

microorganism FAD(FMN)-dependent dehydrogenases;

- Ascorutinum is recommended to use as a more effective drug in

comparison with ascorbic acid for patients with reduced immunity and frequent

colds. Vitamin C (Ascorbic acid) is involved in the hydroxylation of prolyl- and

lysyl residues by prolyl 3(4)-hydroxylase and lysyl 5-hydroxylase during collagen

synthesis. Effect of the vitamin C is enhanced by vitamin P, which stabilizes the

11

ground substance of fibrous connective tissue in way of hyaluronidase inhibition.

Ascorutinum can be recommended in case of bleeding gums, petechial

hemorrhages;

- Sulfonamide drugs are folic acid antivitamin. They are structurally

resemble paraaminobenzoic acid and due to this similarity it is displaced from its

complex with the enzyme synthesizing folic acid. This leads to the inhibition of

bacterial growth. This mechanism of action of sulfonamides allows their use as

antibacterial agents;

- Pregnant women with a history of several miscarriages is assigned the

therapy including α-tocopherol (vitamin E) vitamin supplements using, It

contributes to the childbearing. Furthermore, tocopherol acetate, vitamin

preparation is usually given in the course of radiation therapy, since this substance

has a distinct radioprotective membrane stabilizing action due to its antioxidant

activity;

- Derivatives of pyridoxine (vitamin B6) are used as neurotrophic agents for

the correction of mental retardation in childre; in cases of mental disorders in

adults; as neuroprotective agents in rehabilitation of patients with stroke and other

pathological conditions. The positive effects of pyridoxine is explained by its use

as a precursor of PLP that is prosthetic group of the enzyme glutamate

decarboxylase in neurons. The enzyme carries out inhibitory neurotransmitter

GABA formation.

- Cabbage and potato juices rich in vitamin U are recommended to drink for

patient with duodenal ulcer after the therapy course. Whether taken as a

supplement or from foods, vitamin U has been shown to be able to treat a variety

of gastrointestinal conditions, including ulcerative colitis, acid reflux, and peptic

ulcers. It may also be able to treat skin lesions, improve the symptoms of diabetes,

and strengthen the immune system. Some studies show that it can also help prevent

liver damage by protecting the organ from the effects of high doses of

acetaminophen. Additionally, it may be able to reduce allergies and sensitivities to

cigarette smoke and improve cholesterol levels.

12

The aforecited examples are only a small part of the use of vitamins and

their derivatives in medicine.

Therefore, knowledge of the biochemical basis of vitaminology is of great

importance for future doctors.

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test tasks: Explanations:

1. Examination of a patient with

frequent hemorrhages from internals

and mucous membranes revealed

proline and lysine being a part of

collagen fibers. What vitamin

absence caused disturbance of their

hydroxylation?

A. Vitamin A

B. Thiamine

C. Vitamin K

D. Vitamin E

E. Vitamin C

2. Examination of a man who hadn’t

been consuming fats but had been

getting enough carbohydrates and

proteins for long time revealed

dermatitis, poor wound healing,

vision impairment. What is the

probable cause of metabolic

disorder?

A. Lack of vitamins PP, H

13

№ Test tasks: Explanations:

B. Lack of oleic acid

C. Lack of linoleic acid, vitamins A,

D, E, K.

D. Lack of palmitic acid

E. Low caloric value of diet

3. A woman who has been keeping to a

clean-rice diet for a long time was

diagnosed with polyneuritis

(beriberi). What vitamin deficit

results in development of this

disease?

A. Folic acid

B. Thiamine

C. Ascorbic acid

D. Riboflavin

E. Pyridoxine

4. A patient suffers from vision

impairment hemeralopia (night

blindness). What vitamin preparation

should be administered the patient in

order to restore his vision?

A. Pyridoxine

B. Retinol acetate

C. Vicasol

D. Thiamine chloride

E. Tocopherol acetate

14

№ Test tasks: Explanations:

5. There is disturbed process of Ca2+

absorption through intestinal wall

after the removal of gall bladder in

patient. What vitamin will stimulate

this process?

A. K

B. C

C. D3

D. PP

E. B12

6. A 6 y.o child was administered

vicasol to prevent postoperative

bleeding. Vicasol is a synthetic

analogue of vitamin K. Name post-

translation changes of blood

coagulation factors that will be

activated by vicasol:

A. Carboxylation of glutamic acid

residues

B. Polymerization

C. Partial proteolysis

D. Glycosylation

E. Phosphorylation of serine radicals

7. Most participants of Magellan

expedition to America died from

avitaminosis. This disease declared

itself by general weakness,

subcutaneous hemmorhages, falling

15

№ Test tasks: Explanations:

of teeth, gingival hemmorhages.

What is the name of this

avitaminosis?

A. Biermer's anemia

B. Polyneuritis

(beriberi)

C. Pellagra

D. Rachitis

E. Scurvy

8. A patient with hypochromic anemia

has splitting hair and loss of hair,

increased nail bottling and taste

alteration. What is the mechanism of

the development of these symptoms?

A. Deficiency of vitamin B12

B. Decreased production of thyroid

hormones

C. Deficiency of vitamin K

D. Decreased production of

parathyrin

E. Deficiency of iron-containing

enzymes

9. The structural analogue of vitamin

B2 is administered (acriсhine) in a

case of enterobiasis. The disorder of

which enzyme synthesis is caused by

this medicine in microorganisms?

A. NAD-dependent dehydrogenases

16

№ Test tasks: Explanations:

B. Cytochrome oxidases

C. FAD-dependent dehydrogenases

D. Peptidases

E. Aminotransferases

10. A patient who was previously ill

with mastectomy as a result of breast

cancer was prescribed radiation

therapy. What vitamin preparation

has marked radioprotective action

caused by antioxidant activity?

A. Tocopherol acetate

B. Riboflavin

C. Folic acid

D. Ergocalciferol

E. Thiamine chloride

11. There is an inhibited coagulation in

the patients with bile ducts

obstruction, bleeding due to the low

level of absorption of vitamin. What

vitamin is in deficiency?

A. K

B. E

C. D

D. A

E. Carotene

12. Concentration of pyruvate is

increased in the patient's blood, the

17

№ Test tasks: Explanations:

most of which is excreted with urine.

What avitaminosis has the patient?

A. Avitaminosis B1

B. Avitaminosis B2

C. Avitaminosis E

D. Avitaminosis B9

E. Avitaminosis B3

13. Hydroxylation of endogenous

substrates and xenobiotics requires a

donor of protons. Which of the

following vitamins can play this

role?

A. Vitamin C

B. Vitamin E

C. Vitamin P

D. Vitamin A

E. Vitamin B6

14. A 2-year-old child has got intestinal

dysbacteriosis, which results in

hemorrhagic syndrome. What is the

most likely cause of hemorrhage of

the child?

A. Activation of tissue

thromboplastin

B. PP hypovitaminosis

C. Fibrinogen deficiency

18

№ Test tasks: Explanations:

D. Vitamin K insufficiency

E. Hypocalcemia

15. A 10-year-old girl often experiences

acute respiratory infections with

multiple hemorrages in the places of

clothes friction. Hypovitaminosis of

what vitamin is in this girl organism?

A. A

B. B2

C. B1

D. B6

E. С

16. A doctor recommends a patient with

duodenal ulcer to drink cabbage and

potato juices after the therapy

course. Which substances contained

in these vegetables help to heal and

prevent the ulcers?

A. Vitamin U

B. Vitamin B5

C. Vitamin K

D. Vitamin B1

E. Vitamin C

17. Ultraviolet radiation can be used in

the form of physical treatments.

What vitamin formation is activated

under UV light in the skin:

A. Vitamin B6

19

№ Test tasks: Explanations:

B. Vitamin A

C. Vitamin E

D. Vitamin C

E. Vitamin D3

18. A 9-month-old infant is fed with

artifical formulas with unbalanced

vitamin B6 concentration. The infant

presents with pellagral dermatitis,

convulsions, anaemia. Convulsions

development might be caused by the

disturbed formation of:

A. Dopamine

B. Histamine

C. Serotonin

D. DOPA

E. GABA

19. During examination of an 11-month-

old infant a pediatrician revealed

osteoectasia of the lower extremities

and delayed mineralization of cranial

bones. Such pathology is usually

provoked by the deficit of the

following vitamin:

A. Thiamin

B. Riboflavin

C. Bioflavonoids

20

№ Test tasks: Explanations:

D. Pantothenic acid

E. Cholecalciferol

20. A patient presents with twilight

vision impairment. Which of the

following vitamins should be

administered?

A. Cyanocobalamin

B. Ascorbic acid

C. Nicotinic acid

D. Retinol acetate

E. Pyridoxine hydrochloride

21. After the disease a 16-year-old boy

is presenting with decreased function

of protein synthesis in the liver as a

result of vitamin K deficiency. This

may cause disorder of:

A. Erythropoietin production

B. Erythrocyte sedimentation rate

C. Blood coagulation

D. Osmotic blood pressure

E. Anticoagulant production

22. In clinical practice tuberculosis is

treated with izoniazid preparation –

that is an antivitamin able to

penetrate into the tuberculosis

21

№ Test tasks: Explanations:

bacillus. Tuberculostatic effect is

induced by the interference with

replication processes and oxidation-

reduction reactions due to the

buildup of pseudo-coenzyme:

A. FMN

B. NAD

C. CoQ

D. FAD

E. TPP

23. Some infections diseases caused by

bacteria are treated with

sulfanilamides, which block the

synthesis of bacteria growth factor.

What is the mechanism of their

action?

A. They inhibit the absorption of

folic acid

B. They are allosteric enzyme

inhibitors

C. They are allosteric enzymes

D. They are antivitamins of

paraaminobenzoic acid

E. They are involved in red-ox

processes

24. A 20-year old male patient

complains of general weakness,

rapid fatigability, irritability,

22

№ Test tasks: Explanations:

decreased performance, bleeding

gums, petechiae on the skin. What

vitamin deficiency may be caused of

these changes?

A. Riboflavin

B. Ascorbic acid

C. Retinol

D. Thiamine

E. Folic acid

25. A number of disorders can be

diagnosed by evaluation activity of

blood transaminases. What vitamin

is one of cofactors for these

enzymes?

A. B6

B. B1

C. B5

D. B2

E. B8

26. Symptoms of pellagra (vitamin PP

deficiency) is particularly

pronounced in patients with low

protein diet, because nicotine amide

precursor in humans is one of the

essential amino acids, namely:

A. Lysine

23

№ Test tasks: Explanations:

B. Threonine

C. Tryptophan

D. Arginine

E. Histidine

27. A patient complains of

photoreception disorder and frequent

acute viral diseases. He has been

prescribed a vitamin that affects

photoreception processes by

producing rhodopsin, the

photosensitive pigment. What

vitamin is it?

A. Cyanocobalamin

B. Tocopherol acetate

C. Pyridoxine hydrochloride

D. Thiamine

E. Retinol acetate

28. A 36-year-old female patient has a

history of B2-hypovitaminosis. The

most likely cause of specific

symptoms (epithelial, mucosa,

cutaneous, corneal lesions) is the

deficiency of:

A. Cytochrome oxidase

B. Cytochrome B

C. Cytochrome A1

D. Cytochrome C

E. FAD or FMN

24

№ Test tasks: Explanations:

29. A patient, who has been suffering for

a long time from intestine

disbacteriosis, has increased

hemorrhaging caused by disruption

of posttranslational modification of

blood coagulation factors II, VII, IX

and X in the liver. What vitamin

deficiency is the cause of this

condition?

A. K

B. B12

C. B9

D. C

E. P

30. A 6-year-old child suffers from

delayed growth, disrupted

ossification processes,

decalcification of teeth. What can be

the cause?

A. Vitamin D deficiency

B. Hyperthyroidism

C. Vitamin C deficiency

D. Decreased glucagon production

E. Insulin deficiency

31 A patient is diagnosed with chronic

atrophic gastritis attended by

deficiency of Castle`s (intrinsic)

25

№ Test tasks: Explanations:

factor. What type of anemia does the

patient have?

A. B12-deficiency anemia

B. Iron-deficiency anemia

C. Hemolytic anemia

D. Protein-deficiency anemia

E. Iron refractory anemia

32 During regular check-up a child is

detected with interrupted

mineralization of bones. What

vitamin deficiency can be the cause?

A. Calciferol

B. Riboflavin

C. Tocopherol

D. Folic acid

E. Cobalamin

33 Point out the vitamin, whose

deficiency leads to pellagra:

A. Vitamin P

B. Vitamin A

C. Vitamin C

D. Vitamin B3

E. Vitamin B2

34 The avitaminosis of ascorbic acid is

named as:

A. Cushing`s syndrome

26

№ Test tasks: Explanations:

B. Addison`s disease

C. Kwashiorkor

D. Hemolytic anemia

E. Scurvy

35 Find out the vitamin whose

deficiency is associated with

disturbed transamination of amino

acids:

A. Pyridoxine

B. Rutin

C. Thiamine

D. Folic acid

E. Ascorbic acid

36 Choose the vitamin, whose

antivitamin is named as dicoumarol:

A. Vitamin A

B. Vitamin B6

C. Vitamin C

D. Vitamin D

E. Vitamin K

37 Choose the vitamin, which is a

powerful natural antioxidant:

A. Retinal

B. Tocopherol

C. Ergocalciferol

D. Riboflavin

27

№ Test tasks: Explanations:

E. Pyridoxine

38 Name the blood plasma index

whose low value will prove the

deficiency of vitamin K in patient:

A. Urea

B. Albumins

C. Immunoglobulin G

D. Prothrombin

E. C-reactive protein

39 Name the active form of vitamin

whose level in the blood is depended

on the secretion rate of parathyroid

hormone:

A. Ascorbic acid

B. Calcitriol

C. Thiamine

D. Tocopherol

E. Naphtoquinone

40 Choose the vitamin, whose precursor

is named as β-carotene:

A. Vitamin C

B. Vitamin D

C. Vitamin A

D. Vitamin B12

E. Vitamin P

28

BIOCHEMISTRY OF MUSCULAR AND CONNECTIVE TISSUES

(Ivanchenko D. H., Tikhonovska M. A.)

INFORMATIONAL MATERIAL

Muscular tissue

Muscle tissue account for 40-42 % of body mass.

Muscular tissues consist of elongated cells called muscle fibers or myocytes that

can use ATP to generate force. As a result, muscular tissues produce body

movements, maintain posture, and generate heat. They also provides protection.

Based on their location and certain structural and functional features, muscular

tissues are classified into three types: skeletal, cardiac, and smooth (Table 1).

Table 1. Characteristic of different types of muscular tissues

Description Location Function

Skeletal muscle tissue

Long, cylindrical, striated fibers (striations

are alternating light and dark bands within

fibers that are visible under a light

microscope). Skeletal muscle fibers vary

greatly in length, from a few centimeters in

short muscles to 30-40 cm in longest

muscles. A muscle fiber is a roughly

cylindrical, multinucleated cell with nuclei

at periphery. Skeletal muscle is considered

voluntary because it can be made to

contract or relax by conscious control.

Usually attached

to bones by

tendons.

Motion,

posture, heat

production,

protection.

Cardiac muscle tissue

Branched, striated fibers with usually only

one centrally located nucleus (occasionally

two). Attach end to end by transverse

Heart wall. Pumps blood to

all parts of

body.

29

thickenings of plasma membrane called

intercalated discs (intercalate – to insert

between), which contain desmosomes and

gap junctions. Desmosomes strengthen

tissue and hold fibers together during

vigorous contractions. Gap junctions

provide route for quick conduction of

electrical signals (muscle action potentials)

throughout heart. Involuntary (not

conscious) control.

Smooth muscle tissue

Fibers usually involuntary, nonstriated

(lack striations, hence the term smooth).

Smooth muscle fiber is a small spindle-

shaped cell thickest in middle, tapering at

each end, and containing a single, centrally

located nucleus. Gap junctions connect

many individual fibers in some smooth

muscle tissues (for example, in wall of

intestines). Can produce powerful

contractions as many muscle fibers

contract in unison. Where gap junctions are

absent, such as iris of eye, smooth muscle

fibers contract individually, like skeletal

muscle fibers.

Iris of eyes; walls

of hollow internal

structures such as

blood vessels,

airways to lungs,

stomach,

intestines,

gallbladder,

urinary bladder,

and uterus.

Motion

(constriction of

blood vessels

and airways,

propulsion of

foods through

gastrointestinal

tract,

contraction of

urinary bladder

and

gallbladder).

Skeletal muscle tissue is so named because most skeletal muscles move bones of

the skeleton. A few skeletal muscles attach to and move the skin or other skeletal

muscles. Skeletal muscle tissue is striated: Alternating light and dark protein bands

(striations) are seen when the tissue is examined with a microscope. Skeletal

30

muscle tissue works mainly in a voluntary manner. Its activity can be consciously

controlled by neurons (nerve cells) that are part of the somatic (voluntary) division

of the nervous system. Most skeletal muscles also are controlled subconsciously to

some extent. For example, your diaphragm continues to alternately contract and

relax without conscious control so that you don’t stop breathing. Also, you do not

need to consciously think about contracting the skeletal muscles that maintain your

posture or stabilize body positions.

Only the heart contains cardiac muscle tissue, which forms most of the heart wall.

Cardiac muscle is also striated, but its action is involuntary. The alternating

contraction and relaxation of the heart is not consciously controlled. Rather, the

heart beats because it has a pacemaker that initiates each contraction. This built-in

rhythm is termed autorhythmicity. Several hormones and neurotransmitters can

adjust heart rate by speeding or slowing the pacemaker.

Smooth muscle tissue is located in the walls of hollow internal structures, such as

blood vessels, airways, and most organs in the abdominopelvic cavity. It is also

found in the skin, attached to hair follicles. Under a microscope, this tissue lacks

the striations of skeletal and cardiac muscle tissue. For this reason, it looks non-

striated, which is why it is referred to as smooth. The action of smooth muscle is

usually involuntary, and some smooth muscle tissue, such as the muscles that

propel food through your gastrointestinal tract, has autorhythmicity. Both cardiac

muscle and smooth muscle are regulated by neurons that are part of the autonomic

(involuntary) division of the nervous system and by hormones released by

endocrine glands.

Functions of Muscular Tissue. Through sustained contraction or alternating

contraction and relaxation, muscular tissue has four key functions: producing body

movements, stabilizing body positions, storing and moving substances within the

body, and generating heat.

1. Producing body movements. Movements of the whole body such as walking

and running, and localized movements such as grasping a pencil, keyboarding, or

31

nodding the head as a result of muscular contractions, rely on the integrated

functioning of skeletal muscles, bones, and joints.

2. Stabilizing body positions. Skeletal muscle contractions stabilize joints and

help maintain body positions, such as standing or sitting. Postural muscles contract

continuously when you are awake; for example, sustained contractions of your

neck muscles hold your head upright when you are listening intently to your

anatomy and physiology lecture.

3. Storing and moving substances within the body. Storage is accomplished by

sustained contractions of ringlike bands of smooth muscle called sphincters, which

prevent outflow of the contents of a hollow organ. Temporary storage of food in

the stomach or urine in the urinary bladder is possible because smooth muscle

sphincters close off the outlets of these organs. Cardiac muscle contractions of the

heart pump blood through the blood vessels of the body. Contraction and

relaxation of smooth muscle in the walls of blood vessels help adjust blood vessel

diameter and thus regulate the rate of blood flow. Smooth muscle contractions also

move food and substances such as bile and enzymes through the gastrointestinal

tract, push gametes (sperm and oocytes) through the passageways of the

reproductive systems, and propel urine through the urinary system. Skeletal muscle

contractions promote the flow of lymph and aid the return of blood in veins to the

heart.

4. Generating heat. As muscular tissue contracts, it produces heat, a process

known as thermogenesis. Much of the heat generated by muscle is used to

maintain normal body temperature. Involuntary contractions of skeletal muscles,

known as shivering, can increase the rate of heat production.

Microscopic Anatomy of a Skeletal Muscle Fiber. The most important

components of a skeletal muscle are the muscle fibers themselves. The diameter of

a mature skeletal muscle fiber ranges from 10 to 100 μm. The typical length of a

mature skeletal muscle fiber is about 10 cm, although some are as long as 30 cm.

Because each skeletal muscle fiber arises during embryonic development from the

fusion of a hundred or more small mesodermal cells called myoblasts (Fig. 1 a),

32

each mature skeletal muscle fiber has a hundred or more nuclei. Once fusion has

occurred, the muscle fiber loses its ability to undergo cell division. Thus, the

number of skeletal muscle fibers is set before you are born, and most of these cells

last a lifetime.

Figure 1. Microscopic organization of skeletal muscle.

Sarcolemma, Transverse Tubules, and Sarcoplasm. The multiple nuclei of a

skeletal muscle fiber are located just beneath the sarcolemma, the plasma

33

membrane of a muscle cell (Fig. 1 b, c). Thousands of tiny invaginations of the

sarcolemma, called transverse (T) tubules, tunnel in from the surface toward the

center of each muscle fiber. Because T tubules are open to the outside of the fiber,

they are filled with interstitial fluid. Muscle action potentials travel along the

sarcolemma and through the T tubules, quickly spreading throughout the muscle

fiber. This arrangement ensures that an action potential excites all parts of the

muscle fiber at essentially the same instant.

Within the sarcolemma is the sarcoplasm, the cytoplasm of a muscle fiber.

Sarcoplasm includes a substantial amount of glycogen, which is a large molecule

composed of many glucose molecules. Glycogen can be used for synthesis of ATP.

In addition, the sarcoplasm contains a red-colored protein called myoglobin. This

protein, found only in muscle, binds oxygen molecules that diffuse into muscle

fibers from interstitial fluid. Myoglobin releases oxygen when it is needed by the

mitochondria for ATP production. The mitochondria lie in rows throughout the

muscle fiber, strategically close to the contractile muscle proteins that use ATP

during contraction so that ATP can be produced quickly as needed (Fig. 1 c).

Myofibrils and Sarcoplasmic Reticulum. At high magnification, the sarcoplasm

appears stuffed with little threads. These small structures are the myofibrils, the

contractile organelles of skeletal muscle (Fig. 1 c). Myofibrils are about 2 μm in

diameter and extend the entire length of a muscle fiber. Their prominent striations

make the entire skeletal muscle fiber appear striped (striated).

A fluid-filled system of membranous sacs called the sarcoplasmic reticulum or

SR encircles each myofibril (Fig. 1 c). This elaborate system is similar to smooth

endoplasmic reticulum in nonmuscular cells. Dilated end sacs of the sarcoplasmic

reticulum called terminal cisterns butt against the T tubule from both sides. A

transverse tubule and the two terminal cisterns on either side of it form a triad. In a

relaxed muscle fiber, the sarcoplasmic reticulum stores calcium ions (Ca2+

).

Release of Ca2+

from the terminal cisterns of the sarcoplasmic reticulum triggers

muscle contraction.

34

Filaments and the Sarcomere. Within myofibrils are smaller protein structures

called filaments or myofilaments (Fig. 1 c). Thin filaments are 8 nm in diameter

and 1-2 μm long and composed mostly of the protein actin, while thick filaments

are 16 nm in diameter and 1-2 μm long and composed mostly of the protein

myosin. Both thin and thick filaments are directly involved in the contractile

process. Overall, there are two thin filaments for every thick filament in the regions

of filament overlap. The filaments inside a myofibril do not extend the entire length

of a muscle fiber. Instead, they are arranged in compartments called sarcomeres,

which are the basic functional units of a myofibril (Fig. 2 a).

Figure 2. The arrangement of filaments within a sarcomere.

Narrow, plate-shaped regions of dense protein material called Z discs separate one

sarcomere from the next. Thus, a sarcomere extends from one Z disc to the next Z

disc.

The extent of overlap of the thick and thin filaments depends on whether the

muscle is contracted, relaxed, or stretched. The pattern of their overlap, consisting

35

of a variety of zones and bands (Fig. 2 b), creates the striations that can be seen

both in single myofibrils and in whole muscle fibers. The darker middle part of the

sarcomere is the A band, which extends the entire length of the thick filaments

(Fig. 2 b). Toward each end of the A band is a zone of overlap, where the thick and

thin filaments lie side by side. The I band is a lighter, less dense area that contains

the rest of the thin filaments but no thick filaments (Fig. 2 b), and a Z disc passes

through the center of each I band. A narrow H zone in the center of each A band

contains thick but not thin filaments. A mnemonic that will help you to remember

the composition of the I and H bands is as follows: the letter I is thin (contains thin

filaments), while the letter H is thick (contains thick filaments). Supporting

proteins that hold the thick filaments together at the center of the H zone form the

M line, so named because it is at the middle of the sarcomere. Table 2 summarizes

the components of the sarcomere.

Table 2. Components of a Sarcomere

Component Description

Z discs Narrow, plate-shaped regions of dense material that separate one

sarcomere from the next.

A band Dark, middle part of sarcomere that extends entire length of thick

filaments and includes those parts of thin filaments that overlap

thick filaments.

I band Lighter, less dense area of sarcomere that contains remainder of

thin filaments but no thick filaments. A Z disc passes through

center of each I band.

H zone Narrow region in center of each A band that contains thick

filaments but no thin filaments.

M line Region in center of H zone that contains proteins that hold thick

filaments together at center of sarcomere.

Muscle Proteins. Myofibrils are built from three kinds of proteins: (1)

contractile proteins, which generate force during contraction; (2) regulatory

36

proteins, which help switch the contraction process on and off; and (3) structural

proteins, which keep the thick and thin filaments in the proper alignment, give the

myofibril elasticity and extensibility, and link the myofibrils to the sarcolemma and

extracellular matrix.

The two contractile proteins in muscle are myosin and actin, components of

thick and thin filaments, respectively. Myosin is the main component of thick

filaments and functions as a motor protein in all three types of muscle tissue. Motor

proteins pull various cellular structures to achieve movement by converting the

chemical energy in ATP to the mechanical energy of motion, that is, the production

of force. In skeletal muscle, about 300 molecules of myosin form a single thick

filament. Each myosin molecule is shaped like two golf clubs twisted together (Fig.

3 a). The myosin tail (twisted golf club handles) points toward the M line in the

center of the sarcomere. Tails of neighboring myosin molecules lie parallel to one

another, forming the shaft of the thick filament. The two projections of each

myosin molecule (golf club heads) are called myosin heads. The heads project

outward from the shaft in a spiraling fashion, each extending toward one of the six

thin filaments that surround each thick filament.

Figure 3. Structure of thick and thin filaments.

Limited proteolysis can be a powerful tool in probing the activity of large

proteins. The treatment of myosin with trypsin and papain results in the formation

of four fragments: two S1 fragments; an S2 fragment, also called heavy

meromyosin (HMM); and a fragment called light meromyosin (LMM; Fig. 4). Each

S1 fragment corresponds to one of the heads from the intact structure and includes

850 amino-terminal amino acids from one of the two heavy chains as well as one

37

copy of each of the light chains. Examination of the structure of an S1 fragment at

high resolution reveals the presence of a P-loop NTPase-domain core that is the

site of ATP binding and hydrolysis.

Thin filaments are anchored to Z discs (Fig. 2 b). Their main component is the

protein actin. Filamentous actin, or F-actin, is a twisted strand composed of two

rows of 300–400 individual globular molecules of G-actin (Fig. 3 b). On each

actin molecule is a myosin-binding site, where a myosin head can attach.

Figure 4. Myosin dissection.

Smaller amounts of two regulatory proteins – tropomyosin and troponin –

are also part of the thin filament. In relaxed muscle, myosin is blocked from

binding to actin because strands of tropomyosin cover the myosin-binding sites on

actin. The tropomyosin strands in turn are held in place by troponin molecules.

You will soon learn that when calcium ions bind to troponin, it undergoes a change

in shape; this change moves tropomyosin away from myosin-binding sites on actin

and muscle contraction subsequently begins as myosin binds to actin.

Besides contractile and regulatory proteins, muscle contains about a dozen

structural proteins, which contribute to the alignment, stability, elasticity, and

extensibility of myofibrils. Several key structural proteins are titin, α-actinin,

myomesin, nebulin, and dystrophin. Titin is the third most plentiful protein in

skeletal muscle (after actin and myosin). This molecule’s name reflects its huge

size. With a molecular mass of about 3 million daltons, titin is 50 times larger than

an average-sized protein. Each titin molecule spans half a sarcomere, from a Z disc

to an M line (Fig. 2 b), a distance of 1 to 1.2 μm in relaxed muscle. Each titin

38

molecule connects a Z disc to the M line of the sarcomere, thereby helping

stabilize the position of the thick filament. The part of the titin molecule that

extends from the Z disc is very elastic. Because it can stretch to at least four times

its resting length and then spring back unharmed, titin accounts for much of the

elasticity and extensibility of myofibrils. Titin probably helps the sarcomere return

to its resting length after a muscle has contracted or been stretched, may help

prevent overextension of sarcomeres, and maintains the central location of the A

bands.

The dense material of the Z discs contains molecules of α-actinin, which bind to

actin molecules of the thin filament and to titin. Molecules of the protein

myomesin form the M line. The M line proteins bind to titin and connect adjacent

thick filaments to one another. Myosin holds the thick filaments in alignment at the

M line. Nebulin is a long, nonelastic protein wrapped around the entire length of

each thin filament. It helps anchor the thin filaments to the Z discs and regulates

the length of thin filaments during development. Dystrophin links thin filaments of

the sarcomere to integral membrane proteins of the sarcolemma, which are

attached in turn to proteins in the connective tissue extracellular matrix that

surrounds muscle fibers. Dystrophin and its associated proteins are thought to

reinforce the sarcolemma and help transmit the tension generated by the

sarcomeres to the tendons.

Sliding Filaments and Muscle Contraction. Muscle contraction occurs because

myosin heads attach to and ―walk‖ along the thin filaments at both ends of a

sarcomere, progressively pulling the thin filaments toward the M line (Fig. 5). As a

result, the thin filaments slide inward and meet at the center of a sarcomere. They

may even move so far inward that their ends overlap (Fig. 5 c). As the thin

filaments slide inward, the Z discs come closer together, and the sarcomere

shortens. However, the lengths of the individual thick and thin filaments do not

change. Shortening of the sarcomeres causes shortening of the whole muscle fiber,

which in turn leads to shortening of the entire muscle.

39

Figure 5. Sliding filament mechanism of muscle contraction, as it occurs in two

adjacent sarcomeres.

The Contraction Cycle. At the onset of contraction, the sarcoplasmic reticulum

releases Ca2+

into the sarcoplasm. There, they bind to troponin. Troponin then

moves tropomyosin away from the myosin-binding sites on actin. Once the binding

sites are ―free,‖ the contraction cycle – the repeating sequence of events that causes

the filaments to slide – begins. The contraction cycle consists of four steps (Fig. 6):

1. ATP hydrolysis. The myosin head includes an ATP-binding site and an ATPase,

an enzyme that hydrolyzes ATP into ADP (adenosine diphosphate) and a

phosphate group. This hydrolysis reaction reorients and energizes the myosin head.

40

Notice that the products of ATP hydrolysis – ADP and a phosphate group – are

still attached to the myosin head.

2. Attachment of myosin to actin to form cross-bridges. The energized myosin

head attaches to the myosin-binding site on actin and releases the previously

hydrolyzed phosphate group. When the myosin heads attach to actin during

contraction, they are referred to as cross-bridges.

3. Power stroke. After the cross-bridges form, the power stroke occurs. During the

power stroke, the site on the cross-bridge where ADP is still bound opens. As a

result, the cross-bridge rotates and releases the ADP. The cross-bridge generates

force as it rotates toward the center of the sarcomere, sliding the thin filament past

the thick filament toward the M line.

4. Detachment of myosin from actin. At the end of the power stroke, the cross-

bridge remains firmly attached to actin until it binds another molecule of ATP. As

ATP binds to the ATP-binding site on the myosin head, the myosin head detaches

from actin.

Figure 6. The contraction cycle.

The contraction cycle repeats as the myosin ATPase hydrolyzes the newly bound

molecule of ATP, and continues as long as ATP is available and the Ca2+

level near

41

the thin filament is sufficiently high. The cross-bridges keep rotating back and forth

with each power stroke, pulling the thin filaments toward the M line. Each of the

600 cross-bridges in one thick filament attaches and detaches about five times per

second. At any one instant, some of the myosin heads are attached to actin,

forming cross-bridges and generating force, and other myosin heads are detached

from actin, getting ready to bind again.

As the contraction cycle continues, movement of cross-bridges applies the force

that draws the Z discs toward each other, and the sarcomere shortens. During a

maximal muscle contraction, the distance between two Z discs can decrease to half

the resting length. The Z discs in turn pull on neighboring sarcomeres, and the

whole muscle fiber shortens. Some of the components of a muscle are elastic: They

stretch slightly before they transfer the tension generated by the sliding filaments.

The elastic components include titin molecules, connective tissue around the

muscle fibers (endomysium, perimysium, and epimysium), and tendons that attach

muscle to bone. As the cells of a skeletal muscle start to shorten, they first pull on

their connective tissue coverings and tendons. The coverings and tendons stretch

and then become taut, and the tension passed through the tendons pulls on the

bones to which they are attached. The result is movement of a part of the body.

Muscles energy supply. Muscle’s major fuels are glucose from glycogen, fatty

acids, and ketone bodies. Rested, well-fed muscle, in contrast to brain, synthesizes

a glycogen store comprising 1 to 2% of its mass. The glycogen serves muscle as a

readily available fuel depot since it can be rapidly converted to glucose-6-

phosphate for entry into glycolysis.

Muscle cannot export glucose because it lacks glucose-6-phosphatase.

Nevertheless, muscle serves the body as an energy reservoir because, during the

fasting state, its proteins are degraded to amino acids, many of which are converted

to pyruvate, which in turn, is transaminated to alanine. The alanine is then exported

via the bloodstream to the liver, which transaminates it back to pyruvate, a glucose

precursor. This process is known as the glucose-alanine cycle.

42

Since muscle does not participate in gluconeogenesis, it lacks the machinery that

regulates this process in such gluconeogenic organs as liver and kidney. Muscle

does not have receptors for glucagon, which stimulates an increase in blood

glucose levels. However, muscle possesses epinephrine receptors (β-adrenergic

receptors), which through the intermediacy of cAMP control the

phosphorylation/dephosphorylation cascade system that regulates glycogen

breakdown and synthesis. This is the same cascade system that controls the

competition between glycolysis and gluconeogenesis in liver in response to

glucagon.

Heart muscle and skeletal muscle contain different isozymes of PFK-2/FBPase-2.

The heart muscle isozyme is controlled by phosphorylation oppositely to that in

liver, whereas skeletal muscle PFK-2/FBPase-2 is not controlled by

phosphorylation at all. Thus the concentration of F2,6P rises in heart muscle but

falls in liver in response to an increase in [cAMP]. Moreover the muscle isozyme

of pyruvate kinase, which, it will be recalled, catalyzes the final step of glycolysis,

is not subject to phosphorylation/dephosphorylation as is the liver isozyme. Thus,

whereas an increase in liver cAMP stimulates glycogen breakdown and

gluconeogenesis, resulting in glucose export, an increase in heart muscle cAMP

activates glycogen breakdown and glycolysis, resulting in glucose consumption.

Consequently, epinephrine, which prepares the organism for action (fight or flight),

acts independently of glucagon which, acting reciprocally with insulin, regulates

the general level of blood glucose.

Muscle contraction is driven by ATP hydrolysis and is therefore ultimately

dependent on respiration. Skeletal muscle at rest utilizes ~30% of the O2 consumed

by the human body. A muscle’s respiration rate may increase in response to a

heavy workload by as much as 25-fold. Yet, its rate of ATP hydrolysis can

increase by a much greater amount. The ATP is initially regenerated by the

reaction of ADP with phosphocreatine as catalyzed by creatine kinase:

Phosphocreatine + ADP ↔ creatine + ATP

43

(phosphocreatine is resynthesized in resting muscle by the reversal of this

reaction). Under conditions of maximum exertion, however, such as occurs in a

sprint, a muscle has only an ~5-s supply of phosphocreatine. It must then shift to

ATP production via glycolysis of glucose-6-phosphate resulting from glycogen

breakdown, a process whose maximum flux greatly exceeds those of the citric acid

cycle and oxidative phosphorylation. Much of this glucose-6-phosphate is

therefore degraded anaerobically to lactate which, in the Cori cycle, is exported via

the bloodstream to the liver, where it is reconverted to glucose through

gluconeogenesis. Gluconeogenesis requires ATP generated by oxidative

phosphorylation. Muscles thereby shift much of their respiratory burden to the

liver and consequently also delay the O2-consumption process, a phenomenon

known as oxygen debt. The source of ATP during exercise of varying duration is

summarized in Fig. 7.

Figure 7. Source of ATP during exercise in humans.

Biosynthesis of creatine. Creatine is present in the tissues (muscle, brain,

blood etc.) as the high energy compound, phosphocreatine and as free creatine.

Three amino acids – glycine, arginine and methionine – are required for creatine

formation (Fig. 8). The first reaction occurs in the kidney. lt involves the transfer

of guanidino group of arginine to glycine, catalyzed by arginine-glycine

44

transamidinase to produce guanidoacetate (glycocyamine). S-Adenosylmethionine

(active methionine) donates methyl group to glycocyamine to produce creatine.

This reaction occurs in liver. Creatine is reversible phosphorylated to

phosphocreatine (creatine phosphate) by creatine kinase. It is stored in muscle as

high energy phosphate.

Creatinine is an anhydride of creatine. It is formed by spontaneous

cyclization of creatine or creatine phosphate. Creatinine is excreted in urine.

Figure 8. Creatine metabolism.

Clinical significance of creatine and creatinine determination. The normal

concentrations of creatine and creatinine in human serum and urine are:

45

Serum: creatine – 0.2-0.6 mg/dL, creatinine – 0.6-1 mg/dL;

Urine: creatine – 0-50 mg/day, creatinine – 1-2 g/day.

Estimation of serum creatinine (along with blood urea) is used as a

diagnostic test to assess kidney function. Seru m creati n i ne concentration is not

influenced by endogenous and exogenous factors, as is the case with urea. Hence,

some workers consider serum creatinine as a more reliable indicator of renal

function.

The amount of creatinine excreted is proportional to total creatine phosphate

content of the body and, in turn, the muscle mass. The daily excretion of creatinine

is usually constant. Creatinine coefficient is defined as the mg of creatinine and

creatine (put together) excreted per kg body weight per day. For a normal adult

man, the value is 24-26 mg, while for a woman, it is 20-22 mg.

Increased output of creatine in urine is referred to as creatinuria. Creatinuria is

observed in muscular dystrophy, diabetes mellitus, hyperthyroidism, starvation etc.

Connective tissue

Cells are the basis units of life. Most mammalian cells are located in tissues, where

they are surrounded by a complex of extra-cellular matrix, often referred to as

―connective tissue‖. Extra-cellular matrix contains three major classes of

biomolecules:

1. Structural proteins: collagen, elastin and fibrillin.

2. Certain specialized proteins, such as fibrillin, fibronectin, and lamilin.

3. Proteoglycans, which consist of long chains of repeating disaccharides

fragments (glucosoaminoglycans or mucopolysaccharides) attached to specific

core proteins.

Collagen. Collagen, which is present in all multicellular organisms, is not

one protein but diversity a family of structurally related proteins. It is the most

abundant protein in mammals and is present in most organs of the body, where it

serves to hold cells together in discrete units. It is also the major fibrous element of

skin, bones, tendons, cartilage, blood vessels and teeth. The different collagen

proteins have very diverse functions. The extremely hard structures of bones and

46

teeth contain collagen and a calcium phosphate polymer. In tendons, collagen

forms rope-like fibers of high tensile strength, while in the skin collagen forms

loosely woven fibers that can expand in all directions. The different types of

collagen are characterized by different polypeptide compositions (Table 3). Each

collagen is composed of three polypeptide chains, which may be all identical (as in

types II and III) or may be of two different chains (types I, IV and V). A single

molecule of type I collagen has a molecular mass of 285 kDa, a width of 1.5 nm

and a length of 300 nm.

Table 3. Types of collagen.

Type Polypeptide composition Distribution

I [α1(I)]2α2(I) Skin, bone, tendon, cornea, blood vessels

II [α1(II)]3 Cartilage, intervertebral disk

III [α1(III)]3 Fetal skin, blood vessels

IV [α1(IV)]2α2(IV) Basement membrane

V [α1(V)]2α2(V) Placenta, skin

VII [α1(VII)]3 Beneath stratified squamous epithelia

IX α1(IX)α2(IX)α3(IX) Cartilage

XII [α1(XII)]3 Tendon, ligaments, some other tissues

Collagen has a distinctive amino acid composition: nearly one-third of its residues

are Gly; another 15 to 30% of them are Pro and 4-hydroxyprolyl (Hyp) residues:

3-Hydroxyprolyl and 5-hydroxylysyl (Hyl) residues also occur in collagen but in

smaller amounts. Radioactive labeling experiments have established that these

nonstandard hydroxylated amino acids are not incorporated into collagen during

47

polypeptide synthesis: If 14

C-labeled 4-hydroxyproline is administered to a rat, the

collagen synthesized is not radioactive, whereas radioactive collagen is produced if

the rat is fed 14

C-labeled proline. The hydroxylated residues appear after the

collagen polypeptides are synthesized, when certain Pro residues are converted to

Hyp in a reaction catalyzed by the enzyme prolyl hydroxylase.

The amino acid sequence of bovine collagen α1(I), which is similar to that of

other collagens, consists of monotonously repeating triplets of sequence Gly-X-Y

over a continuous 1011-residue stretch of its 1042-residue polypeptide chain (Fig.

9). Here X is often Pro (~28%) and Y is often Hyp (~38%). The restriction of Hyp

to the Y position stems from the specificity of prolyl hydroxylase. Hyl is similarly

restricted to the Y position.

Figure 9. The amino acid sequence at the C-terminal end of the triple helical region

of the bovine α1(I) collagen chain.

Each of the three polypeptide chains in collagen is some 1000 residues long

and they each fold up into a helix that has only 3.3 residues per turn, rather than the

3.6 residues per turn of an α-helix. This secondary structure is unique to collagen

and is often called the collagen helix. The three polypeptide chains lie parallel and

wind round one another with a slight right-handed, rope-like twist to form a triple-

helical cable (Fig. 10). Every third residue of each polypeptide passes through the

center of the triple helix, which is so crowded that only the small side chain of Gly

can fit in. This explains the absolute requirement for Gly at every third residue.

The residues in the X and Y positions are located on the outside of the triple-

48

helical cable, where there is room for the bulky side-chains of Pro and other

residues. The three polypeptide chains are also staggered so that the Gly residue in

one chain is aligned with the X residue in the second and the Y residue in the third.

The triple helix is held together by an extensive network of hydrogen bonds, in

particular between the primary amino group of Gly in one helix and the primary

carboxyl group of Pro in position X of one of the other helices. In addition, the

hydroxyl groups of Hyp residues participate in stabilizing the structure. The

relatively inflexible Pro and Hyp also confer rigidity on the collagen structure.

Figure 10. Arrangement of the three polypeptide chains in collagen.

Collagen synthesis. Collagen biosynthesis and secretion follow the normal

pathway for a secreted protein. The collagen α-chains are synthesized as longer

precursors, called pro-α-chains, by ribosomes attached to the endoplasmic

reticulum (ER). The pro-α-chains undergo a series of covalent modifications and

fold into triple-helical procollagen molecules before their release from cells (Fig.

11).

49

Figure 11. Major events in biosynthesis of fibrillar collagens.

After the secretion of procollagen from the cell, extracellular peptidases (e.g., bone

morphogenetic protein-1) remove the N-terminal and C-terminal propeptides. In

regard to fibrillar collagens, the resulting molecules, which consist almost entirely

of a triple-stranded helix, associate laterally to generate fibrils with a diameter of

50-200 nm. In fibrils, adjacent collagen molecules are displaced from one another

by 67 nm, about one-quarter of their length. This staggered array produces a

striated effect that can be seen in electron micrographs of collagen fibrils. The

unique properties of the fibrous collagens (e.g., types I, II, III) are mainly due to

the formation of fibrils.

Short non-triple-helical segments at either end of the collagen chains are of

particular importance in the formation of collagen fibrils. Lysine and

hydroxylysine side chains in these segments are covalently modified by

extracellular lysyl oxidases to form aldehydes in place of the amine group at the

50

end of the side chain. These reactive aldehyde groups form covalent crosslinks

with lysine, hydroxylysine, and histidine residues in adjacent molecules. These

cross-links stabilize the side-by-side packing of collagen molecules and generate a

strong fibril. The removal of the propeptides and covalent cross-linking take place

in the extracellular space to prevent the potentially catastrophic assembly of fibrils

within the cell.

The post-translational modifications of pro-α-chains are crucial for the formation

of mature collagen molecules and their assembly into fibrils. Defects in these

modifications have serious consequences, as ancient mariners frequently

experienced. For example, ascorbic acid (vitamin C) is an essential cofactor for the

hydroxylases responsible for adding hydroxyl groups to proline and lysine residues

in pro-α-chains. In cells deprived of ascorbate, as in the disease scurvy, the pro-α-

chains are not hydroxylated sufficiently to form stable triple-helical procollagen at

normal body temperature, and the procollagen that forms cannot assemble into

normal fibrils. Without the structural support of collagen, blood vessels, tendons,

and skin become fragile. Because fresh fruit in the diet can supply sufficient

vitamin C to support the formation of normal collagen, early British sailors were

provided with limes to prevent scurvy, leading to their being called ―limeys‖.

When the collagen polypeptides are synthesized they have additional amino

aggregation acid residues (100-300) on both their N and C termini that are absent

in the mature collagen fiber (Fig. 12). These extension peptides often contain Cys

residues, which are usually absent from the remainder of the polypeptide chain.

The extension peptides help to correctly align the three polypeptides as they come

together in the triple helix, a process that may be aided by the formation of

disulfide bonds between extension peptides on neighboring polypeptide chains.

The extension peptides also prevent the premature aggregation of the procollagen

triple helices within the cell. On secretion out of the fibroblast the extension

peptides are removed by the action of extracellular peptidases. The resulting

tropocollagen molecules then aggregate together in a staggered head-to-tail

arrangement in the collagen fiber (Fig. 12).

51

Figure 12. Role of the extension peptides in the folding and secretion of

procollagen.

Elastin. In contrast to collagen, which forms fibers that are tough and have

high tensile strength, elastin is a connective tissue protein with rubber-like

properties. Elastic fibers composed of elastin and glycoprotein microfibrils are

found in the lungs, the walls of large arteries, and elastic ligaments. They can be

stretched to several times their normal length, but recoil to their original shape

when the stretching force is relaxed.

Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin,

which is a linear polypeptide composed of about 700 amino acids that are primarily

small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich

in proline and lysine, but contains only a little hydroxyproline and hydroxy lysine.

Tropoelastin is secreted by the cell into the extracellular space. There it interacts

with specific glycoprotein microfibrils, such as fibrillin, which function as a

scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the

52

tropoelastin poly peptides are oxidatively deaminated by lysyl oxidase, forming

allysine residues. Three of the allysyl side chains plus one unaltered lysyl side

chain from the same or neighboring polypeptides form a desmosine cross-link (Fig.

13). This produces elastin – an extensively interconnected, rubbery network that

can stretch and bend in any direction when stressed, giving connective tissue

elasticity (Fig. 14). Mutations in the fibrillin-1 protein are responsible for Marfan

syndrome – a connective tissue disorder characterized by impaired structural

integrity in the skeleton, the eye, and the cardiovascular system. With this disease,

abnormal fibrillin protein is incorporated into microfibrils along with normal

fibrillin, inhibiting the formation of functional microfibrils.

Figure 13. Desmosine structure. Figure 14. Elastin fibers in relaxed and

stretched conformations.

53

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test: Explanation:

1. A 30 y.o. woman had been ill for a

year when she felt pain in the area of

joints for the first time, they got

swollen, and skin above them

became reddened. Provisional

diagnosis is rheumatoid arthritis.

One of the most probable causes of

this disease is a structure alteration

of a connective tissue protein:

A. Ovoalbumin

B. Collagen

C. Myosin

D. Troponin

E. Mucin

2 Increased fragility of vessels, enamel

and dentine destruction resulting

from scurvy are caused by disorder

of collagen maturation. What stage

of procollagen modification is

disturbed under this avitaminosis?

A. Hydroxylation of proline

B. Detaching of N-ended peptide

C. Formation of polypeptide chains

D. Glycosylation of hydroxylysine

residues

54

№ Test: Explanation:

E. Removal of C-ended peptide from

procollagen

3 The high levels of creatine kinase

(MB-isozyme) and lactate

dehydrogenase LDH1 activity were

revealed. Point out the most probable

pathology in the patient:

A. Hepatitis

В. Myocardium infarction

С. Osteoartritis

D. Pancreatitis

Е. Cholecystitis

4 Name the polysaccharide

represented in connective tissue:

A. Collagen

B. Elastin

C. Laminin

D. Hyaluronic acid

Е. Fibrillin

5 It is established that there is specific

system of energy supply in muscular

55

№ Test: Explanation:

cell. Point out this system:

A. Renin-angiotensinogen system

B. Creatine phosphate kinase system

C. Adenylate cyclase system

D. Translation system of a cell

Е. Palmitate synthetase complex

6 A patient with serious damage of

muscular tissue was admitted to the

trauma department. What

biochemical urine index will be

increased in this case?

A. Glucose

B. Common lipids

C. Uric acid

D. Creatinine

E. Mineral salts

7 A 46-year-old female patient has a

continuous history of progressive

muscular (Duchenne`s) dystrophy.

Which blood enzyme activity

changes will be of diagnostic value

in this case?

A. Lactate dehydrogenase

56

№ Test: Explanation:

B. Glutamate dehydrogenase

C. Adenylate cyclase

D. Pyruvate dehydrogenase

E. Creatine phosphokinase

8 A 53-year-old male patient is

diagnosed with Paget’s disease. The

concentration of oxyproline in daily

urine is sharply increased, which

primarily means intensified

disintegration of:

A. Albumin

B. Hemoglobin

C. Collagen

D. Fibrinogen

E. Keratin

9 It is established, that muscular

contraction depends on Са2+

concentration. Point out the protein

that is able to conjugate Са2+

ion

during muscular contraction:

A. Ceruloplasmin

B. С-reactive protein

C. Myosin

D. Ferritin

Е. Troponin

57

№ Test: Explanation:

10 The collagen triple helix structure is

not found in

A. Cytoplasm

B. Golgi apparatus

C. Lumen of endoplasmic reticulum

D. Intracellular vesicles

E. All positions are right

11 Choose the product of

guanidoacetate transmethylation

from following list:

A. Chlorine

B. Hydroxyproline

C. Creatinine

D. Creatine

E. Glutathione

12 Point out compounds required for

creatine synthesis:

A. Glycine, arginine and methionine

B. Glycine and methionine

C. Ornithine and glycine

D. Thymine and ornithine

E. Glycine, cysteine and glutamine

58

№ Test: Explanation:

13 Triple helix is seen in one compound

listed bellow. Choose it:

A. Collagen

B. Fibrinogen

C. Histones

D. Serum amylase

E. F-actin

14 Name the immediate source of

energy for muscular contraction.

A. Glycogen

B. ATP

C. Creatine phosphate

D. Glucose

E. Pyruvate

15 What does cardiac muscle prefer as

source of energy?

A. Fatty acids

B. Glucose

C. Ketone bodies

D. Glycogen

E. Fructose

16 Hydroxylation of proline to

59

№ Test: Explanation:

hydroxyproline in collagen synthesis

requires all except one. Point out it.

A. Pyridoxal phosphate

B. Ascorbic acid

C. O2

D. Specific hydroxylase

E. Iron ion

17 What is the product of guanidoacetic

acid transmethylation?

A. Acetylcholine

B. Choline

C. Creatinine

D. N-methyl nicotinamide

E. Creatine

18 Creatine is formed metabolically

using one compound listed below.

Choose it:

A. Tryptophan

B. Phenylalanine

C. Lysine

D. Valine

E. Leucine

60

№ Test: Explanation:

19 Three residues (Gly-X-Y-) are

repeated many times, and it is the

absolute requirement for formation

of the triple helix of collagen

molecule type 1. What amino acid

and its derivative mainly is

represented as letters X and Y?

A. Proline

B. Tryptophan

C. Lysine

D. Valine

E. Leucine

20 Name biochemical tests used for

diagnostics of muscular dystrophy

development:

A. Creatine content in the blood

plasma and urine

B. Creatinine content in the blood

plasma

C. Ctreatine phosphate kinase

activity in the blood plasma

D. Myofibril proteins content in

tissue homogenate obtained due to

biopsy method

E. All that is placed above

61

BIOCHEMISTRY OF NERVOUS TISSUE

(Krisanova N. V.)

INFORMATIONAL MATERIAL

INTRODUCTION

The nervous system, a network of neurons in active communication, reaches

its ultimate development in the 1.5 kg human brain. The human brain contains

~1011

neurons. Each of these neurons interconnects through synapses with

hundreds or thousands of other neurons. The number of connections is estimated to

be as many as 60,000 with each Purkinje cell of the human cerebellum. There may

be many more than 1014

synapses in the human brain.

Figure 1. Schematic picture of neurons

In addition to neurons, the brain contains 5–10 times as many glial cells of

several types. The neuroglia occupy 40% of the volume of brain and spinal cord in

62

the human. Some glial cells seem to bridge the ace between neurons and blood

carrying capillaries. Others synthesize myelin. Some are very irregular in shape.

Figure 1 shows all the compartments of neurons (two) which are involved in

transmission of nerve impulse:

The brain, which must function in a chemically stable environment, is protected by

a tough outer covering, the arachnoid membrane, and by the blood–brain

barrier and the blood–cerebrospinal barrier. Both of these barriers consist of

tight junctions. They are formed between the endothelial cells of the cerebral

capillaries and between the epithelial cells that surround the capillaries of the

choroid plexus. The choroid plexus consists of capillary beds around portions of

the fluid-filled ventricles deep in the interior of the brain. They serve as a kind of

―kidney‖ for the brain assisting in bringing nutrients in from the blood and helping

to keep dangerous compounds out.

NERVOUS TISSUE REGULATES AND CONTROLS BODY FUNCTIONS.

ITS MAIN FUNCTIONS ARE:

• Metabolic (promoted in neurons and neuroglia)

• Generation of nerve impulses

• Transmission of nerve impulse

• Remembering and keeping of information

• Creation of emotions and models of behavior

• Thinking

IT IS COMPOSED OF: NEURONS, THE NEUROGLIAL CELLS,

THE MICROGLIAL CELLS

Any type of a cell is important for all the functions described before, but

differences we have to underline, first of all for astrocytes:

they are involved in the physical structuring of the brain. Astrocytes get their

name because they are "star-shaped". They are the most abundant glial cells in the

brain that are closely associated with neuronal synapses. They regulate the

transmission of electrical impulses within the brain.

63

astrocytes contain glycogen and are capable of glycogenesis. Astrocytes can fuel

neurons with glucose during periods of high rate of glucose consumption and

glucose shortage.

they provide neurons with nutrients such as lactate.

astrocytes in tight junctions with basal lamina of the cerebral endothelial cells

play substantial role in maintaining the blood-brain barrier

astrocytes express plasma membrane transporters such as glutamate transporters

for several neurotransmitters, including Glutamate, ATP, and GABA

they are involved in regulation of ion concentration in the extracellular space

(potassium ions, mostly).

Microglia

Neuron

Astrocyte

Capillary

Ependymo-

cyte

Myelinated

axon

Oligodendro

cyte

Figure 2. Structural organization of nervous tissue

Oligodendrocytes are very important in the promotion of myelination of axons to

create myelin sheaths which reduce ion leakage and decrease the capacitance of the

cell membrane. Myelin also increases impulse speed, as saltatory propagation of

action potentials occurs at the nodes of Ranvier in between Schwann cells (in

peripheral nervous system) and oligodendrocytes (in CNS). Metabolic activity of

them is associated with creation of myelin components.

64

Ependymocytes. Lining the cerebrospinal fluid (CSF)-filled ventricles, the

ependymal cells play an important role in the production of substances for CSF and

regulation of CSF composition.

Microglial cells. In the case where infectious agents are directly introduced to the

brain or cross the blood–brain barrier, microglial cells must react quickly to

decrease inflammation and destroy the infectious agents before they damage the

sensitive neural tissue. Due to the unavailability of antibodies from the rest of the

body (few antibodies are small enough to cross the blood–brain barrier), microglia

must be able to recognize foreign bodies, swallow them, and act as antigen-

presenting cells activating T-cells.

CHEMICAL COMPOSITION OF BRAIN TISSUE

PROTEINS

Account for about 40% of the dry weight of the brain

Over 100 soluble fractions have been isolated from the brain tissue

The grey matter is more rich in water-soluble proteins than the white

matter

There are both simple and conjugated proteins

SIMPLE PROTEINS:

Neuroalbumins (90% of soluble proteins)

Neuroglobulins (5%)

Histones

Neuroscleroproteins: collagens, elastins, stromatins (5%)

CONJUGATED PROTEINS

Lipoproteins

Proteo-lipid complexes (in myelin substance, mainly)

Phosphoproteins (2%)

Nucleoproteins

Glycoproteins

Chromoproteins

65

SPECIFIC PROTEINS

S-100 protein (Moore protein): Ca2+-binding protein, contains a large number

of Glu and Asp , occurs chiefly in the neuroglia – 90%, in the neurons – 10% . Its

concentration rises in the brain of animal subjected to training.

14-3-2 protein: Is acidic too, occurs chiefly in the neurons, functions of it are

unclear.

Glial Fibrous Acidic Protein (GFAP): placed mainly in astrocytes, specific for

CNS, the biggest content is observed in differentiation of astrocytes.

N-CAM (neural cells adhesion molecule; NG-CAM (neuralglial cells adhesion

molecule); MAG (myelin-associated glycoprotein) : neurospecific glycoproteins

participated in formation of myelin substance, in processes of cells adhesion, etc.

ENZYMES

Lactate dehydrogease

C- izoform of Aldolase

BB- izoform of Creatine phospho kinase (CPK)

Hexokinase

Cholinesterase

Monoaminoxidase

Acidic phosphatase

Gamma-izoform of enolase

MAIN LIPIDS

Phosphoglycerides

Cholesterol

Sphingomyelins

Cerebrosides

Gangliosides

66

CHEMICAL COMPOSITION OF MYELIN SUBSTANCE

Myelin is a dielectric material that forms a layer, the myelin sheath, usually around

only the axon of a neuron. Myelin is an outgrowth of a type of glial cell. Its

composition is:

~ 40 % water

the dry mass of myelin is ~ 70-85 % of lipids (sphingomyelins,

cholesterol,cerebrosides); the primary lipid of myelin is galactocerebroside

the dry mass of myelin is 15-30 % proteins

Some of the proteins make up myelin are:

Myelyn basic protein (MBP)

Myelin oligodendrocyte glycoprotein (MOG)

Proteolipid protein (PLP) - Folch complex

ENERGY REQUIREMENTS OF BRAIN TISSUE ARE PROMOTED BY:

Aerobic oxidation of glucose (90%) up to СО2 and Н2О

Non-oxidative phase of HMP shunt using other monosaccharides, and

Glycolysis

Keto acids and products of neurotransmitters utilization across catabolic

pathways for them

Lactate utilized by mitochondrial Lactate dehydrogenase to form pyruvate (in

glial cells and neurons)

Ketone bodies destruction

Fatty acids oxidation in glial cells

Branched chain amino acids destruction

It should be noted that Glucose is transported across the cell membrane by

specific saturable transport system, which includes two types of glucose

transporters: 1) sodium dependent glucose transporters (SGLTs) which transport

glucose against its concentration gradient and 2) sodium independent glucose

transporters (GLUTs), which transport glucose by facilitative diffusion in its

concentration gradient. In the brain, both types of transporters are present with

67

different function, affinity, capacity, and tissue distribution. GLUT1 occurs in

brain in two isoforms. The more glycosylated GLUT1 is produced in brain

microvasculature and ensures glucose transport across the blood brain barrier. The

less glycosylated form is localized in astrocytic end-feet and cell bodies and is not

present in axons, neuronal synapses or microglia. Glucose transported to astrocytes

by GLUT1 is metabolized to lactate serving to neurons as energy source. GLUT2

is present in hypothalamic neurons and serves as a glucose sensor in regulation of

food intake. In neurons of the hippocampus, GLUT2 is supposed to regulate

synaptic activity and neurotransmitter release. GLUT3 is the most abundant

glucose transporter in the brain having five times higher transport capacity than

GLUT1. It is present mostly in axons and dendrites. Its density and distribution

correlate well with the local cerebral glucose demands. GLUT5 is predominantly

fructose transporter. In brain, GLUT5 is the only hexose transporter in microglia,

whose regulation is not yet clear. It is not present in neurons. GLUT4 and GLUT8

are insulin-regulated glucose transporters in neuronal cell bodies in the cortex and

cerebellum, but mainly in the hippocampus, where they maintain hippocampus-

dependent cognitive functions. Insulin translocates GLUT4 from cytosol to plasma

membrane to transport glucose into cells, and GLUT8 from cytosol to rough

endoplasmic reticulum to recover redundant glucose to cytosol after protein

glycosylation.

SYNTHESIS OF ATP IN BRAIN TISSUE IS DUE TO:

• Oxidative phosphorylation (95%)

• Substrate phosphorylation (ВВ-isoform of CPK, pyruvate kinase,

phosphoglycerate kinase) (5%)

It should be noted that content of glycogen is too small in brain tissue, and its

utilization to give energy as the result usually is discussed under special states

beginning for neurons (like hypoxia) according the way shown in figure 3. But in

glial cells its utilization is up to lactate.

68

Production of ATPProduction of ATP

Acetyl-CoA

Lactate

(glial)

Glycolysis

Glucose

Pyruvate

Branched Chain AA

Ile, Leu, Val

Krebscycle

oxidative

phosphorylation

Glycogen

Glycogenolysis

Lactate

No O2

Ketone bodies

Energy suppliersduring prolonged starvation

Constantly required

Small amount

Figure 3. Main metabolic pathways to promote energy requirements of brain tissue.

MAIN NEUROTRANSMITTERS

Acetylcholine. It is produced due to the action of acetyl-CoA Choline

transferase from acetyl-CoA and specific alcohol choline. Its destruction is

made by acetylcholine esterase to form free acetic acid and choline.

Amino acids: Glutamate, Aspartate, Glycine, D-Serine, dihydroxy phenyl

alanine (DOPA)

Biogenic amines: dopamine, norepinephrine, epinephrine, histamine,

serotonin, gamma-amiobutyric acid (GABA) . They are produced mostly

due to alpha-decarboxylation from amino acids.

Purine derivatives: ATP, ADP, AMP, adenosine

69

Structures and functions of some of them is placed in figure 4.

Figure 4. Structure and function of neurotransmitters

Feature of

action

Main function

excitation suppression

neuromediator

Glutamate

Acetylcholine

GABA

Glycine

neuromodulator

Nor-epinephrine

Serotonin

Adenosine

Dopamine

Amino acids may be precursors for formation of neurotransmitters. α-

Decarboxylation of histidine gives histamine:

HN N

CH2

CH

NH2

COOH

HN N

CH2

CH2

NH2

Decarboxylase

Histidine Histamine

CO2

Biological role of histamine

70

Histamine is vasodilator. This fact markedly differentiates its action from

other biogenic amines on blood vessels.

Histamine facilitates the afflux of leukocytes during the inflammation of

tissue and activates thereby the defence function of the organism.

Histamine stimulates the gastric juice secretion.

Histamine is directly involved in the effects of sensitization and

desensitization.

In the figure 5 it is shown how other neurotransmitters are produced from

corresponded amino acids.

Figure 5. Synthesis of some neurotransmitters: PLP – pyridoxal phosphate;

deCO2ase - decarboxylase.

GABA. Formation of this substance in CNS (reaction is shown below) is very

important for braking of super-excitation. Synthetic derivatives of this substance

are used in therapy of epilepsy.

HOOCCH2

CH2

CH

NH2

COOHHOOC

CH2

CH2

CH2

GlutamateGABA

Glutamate -Decarboxylase

GABA - -aminobutyric acid

vit B6 NH2

CO2

In figure 6 amino acids phenyl alanine and tyrosine may be discussed as

precursors for dihydroxy phenyl alanine (DOPA), dopamine, norepinephrine and

71

epinephrine. All enzymes for hydroxylation (*, figure 5) have special prosthetic

group: Tetrahydrobiopterin (THBP). This cofactor is oxidized to dihydrobiopterin

during the hydroxylation of corresponding substrate and must be regenerated by

another enzyme, dihydrobiopterin reductase, which uses NADPH as donor of

protons and electrons.

H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

OH

H2N CH C

CH2

OH

O

OH

OH

NH2

CH2

H2CHO

HO

Phenylalanine

TyrosineDihydroxyphenylalanine

Dopamine

Phenylalanine

4-monooxygenase*Tyrosine

3-monooxygenase*

Decarboxylase

CO2

1 / 2 O2 1 / 2 O2

NH2

CH2CHHO

HO

NH

CH2CHHO

HO

1 / 2 O2

Hydroxylase

S-Adenosyl methionine

S-Adenosyl homocysteine

H3C

Norepinephrine

Epinephrine

Methyltransferase

OH

OH

Figure 6. A production ways for DOPA, dopamine, norepinephrine and

epinephrine.

Decarboxylation of amino acids is catalyzed by amino acid alpha-

decarboxylases (Pyridoxal phosphate – the non-protein part). This enzyme subclass

is abundant in the adrenal glands and CNS.

Directed alpha-decarboxylation of tryptophan gives tryptamine, and 5-hydroxy

tryptophan decarboxylation is finished by formation of serotonin (figure 6).

Serotonin takes part in:

1) exhibition of vasoconstrictive action;

2) regulation of arterial pressure, body temperature, respiration, renal filtration;

72

3) serotonin may be (some scientists proposed) a causative factor in the

development of allergy, dumping syndrome, carcinoid syndrome, hemorrhagic

diatheses,

4) stimulation of smooth muscle contraction.

Serotonin and Tryptamine are considered as neuromedulators of CNS.

H2N CH C

CH2

OH

O

HN

H2N CH C

CH2

OH

O

HN

OH

H2N CH2

CH2

HN

OH

1/2 O2

Hydroxylase

Tryptophan

5-Hydroxytryptophan

Serotonin

H2N CH2

CH2

HN

Tryptamine

CO2

Decarboxylase

CO2

Decarboxylase

Figure 7. Reactions to form neuromodulators Tryptamine and Serotonin.

MAIN WAYS TO UTILIZE NEUROTRANSMITTERS

1. Diffusion: the neurotransmitter drifts away, out of the synaptic cleft

2. Enzymatic degradation (deactivation): a specific enzyme changes the

structure of the neurotransmitter so it is not recognized by the receptor

3. Glial cells: astrocytes remove neurotransmitters from the synaptic cleft

4. Reuptake: the whole neurotransmitter molecule is taken back into the axon

terminal that released it.

73

For acetylcholine p.p.2, 4 may be considered, its destruction is due to acetylcholine

esterase to form free acidic acid and free choline.

As example in the figure 8 formation of gamma-amino butyric acid (GABA)

in GABA-neuron, its utilization in GABA-shunt across succinic acid and alpha-

ketoglutarate to form Glutamate, Glutamine in glial cell, and the use of Glutamine

to form Glutamate as neurotransmitter in Glutamate neuron are shown.

Figure 8. Metabolism of Glutamine, Glutamate and GABA in nervous tissue cells.

THE UTILIZATION OF NEUROTRANSMITTERS BY

MONOAMINOOXIDASE (MAO)

MAO is in most cell types in the body

There are two types of MAO: A and B

74

Both are found in neurons and astroglia and bound to the outer membrane of

mitochondria

Serotonin, nor-epinephrine, epinephrine are mainly broken down by MAO-A

Both forms break down dopamine equally.

The reaction for MAO is oxidative deamination type, it is shown below:

R CH2

NH2

H2O NH3

R C

O

H

R C

O

OHFADÍ 2

Î 2

Í 2Î 2 1/2 Î 2 + Í 2Î

FAD+

Amino cid metabolism and neurotransmitters utilization is in close relation with

toxic ammonia formation, its utilization pathways in nervous tissue are shown in

figure 9.

AmmoniaAmmonia utilizationutilization waysways

inin thethe nervousnervous tissuetissue

NHNH33++αα--ketoglutarate+NADPHketoglutarate+NADPH

GluGlu + NADP+ NADP++

NHNH33 + + GluGlu + ATP + ATP

GlnGln + ADP + Pi+ ADP + Pi

Asp + Asp + GlnGln + ATP + ATP

AsnAsn + + GluGlu + AMP + + AMP + PPiiPPii

GDHGDH

GlnGln synthasesynthase

AsnAsn synthasesynthase

Figure 9. Reductive amination of alpha-ketoglutarate, synthesis of glutamine (Gln)

and asparagine (Asn).

First two reactions (fig.9) are the most important for nervous tissue to utilize

ammonia: reductive amination of alpha-ketoglutarate catalyzed by glutamate

dehydrogenese (GDH) and synthesis of glutamine (Gln) from glutamic acid (Glu)

75

with the use of ATP due to glutamine synthase. Toxicity of ammonia partially may

be explained due to decrease of alpha-ketoglutarate use in Citric acid cycle as

energy source for neurons because of its increased content to utilize ammonia in

GDH reaction.

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test tasks: Explanations:

1. Monoamine oxidase inhibitors are

widely used as

psychopharmacological drugs. They

change the level of nearly all

neurotransmitters in synapses, with

the following neurotransmitter being

the exception:

A. Acetylcholine

B. Serotonin

C. Dopamine

D. Noradrenalin

E. Adrenalin

2. Decarboxylation of glutamate

induces production of gamma-

aminobutyric acid (GABA)

neurotransmitter. After breakdown,

GABA is converted into a

metabolite of the Citric acid cycle,

that is:

A. Fumarate

B. Succinate

C. Oxaloacetate

76

№ Test tasks: Explanations:

D. Malate

E. Citric acid

3. An unconscious patient was taken by

ambulance to the hospital. On

objective examination the patient

was found to have no reflexes,

periodical convulsions, irregular

breathing. After laboratory

examination the patient was

diagnosed with hepatic coma.

Disorders of the central nervous

system develop due to the

accumulation of the following

metabolite:

A. Urea

B. Histamine

C. Glutamine

D. Ammonia

E. Bilirubin

4. Disruption of nerve fiber

myelinogenesis causes neurological

disorders and mental retardation.

These symptoms are typical for

hereditary and acquired alterations in

the metabolism of:

A. Phosphatidic acid

B. Cholesterol

C. Sphingolipids

77

№ Test tasks: Explanations:

D. Neutral fats

E. Higher fatty acids

5. Depressions and emotional insanities

result from the deficit of

noradrenalin, serotonin and other

biogenic amines in the brain. Their

concentration in the synapses can be

increased by means of the

antidepressants that inhibit the

following enzyme:

A. Phenylalanine-4-monooxygenase

B. Monoamino oxidase

C. D-amino-acid oxidase

D. L-amino-acid oxidase

E. Diamine oxidase

6. It is known that the monoamino

oxidase (MAO) enzyme plays an

important part in the metabolism of

catecholamine neurotransmitters. In

what way this enzyme inactivates

these neurotransmitters

(norepinephrine, epinephrine,

dopamine)?

A. Oxidative deamination

B. Carboxylation

C. Addition of an amino group

D. Removal of methyl group

E. Hydrolysis

78

№ Test tasks: Explanations:

7. An inhibitory mediator is formed by

the decarboxylation of glutamate in

the CNS. Name it:

A. Asparagine

B. Serotonine

C. Histamine

D. GABA

E. Glutathione

8. A dizziness, memory impairment

and periodical convulsions are

observed in patient. It was revealed

that these changes were caused by a

deficiency of a product of glutamic

acid decarboxylation. Name this

product:

A. TDP

B. GABA

C. THFA

D. Pyridoxal phosphate

E. ATP

9. During hypersensitivity test a patient

got subcutaneous injection of an

antigen which caused reddening of

skin, edema, pain as a result of

histamine action .This biogenic

79

№ Test tasks: Explanations:

amine generated as a from histidine

amino acid across:

A. Methylation

B. Isomerization

C. Phosphorylation

D. Decarboxylation

E. Deamination

10. A patient diagnosed with carcinoid

of bowels was admitted to the

hospital. Analysis revealed high

production of serotonin. It is known

that this substance is formed of

tryptophan amino acid. What

biochemical mechanism underlies

this process?

A. Decarboxylation

B. Microsomal oxidation

C. Transamination

D. Desamination

E. Formation of paired compounds

11. Cerebral trauma caused the increase

of ammonia formation. What amino

acid takes part in removal of

ammonia from cerebral tissue?

A. Tryptophan

B. Lysine

C. Glutamic acid

D. Valine

80

№ Test tasks: Explanations:

E. Туrosine

12. Glutamate decarboxylation results in

the formation of inhibitory

transmitter in CNS. Name it:

A. Glutathione

B. Gamma amino butyric acid

C. Serotonin

D. Histamine

E. Asparagine

13. Ammonia is a very toxic substance,

especially for the nervous system.

What substance takes the most

active part in ammonia

detoxification in the brain tissue?

A. Lysine

B. Glutamic acid

C. Histidine

D. Proline

E. Alanine

14. Neurotransmitter serotonin is

derived from one amino acid.

Choose it:

A. Phenylalanine

B. Serine

C. Tryptophan

D. Cysteine

E. Proline

15. In the brain ammonia is converted to

81

№ Test tasks: Explanations:

product from following list. Point

out it:

A. Aspartate

B. Glutamine

C. Alanine

D. Histidine

E. Urea

16. The brain contains relatively high

amounts of all compounds from the

following list except one. Point out

it:

A. Glutamine

B. N-Acetylaspartate

C. Gamma-aminobutyric acid

(GABA)

D. Glycogen

E. Proteolipid

17. Point out the main pathways of

catabolism in brain:

A. Glycolysis and Citric Acid Cycle

B. Glycogenolysis and Glycogenesis

C. Glycogenolysis and Citric Acid

Cycle

D. Embden-Meyerhof pathway and

HMP shunt

E. Oxidation of fatty acids and

ketogenesis

18. This enzyme may be promoter of

82

№ Test tasks: Explanations:

Glutamate utilization in direct

reaction, and in opposite reaction it

can utilize toxic ammonia in nervous

tissue. Name it:

A. Glutaminase

B. Glutamine oxidase

C. Glutamate dehydrogenase

D. Glutamate decarboxylase

E. Glutamate reductase

19. Tetrahydrobiopterin (THBP) is in

need for hydroxylation reactions of

some amino acids to produce

neurotransmitters in nervous tissue.

Name these neurotransmitters:

A. Dopamine, Serotonin

B. Glycine, Glutamate

C. GABA, Glycine

D. Histamine, Tryptamine

GABA, Histamine

20. Insulin-regulated glucose trans-

porters are found in neuronal cell

bodies in the brain cortex of humans.

Name them:

A. GLUT4, GLUT8

B. GLUT1

C. GLUT2

D. GLUT1, GLUT2

E. GLUT5

83

BIOCHEMICAL FUNCTIONS OF THE LIVER

AT HEALTHY AND DISEASED PEOPLE

(Rudko N.P., Ivanchenko D. G.)

INFORMATIONAL MATERIAL

The liver is the largest parenchymal organs. It is a vital organ that supports

nearly every other organ in the body in some facet. Without a healthy liver, a

person cannot survive. Liver performs multiple critical functions:

1. Maintenance of blood glucose level.

2. Regulation of blood lipid levels:

- fatty acid synthesis, lipoprotein formation (VLDL, HDL) and transformation

(ChM).

3. Synthesis of ketone bodies (for using by other tissues).

4. Synthesis of plasma proteins:

- albumins (100%), α-globulins (85%), β-globulins (50%) (there are

components of blood clotting system and other plasma enzymes among

them).

5. Exocrine secretion:

- bile acid synthesis and bile secretion (help in emulsification of fats in the

intestine).

6. Excretion (blood filtering):

- RBCs phagocytosed by Kupffer cells, waste product bilirubin is conjugated

by hepatocytes and secreted in bile.

7. Biotransformation (Detoxification):

- ammonia to urea;

- xenobiotics to more soluble and easily excretable compounds;

- hormones and biogenic amines to inactive forms.

84

The role of the liver in the metabolism of carbohydrates

Carbohydrates are very important nutrients for humans. 200g/day is total intake

as a requirement. Main metabolic form of carbohydrate is glucose. Only 10g of

glucose present in blood plasma and 300g in liver.

Blood glucose must be replenished constantly (most of glucose (80%)

consumed daily is utilized by RBCs and brain). Hypoglycemia and coma may

result if concentration of glucose in the blood is less 45mg/dL.

The liver plays a key role in maintaining the physiological concentration of

glucose in the blood.

The total amount of glucose entering the liver after absorption from intestine

is distributed in the following way: 60% - for oxidative process (NADH, NADPH

generation), 30% - for fatty acid synthesis, 10-15% of that is used for the synthesis

of glycogen.

Hyperglycemia state

Excessive dietary intake of glucose increases in the intensity of all metabolic

pathways of its transformation in hepatocytes. So, most glucose is involved into

aerobic glycolysis: glucose →…→ 2 pyruvates. Further splitting of pyruvates

requires a large amount of CoA, which is also used for the oxidation of fatty acids.

As a result mobilization of lipids from fat depots and oxidation of higher fatty

acids (HFA) in the liver is decreased.

Glycogenesis - conversion of glucose to glycogen for storage (the major

pathway in hepatocytes at hyperglycemia). High activity of glycogenesis in the

liver from blood glucose is due to:

- glucose transporter type GLUT-2. The transporter provides glucose permeability

of liver cells if high concentrations is in portal blood. Liver cell do not require

insulin for amino acid or glucose uptake;

- glucokinase. It is glucose specific enzyme in liver (but in pancreas, gut, brain

too). It catalyzes glucose conversion to glucose 6-phosphate. It acts as a glucose

sensor, triggering shifts in metabolism or cell function in response to rising or

85

falling levels of glucose. Glucokinase has a low affinity for glucose (high Km);

it is not inhibited by glucose 6-phosphate; and it is inducible by insulin.

Hypoglycemia state

Glycogenolysis - degradation of liver glycogen stores to glucose. At physiological

hypoglycemia glycogen breakdown by the action of glycogen phosphorylase is

activated in the liver. The resulting glucose-6-phosphate can be spent in three

areas:

1) cleaved by glucose-6-phosphatase to form glucose and phosphoric acid;

2) the pentose phosphate pathway;

3). by way of glycolysis to form pyruvic acid and lactate.

A product formed from glucose-6-phosphate mainly is glucose. This leads to

the exit of free glucose from liver to bloodstream.

Hepatic glycogen not sufficient during 12 hr fast. During sleep there is shift

from glycogenolysis to de novo synthesis of glucose in liver - gluconeogenesis. It

is essential during fasting or starvation.

Substrates for gluconeogenesis are:

- glucogenic amino acids mainly coming from muscle proteins (starvation);

- lactate (from RBC & muscle after intensive physical activity) and pyruvate;

- glycerol from backbone of the fats and glycerophospholipids. Aside from

propionyl-CoA produced by oxidation of rare, odd-chain fatty acids, glycerol is

the only portion of the fat molecule that can be made directly into glucose by

animal fat. Acetyl CoA (formed from even-chain fatty acids) cannot be termed

glucogenic, since the conversion back to pyruvate is not possible due to

irreversible nature of the reaction. In plants the glyoxylate cycle produces four-

carbon dicarboxylic acids that can enter gluconeogenesis. The existence of

glyoxylate cycles in humans has not been established, and it is widely held that

fatty acids cannot be converted to glucose in humans directly. (These substrates

are usual for fasting).

86

In humans the main gluconeogenic precursors are lactate, glycerol (which is

a part of the triacylglycerol molecule), alanine and glutamine. Altogether, they

account for over 90% of the overall gluconeogenesis.

Even-chain fatty acids can’t be converted into glucose in animals. Only odd-

chain fatty acids can be oxidized to yield propionyl-CoA, a precursor for succinyl-

CoA, which can be converted to pyruvate and enter into gluconeogenesis.

The role of the liver in the metabolism of lipids

Most of the nutrients entering the liver follow metabolic pathways to lipids

rather than glycogen, ex: glucose----> acetyl CoA---> triacylglycerols, cholesterol.

Triacylglycerols can be stored in the liver or released into the blood plasma to

travel to adipose tissue. Lipids travel in the bloodstream in the form of lipoproteins

(complexes of triacylglycerols, phospholipids, cholesterol and proteins);

LIPOPROTEINS:

- Very Low Density Lipoproteins (VLDL): liver forms and releases lipids in the

form of VLDL. They exist average about 3 hours in the circulatory system;

they deliver their main elements triacylglycerols to cells (first of all they are

adipocytes, skeletal and cardiac muscles). They lost triacylglycerols then

become Low Density Lipoproteins.

- Low Density Lipoproteins (LDL): cholesterol that they contain may be taken

up by a variety of cells including those of arterial walls; ultimately they return

to the liver.

- Chylomicrones (ChM): blood rich with ChM following a meal; they are

formed and released from intestine and have a relatively short life span in the

blood stream - about 8 minutes. Their remnant forms are trapped by

hepatocytes for subsequent utilization.

- High Density Lipoproteins (HDL): They are synthesized primarily in the liver;

they facilitate the uptake of lipids and activation of lipoprotein lipase; HDL

particles collect liberated cholesterol in the blood and carry it back to the liver

for excretion as bile acids or recycled as bile.

87

Liver taking part in the transformation of the lipoproteins carries cholesterol

metabolism regulation.

The liver plays a key role in the regulation of cholesterol metabolism. The

initial substrate for cholesterol synthesis is acetyl-CoA. As acetyl-CoA is formed

by the decomposition of glucose and fatty acids excess meal containing

carbohydrates and fats stimulates the synthesis of cholesterol (due to increased

availability of the substrate for the synthesis of cholesterol - acetyl-CoA).

FORMATION OF BILE

Bile consists of watery mixture of organic and inorganic compounds.

Lecithin and bile salts are quantitatively the most important organic components of

bile. Bile can either pass directly from the liver where it is formed into the

duodenum through the common bile duct, or the stored in the gallbladder.

Daily bile secretion is 0.6 L, pH 6.9–7.7

Bile composition:

1) bile acids

- are synthesized from cholesterol (0.5 gram/day);

- are combined with amino acids like glycine or taurine before secretion;

- form a water soluble form called bile salts

2) cholesterol and lecithin (phospholipid)

- normally the cholesterol secreted in bile is kept in solution by the detergent

action of lecithin;

- excessive secretion of cholesterol by the liver or over concentration of bile

in the gall bladder can cause precipitation of cholesterol from solution and

formation of aggregates called gallstones

3) bilirubindiglucuronides (bile pigment)

4) detoxified chemicals

5) NaHCO3

88

Components Function or substrate

Water Solvent

HCO3- Neutralizes gastric juice

Bile salts Facilitate lipid digestion

Phospholipids Facilitate lipid digestion

Bile pigments Waste product

Cholesterol Waste product

SYNTHESIS OF KETONE BODIES

The ketone bodies are produced from acetyl CoA which comes from three

sources: glucose, fatty acids and amino acids. These ketone bodies cannot be

utilized by the liver because hepatocytes lack β-ketoacyl-CoA transferase

(thiophorase) which converts acetoacetate to acetoacetyl-CoA and hence these

ketone bodies are supplied to the peripheral tissues for oxidation. Brain can use

ketone bodies if glucose supplies fall: prolonged starvation (>1 week of fasting),

glycogen and glucogenic substrates are exhausted. Ketogenesis can provide energy

to body in prolonged energy needs. The ketone bodies are synthesized in the liver

even under normal conditions.

In normal metabolism, some ketone bodies are continuously produced and

broken down in energy production. The normal blood level of ketone bodies

seldom exceeds 3 mg/100 mL of blood. In diabetes, however, the liver produces

large quantities of ketone bodies, releasing them into the blood-stream for delivery

to other tissues. This causes a substantial increase in the level of ketone bodies in

the blood of untreated diabetics.

The role of the liver in the metabolism of proteins

The most critical aspects of protein metabolism that occur in the liver are:

1) The liver is central to the metabolism of amino acids, as it actively proceed

their chemical modification processes:

- deamination and transamination of amino acids, followed by

conversion of the non-nitrogenous part of those molecules to glucose or

89

lipids. Several of the enzymes used in these pathways (for example, alanine

and aspartate aminotransferases) are commonly assayed in serum to assess

liver damage;

- removal of ammonia from the body by synthesis of urea. Ammonia is

very toxic and if not rapidly and efficiently removed from the circulation,

will result in central nervous system disease.

- synthesis of non-essential amino acids.

2) Hepatocytes use the amino acids coming from the digestive tract for

synthesis of own proteins, but most of them used for synthesis of plasma

proteins. In the liver are synthesized protein-transporters for: lipids

(apolipoproteins, albumin), steroid, thyroid hormones, iones (Cu2+

, Fe2+

,

Ca2+

); components for blood clotting system: prothrombin, fibrinogen,

proconvertin, proaccelerin, Stuart-Prower factor etc.

Specific enzymes for the liver are: arginase, ornithine carbamoyltransferase,

histidase, sorbitol dehydrogenase, urocaninase, α-antitrypsin.

The role of the liver in pigment metabolism

Bilirubin must be conjugated to a water-soluble substance. This increase its

water solubility, decreases its lipid solubility and makes easier its excretion.

Conjugation is accomplished by attaching two molecules of glucuronic acid to it in

two step process.

The enzyme is UDP-glucuronyl transferase. The substrates are: bilirubin (or

bilirubin monoglucuronide), UDP-glucuronic acid. Glucuronide synthesis is the

rate-determining step in hepatic bilirubin metabolism. Drugs such as

phenobarbital, for example, can induce both conjugate formation and the transport

process.

The bilirubin glucuronides are then excreted by active transport into the bile,

where they form what are known as the bile pigments.

CAUSES OF INCREASED BILIRUBIN

1. Abnormal metabolism of bilirubin:

90

- overproduction of bilirubin due to hemolysis. Ex: incompatible blood

transfusion

2. Hepatic cell (liver) disease:

- impaired uptake of bilirubin by the liver cells. Ex: hepatitis and cirrhosis

3. Obstruction to outflow of bilirubin:

- liver damage (scarred bile ducts)

- gallstones, inflammation of bile ducts

Levels of bilirubin in blood are normally below 1.0 mg% (17 µmol/L) and

levels over 2.5-3mg% (34-51µmol/L) typically results in JAUNDICE.

HEPATITIS: - inflammation of the liver, may be caused by viral or toxic

factors. Viral hepatitis produced by 3 known viral agents known as hepatitis A, B

and C

CIRRHOSIS: condition in which necrosis of the liver leads to a proliferation

of fibrous connective tissue (fibrosis). It can be caused by alcoholism, hepatitis

infection, obstructed bile flow and back pressure from elevated hepatic vein

pressure. Blood ammonia levels are frequently elevated since normally ammonia is

converted to urea by the liver.

The role of the liver in the harmful substance detoxication

Alcohol is metabolized usually by liver the rate of about 50 ml of spirits (a

typical drink-size serving of beer, wine, or spirits) every 90 minutes. It takes

approximately 90 minutes for a healthy liver to metabolize a 30 ml of pure ethanol.

But diseased liver with conditions such as hepatitis, cirrhosis, cancer, and

gallbladder disease are likely to result in a slower rate of metabolism.

Ethanol's acute effects are due largely to its nature as a central nervous

system depressant, and are dependent on blood alcohol concentrations. As drinking

increases, people become sleepy, or fall into a stupor. After a very high level of

consumption, the respiratory system becomes depressed and the person will stop

breathing. Comatose patients may aspirate their vomit (resulting in vomitus in the

lungs, which may cause "drowning" and later pneumonia if survived). In addition

to respiratory failure and accidents caused by effects on the central nervous system,

91

alcohol causes significant metabolic derangements. Hypoglycemia occurs due to

ethanol's inhibition of gluconeogenesis, especially in children, and may cause

lactic acidosis, ketoacidosis, and acute renal failure. Metabolic acidosis is

compounded by respiratory failure. Patients may also present with hypothermia.

What is the biochemical basis of these health problems? Ethanol cannot be

excreted and must be metabolized, primarily by the liver. This metabolism occurs

by two pathways. The first pathway comprises two steps. The first step, catalyzed

by the enzyme alcohol dehydrogenase (Alcohol DH), takes place in the cytoplasm:

Alcohol DH

CH3-CH2-OH + NAD+ −−−−→ CH3CHO + NADH + H+ Ethanol Acetaldehyde

The second step, catalyzed by aldehyde dehydrogenase (Aldehyde DH), takes

place in mitochondria:

Aldehyde DH

CH3CHO + NAD+ + H2O −−−−→ CH3-COO- + NADH + H+

Acetaldehyde Acetate

Desulfiram is widely used in medical practice to prevent alcoholism, it

inhibits aldehyde dehydrogenase. Increased level of acetaldehyde causes aversion

to alcohol.

1. High level of ethanol consumption leads to an accumulation of NADH.

- This high concentration of NADH inhibits gluconeogenesis by preventing

the oxidation of lactate to pyruvate. In fact, the high concentration of NADH

will cause the reverse reaction to predominate, and lactate will accumulate.

The consequences may be hypoglycemia and lactic acidosis.

- NADH glut also inhibits fatty acid oxidation. The metabolic purpose of

fatty acid oxidation is to generate NADH for energy (ATP) generation by

oxidative phosphorylation, but an alcohol consumer's NADH needs are met

by ethanol metabolism. In fact, the excess NADH signals that conditions are

right for fatty acid synthesis. Hence, triacylglycerols are accumulated in the

liver, leading to a condition known as ―fatty liver.‖

92

2. The second pathway for ethanol metabolism is called the ethanol inducible

microsomal ethanol-oxidizing system (MEOS). This is cytochrome P450-

dependent pathway.

- It generates acetaldehyde and subsequently acetate herewith oxidizing

biosynthetic reducing power NADPH to NADP+. Moreover, because the

system consumes NADPH, the antioxidant glutathione cannot be

regenerated, exacerbating the oxidative stress.

-MEOS uses oxygen, this pathway generates free radicals that damage

tissues.

Under these conditions demands in NADPH as fuel for functioning of

antioxidant system like glutathione dependent increases. But NADPH

consumed by MEOS and it causes its deficiency.

What are the effects of the other metabolites of ethanol? Liver mitochondria

can convert acetate into acetyl CoA in a reaction requiring ATP. The enzyme is the

thiokinase that normally activates short-chain fatty acids.

Thiokinase

CH3-COO-

+ CoA + ATP −−−−→ CH3-CO˜CoA + ADP + Pi Acetate Acetyl CoA

However, further processing of the acetyl CoA by Krebs cycle is blocked,

because accumulated NADH inhibits two important regulatory enzymes: isocitrate

dehydrogenase and α-ketoglutarate dehydrogenase. This cause accumulation of

acetyl CoA and has several consequences. First, ketone bodies will form and be

released into the blood, exacerbating the acidic condition already resulting from

the high lactate concentration. The processing of the acetate in the liver becomes

inefficient, leading to a buildup of acetaldehyde. This very reactive compound

forms covalent bonds with many important functional groups in proteins,

impairing protein function. If ethanol is consistently consumed at high levels, the

acetaldehyde can significantly damage the liver, eventually leading to cell death.

93

Thus liver damage from excessive ethanol consumption occurs in three

stages. The first stage is the aforementioned development of fatty liver. In the

second stage—alcoholic hepatitis—groups of cells die and inflammation results.

This stage can itself be fatal. In stage three – cirrhosis - fibrous structure and scar

tissue are produced around the dead cells. Cirrhosis impairs many of the liver's

biochemical functions. The cirrhotic liver is unable to convert ammonia into urea,

and blood levels of ammonia rise. Ammonia is toxic to the nervous system and can

cause coma and death. Cirrhosis of the liver arises in about 25% of alcoholics, and

about 75% of all cases of liver cirrhosis are the result of alcoholism. Viral hepatitis

is a nonalcoholic cause of liver cirrhosis.

Liver inactivates many flowing substances. This process can be done in

different ways: by oxidation, destruction and connection with other substances.

One of the main mechanisms of detoxification - the so-called protective synthesis,

i.e. the transformation of toxic metabolic products in more complex non-toxic

complexes, which are excreted from the body. By type of such synthesis is the

forms hippuric acid by combining benzoic acid with glycine. In normal conditions

are formed and excreted in the urine insignificant amount of hippuric acid (0,1-1,0

g / day), its quantity increases by eating fruit with peel containing benthological

sodium.

The synthesis of hippuric acid is a physiological basis of the sample with the

load benzalkonium sodium proposed by the Quick and modified A. Ya. by Pytel in

1945. Clinical-anatomical mapping by L. A. Vinnik (1956) showed the existence

of parallelism between the degree of anatomical lesion of the liver cells and

reduced synthesis of hippuric acid in the formulation of samples of Quick - Pytel.

A test for assessing the detoxification function of liver is Quick`s test:

measurement of hippuric acid in the urine.

Hippuric acid is produced in the liver in two reactions:

94

1. This reaction is catalyzed by Xenobiotic medium-chain fatty acid:CoA ligase:

Benzoic acid Benzoyl-CoA

2. The reaction is catalyzed by Glycine transferase:

Load by sodium benzoate is given in the amount of 6 grams dissolved in 250

ml of water per os. The amount of hippuric acid excreted in the urine during the

first 4 hours after the loading is estimated. 6 g of sodium benzoate should yield 7,5

g of hippuric acid. In the healthy persons, more than 60 % sodium benzoate

equivalent to 4,5 g of hippuric acid is excreted in urine. A reduction in hippuric

acid excretion indicates hepatic damage. The definition is not only a total for 4

hours removing the hippuric acid, but defining it in each time portion allows you to

more accurately assess the degree of disturbance antitoxic function of the liver.

Normal curve excretion the hippuric acid has a rapid rise in the 1-St and 2-nd hour

followed a sharp decline to the 4th hour. Slow increase excretion by the end of the

4-hour research indicates severe violation of antitoxic function of the liver. In

addition, this type of curve total number derived the hippuric acid, as a rule,

considerably reduced. The total number of the hippuric acid in cases of the most

CoASH

95

severe liver injury can be reduced to 35-20%. Defeat moderate give the decrease

excretion to 60%.

Laboratory tests for to estimate liver function

Liver function tests are a battery of tests that give your doctor an idea of how

well your liver is working. From these studies, your doctor can identify possible

liver disease, medication stress on liver function, or infections of the liver such as

hepatitis. There are several different tests that comprise LFT's.

What do you understand as Liver Function Tests (LFT)? They:

- are crude indices of hepatic structure, cellular integrity, and function;

- are based on measurements of substances released from damaged hepatic cells

into the blood;

- are measurements of blood components that gives an idea of the existence, extent

and type of liver damage;

- provide useful information regarding the presence and severity of hepatobiliary

injury or impairment of liver function.

Biochemical parameters in LFT are:

3) bilirubin (conjugated and unconjugated);

4) aminotransferases (ALT, AST);

5) alkaline phosphatase (ALP);

6) serum albumin and total protein.

The biochemical parameters assist in differentiating:

OBSTRUCTION TO THE BILIARY TRACT

7) indices of cholestasis, blockage of bile flow are indicated by 1) serum total

bilirubin concentration and 2) serum alkaline phosphatase activity;

ACUTE HEPATOCELLULAR DAMAGE

8) serum aminotransferase (ALT & AST) activities are measure of the integrity

of hepatocytes,

9) ALT & AST levels in plasma/serum are sensitive index of hepatocellular

damage,

96

10) ALT & AST are located mainly in the peri-portal hepatocytes, thus do not

give reliable indication of centri-lobular liver damage;

CHRONIC LIVER DISEASE

11) serum albumin concentration is a crude measure of the synthetic capacity of

the liver, although it is affected by many other factors.

Liver is highly compartmentalized, therefore no single biochemical test can

be used to fully access functional state of liver. What are the criteria used to select

parameters in LFT?

1. Tests based on substances produced or synthesized by liver. Example: albumin,

cholinesterase, coagulation factors.

2. Tests based on substances released from damaged hepatocytes. Tests separated

into two groups:

12) endogenous compounds released by damaged hepatocytes. Examples:

enzymes such as AST and ALT;

13) endogenous compounds synthesized at increased rate or released by

canalicular membrane, bile duct epithelium and endothelium of central and

periportal veins. Examples: ALP, gamma glutamyl transpeptidase (GGTP or

γGT), 5’nucleotidase.

3. Test based on substances cleared from plasma by liver. They can be separated

into two groups:

14) endogenous metabolites. Examples: bilirubin, bile acids, ammonia;

15) exogenous compounds. Examples: benzoic acid, indole, aminopyrine,

lidocaine, indocyanine green, caffeine

What is the diagnostic significance of Aspartate Aminotransferase?

AST was formerly called Serum Glutamate Oxaloacetate Transaminase

(SGOT). Its activity is high in heart muscle, liver, skeletal muscle, but found in

lesser degree in kidneys, pancreas, RBC Damage tissues releases AST in blood:

serum/plasma level rises. AST elevation is directly related to extent of cellular

damage or injury. Elevation of AST in plasma depends on length of time that the

97

blood is drawn after damage or injury because AST is cleared from the blood in a

few days. AST level in plasma is elevated 8 hours after cellular injury, peak at 24

to 36 hours, and return to normal in 3 to 7 days.

AST level is persistently elevated in chronic hepatocellular disease.

In acute hepatitis, AST can be elevated as much as 20 times the normal

value.

In Acute Extra-hepatic Obstruction (e.g., Gallstone), AST levels quickly

rise to 10 times the normal and swiftly fall.

In Cirrhotic patients level of AST depends on the amount of active

inflammation.

Factors that interfere with serum AST include: pregnancy – can cause

decreased levels of AST; exercise – can cause increased levels of AST; drugs such

as anti-hypertensives, cholinergic agents, coumarin-type anticoagulants, oral

contraceptives, opiates, salicylates, hepatotoxic medications.

What is the diagnostic significance of Alanine Aminotransferase ?

ALT was formerly called Serum Glutamate-Pyruvate Transaminase

(SGPT). It is mainly in liver, lesser quantities are in kidneys, heart and skeletal

muscle. Liver dysfunction or injury causes elevation of ALT level in blood.

ALT is a sensitive and specific indicator of hepatocellular disease. Plasma

ALT level is more liver-specific than AST. ALT elevation is directly related to

extent of cellular damage or injury. Elevation of ALT in plasma depends on

length of time that the blood is drawn after damage or injury because ALT is

cleared from the blood in a few days. ALT level in plasma is elevated 8 hours

after cellular injury, peak at 24 to 36 hours, and return to normal in 3 to 7 days.

AST is released more than ALT in chronic hepatocellular disease (cirrhosis).

Large number of drugs can increase serum level of ALT.

What is the diagnostic significance of Alkaline Phosphatase (ALP)?

98

ALP activity is increased in an alkaline (pH of 9 to 10) medium. It is highest

in liver, biliary tract epithelium, bone, placenta. ALP is in Kupffer’s cells lining

Biliary collecting system. Plasma/Serum ALP level use to detect disorders in liver

and bond. In Liver disease, increase plasma ALP is due to increased synthesis by

cells lining the bile canaliculi, usually in response to cholestasis, which may be

either intra-hepatic or extra-hepatic. Levels of ALP in plasma/serum are greatly

increased in both extra-hepatic and intra-hepatic obstructive biliary disease and

cirrhosis. Hepatic tumors, hepatotoxic drugs and hepatitis, cause smaller elevations

in serum ALP levels.

What are some of the extra-hepatic sources of ALP? Bone is the most

frequent extra-hepatic source of ALP. New bone growth is associated with

elevated levels of ALP, but and healing fractures, rheumatoid arthritis,

hyperparathyroidism also.

How are the isoenzymes of ALP used in diagnosis? They are used to

distinguish between liver and bone diseases. Isoenzymes are most easily

differentiated by heat Stability test and by electrophoresis. ALP isoenzyme

produced in Liver (ALP 1) is heat stable but ALP isoenzyme produced in bone

(ALP 2) is inactivated by heat. Detection of isoenzymes helps differentiate the

source of the pathologic condition associated with elevated total ALP. ALP 1 is

expected to be higher in liver disease.

Sources of elevated ALP can be determined by analyzing 5` nucleotidase in

the same serum sample. 5`nucleotidase is produced predominantly in the liver. If

both total ALP and 5`nucleotidase are elevated, then it is liver disease. If

5`nucleotidase is normal and ALP is elevated then bone is the most probable

source of the elevated ALP.

What is the diagnostic significance of Gamma Glutamyl Transpeptidase

(GGTP or γGT)?

GGTP participates in the transfer of amino acids and peptides across cellular

membrane and possibly participates in glutathione metabolism. Its level is very

99

high in liver and biliary tract. Lesser concentrations are in kidney, spleen, heart,

intestine, brain, and prostate gland. Men may have higher GGTP levels than

women because of the additional levels in the prostate.

Test for GGTP is used to detect liver cell dysfunction. GGTP test is highly

accurate in indicting cholestasis. GGTP is the most sensitive liver enzyme for

detecting biliary obstruction, cholangitis, or cholecystitis. Elevation of GGTP

parallels that of ALP in liver disease. GGTP is not increased in bone disease.

What is the diagnostic significance of albumin in blood?

Albumin is the major protein synthesized within the liver, thus can be use to

assess hepatic function. Estimation of Pre-albumin is a better assessment of liver

function. When disease affects the liver, the hepatocytes lose ability to synthesize

albumin, and the serum albumin level is diminished because the half-life of

albumin is 12 to 18 days, severe impairment of hepatic albumin synthesis may not

be recognized for several weeks or even months. Hypoalbuminaemia is a feature of

advanced chronic liver disease and severe acute liver damage. Albumin level is

low in some cases of chronic liver disease, but globulin level is high given a

normal total protein level. Reason for this might be that the liver cannot produce

albumin, thus accounting for the low albumin level, whereas the globulins are

mostly made in the reticuloendothelial system and therefore their levels tend to

increase. These changes can however be detected by measuring the

albumin/globulin (A/G) ratio or performs protein electrophoresis.

What is the significant of prothrombin time in LFT?

Prothrombin time is a measure of the activities of certain coagulation factors

synthesized by the liver. It is used as an indicator of hepatic synthetic function.

Prothrombin has a very short half-life, and an increased prothrombin time may be

the earliest indicator of hepatocellular damage.

100

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test tasks: Explanations:

1. A patient with symptoms of acute

alcohol poisoning was brought to

the hospital. What carbohydrates

metabolism changes are typical for

this condition?

A. The anaerobic glucose

metabolism predominates in muscles

B. The gluconeogenesis is increased

in the liver

C. The breakage of glycogen is

increased in the liver

D. The gluconeogenesis velocity in

the liver is decreased

E. The anaerobic breakage of

glucose is increased in muscles

2. A patient suffers from hepatic

cirrhosis. Examination of which of

the following substances excreted

by urine can characterize the state of

antitoxic function of liver?

A. Uric acid

B. Сreatinine

C. Ammonium salts

D. Hippuric acid

E. Amino acids

101

№ Test tasks: Explanations:

3. A patient has been admitted to the

contagious isolation ward with signs

of jaundice caused by hepatitis virus.

Which of the symptoms given below

is strictly specific for hepatocellular

jaundice?

A. Bilirubinuria

B. Increase of ALT, AST level

C. Heperbilirubinemia

D. Cholemia

E. Urobilinuria

4. A patient suffering from chronic

hepatitis. Antitoxic liver function

evaluation is as follows: sodium

benzoate dissolved in water is orally

given to the patient and the amount

of certain substance excreted with

urine is estimated. What chemical

compound measurement is an test

for assessing the detoxification

function of liver:

A. Phenylacetic acid

B. Citric acid

C. Oxalic acid

D. Valerian acid

E. Hippuric acid

102

№ Test tasks: Explanations:

5. There is impairment of the liver

function in a patient. What

biochemical parameters necessary to

determine for evaluation of the liver

in the blood:

A. Aldolase

B. Lipase

C. AlAT (ALT)

D. LDH1

E. Creatine kinase

6. Select the function that is not

inherent for the liver:

A. The distribution of substances

entering the body from the

gastrointestinal tract

B. Bile formation

C. Synthesis of substances used by

other tissues (creatine, ketones, etc.)

D. Synthesis of cortisol and

aldosterone

E. Inactivation of toxic substances

7. What enzyme is activated in liver at

physiological hypoglycemia:

A. Glycogen phosphorylase

B. Glutaminase

C. Glutamate decarboxylase

D. Creatine kinase

103

№ Test tasks: Explanations:

E. Glycogen synthase

8. Gluconeogenesis is active in the

liver of human. What substance

cannot be used as a substrate for the

metabolic pathway:

A. Alanine

B. Lactate

C. Acetyl-CoA

D. Pyruvate

E. Glycerol

9. Glycogen breakdown in the liver

leads to the formation of glucose-6-

phosphate. Further conversion of

this metabolite is multivariate. Select

a product formed from glucose-6-

phosphate mainly:

A. Pyruvate

B. Lactate

C. Alanine

D. Ribose-5-phosphate

E. Glucose

10. Mobilization of lipids from fat

depots and oxidation of higher fatty

acids (HFA) in the liver is decreased

at the use of excessive amounts of

carbohydrates. What is the basis of

104

№ Test tasks: Explanations:

such changes:

A. Reduced absorption of HFA in

the intestine

B. Reduced HFA delivery to the

liver

C. Deficiency of CoA

D. NADPH deficit

E. Biotin deficiency

11. Liver plays a key role in the

regulation of lipid metabolism in the

body. Only one of following

functions is not characteristic of

liver. Point out it:

A. Synthesis of bile acids

B. Synthesis of cholecalciferol

C. Formation of lipoproteins

D. The regulation of cholesterol

metabolism

E. Synthesis of ketone bodies

12. Most amino acids originating from

food are used by the liver for:

A. Synthesis of blood proteins

B. Synthesis of creatine

C. Synthesis of urea

D. Synthesis of uric acid

E. Synthesis of bile acids

105

№ Test tasks: Explanations:

13. Point out the conjugation agent used

for conjugated bilirubin formation in

hepatocytes:

A. Glycine

B. Cysteine

C. UDP-glucuronic acid

D. PAPS

E. Acetyl-CoA

14. Find the protein name that is

synthesized in the liver, only:

A. Albumin of blood plasma

B. Actin

C. Myosin

D. Tropomyosin

E. All the names above are right

answers

15. Point out the amino acid that is

conjugative agent at Quick`s test:

A. Lactic acid

B. Glycine

C. Valine

D. Leucine

E. Histidine

106

№ Test tasks: Explanations:

16. Find out the enzyme name which is

specific for liver tissue, only:

A. Succinate dehydrogenase

B. Arginase

C. Alanine amino transferase

D. Aspartate amino transferase

E. Isocitrate dehydrogenase

17. This lipoprotein class is synthesized

in the liver, and is in need for the

transport of triacylglycerols and

cholesterol from the liver to tissues.

Name it:

A. IDL

B. HDL

C. LDL

D. VLDL

E. Chylomicrons

18. Point out the enzyme whose activity

is decreased in the blood plasma at

liver cirrhosis in patient:

A. Glutamine synthetase

B. Glutamate dehydrogenase

C. Alanine amino transferase

D. Choline esterase

107

№ Test tasks: Explanations:

E. UDP - glucoronyl transferase

19. Disulfiram is widely used in medical

practice to prevent alcoholism, it

inhibits aldehyde dehydrogenase.

Increased level of what metabolite

causes aversion to alcohol?

A. Acetaldehyde

B. Ethanol

C. Malonyl aldehyde

D. Propionic aldehyde

E. Methanol

20. One of liver functions is

maintenance of glucose

concentration in the blood. Point out

the carbohydrate metabolic pathway

in the liver that provides realization

of this function at exception of diet

carbohydrates:

A. Aerobic oxidation of glucose

B. Anaerobic oxidation of glucose

C. Gluconeogenesis

D. Pentose phosphate cycle

E. Glycogenesis

108

XENOBIOTIC TRANSFORMATION IN HUMANS.

MICROSOMAL OXIDATION

(Rudko N.P., Aleksandrova K. V.)

INFORMATIONAL MATERIAL

Biological basis for xenobiotic metabolism:

- to convert lipid-soluble, non-polar, non-excretable forms of chemicals to

water-soluble, polar forms that are excretable in bile and urine;

- the transformation process may take place as a result of the interaction of the

toxic substance with enzymes found primarily in the cell endoplasmic

reticulum, cytoplasm, and mitochondria;

- the liver is the primary organ where biotransformation occurs.

Xenobiotics: the definition. The term xenobiotic is derived from the Greek

words ξένος (xenos) = foreigner, and βίος (bios) = life, plus the Greek suffix for

adjectives -τικός, -ή, -ό (tic). A xenobiotic is a chemical which is found in an

organism but which is not normally produced or expected to be present in it. It can

also cover substances which are present in much higher concentrations than are

109

usual Specifically, drugs such as antibiotics are xenobiotics in humans because the

human body does not produce them itself, nor are they part of a normal diet.

However, the term xenobiotics is very often used in the context of pollutants

such as dioxins and polychlorinated biphenyls and their effect on the alive

organism, because xenobiotics are understood as substances foreign to an entire

biological system, i.e. artificial substances, which did not exist in nature before

their synthesis by humans. So a xenobiotic is a chemical compound foreign to a

given biologic system. With respect to animals and humans, xenobiotics include

drugs, drug metabolites, and environmental compounds, such as pollutants that are

not produced by the body. In the environment, xenobiotics include synthetic

pesticides, herbicides, and industrial pollutants that would not be found in nature.

The most common classification in the modern science of xenobiotics is

reduced to the following groups:

1) chemicals (mercury, lead, cadmium, etc.);

2) the radionuclides;

3) drugs;

4) the substances used in plant cultivation and animal husbandry (pesticides,

insecticides, herbicides, nitrates, etc.);

5) polycyclic aromatic and chlorinated hydrocarbons;

6), dioxins and dioxin-like substances;

7) the metabolites of microorganisms

Current conceptions about the mechanism of xenobiotic toxic action.

The rate at which metabolism of toxic substances occurs is dependent on a

variety of factors that can be categorized into two groups:

- factors that affect the metabolic processes directly, and

- factors that affect the transport of toxic substance to tissues where

metabolism occurs.

Biotransformation is affected by the species of the test animal, age, sex,

nutritional status, disease, enzyme induction or inhibition, and genetics. Newborn

babies and young infants are more susceptible to a variety of chemicals such as

110

pesticides because the cytochrome P450 enzymes important in pesticide

detoxification reaction are not well developed.

A balanced diet will provide the necessary protein as well as essential metals

and minerals such as copper, zinc, and calcium to assist normal cellular enzymatic

activities associated with biotransformation. Protein deficient diets can result in a

decrease in protein synthesis, thus affecting the synthesis of enzymes involved in

the metabolic reactions used in detoxification.

Cirrhosis of the liver is often caused by excessive drinking of alcohol.

During the disease process the liver cells are damaged and replaced by connective

tissue. If enough cells are killed, the ability of the liver to metabolize toxic

substances is dramatically reduced. Cirrhosis can also be caused by repeated

exposure to arsenic or to high levels of vitamin A. Exposure to chemicals such as

carbon tetrachloride and vinyl chloride may result in liver cell damage and

decrease metabolism of toxic substances. These two substances are also associated

with the development of liver cancer.

Kidneys are damaged by absorption and concentration of heavy metals (e.g.,

Hg, Cd, etc.) in the cells of the proximal convoluted tubules of the nephron.

Absorption rate, perfusion rate, plasma protein binding, and storage will

affect the rate at which a toxic substance is delivered to the tissue where

metabolism occurs. The perfusion rate of a given tissue is important in determining

how quickly a toxic substance will be transformed. Organs such as the liver and

kidneys have a high perfusion rate relative to other tissue types. These organs have

the potential to extract and detoxify larger quantities of toxicants from the blood.

Toxic substances bound to proteins in the blood do not easily move across

cell membranes. In many cases this slows the rate at which the toxicant is

metabolized because the substance may not be readily absorbed by the tissue

where detoxification occurs. The faster a substance is eliminated from the body,

the more unlikely a biological effect will be.

The primary organs involved in xenobiotic excretion are the kidneys, liver,

and lung.

111

Excretion by the liver

Many toxic substances are stored and detoxified in the liver. The toxic

substances are then excreted into the bile. Bile is produced in the liver by the

hepatic cells. This mechanism is important in removing large protein-bound

toxicants such as heavy metals. Excretion of the toxicant from the liver to the

intestinal tract will usually result in the substance being removed in the feces.

However, the intestinal bacteria are also capable of producing enzymes that cause

the detoxified substance to become less water soluble. As a result, the toxic

substance may be reabsorbed from the digestive tract. The process of excreting

toxic substances from the liver and their subsequent reabsorption from the

digestive tract is referred to as enterohepatic circulation. Gluconated polycyclic

aromatic hydrocarbons and glutathione conjugates of trichloroethylene are

reabsorbed by this mechanism and therefore retained. Detoxification of toxic

substances before they reach the other portions of the systemic circulation is

referred to as the ―first pass effect.‖ This effect can decrease the systemic toxicity

of those substances absorbed from the digestive tract.

Excretion by the kidneys

The kidneys receive 25 percent of the cardiac output. The high perfusion rate

not only results in significant exposure to circulating toxic substances, but also

facilitate the excretion of the toxicants. Toxic substances enter the kidneys as a

result of active and passive transport mechanisms present in the glomerulus and the

nephron tubules. Several heavy metals such as cadmium, lead and mercury are

excreted by the kidneys. These metals are bound to plasma protein. The protein

metal complex has a low molecular weight and is able to pass through the

glomerulus to the nephron. However, this complex may be reabsorbed by active

transport mechanisms in the proximal convoluted tubules.

Excretion by the lungs

Excretion of volatile toxic gases, such as those associated with organic

compounds, occurs in the lungs. The transfer of gases from the blood to the lungs

is influenced by concentration gradients and by their solubility in water. Ethylene

112

which is only slightly soluble in water will readily diffuse from the blood into the

lungs and will therefore be easily removed. Chloroform however, is more water

soluble and will not diffuse as easily from the blood into the lungs.

Interaction with these enzymes may change the toxicant to either a less or a

more toxic form . Generally, biotransformation occurs in two phases.

PHASE I BIOTRANSFORMATION

- Phase I involves catabolic reactions that break down the toxicant into

various components. Catabolic reactions include oxidation, reduction, and

hydrolysis . Oxidation occurs when a molecule combines with oxygen, loses

hydrogen, or loses one or more electrons. Reduction occurs when a molecule

combines with hydrogen, loses oxygen, or gains one or more electrons. Hydrolysis

is the process in which a chemical compound is split into smaller molecules by

reacting with water. In most cases these reactions make the chemical less toxic,

more water soluble, and easier to excrete.

Phase I is mainly microsomal oxidation. The cytochrome P450 mixed

function oxidase system plays a major role in the metabolism of xenobiotics. The

biological effectiveness and the potential toxicity of many drugs are strongly

influenced by their metabolism, much of which is accomplished by P450-

dependent monoxygenase systems.

113

Although several enzyme systems participate in phase I metabolism of

xenobiotics, perhaps the most notable pathway is the monooxygenation function

catalyzed by the cytochrome P450s (CYPs). The CYPs detoxify and/or bioactivate

a vast number of xenobiotic chemicals and conduct functionalization reactions that

include N- and O-dealkylation, aliphatic and aromatic hydroxylation, N- and S-

oxidation, and deamination. Examples of toxicants metabolized by this system

include nicotine and acetaminophen, as well as the procarcinogenic substances,

benzene and polyaromatic hydrocarbons. The discovery of the CYPs dates back to

1958 when Martin Klingenberg initially reported his observation of a carbon

monoxide (CO)–binding pigment present in rat liver microsomes that was

characterized by absorbance spectra at 450 nm. The spectra remained an anomaly

until the work of Ryo Sato and Tsuneo Omura, published in 1962, provided the

critical evidence that the CO chromaphore was a hemoprotein. They further

described the properties of the protein, ascribing the term, CYP. Prior to these

events, research conducted by Alan Conney and the Millers in the United States

and H. Remmer in Germany demonstrated that rates of hepatic drug metabolism

could be induced or enhanced by pretreatment of animals with several types of

compounds, including phenobarbital (PB) and 3-methylcholanthrene (3-MC);

114

however, the identities of the enzymes responsible for these biotransformation

events were not known at the time. Also in the 1950’s, the work of R. T. Williams

from the United Kingdom greatly expanded our scope of xenobiotic metabolism,

elucidating the chemistries and reactions of a great many compounds. His

achievements are summarized in a book that he authored in 1959, entitled,

―Detoxication mechanisms: the metabolism and detoxication of drugs, toxic

substances and other organic compounds‖. In his book, he established the terms

phase I and phase II biotransformation, which are still used today, to denote the

biphasic nature of metabolism and together account for a large extent of chemical

detoxication that occurs in mammalian organisms. Certainly, many other scientists

have also contributed importantly in the area of phase I xenobiotic metabolism

over the past 50 years, so, with apologies, space limitations have precluded their

mention here. Suffice it to say that today, 57 functional P450 genes have been

identified in the human, and as of 2009, over 11,000 individual P450s have been

identified at the primary sequence level across all known species of organism!

The P450 catalytic cycle

1. The substrate binds to the active site of the enzyme, in close proximity to the

haem group, on the side opposite to the peptide chain. The bound substrate

induces a change in the conformation of the active site, often displacing a

water molecule from the distal axial coordination position of the haem iron,

and sometimes changing the state of the haem iron from low-spin to high-

spin. If no reducing equivalents are available, this complex may remain

stable.

2. The change in the electronic state of the active site favors the transfer of an

electron from NADPH via cytochrome P450 reductase or another associated

reductase. This takes place by way of the electron transfer chain, as

described above, reducing the ferric haem iron to the ferrous state.

3. Molecular oxygen binds covalently to the distal axial coordination position

of the haem iron. The cysteine ligand is a better electron donor than

histidine, which is normally found in haem-containing proteins. As a

115

consequence, the oxygen is activated to a greater extent than in other haem

proteins. However, this sometimes allows the iron-oxygen bond to

dissociate, causing the so-called "decoupling reaction", which releases a

reactive superoxide radical and interrupts the catalytic cycle.

4. A second electron is transferred via the electron-transport system, from

either cytochrome P450 reductase, ferredoxins, or cytochrome b5, reducing

the dioxygen adduct to a negatively charged peroxo group. This is a short-

lived intermediate state.

5. The peroxo group formed in step 4 is rapidly protonated twice by local

transfer from water or from surrounding amino-acid side-chains, releasing

one water molecule, and forming a highly reactive iron (V)-oxo species.

6. Depending on the substrate and enzyme involved, P450 enzymes can

catalyse any of a wide variety of reactions. A hypothetical hydroxylation is

shown in this illustration. After the product has been released from the

active site, the enzyme returns to its original state, with a water molecule

returning to occupy the distal coordination position of the iron nucleus.

116

Because most CYPs require a protein partner to deliver one or more

electrons to reduce the iron (and eventually molecular oxygen), CYPs are part of

P450-containing systems of proteins. Five general schemes are known:

CPR/cyb5/P450 systems employed by most eukaryotic microsomal (i.e.,

not mitochondrial) CYPs involve the reduction of cytochrome P450

reductase (variously CPR, POR, or CYPOR) by NADPH, and the transfer of

reducing power as electrons to the CYP. Cytochrome b5 (cyb5) can also

contribute reducing power to this system after being reduced by cytochrome

b5 reductase (CYB5R).

FR/Fd/P450 systems, which are employed by mitochondrial and some

bacterial CYPs.

CYB5R/cyb5/P450 systems in which both electrons required by the CYP

come from cytochrome b5.

FMN/Fd/P450 systems originally found in Rhodococcus sp. in which a

FMN-domain-containing reductase is fused to the CYP.

P450 only systems, which do not require external reducing power. Notable

ones include CYP5 (thromboxane synthase), CYP8, prostacyclin synthase,

and CYP74A (allene oxide synthase).

CYPs are the major enzymes involved in drug metabolism, accounting for

~75% of the total metabolism. Most drugs undergo deactivation by CYPs, either

directly or by facilitated excretion from the body. Also, many substances are

bioactivated by CYPs to form their active compounds.

Human CYPs are primarily membrane-associated proteins, located either in

the inner membrane of mitochondria or in the endoplasmic reticulum of cells.

CYPs metabolize thousands of endogenous and exogenous chemicals. Some CYPs

metabolize only one (or a very few) substrates, such as CYP19 (aromatase), while

others may metabolize multiple substrates. Both of these characteristics account for

their central importance in medicine. Cytochrome P450 enzymes are present in

most tissues of the body, and play important roles in hormone synthesis and

breakdown (including estrogen and testosterone synthesis and metabolism),

117

cholesterol synthesis, and vitamin D metabolism. Cytochrome P450 enzymes also

function to metabolize potentially toxic compounds, including drugs and products

of endogenous metabolism such as bilirubin, principally in the liver.

Many drugs may increase or decrease the activity of various CYP isozymes

either by inducing the biosynthesis of an isozyme (enzyme induction) or by

directly inhibiting the activity of the CYP (enzyme inhibition). This is a major

source of adverse drug interactions, since changes in CYP enzyme activity may

affect the metabolism and clearance of various drugs. For example, if one drug

inhibits the CYP-mediated metabolism of another drug, the second drug may

accumulate within the body to toxic levels. Hence, these drug interactions may

necessitate dosage adjustments or choosing drugs that do not interact with the CYP

system. Such drug interactions are especially important to take into account when

using drugs of vital importance to the patient, drugs with important side-effects and

drugs with small therapeutic windows, but any drug may be subject to an altered

plasma concentration due to altered drug metabolism.

A classic example includes anti-epileptic drugs. Phenytoin, for example,

induces CYP1A2, CYP2C9, CYP2C19, and CYP3A4. Substrates for the latter may

be drugs with critical dosage, like amiodarone or carbamazepine, whose blood

plasma concentration may either increase because of enzyme inhibition in the

former, or decrease because of enzyme induction in the latter.

Naturally occurring compounds may also induce or inhibit CYP activity. For

example, bioactive compounds found in grapefruit juice and some other fruit

juices, including bergamottin, dihydroxybergamottin, and paradisin-A, have been

found to inhibit CYP3A4-mediated metabolism of certain medications, leading to

increased bioavailability and, thus, the strong possibility of overdosing. Because of

this risk, avoiding grapefruit juice and fresh grapefruits entirely while on drugs is

usually advised.

Other examples:

Saint-John's wort, a common herbal remedy induces CYP3A4, but also

inhibits CYP1A1, CYP1B1, CYP2D6, and CYP3A4.

118

Tobacco smoking induces CYP1A2 (example CYP1A2 substrates are

clozapine, olanzapine, and fluvoxamine)

At relatively high concentrations, starfruit juice has also been shown to

inhibit CYP2A6 and other CYPs.[18]

Watercress is also a known inhibitor of

the Cytochrome P450 CYP2E1, which may result in altered drug

metabolism for individuals on certain medications (ex., chlorzoxazone).

A subset of cytochrome P450 enzymes play important roles in the synthesis

of steroid hormones (steroidogenesis) by the adrenals, gonads, and peripheral

tissue:

CYP11A1 (also known as P450scc or P450c11a1) in adrenal mitochondria

effects ―the activity formerly known as 20,22-desmolase‖ (steroid 20α-

hydroxylase, steroid 22-hydroxylase, cholesterol side-chain scission).

CYP11B1 (encoding the protein P450c11β) found in the inner mitochondrial

membrane of adrenal cortex has steroid 11β-hydroxylase, steroid 18-

hydroxylase, and steroid 18-methyloxidase activities.

CYP11B2 (encoding the protein P450c11AS), found only in the

mitochondria of the adrenal zona glomerulosa, has steroid 11β-hydroxylase,

steroid 18-hydroxylase, and steroid 18-methyloxidase activities.

CYP17A1, in endoplasmic reticulum of adrenal cortex has steroid 17α-

hydroxylase and 17, 20-lyase activities.

CYP21A1 (P450c21) in adrenal cortex conducts 21-hydroxylase activity.

CYP19A (P450arom, aromatase) in endoplasmic reticulum of gonads, brain,

adipose tissue, and elsewhere catalyzes aromatization of androgens to

estrogens.

PHASE II BIOTRANSFORMATION

Phase II biotransformation is catalyzed often by the ―transferase‖ enzymes

that perform conjugating reactions. Included in the phase II reaction schemes are

glucuronidation, sulfation, methylation, acetylation, glutathione conjugation, and

amino acid conjugation. The products of phase II conjugations are typically more

hydrophilic than the parent compounds and therefore usually more readily

119

excretable. Specific families of phase II xenobiotic-metabolizing enzymes include

the UDP-glucuronosyltransferases (UGTs), sulfotransferases (STs), N-

acetyltransferases (arylamine N-acetytransferase; NATs), and glutathione S-

transferases (GSTs) and various methyltransferases, such as thiopurine S-methyl

transferase and catechol O-methyl transferase. Perhaps of particular note, the

UGTs conduct glucuoronidation reactions, principally with electron-rich

nucleophilic heteroatoms, such as O, N, or S, present in aliphatic alcohols and

phenols. This microsomal system is a principal player in phase II metabolism and

is known for its high metabolic capacity but relatively low affinity for xenobiotic

substrates.

There are > 10 UGTs in humans. STs also constitute a large multigene

family of cytosolic enzymes that catalyze the sulfation of primarily aliphatic

alcohols and phenols and represent another important phase II pathway noted for

its high affinity for xenobiotic substrates but low capacity. The GSTs function as

cytosolic dimeric isoenzymes of 45–55 kDa size that have been assigned to at least

four classes: alpha, mu, pi, theta, and zeta; humans possess > 20 distinct GST

family members. There are two NATs, NAT1 and NAT2, that possess different but

overlapping substrate specificities and can function to both activate and deactivate

arylamine and hydrazine drugs and carcinogens. In concert with the phase I

enzymatic machinery, the phase II enzymes coordinately metabolize, detoxify, and

at times bioactivate xenobiotic substrates.

Example of indole biotransformation in two phases:

120

Indole and its derivatives are highly toxic to microorganisms and animals

and are considered mutagens and carcinogens. Experimental evidences showed

that indole caused glomerular sclerosis, hemolysis, improper oviduct functioning,

and chronic arthritis. Indol and its derivative like indole-3-acetic acid induced

neuroepithelial cell apoptosis in embryos; 6-hydroxyskatol, a metabolite of 3-

methylindole generated in the human intestine, has possible psychotropic effects.

Human beings can be exposed to indole via ambient air, tobacco smoke,

food, and skin contact with vapors and other products, such as perfumes that

contain indole. There is revealed a correlation between increasing of

encephalopathy and substances absorbed by the bloodstream from the intestines.

Indol and its derivatives are the substances that are formed in the intestines can

cause endotoxemia.

OTHER CASES OF HARMFUL COMPOUND BIOTRANSFORMATION

During the P450 catalytic cycle small amounts of reactive oxygen species

(ROS) such as superoxide radical anion and H2O2 are produced and cytochrome

P450 enzymes are a significant source of ROS in biological systems, especially

tissues like the liver where P450 is present in high amounts. Several factors

determine the generation of ROS by P450s including the specific form of P450,

entry of the second electron into the P450 cycle, the presence of substrate and

nature of the substrate. The toxicity of many reagents is due, in part, to increased

production of ROS when they are metabolized by cytochrome P450s e.g. CCL4,

121

halogenated hydrocarbons, benzene, acetaminophen, anesthetics, nitrosamines etc.

CYP2E1 appears to be significant generator of ROS and this may play a role in

alcohol-induced liver toxicity.

There are several enzyme systems that catalyze reactions to neutralize free

radicals and reactive oxygen species. These enzymes include:

superoxide dismutase

glutathione peroxidise

glutathione reductase

catalases

These form the body’s endogenous defence mechanisms to help protect

against free radical-induced cell damage. The antioxidant enzymes – glutathione

peroxidase, catalase, and superoxide dismutase (SOD) – metabolize oxidative toxic

intermediates. These enzymes also require co-factors such as selenium, iron,

copper, zinc, and manganese for optimum catalytic activity. It has been suggested

that an inadequate dietary intake of these trace minerals may compromise the

effectiveness of these antioxidant defense mechanisms. The consumption and

absorption of these important trace minerals may decrease with aging.

Glutathione: enzymes and system

Glutathione, an important water-soluble antioxidant, is synthesized from the

amino acids glycine, glutamate, and cysteine. Glutathione can directly neutralize

ROS such as lipid peroxides, and also plays a major role in xenobiotic metabolism.

When an individual is exposed to high levels of xenobiotics, more glutathione is

utilized for conjugation. Conjugation with glutathione renders the toxin neutral and

makes it less available to serve as an antioxidant. Research suggests that

glutathione and vitamin C work interactively to neutralize free radicals. These two

also have a sparing effect upon each other.

The glutathione system includes glutathione, glutathione reductase,

glutathione peroxidases and glutathione ''S''-transferases. Of these glutathione

peroxidase is an enzyme containing four selenium-cofactors that catalyzes the

breakdown of hydrogen peroxide and organic hydroperoxides. Glutathione ''S''-

122

transferases show high activity with lipid peroxides. These enzymes are at

particularly high levels in the liver.

Lipoic acid

This is another important endogenous antioxidant. It is categorized as ―thiol‖

or ―biothiol‖. These are sulfur-containing molecules that catalyze the oxidative

decarboxylation of alpha-keto acids, such as pyruvate and alphaketoglutarate, in

the Krebs cycle. Lipoic acid and its reduced form, dihydrolipoic acid (DHLA),

neutralize the free radicals in both lipid and aqueous domains and as such has been

called a ―universal antioxidant.‖

Superoxide dismutase

Superoxide dismutases (SODs) are a class of enzymes that catalyse the

breakdown of the superoxide anion into oxygen and hydrogen peroxide. These

enzymes are present in almost all aerobic cells and in extracellular fluids. SODs

contain metal ion cofactors that, depending on the isozyme, can be copper, zinc,

manganese or iron. For example, in humans copper/zinc SOD is present in the

cytosol, while manganese SOD is present in the mitochondrion. The mitochondrial

SOD is most biologically important of these three.

Catalases

Catalases are enzymes that catalyse the conversion of hydrogen peroxide to

water and oxygen, using either an iron or manganese cofactor. This is found in

peroxisomes in most eukaryotic cells. Its only substrate is hydrogen peroxide. It

follows a ping-pong mechanism. Here, its cofactor is oxidised by one molecule of

hydrogen peroxide and then regenerated by transferring the bound oxygen to a

second molecule of substrate.

Peroxiredoxins

There are peroxidases that catalyze the reduction of hydrogen peroxide, organic

hydroperoxides, as well as peroxynitrite. These may be of three basic types -

typical 2-cysteine peroxiredoxins; atypical 2-cysteine peroxiredoxins; and 1-

cysteine peroxiredoxins. Peroxiredoxins seem to be important in

antioxidant metabolism.

123

Rhodanase

Sodium thiosulfate (Na2S2O3) is used as an antidote to cyanide poisoning.

Thiosulfate acts as a sulfur donor for the conversion of cyanide to thiocyanate

(which can then be safely excreted in the urine), catalyzed by the enzyme

rhodanase.

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test tasks: Explanations:

1. Study of conversion of a food

colouring agent revealed that

utilization of this xenobiotic takes

place only in one phase –

microsomal oxidation (modification

phase). Name an enzyme of this

phase:

A. Cytochrome aa3

B. Cytochrome C oxidase

C. Cytochrome P-450

D. Cytochrome C1

E. Cytochrome b

2. In course of metabolic process active

forms of oxygen including

superoxide anion radical are formed

in the human body. By means of

what enzyme is this anion

inactivated?

A. Catalase

B. Glutathione reductase

124

№ Test tasks: Explanations:

C. Peroxidase

D. Superoxide dismutase

E. Glutathione peroxidase

3. A patient with encephalopathy was

admitted to the neurological in

patient department. There was

revealed a correlation between

increasing of encephalopathy and

substances absorbed by the

bloodstream from the intestines.

What substances that are formed in

the intestines can cause

endotoxemia?

A. Indole

B. Ornithine

C. Acetacetate

D. Butyrate

E. Biotin

4. Point out the main place for the

location of microsomal oxidation in

a cell:

A. Nucleus

B. Cytoplasm

C. EPR, smooth part

D. EPR, rough part

E. Lysosomes

125

№ Test tasks: Explanations:

5. Point out the enzyme of

monooxygenase chain as a final

electron acceptor from NADPН:

A. Cytochrome b5

B. Cytochrome b

C. Cytochrome P450

D. Cytochrome c1

E. Cytochrome aa3

6. Find the enzyme participating in the

function of the microsomal

monooxygenase chain:

A. НАДН - dehydrogenase

B. Cytochrome b

C. Cytochrome c1

D. Cytochrome c

E. Cytochrome P450

7. Point out the liver enzyme

participating in the neutralization of

xenobiotics, their metabolites and

harmful endogenous products:

A. Glutamine synthetase

B. Glutamate dehydrogenase

C. Alanine amino transferase

126

№ Test tasks: Explanations:

D. Carbomoyl phosphate synthetase

E. UDP - glucoronyl transferase

8. Find the correct definition of the

term "xenobiotic":

A. A substance that is an obligatory

component of food products

B. A substance that is unnatural for

humans

C. A substance that is synthesized in

small quantities in humans

D. A substance that regulates

metabolism in organism

E. A substance that is a terminal

product of metabolism

9. Point out the tripeptide participating

in the conjugation of some harmful

products in the liver:

A. Glutathione

B. Methionine

C. Trialanine

D. Oxytocin

E. Prolylproline

127

№ Test tasks: Explanations:

10. In course of metabolic process active

forms of oxygen including hydrogen

peroxide are formed in the human

body. By means of what enzyme is

this compound inactivated?

A. Catalase

B. Glutathione reductase

C. Peroxidase

D. Superoxide dismutase

E. Glutathione peroxidase

11. Point out the donor of sulfate group

in the conjugation phase of

xenobiotics transformation:

A. Glutathione

B. UDP-glucuronic acid

C. Adenosine 3 -phosphate-5 -

phosphosulfate (PAPS)

D. Acetyl-CoA

E. S-adenosylmethionine

12. All of the following may have a

physiological antioxidant role except

A. Lipoic acid

B. Vitamin C

C. Selenium

128

№ Test tasks: Explanations:

D. Iron

E. Vitamin E

13. Point out the chemical nature of

prosthetic group of cytochrome

P450:

A. Nucleotide

B. Fe3+

C. Fe2+

D. Phosphate

E. Haem

14. Choose the exogenous factor (the

drug) that can induce the UDP-

glucuronosyltransferase gene

expression in the liver:

A. Calcitriol

B. Thyroxine

C. Riboxin

D. Phenobarbital

E. Thiamine diphosphate

15. Choose one wrong continuation of a

phrase: Phase I of xenobiotics

transformation:

A. Is carried out by enzymes of

endoplasmic reticulum

129

№ Test tasks: Explanations:

B. Demands presence of NADPH

C. Results in increase of polarity of a

substance

D. Occurs in anaerobic conditions

E. Proceeds at participation of

cytochrome Р450

16. Point out the main enzyme in

monooxygenase system of EPR

responsible for modification of

xenobiotics:

A. Glucuronyl transferase

B. Cytochrome P450

C. NADH reductase

D. Glutathione S-transferase

E. Cytochrome C oxidase

17. Choose the correct statement about

hepatic monooxygenases linked with

cytochrome P450 enzyme.

A. Located mainly in smooth EPR

B. Catalyzes oxidation, reduction

and hydrolysis reactions at the

same time

C. They are inducible

D. Their action always causes the

detoxification of xenobiotics

E. Positions A, C are correct

130

№ Test tasks: Explanations:

18. Which of following cytochrome

participates in drug metabolism?

A. Cytochrome aa3

B. Cytochrome c1

C. Cytochrome P450

D. Cytochrome c

E. Cytochrome b

19. Point out the conjugation agent that

is in need to detoxify heterocyclic

alcohols in the liver:

A. Glucose

B. Methionine

C. Valine

D. PAPS

E. Histidine

20. Benzoic acid causes the toxic effect

at its accumulation in the liver.

Choose the main conjugative agent

to detoxify it:

A. Glycine

B. PAPS

C. S-adenosyl methionine

D. Glutathione

E. Acetyl-CoA

131

BIOCHEMISTRY OF BLOOD TISSUE. PROTEINS OF BLOOD PLASMA.

NON-PROTEIN COMPONENTS OF BLOOD PLASMA AT HEALTHY

AND DISEASED PEOPLE

(Levich S. V.)

INFORMATIONAL MATERIAL

Blood is a body fluid in humans and other animals that delivers necessary

substances such as nutrients and oxygen to the cells and transports metabolic waste

products away from those same cells

Functions

Blood has three main functions: transport, protection and regulation.

Transport

Blood transports the following substances:

Gases, namely oxygen (O2) and carbon dioxide (CO2), between the lungs and

rest of the body

Nutrients from the digestive tract and storage sites to the rest of the body

Waste products to be detoxified or removed by the liver and kidneys

Hormones from the glands in which they are produced to their target cells

Heat to the skin so as to help regulate body temperature

Protection

Blood has several roles in inflammation:

Leukocytes, or white blood cells, destroy invading microorganisms and cancer

cells

Antibodies and other proteins destroy pathogenic substances

Platelet factors initiate blood clotting and help minimise blood loss

Regulation

Blood helps regulate:

pH by interacting with acids and bases

Water balance by transferring water to and from tissues

132

Composition of blood

Blood is classified as a connective tissue and consists of two main

components:

1. Plasma, which is a clear extracellular fluid

2. Formed elements, which are made up of the blood cells and platelets

The formed elements are so named because they are enclosed in a plasma

membrane and have a definite structure and shape. All formed elements are cells

except for the platelets, which are tiny fragments of bone marrow cells.

Formed elements are:

Erythrocytes, also known as red blood cells (RBCs)

Leukocytes, also known as white blood cells (WBCs)

Platelets

Human RBCs do not contain mitochondria, so the main pathway for ATP

production in these cells is anaerobic glycolysis.

Platelets play important role in blood clotting. Deficiency of VIII factor lead

to hereditary coagulopathy caused by blockage of thromboplastin formation

Leukocytes are further classified into two subcategories called granulocytes which

consist of neutrophils, eosinophils and basophils; and agranulocytes which consist

of lymphocytes and monocytes. Lymphocytes synthesize interferon – universal

antiviral agents as a response to viral invasion.

The formed elements can be separated from plasma by centrifuge, where a

blood sample is spun for a few minutes in a tube to separate its components

according to their densities. RBCs are denser than plasma, and so become packed

into the bottom of the tube to make up 45% of total volume. This volume is known

as the haematocrit. WBCs and platelets form a narrow cream-coloured coat known

as the buffy coat immediately above the RBCs. Finally, the plasma makes up the

top of the tube, which is a pale yellow colour and contains just under 55% of the

total volume.

133

Anemia

Anemia, also spelled anaemia, is usually defined as a decrease in the

amount of red blood cells (RBCs) or hemoglobin in the blood. It can also be

defined as a lowered ability of the blood to carry oxygen. When anemia comes on

slowly, the symptoms are often vague and may include: feeling tired, weakness,

shortness of breath or a poor ability to exercise. Anemia that comes on quickly

often has greater symptoms, which may include: confusion, feeling like one is

going to pass out, loss of consciousness, or increased thirst. Anemia must be

significant before a person becomes noticeably pale. Additional symptoms may

occur depending on the underlying cause.

There are three main types of anemia: that due to blood loss, that due to

decreased red blood cell production, and that due to increased red blood cell

breakdown. Causes of blood loss include trauma and gastrointestinal bleeding,

among others. Causes of decreased production include iron deficiency, a lack of

vitamin B12, thalassemia, and a number of neoplasms of the bone marrow. Causes

of increased breakdown include a number of genetic conditions such as sickle cell

anemia, infections like malaria, and certain autoimmune diseases. It can also be

classified based on the size of red blood cells and amount of hemoglobin in each

cell. If the cells are small, it is microcytic anemia. If they are large, it is macrocytic

anemia while if they are normal sized, it is normocytic anemia. Diagnosis in men is

based on a hemoglobin of less than 130 to 140 g/L (13 to 14 g/dL), while in

women, it must be less than 120 to 130 g/L (12 to 13 g/dL). Further testing is then

required to determine the cause.

Signs and symptoms

Anemia goes undetected in many people and symptoms can be minor. The

symptoms can be related to an underlying cause or the anemia itself. Most

commonly, people with anemia report feelings of weakness, or fatigue, general

malaise, and sometimes poor concentration. They may also report dyspnea

(shortness of breath) on exertion. In very severe anemia, the body may compensate

for the lack of oxygen-carrying capability of the blood by increasing cardiac

134

output. The patient may have symptoms related to this, such as palpitations, angina

(if pre-existing heart disease is present). There may be signs of specific causes of

anemia, e.g., koilonychia (in iron deficiency), jaundice (when anemia results from

abnormal break down of red blood cells — in hemolytic anemia), bone deformities

(found in thalassemia major) or leg ulcers (seen in sickle-cell disease). In severe

anemia, there may be signs of a hyperdynamic circulation: tachycardia (a fast heart

rate), bounding pulse, flow murmurs, and cardiac ventricular hypertrophy

(enlargement). There may be signs of heart failure. Pica, the consumption of non-

food items such as ice, but also paper, wax, or grass, and even hair or dirt, may be

a symptom of iron deficiency, although it occurs often in those who have normal

levels of hemoglobin.

Causes

Figure shows normal red blood cells flowing freely in a blood vessel. The

inset image shows a cross-section of a normal red blood cell with normal

hemoglobin.

The causes of anemia may be classified as impaired red blood cell (RBC)

production, increased RBC destruction (hemolytic anemias), blood loss and fluid

overload (hypervolemia). Several of these may interplay to cause anemia

eventually. Indeed, the most common cause of anemia is blood loss, but this

usually does not cause any lasting symptoms unless a relatively impaired RBC

production develops, in turn most commonly by iron deficiency.

Impaired production

Disturbance of proliferation and differentiation of stem cells

o Pure red cell aplasia

o Aplastic anemia affects all kinds of blood cells. Fanconi anemia is a hereditary

disorder or defect featuring aplastic anemia and various other abnormalities.

o Anemia of renal failure by insufficient erythropoietin production

o Anemia of endocrine disorders

Disturbance of proliferation and maturation of erythroblasts

o Pernicious anemia is a form of megaloblastic anemia due to vitamin B12

135

deficiency dependent on impaired absorption of vitamin B12. Lack of dietary B12

causes non-pernicious megaloblastic anemia

o Anemia of folic acid deficiency, as with vitamin B12, causes megaloblastic

anemia

o Anemia of prematurity, by diminished erythropoietin response to declining

hematocrit levels, combined with blood loss from laboratory testing, generally

occurs in premature infants at two to six weeks of age.

o Iron deficiency anemia, resulting in deficient heme synthesis

o Thalassemias, causing deficient globin synthesis

o Congenital dyserythropoietic anemias, causing ineffective erythropoiesis

o Anemia of renal failure (also causing stem cell dysfunction)

Other mechanisms of impaired RBC production

o Myelophthisic anemia or myelophthisis is a severe type of anemia resulting

from the replacement of bone marrow by other materials, such as malignant tumors

or granulomas.

o Myelodysplastic syndrome

o anemia of chronic inflammation

Increased destruction

Anemias of increased red blood cell destruction are generally classified as

hemolytic anemias. These are generally featuring jaundice and elevated lactate

dehydrogenase levels. Glutathione peroxidase deficiency and low concentration of

reduced glutathione also lead to the RBCs restruction.

Blood loss

Anemia of prematurity from frequent blood sampling for laboratory testing,

combined with insufficient RBC production

Trauma or surgery, causing acute blood loss

Fluid overload

Fluid overload (hypervolemia) causes decreased hemoglobin concentration and

apparent anemia:

General causes of hypervolemia include excessive sodium or fluid intake, sodium

136

or water retention and fluid shift into the intravascular space.

Anemia of pregnancy is induced by blood volume expansion experienced in

pregnancy.

Blood plasma

Blood plasma is a mixture of proteins, enzymes, nutrients, wastes, hormones

and gases. The specific composition and function of its components are as follows:

Proteins

These are the most abundant substance in plasma by weight and play a part

in a variety of roles including clotting, defence and transport. Collectively, they

serve several functions:

They are an important reserve supply of amino acids for cell nutrition. Cells

called macrophages in the liver, gut, spleen, lungs and lymphatic tissue can break

down plasma proteins so as to release their amino acids. These amino acids are

used by other cells to synthesise new products.

Plasma proteins also serve as carriers for other molecules. Many types of small

molecules bind to specific plasma proteins and are transported from the organs that

absorb these proteins to other tissues for utilisation. The proteins also help to keep

the blood slightly basic at a stable pH. They do this by functioning as weak bases

themselves to bind excess H+ ions. By doing so, they remove excess H+ from the

blood which keeps it slightly basic.

The plasma proteins interact in specific ways to cause the blood to coagulate,

which is part of the body’s response to injury to the blood vessels (also known as

vascular injury), and helps protect against the loss of blood and invasion by foreign

microorganisms and viruses.

Plasma proteins govern the distribution of water between the blood and tissue

fluid by producing what is known as a colloid osmotic pressure.

There are three major categories of plasma proteins, and each individual type of

proteins has its own specific properties and functions in addition to their overall

collective role:

Albumin: This is the most abundant class of plasma proteins (2.8 to 4.5

137

gm/100ml) with highest electrophoretic mobility. It is soluble in water ad is

precipitated by fully saturated ammonium sulphate. Albumin is synthesized in liver

and consists of a single polypeptide chain of 610 amino acids having a molecular

weight of 69,000. It is rich in some essential amino acids such as lysine, leucine,

valine, phenylalanine, threonine, arginine and histidine. The acidic amino acids

like aspartic acid and glutamic acid are also concentrated in albumin. The presence

of these residues makes the molecule highly charged with positive and negative

charge. Besides having a nutritive role, albumin acts as a transport carrier for

various biomolecules such s fatty acids, trace elements and drugs. Another

important role of albumin is in the maintenance of osmotic pressure and fluid

distribution between blood and tissues.

Globulins, which can be subdivided into three classes from smallest to

largest in molecular weight into alpha, beta and gamma globulins. The globulins

include high density lipoproteins (HDL), an alpha-1 globulin, and low density

lipoproteins (LDL), a beta-1 globulin. HDL functions in lipid transport carrying

fats to cells for use in energy metabolism, membrane reconstruction and hormone

function. HDLs also appear to prevent cholesterol from invading and settling in the

walls of arteries. LDL carries cholesterol and fats to tissues for use in

manufacturing steroid hormones and building cell membranes, but it also favours

the deposition of cholesterol in arterial walls and thus appears to play a role in

disease of the blood vessels and heart. HDL and LDL therefore play important

parts in the regulation of cholesterol and hence have a large impact on

cardiovascular disease.

By electrophoresis plasma globulins are separated into α1, α2,β and ¥-

globulins are synthesized in liver, whereas ¥-globulins are formed in the cells of

reticulo-endothelial system. The average normal serum globulin (total)

concentration is 2.5 gm / 100 ml (Howe method) or 3.53 gm/100 ml by

electrophoresis.

138

α1-Globulin: This fraction includes several complex proteins containing

carbohydrates and lipids. These are, orosomucoid, α1-glycoprotein and α-

lipoproteins. The normal serum level of α1-globulin is 0.42 gm/100 ml.

Orosomucoid is rich in carbohydrates. It is water-soluble, heat stable and has

a molecular weight of 44,000. It serves to transport hexosamine complexes to

tissues.

Lipoproteins are soluble complexes which contain non-covalently bound

lipid. These proteins act mainly as transport carrier to different types of lipids in

the body. Increasing of low-density lipoprotein fraction (LDL) could cause

hyperlipoproteinemia type IIa.

1-antitrypsin (1-proteinase inhibitor) – glycoprotein with a molecular

weight 55 kDa. Its concentration in blood plasma is 2-3 г/л. The main biological

property of this inhibitor is its capacity to form complexes with proteinases

oppressing proteolitic activity of such enzymes as trypsin, chemotrypsin, plasmin,

trombin. The content of 1-antitrypsin is markedly increased in inflammatory

processes. The inhibitory activity of 1-antitrypsin is very important in pancreas

necrosis and acute pancreatitis because in these conditions the proteinase level in

blood and tissues is sharply increased. The congenital deficiency of 1-antitrypsin

results in the lung emphysema.

α2-Globulins: This fraction also contains complex proteins such as α2-

glycoproteins, plasminogen, prothrombin, haptoglobulin, ceruloplasmin (transports

Cu) and α2-macroglobulin. The normal serum value of this fraction is 0.67

gm/100ml.

Plasminogen and prothrombin are in the inactive precursors of plasmin and

thrombin respectively. Both these proteins play an important role in blood clotting.

Haptoglobulins are also glycoproteins having a molecular weight of 85,000.

These are synthesized in liver and can bind with any free hemoglobin that may

arise in plasma due to lysis of erythrocytes and thus prevent excretion of Hb and

iron associated with it.

Ceruloplasmin - glycoprotein of the 2-globulin fraction. It can bind the

139

copper ions in blood plasma. Up to 3 % of all copper contents in an organism and

more than 90 % copper contents in plasma is included in ceruloplasmin.

Ceruloplasmin has properties of ferroxidase oxidizing the iron ions. The decrease

of ceruloplasmin in organism (Wilson disease) results in exit of copper ions from

vessels and its accumulation in the connective tissue that shows by pathological

changes in a liver, main brain, cornea.

2-Macroglobulin - protein of 2-globulin fraction, universal serum

proteinase inhibitor. Its contents (2,5 g/l) in blood plasma is highest comparing to

another proteinase inhibitors.

The biological role of 2-macroglobulin consists in regulation of the tissue

proteolysis systems which are very important in such physiological and

pathological processes as blood clotting, fibrinolysis, processes of immunodefence,

functionality of a complement system, inflammation, regulation of vascular tone

(kinine and renin-angiothensine system).

β-Globulins: This fraction of plasma proteins contain these different β-

lipoproteins which are very rich in lipid content. It also contains transferrin

(siderophilin) which transports non-heme iron in plasma. The normal serum value

of β-globulins is 0.91 gm/100ml.

Transferrin is an iron transport protein. In plasma it can be saturated even up

to 33% with iron. It has a low content of carbohydrate.

γ-Globulins:

Immunoglobulins (Ig A, Ig G, Ig E, Ig M) - proteins of -globulin fraction of

blood plasma executing the functions of antibodies which are the main effectors of

humoral immunity. They appear in the blood serum and certain cells of a

vertebrate in response to the introduction of a protein or some other

macromolecule foreign to that species.

Immunoglobulin molecules have bindind sites that are specific for and

complementary to the structural features of the antigen that induced their

formation. Antibodies are highly specific for the foreign proteins that evoke their

formation.

140

Molecules of immunoglobulins are glycoproteins. The protein part of

immunoglobulins contain four polipeptide chains: two heavy H-chains and two

light L-chains.

Fibrinogen: It is a fibrous protein with a molecular weight of 340,000. It has

6 polypeptide chains which are held together by disulphide linkages. Fibrinogen

plays an important role in clothing of blood where it is converted to fibrin by

thrombin.

In addition to the above mentioned proteins, the plasma contains a number

of enzymes such as acid phosphatase and alkaline phosphatase which have great

diagnostic value.

Functions of Plasma Proteins:

1. Protein Nutrition: Plasma proteins act as a source of protein for the tissues,

whenever the need arises.

2. Osmotic Pressure and water balance: Plasma proteins exert an osmotic

pressure of about 25 mm of Hg and therefore play an important role in maintaining

a proper water balance between the tissues and blood. Plasma albumin is mainly

responsible for this function due to its low molecular weight and quantitative

dominance over other proteins. During the condition of protein loss from the body

as occurs in kidney diseases, excessive amount of water moves to the tissues

producing edema.

3. Buffering action: Plasma proteins help in maintaining the pH of the body by

acting asampholytes. At normal blood pH they act as acids and accept captions.

4. Transport of Lipids: One of the most important functions of plasma proteins us

to transport lipids and lipid soluble substances in the body. Fatty acids and

bilirubin are transported mainly by albumin, whereas cholesterol and

phospholipids are carried by the lipoproteins present in β-globulins also transport

fat soluble vitamins (A, D, K and E)

5. Transport of other substances: In addition to lipids, plasma proteins also

transport several metals and other substances α2-Globulins transport copper

(Ceruloplasmin), bound hemoglobin (haptoglobin) and thyroxine (glycoprotein)

141

and non-heme iron is transported by transferrin present in β-globulin fraction.

Calcium, Magnesium, some drugs and dyes and several cations and anions are

transported by plasma albumin.

6. Blood Coagulation: Prothrombin present in α2-globulin fraction and fibrinogen,

participate in the blood clotting process as follows.

Causes and consequences of protein content changes in blood plasma.

Hypoproteinemia - decrease of the total contents of proteins in blood

plasma. This state occurs in old people as well as in pathological states

accompanying with the oppressing of protein synthesis (liver diseases) and

activation of decomposition of tissue proteins (starvation, hard infectious diseases,

state after hard trauma and operations, cancer). Hypoproteinemia

(hypoalbuminemia) also occurs in kidney diseases, when the increased excretion of

proteins via the urine takes place.

Hyperproteinemia - increase of the total contents of proteins in blood

plasma. There are two types of hyperproteinemia - absolute and relative.

Absolute hyperproteinemia – accumulation of the proteins in blood. It occurs

in infection and inflammatory diseases (hyperproduction of immunoglobulins),

rheumatic diseases (hyperproduction of C-reactive protein), some malignant

tumors (myeloma) and others.

Relative hyperproteinemia – the increase of the protein concentration but not

the absolute amount of proteins. It occurs when organism loses water (diarrhea,

vomiting, fever, intensive physical activity etc.).

Paraproteinemia, also known as monoclonal gammopathy, is the presence

of excessive amounts of paraprotein or single monoclonal gammaglobulin in the

blood. It is usually due to an underlying immunoproliferative disorder or

hematologic neoplasms, especially multiple myeloma (presence of Bence Jones

protein).

Enzymes

Blood plasma contains many enzymes, which are classified into functional

and non-functional plasma enzymes.

142

Differences between functional and non-functional plasma enzymes

represents in table 1

Table 1

Functional plasma enzymes

Non-functional plasma

enzymes

Concentration in plasma

Present in plasma in higher

concentrations in comparison to

tissues

Normally, present in plasma in

very low concentrations in

comparison to tissues.

Function Have known functions No known functions

The substrates Their substrates are always present

in the blood

Their substrates are absent from

the blood

Site of synthesis Liver Different organs e.g. liver, heart,

brain and skeletal muscles

Effect of diseases Decrease in liver diseases Different enzymes increase in

different organ diseases

Examples

Clotting factors e.g. prothrombin,

Lipoprotein lipase and pseudo-

choline esterase

ALT, AST, CK, LDH, alkaline

phosphatase, acid phosphatase

and amylase,

Sources of non-functional plasma enzymes :

1. Increase in the rate of enzyme synthesis) e.g. bilirubin increases the rate of

synthesis of alkaline phosphatase in obstructive liver diseases.

2. Obstruction of normal pathway e.g. obstruction of bile ducts increases alkaline

phosphatase.

3. Increased permeability of cell membrane as in tissue hypoxia.

4. Cell damage with the release of its content of enzymes into the blood e.g.

myocardial infarction and viral hepatitis.

Medical importance of non-functional plasma enzymes :

Measurement of non-functional plasma enzymes is important for:

1. Diagnosis of diseases as diseases of different organs cause elevation of different

plasma enzymes.

2. Prognosis of the disease; we can follow up the effect of treatment by measuring

plasma enzymes before and after treatment.

Examples of medically important non-functional plasma enzymes :

1. Amylase and lipase enzymes increase in diseases of the pancreas as acute

143

pancreatitis.

2. Creatine kinase (CK) enzyme increases in heart, brain and skeletal muscle

diseases.

3. Lactate dehydrogenase (LDH) enzyme increases in heart, liver and blood

diseases.

4. Alanine transaminase (ALT) enzyme, it is also called serum glutamic pyruvic

transaminase (SGPT). It increases in liver and heart diseases.

5. Aspartate transaminase (AST) enzyme, it is also called serum glutamic

oxalacetic transaminase (SGOT). It increases in liver and heart diseases.

6. Acid phosphatase enzyme increases in cancer prostate.

7. Alkaline phosphatase enzyme increases in obstructive liver diseases, bone

diseases and hyperparathyroidism.

For example, high activity of LDH1,2, aspartate aminotransferase, creatine

phosphokinase (MB isoform) in the blood are caused by myocardial infarction.

Amino acids

These are formed from the break down of tissue proteins or from the

digestion of digested proteins. Significant proteolisys of proteins could lead to the

development of aminoacidemia (increasing of aminoacid content in blood).

Nitrogenous waste

Being toxic end products of the break down of substances in the body, these

are usually cleared from the bloodstream and are excreted by the kidneys at a rate

that balances their production.

Nutrients

Those absorbed by the digestive tract are transported in the blood plasma.

These include glucose, amino acids, fats, cholesterol, phospholipids, vitamins and

minerals.

Gases

Some oxygen and carbon dioxide are transported by plasma. Plasma also

contains a substantial amount of dissolved nitrogen.

Electrolytes

144

The most abundant of these are sodium ions, which account for more of the

blood’s osmolarity than any other solute.

Acid-base balance

The body's acid–base balance is normally tightly regulated by buffering

agents, the respiratory system, and the renal system, keeping the arterial blood pH

between 7.36 and 7.42. Several buffering agents that reversibly bind hydrogen ions

and impede any change in pH exist.

Acid-base imbalance

Acid–base imbalance is an abnormality of the human body's normal

balance of acids and bases that causes the plasma pH to deviate out of the normal

range (7.35 to 7.45). In the fetus, the normal range differs based on which

umbilical vessel is sampled (umbilical vein pH is normally 7.25 to 7.45; umbilical

artery pH is normally 7.18 to 7.38). It can exist in varying levels of severity, some

life-threatening.

An excess of acid is called acidosis or acidaemia and an excess in bases is

called alkalosis or alkalemia. The process that causes the imbalance is classified

based on the etiology of the disturbance (respiratory or metabolic) and the

direction of change in pH (acidosis or alkalosis).

Metabolic acidosis is a condition that occurs when the body produces

excessive quantities of acid or when the kidneys are not removing enough acid

from the body. If unchecked, metabolic acidosis leads to acidemia, i.e., blood pH is

low (less than 7.35) due to increased production of hydrogen ions by the body or

the inability of the body to form bicarbonate (HCO3−) in the kidney. Its causes are

diverse, and its consequences can be serious, including coma and death. Together

with respiratory acidosis, it is one of the two general causes of acidemia.

Metabolic acidosis occurs when the body produces too much acid (for

example, during intensive musle work too much lactate is produced), or when the

kidneys are not removing enough acid from the body. There are several types of

metabolic acidosis. The main causes are best grouped by their influence on the

anion gap.

145

It bears noting that the anion gap can be spuriously normal in sampling

errors of the sodium level, e.g. in extreme hypertriglyceridemia. The anion gap can

be increased due to relatively low levels of cations other than sodium and

potassium (e.g. calcium or magnesium).

Respiratory acidosis is a medical emergency in which decreased ventilation

(hypoventilation) increases the concentration of carbon dioxide in the blood and

decreases the blood's pH (a condition generally called acidosis).

Carbon dioxide is produced continuously as the body's cells respire, and this

CO2 will accumulate rapidly if the lungs do not adequately expel it through

alveolar ventilation. Alveolar hypoventilation thus leads to an increased PaCO2 (a

condition called hypercapnia). The increase in PaCO2 in turn decreases the

HCO3−/PaCO2 ratio and decreases pH.

Acute respiratory acidosis occurs when an abrupt failure of ventilation

occurs. This failure in ventilation may be caused by depression of the central

respiratory center by cerebral disease or drugs, inability to ventilate adequately due

to neuromuscular disease (e.g., myasthenia gravis, amyotrophic lateral sclerosis,

Guillain-Barré syndrome, muscular dystrophy), or airway obstruction related to

asthma or chronic obstructive pulmonary disease (COPD) exacerbation.

Chronic respiratory acidosis may be secondary to many disorders, including

COPD. Hypoventilation in COPD involves multiple mechanisms, including

decreased responsiveness to hypoxia and hypercapnia, increased ventilation-

perfusion mismatch leading to increased dead space ventilation, and decreased

diaphragm function secondary to fatigue and hyperinflation.

Metabolic alkalosis is a metabolic condition in which the pH of tissue is

elevated beyond the normal range (7.35-7.45). This is the result of decreased

hydrogen ion concentration, leading to increased bicarbonate, or alternatively a

direct result of increased bicarbonate concentrations.

The causes of metabolic alkalosis can be divided into two categories, depending

upon urine chloride levels.

146

Chloride-responsive (Urine chloride < 20 mEq/L)

o Loss of hydrogen ions - Most often occurs via two mechanisms, either vomiting

or via the kidney. Vomiting results in the loss of hydrochloric acid (hydrogen and

chloride ions) with the stomach contents. In the hospital setting this can commonly

occur from nasogastric suction tubes. Severe vomiting also causes loss of

potassium (hypokalaemia) and sodium (hyponatremia). The kidneys compensate

for these losses by retaining sodium in the collecting ducts at the expense of

hydrogen ions (sparing sodium/potassium pumps to prevent further loss of

potassium), leading to metabolic alkalosis.

Congenital chloride diarrhea - rare for being a diarrhea that causes alkalosis

instead of acidosis.

Contraction alkalosis - This results from a loss of water in the extracellular

space, such as from dehydration.

Diuretic therapy - loop diuretics and thiazides can both initially cause increase

in chloride, but once stores are depleted, urine excretion will be below < 25

mEq/L.

Posthypercapnia - Hypoventilation (decreased respiratory rate) causes

hypercapnia (increased levels of CO2), which results in respiratory acidosis.

Chloride-resistant (Urine chloride > 20 mEq/L)

Retention of bicarbonate - retention of bicarbonate would lead to alkalosis

Shift of hydrogen ions into intracellular space - Seen in hypokalemia. Due to a

low extracellular potassium concentration, potassium shifts out of the cells. In

order to maintain electrical neutrality, hydrogen shifts into the cells, raising blood

pH.

Alkalotic agents - Alkalotic agents, such as bicarbonate (administrated in cases

of peptic ulcer or hyperacidity) or antacids, administered in excess can lead to an

alkalosis.

Hyperaldosteronism - Renal loss of hydrogen ions occurs when excess

aldosterone (Conn's syndrome) increases the activity of a sodium-hydrogen

exchange protein in the kidney.

147

Respiratory alkalosis is a medical condition in which increased respiration

elevates the blood pH beyond the normal range (7.35-7.45) with a concurrent

reduction in arterial levels of carbon dioxide. This condition is one of the four

basic categories of disruption of acid-base homeostasis

Respiratory alkalosis may be produced as a result of the following causes:

stress, pulmonary disorder, thermal insult, fever, hyperventilation, liver disease.

The presence of only one of the above derangements is called a simple acid–

base disorder. In a mixed disorder more than one is occurring at the same time.

Mixed disorders may feature an acidosis and alkosis at the same time that partially

counteract each other, or there can be two different conditions affecting the pH in

the same direction.

The body's acid–base balance is tightly regulated. Several buffering agents

exist which reversibly bind hydrogen ions and impede any change in pH.

Extracellular buffers include bicarbonate and ammonia, while proteins and

phosphate act as intracellular buffers. The bicarbonate buffering system is

especially key, as carbon dioxide (CO2) can be shifted through carbonic acid

(H2CO3) to hydrogen ions and bicarbonate (HCO3−).

Acid–base imbalances that overcome the buffer system can be compensated

in the short term by changing the rate of ventilation. This alters the concentration

of carbon dioxide in the blood, shifting the above reaction according to Le

Chatelier's principle, which in turn alters the pH. For instance, if the blood pH

drops too low (acidemia), the body will compensate by increasing breathing,

expelling CO2, and shifting the reaction above to the right such that fewer

hydrogen ions are free – thus the pH will rise back to normal. For alkalemia, the

opposite occurs.

Acute-phase proteins

Acute-phase proteins are a class of proteins whose plasma concentrations

increase (positive acute-phase proteins) or decrease (negative acute-phase proteins)

in response to inflammation. This response is called the acute-phase reaction (also

called acute-phase response).

148

In response to injury, local inflammatory cells (neutrophil granulocytes and

macrophages) secrete a number of cytokines into the bloodstream, most notable of

which are the interleukins IL1, IL6 and IL8, and TNFα. The liver responds by

producing a large number of acute-phase reactants. At the same time, the

production of a number of other proteins is reduced; these are, therefore, referred

to as "negative" acute-phase reactants. Increased acute phase proteins from the

liver may also contribute to the promotion of sepsis

Positive acute-phase proteins serve (part of the innate immune system)

different physiological functions for the immune system. Some act to destroy or

inhibit growth of microbes, e.g., C-reactive protein, mannose-binding protein,[2]

complement factors, ferritin, ceruloplasmin, serum amyloid A and haptoglobin.

Others give negative feedback on the inflammatory response, e.g. serpins. Alpha 2-

macroglobulin and coagulation factors affect coagulation, mainly stimulating it.

This pro-coagulant effect may limit infection by trapping pathogens in local blood

clots. Also, some products of the coagulation system can contribute to the innate

immune system by their ability to increase vascular permeability and act as

chemotactic agents for phagocytic cells.

Measurement of acute-phase proteins, especially C-reactive protein, is a

useful marker of inflammation in both medical and veterinary clinical pathology.

It correlates with the erythrocyte sedimentation rate (ESR), however not always

directly. This is due to the ESR being largely dependent on elevation of

fibrinogen, an acute phase reactant with a half-life of approximately one week.

This protein will therefore remain higher for longer despite removal of the

inflammatory stimuli. In contrast, C-reactive protein (with a half-life of 6-8 hours)

rises rapidly and can quickly return to within the normal range if treatment is

employed. For example, in active systemic lupus erythematosus, one may find a

raised ESR but normal C-reactive protein. They may also indicate liver failure.

During rheumatoid arthritis in the blood appear additive glycosaminoglycans as

acute-phase proteins

149

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test: Explanation:

1 12 hours after an acute attack of

retrosternal pain a patient presented

a jump of aspartate aminotransferase

activity in blood serum. What

pathology is this deviation typical

for?

A. Viral hepatitis

B. Diabetes insipidus

C. Collagenosis

D. Diabetes mellitus

E. Myocardial infarction

2 A patient who had been working

hard under condition of elevated

temperature of the environment has

now a changed quantity of blood

plasma proteins. What phenomenon

is the case?

A. Absolute hyperproteinemia

B. Relative hyperproteinemia

C. Absolute hypoproteinemia

D. Disproteinemia

E. Paraproteinemia

150

№ Test: Explanation:

3 62 y.o. woman complains of

frequent pains in the area of her

chest and backbone, rib fractures. A

doctor assumed myelomatosis

(plasmocytoma).What of the

following laboratory characteristics

will be of the greatest diagnostic

importance?

A. Proteinuria

B. Hypoproteinemia

C. Hypoglobunemia

D. Hyperalbuminemia

E. Paraproteinemia

4 Diabetes mellitus causes ketosis as a

result of activated oxidation of fatty

acids. What disorders of acid-base

equilibrium may be caused by

excessive accumulation of ketone

bodies in blood?

A. Metabolic alkalosis

B. Metabolic acidosis

C. Respiratory alkalosis

D. Respiratory acidosis

E. Any changes won't happen

151

№ Test: Explanation:

5 A 63-year-old woman developed

symptoms of rheumatoid arthritis.

Their increase of which blood values

indicators could be most significant

in proving the diagnosis?

A. R-glycosidase

B. Acid phosphatase

C. Lipoproteins

D. General cholesterol

E. Additive glycosaminoglycans

6 Marked increase of activity of MB-

forms of CPK

(creatinephosphokinase) and LDH-1

was revealed by examination of the

patient's blood. What is the most

probable pathology?

A. Myocardial infarction

B. Hepatitis

C. Pancreatitis

D. Rheumatism

E. Cholecystitis

152

№ Test: Explanation:

7 There is high activity of LDH1,2,

aspartate aminotransferase, creatine

phosphokinase in the blood of

patient. In what organs (tissues) the

development of pathological process

is the most probable?

A. In the heart muscle {initial stage

of myocardium infraction}

B. In skeletal muscle {dystrophy,

atrophy}

C. In kidneys and adrenals

D. In liver and kidneys

E. In connective tissue

8 The high level of Lactate

Dehydrogenase (LDH) isozymes

concentration showed the increase

of LDH-1 and LDH-2 in a patient’s

blood plasma. Point out the most

probable diagnosis.

A. Diabetes mellitus

B. Skeletal muscle dystrophy

C. Myocardial infarction

D. Acute pancreatitis

E. Viral hepatitis

153

№ Test: Explanation:

9 Analysis of blood serum of a patient

revealed the increase of alanine

aminotransferase and aspartate

aminotransferase levels. What

cytological changes can cause such a

situation?

A. Disturbance of genetic apparatus

of cells

B. Cellular breakdown

C. Disorder of enzyme systems of

cells

D. Disturbance of cellular

interrelations

E. Disturbed energy supply of cells

10 A worker has decreased buffer

capacity of blood due to exhausting

muscular work. What acidic

substance that came to blood caused

this phenomenon?

A. 3-phosphoglycerate

B. 1,3-bisphosphoglycerate

C. Lactate

D. α-ketoglutarate

E. Pyruvate

154

№ Test: Explanation:

11 Blood sampling for bulk analysis is

recommended to be performed on an

empty stomach and in the morning.

What changes in blood composition

can occur if to perform blood

sampling after food intake?

A. Reduced contents of erythrocytes

B. Increased contents of erythrocytes

C. Increased contents of leukocytes

D. Increased plasma proteins

E. Reduced contents of

thrombocytes

12 Examination of a 43 y.o. anephric

patient revealed anemia symptoms.

What is the cause of these

symptoms?

A. Folic acid deficit

B. Vitamin B12 deficit

C. Reduced synthesis of

erythropoietins

D. Enhanced destruction of

erythrocytes

E. Iron deficit

155

№ Test: Explanation:

13 A 55 y.o. women consulted a doctor

about having continuous cyclic

uterine hemorrhages for a year,

weakness, dizziness. Examination

revealed skin pallor. Hemogram: Hb

– 70 g/L, erythrocytes-3.2 x 1012

/L,

color index – 0.6; leukocytes – 6.0 x

109/L, reticulocytes – 1%,

erythrocyte hypochromia. What

anemia is it?

A. Iron-deficiency anemia

B. B12-folate-deficiency anemia

C. Hemolytic anemia

D. Aplastic anemia

E. Chronic posthemorrhagic anemia

14 Blood plasma of healthy man

contains several dozens of proteins.

During an illness new proteins can

originate named as the ―proteins of

acute phase». Select such protein

from the listed below:

A. Albumin

B. Immunoglobulin G

C. Immunoglobulin E

D. C-reactive protein

E. Prothrombin

156

№ Test: Explanation:

15 A patient complains about dyspnea

provoked by the physical activity.

Clinical examination revealed

anaemia and presence of the para-

protein in the zone of gamma-

globulins. To confirm the myeloma

diagnosis it is necessary to determine

the following index in the patient’s

urine:

A. Ceruplasmin

B. Bilirubin

C. Antitrypsin

D. Bence Jones protein

E. Haemoglobin

16 Examination of 27-year-old patient

revealed pathological changes in

liver and brain. Blood plasma

analysis revealed an abrupt decrease

in the copper concentration, urine

analysis revealed an increased

copper, concentration. The patient

was diagnosed with Wilson’s

degeneration. To confirm the

diagnosis it is necessary to study the

activity of the following enzyme in

blood serum:

A. Leucine aminopeptidase

B. Xanthine oxidase

157

№ Test: Explanation:

C. Alcohol dehydrogenase

D. Ceruloplasmin

E. Carbonic anhydrase

17 After a surgery a 36-year-old woman

was given an intravenous injection

of concentrated albumin solution.

This has induced intensified water

movement in the following direction:

A. From the intercellular fluid to the

capillaries

B. No changes of water movement

will be observed

C. From the intercellular to the cells

D. From the cells to the intercellular

fluid

E. From the capillaries to the

intercellular fluid

18 Electrophoretic study of a blood

serum sample, taken from the patient

with pneumonia, revealed an

increase in one of the protein

fractions. Specify this fraction:

A. γ-globulins

B. Albumins

C. α1-globulins

D. β-globulins

E. α2-globulins

158

№ Test: Explanation:

19 Examination of a 56-year-old female

patient with a history of type 1

diabetes revealed a disorder of

protein metabolism that is

manifested by aminoacidemia in the

laboratory blood test values, and

clinically by the delayed wound

healing and decreased synthesis of

antibodies. Which of the following

mechanisms causes the development

of aminoacidemia?

A. Increased proteolysis

B. Decrease in the concentration

of amino acids in blood

C. Albuminosis

D. Increase in the oncotic

pressure in the blood plasma

E. Increase in low-density

lipoprotein level

20 A 49-year-old male patient with

acute pancreatitis was likely to

develop pancreatic necrosis, while

active pancreatic proteases were

absorbed into the blood stream and

tissue proteins broke up. What

protective factors of the body can

inhibit these processes?

A. Immunoglobulin

159

№ Test: Explanation:

B. Ceruloplasmin, transferrin

C. a2-macroglobulin, a1-antitrypsin

D. Cryoglobulin, interferon

E. Hemopexin, haptoglobin

21 A patient is diagnosed with

hereditary coagulopathy that is

characterized by factor VIII

deficiency. Specify the phase of

blood clotting during which

coagulation will be disrupted in the

given case:

A. Clot retraction

B. Thromboplastin formation

C. Fibrin formation

D. Thrombin formation

22 A 67-year-old male patient

consumes eggs, pork fat, butter, milk

and meat. Blood test results:

cholesterol – 12.3 mmol/l, total

lipids – 8.2 g/l, increased low-

density lipoprotein fraction (LDL).

What type of hyperlipoproteinemia

is observed in the patient?

A. Hyporlipoproteinemia type I.

B. Hyperlipoproteinemia type IV

C. Cholesterol, hyperlipoproteinemia

D. Hyperlipoproteinemia type IIa

E. Hyperlipoproteinemia type IIb

160

№ Test: Explanation:

23 Human red blood cells do not

contain mitochondria. What is the

main pathway for ATP production in

these cells?

A. Creatine kinase reaction

B. Anaerobic glycolysis

C. Cyclase reaction

D. Aerobic glycolysis

E. Oxidative phosphorylation

24 A 28-year-old patient undergoing

treatment in a pulmonological

department has been diagnosed with

pulmonary emphysema caused by

splitting of alveolar septum by

tripsin. The disease is caused by the

congenital deficiency of the

following protein:

A. Alpha-1-proteinase inhibitor

B. Haptoglobin

C. Cryoglobulin

D. Alpha-2-macroglobulin

E. Transferrin

25 Biochemical analysis of an infant`s

erythrocytes revealed evident

glutathione peroxidase deficiency

and low concentration of reduced

glutathione. What pathological

condition can develop in this infant?

161

№ Test: Explanation:

A. Hemolytic anemia

B. Megaloblastic anemia

C. Siclemia

D. Iron-deficiency anemia

E. Pernicous anemia

26 Lymphocytes and other cells of our

body synthesize universal antiviral

agents as a response to viral

invasion. Name this protein factors

A. Interferon

B. Tumor necrosis factor

C. Cytokines

D. Interleukin-2

E. Interleukin-4

162

THE ROLE OF KIDNEYS IN THE REGULATION

OF WATER AND SALTS METABOLISM.

THE NORMAL AND PATHOLOGICAL COMPONENTS OF URINE

(Krisanova N. V.)

INFORMATIONAL MATERIAL

STRUCTURAL ORGANIZATION OF KIDNEY TISSUE

AND ITS FUNCTIONS

Renal tissue is divided in two types:

1) outer or cortical coloured brown-red;

2) inner or medullary coloured lilac-red.

Nephron is the functional unit of renal parenchyma, the two kidneys of human

number about 2 million nephrons. Cortical nephrons are about 85% of the total

number; about 15% of total number is juxtamedullary nephrons whose glomeruli

are located at the boundary between the cortex and medulla of the kidney

Figure 1. A structural unit of kidney tissue - a nephron.

163

The kidney is involved in:

1) the regulation of water and salt balance;

2) the maintenance of acid-base balance and of osmotic pressure of fluid

media of the organism;

3) the removal of terminal products of metabolic processes;

4) the blood pressure control;

5) the stimulation of erythropoiesis, etc;

6) in hormonal control of a lot metabolic pathways..

Filtration at the glomerulus, tubular reabsorption and tubular secretion are

observed in the nephron

Primary urine is formed due to filtration process. The composition of primary

urine is very similar to blood plasm, but there is no any protein in the primary

urine. The pores in the glomerular basal membrane, which are made up by collagen

type IV, have an effective mean diameter of 2.9 nm. This allows all plasma

components with a molecular mass about 15 kDa to pass through the membrane,

that is because proteins are completely unable to enter to primary urine.

Reabsorption. The most of all low-molecular weight plasma components are

transported back into the blood by reabsorption, to prevent losses of valuable

metabolites and electrolytes. In the proximal tubule, organic metabolites (glucose,

amino acids, lactate, ketone bodies) are recovered by secondary active transport.

There are several group-specific transport systems for resorbing amino acids, with

which hereditary disorders can be associated (cystinuria, glycinuria, cystinosis,

Hartnup`s disease). Bicarbonate, sodium ion, phosphate and sulfate are also

resorbed by ATP dependent active mechanisms in the proximal tubule. The later

sections of nephron may only serve for additional water recovery and for regulated

resorption of Na+ and Cl

-. These processes are controlled by hormones.

Secretion helps also to form final urine. Some excreted substances are released

into the urine by active transport in the renal tubules: protons, potassium ions, urea,

creatinine, some drugs (penicillin).

164

These two processes help to keep useful substances and to maintain acid-base

balance for organism. The molecular mechanism for resorption and secretion of

materials by renal tubular cells are quite well understood.

Processes associated with nephron function:

Ultrafiltration of blood at the glomerulus

Tubular reabsorption

Tubular secretion

Primary urine (~170L/day)

Final urine (~2L/day)

Figure 2. Main processes promoted by the function of nephron.

THE FUNCTION OF KIDNEY IN MAINTENANCE OF ACID-BASE

BALANCE IN ORGANISM

The renal tubule cells are capable of secreting protons (H+) from the blood into the

urine against a concentration gradient. Despite the fact that the H+ concentration in

the urine is up to a thousand times higher than in the blood.

To achieve this, carbon dioxide (CO2) is taken up from the blood and together

with water (H2O) and with a help of carbonate dehydratase is converted into

hydrogen carbonate (bicarbonate, HCO3-) and one proton H

+. Formally, this yields

carbonic acid, but it is not released during the reaction. The hydrogen carbonate

returns to the plasma where it contributes to the blood base reserve. The proton is

exported into the urine by secondary active transport in anti-port for Na+. The

driving force for proton excretion is Na+ gradient established by the Na

+, K

+-

ATPase. This integral membrane protein on the basal side (towards the blood) of

tubule cells keeps the Na+ concentration in the tubule cell low, thereby maintaining

Na+ inflow. In addition to this secondary active H+ transport mechanism, there is

a V-type H+-transporting ATPase in the distal tubule and collecting duct.

165

An important function of the secreted H+ ions is to promote HCO3

- reabsorption.

Hydrogen carbonate, the most important buffering base in the blood, passes into

primary urine quantitatively, like all ions. In the primary urine HCO3- reacts with

proton ion to form water and carbon dioxide, which returns by free diffusion to the

tubule cells and from there into the blood. In this way kidneys also influence the

CO2/HCO3- buffering balance in the plasma.

Figure 3. Sodium, bicarbonate ions reabsorption (1, 2) and phosphates buffering

(2) is in close relation to protons secretion.

Approximately 60 mmol of protons are excreted with urine every day. Buffering

systems in the urine catch a large proportion of H+ ions, so that the urine becomes

weakly acidic (down to about pH=4.8). An important buffer in the urine is the

hydrogen phosphate/dihydrogen phosphate system. The conversion of disubstituted

phosphates to mono substituted phosphates is in need to keep sodium ions and

calcium ions for human organism and to remove the accumulated protons from the

blood at acidosis state.

In addition, ammonia also makes a vital contribution to buffering the secreted

protons. Since plasma concentration of ammonia are low, the kidneys release

ammonia from glutamine due to glutaminase action and during oxidative

deamination of glutamate:

1 2

166

Ammonia can diffuse freely into the urine through the tubule membrane while the

ammonium ions that are formed as charged particles and can no longer return to

the cell. Acidic urine therefore promotes ammonia excretion which is normally 30-

50 mmol per day.

At metabolic acidosis glutaminase activity usually is induced and ammonium ions

excretion is increased. At metabolic alkalosis renal excretion of ammonia is

reduced. But the production of the urea

The excess levels of hydrogen ions are considered at humans during starvation and

metabolic acidosis caused by the accumulation of some substances such as lactate,

pyruvate, some ketone bodies, amino acids and others. This condition causes the

stimulation of gluconeogenesis in kidney, and this way is considered as the way for

maintenance of acid-base balance, too. The synthesized glucose is very important

source of ATP (due to oxidative phosphorylation after glucose aerobic oxidation

way) that is used for the active transport mechanism.

METABOLIC PATHWAYS AND ENERGY FORMATION IN KIDNEY

The main energy sources are glucose, fatty acids, ketone bodies, some amino acids.

To a lesser extent lactate, glycerol, and citric acid are used. The endothelial cells in

the proximal tubule are capable of gluconeogenesis from amino acids mainly.

Amino groups of amino acids are used as ammonia for buffering of urine.

Enzymes for protein degradation and the amino acid metabolism occur in the

Glutamine Glytamic acid + NH3

Glutaminase

+ NH3H+

H2O

NH4+

GlutamateGlutamate

dehydrogenase

NAD+

NADH+H+

Alpha-ketoglutarate + NH3

167

kidney at high levels of activity (amino acid oxidases, amino oxidases,

glutaminase). The most important metabolic pathways for kidney tissue are:

• Aerobic oxidation of monosacharides

• Gluconeogenesis

• Hexose Monophosphate Shunt

• Fatty acids oxidation

• Ketone bodies utilization

• Replication;Transcription;Translation

• Transport systems function in cellular membrane

• Antioxidant enzyme systems function

KIDNEYS AND HORMONES

Kidneys have also endocrine function (fig.4). They produce erythropoietin and

calcitriol and play a decisive part in producing the hormone angioteinsin II by

releasing the enzyme rennin.

The activity of calcidiol-1-monooxygenase (hydroxylase) is enhanced by the

hormone PTH. Calcitriol stimulates the resorption of both calcium and phosphates

ions in renal tubules. The proportion of Ca2+

resorbed is over 99%, while for

phosphate the figure is 80-90%. PTH stimulates resorption of Ca2+

but inhibits the

resorption of phosphate.

The erythropoietin is a peptide hormone that is formed predominantly by the

kidneys, but also by the liver. Together with colony-stimulating factors it regulates

the differentiation of stem cells in the bone marrow. Erythropoietin release is

stimulated by hypoxia. The hormone ensures that erythrocyte precursor cells in the

bone marrow are converted to erythrocytes, so that their numbers in the blood

increase. Renal damage leads to reduced erythropoietin release which in turn

results in anemia. Forms of anemia with renal causes can now be successfully

treated using erythropoietin produced by genetic engineering techniques. The

hormone is also administered to dialysis patients.

168

The angiotensin II (A-II) is not secreted by any hormonal gland, it is produced in

the blood from precursor angiotensin I secreted by kidney tissue. Angiotensin I is

produced from angiotensinogen (a plasma glycoprotein in the alpha-2-fraction

synthesized in the liver) due to enzyme rennin in kidney tissue. Angiotensin I is

cleaved by peptidyl dipeptidase A (a membrane enzyme located on the vascular

endothelium in the lungs and other tissues) to form octapeptide A- II. The plasma

level of A II is mainly determined by the rate at which rennin is released by

kidneys. The production of rennin by juxtaglomerular cells is when sodium ion

levels decline in blood plasma or there is a fall in blood pressure.

Hypophysis

Parathyroidal

gland

Adrenal cortex

Vasopressin

PTH

Aldosterone

Erythropoietin

Calcitriol

Rennin

Bone marrow

GIT

Bone tissue

Renal tubules

AngiotensinogenAngiotensin I

Angiotensin II

I

Kidney

Blood

Figure 4.The role of kidney in hormonal regulation

A-II has effects on the kidneys, brain stem, pituitary gland, adrenal cortex, blood

vessel walls and heart via membrane-located receptors.

In the brain stem and at nerve endings in sympathetic nervous system the effects of

A-II lead to increased tonicity (neurotransmitter effect). In pituitary gland A-II

stimulates vasopressin and ACTH secretion. In adrenal cortex stimulation of

aldosterone synthesis and secretion.

All of the effects of angiotensin II lead directly or indirectly to increased blood

pressure as well as increased sodium ions and water retention. This important

169

hormonal system for blood pressure regulation may be pharmacologically

influenced by inhibitors at various points:

using angiotensinogen analogs that inhibit rennin;

using angiotensin I analogs that competitively inhibit peptidyl

dipeptidase A;

using hormone antagonists that block the binding of A-II to its

receptors.

The regulation of sodium, potassium ions, chloride ions content and water volume

in the blood depends on the secretion of some hormones: Aldosterone, Atrial

natriuretic peptide, Vasopressin. Kidney is the main target for them. Controlled

resorption of sodium ions from primary urine is one of the most important

functions of the kidney. Sodium ion resorption is highly effective with more than

97% being resorbed. Several mechanisms are involved: some portion of Na+ ions

is taken up passively in the proximal tubule through the junctions between cells.

There is secondary active transport together with glucose and amino acids, too.

These two pathways are responsible for 60-70% of total Na+ resorption. In

ascending part of Henle`s loop there is another transporter which takes up one Na+

ion and one K+ ion together with two Cl- ions. This symport is also dependent on

the activity of Na+/K+-ATPase, which pumps the Na+ resorbed from primary

urine into the plasma in exchange for K+. This transport system is controlled by

aldosterone an atrial natriuretic peptide.

Water resorption in the proximal tubule is a passive process in which water follows

the osmotically active particles, particularly the Na+. Final regulation of water

excretion takes place in cells of distal tubules and the collecting ducts where the

peptide hormone vasopressin operates.

CLEARANCE OF KIDNEYS

Renal clearance is used as quantitative measure of renal function. It is defined as

plasma volume cleared of a given substance per unit of time. Inulin (fructose

polysaccharide with M=6 kDa) or creatinine is used for the determination of this

170

index. These organic compounds are not reabsorbed in kidneys, and ratio of their

content in the urine to the content in the blood plasma estimates the rate of

glomerular filtration in patient. The index is named as the clearance © of kidney.

You can see the formula of its determination:

C=UCr *V

PCr

ml/min

UCr - concentration of Creatinine in the urine;PCr - concentration of Creatinine in the blood plasm;V -minute diuresis.

Normal clearance index in adults equals 120 ml/min. It is usually determined at

patients which may be potential donors of kidney; in a case of to choose the initial

dose of some toxic drug to treat patients.

INDEXES OF THE BLOOD PLASMA AND URINE TO ESTIMATE KIDNEY

FUNCTION

Creatinine and urea contents in the blood plasma are very important indexes for

glomeruli function estimation. There is the increase of both indexes in the blood

plasma at renal insufficiency in patients.

Urea, as you remember, is a relatively nontoxic substance made by the liver as a

means of disposing of ammonia from protein metabolism.

Urea has MW 60, of which 28 comes from the two nitrogen atoms. Normal blood

urea nitrogen is 8-25 mg/dL (2.9-8.9 mmol/L).

Blood urea levels are quite sensitive indicators of renal disease, becoming

elevated when renal function drops to around 25-50% of normal (remember the

kidney has great functional reserve).

Increased BUN is, by definition, azotemia. It is due either to increased protein

catabolism or impaired kidney function. Increased protein catabolism results from:

a really big protein meal

severe stress (myocardial infarction, high fever, etc.)

upper GI bleeding (blood being digested and absorbed)

171

Impaired kidney function may be "prerenal", "renal", or "postrenal". Prerenal

azotemia results from under-perfusion of the kidney: dehydration, hemorrhage,

shock, congestive heart failure; glomerulonephritis is likely also to be "prerenal" if

mild, since it comprises renal blood flow more than tubular function

Renal azotemia has several familiar causes: acute tubular necrosis, chronic

interstitial nephritis, some glomerulonephritis, etc.

Postrenal azotemia results from obstruction of urinary flow:

prostate trouble, stones, surgical mishaps, tumors .

In acute renal failure, BUN increases around 20 mg/dL each day .

ENZYMES OF BLOOD PLASMA AND URINE TO PROVE SOME

PATHOLOGIC STATES OF KIDNEY

The most active enzymes of kidney are involved in aerobic type of metabolic

processes to produce energy in a form of ATP, and the enzymes used for all type of

transport across the membranes of glomeruli, renal tubules cells.

Glycine amidinotransferase - (first enzyme from creatine synthesis). It is used in

diagnostic of kidney parenchyma damage (its activity is increased in the blood

serum).

N-acetyl-beta-D-glucosaminidase ("glucosaminidase", NAG) is a lysosomal

enzyme (MW 140,000) found in serum and urine. Urinary NAG is a proposed

marker for tubular disease, especially subtle industrial poisoning, acute

pyelonephritis, early acute tubular necrosis, and early transplant rejection.

Lactate dehydrogenase isozymes: In acute renal insufficiency the activity of

LDH1 and LDH2 is observed to increase. LDH1 and LDH2 isozymes are from renal

cortex.LDH4 and LDH5 isozymes found in kidney medulla.

Alanine aminopeptidase isozyme 3 (AAP3) in the blood plasma and urine is

observed as the specific sign of the affected kidney tissue.

Adenosine Deaminase Binding Protein is an enzyme from the brush borders of

the proximal tubule. Like NAG, its presence in urine indicates tubular disease.

172

Urinary alkaline phosphatase in urine comes from the proximal tubular brush

border, detects tubular necrosis too.

THE CHEMICAL COMPOSITION OF URINE OF HEALTHY ADULTS

1. Ions: Na+,

K+. Ca

2+, Mg

2+, SO4

2-, HCO3

-, HPO4

2-, H2PO4

-, PO4

3- and

water.

2. The main nitrogen-containing compounds are ammonia salts and

urea, other ones: uric acid, creatinine, amino acids, hippuric acid, stercobilin,

indican.

3. Nitrogen free organic compounds such as acids: lactic, pyruvic,

citric, oxalic, acetoacetic, and others.

4. Hormone derivatives such as 17-ketosteroids and others.

5. Terminal products of xenobiotics transformation.

6. Some vitamins and their derivatives.

THE PHYSICOCHEMICAL PROPERTIES OF THE URINE OF HEALTHY

AND DISEASED HUMANS

1. Diuresis (urine output)

It is the average volume of urine (ml) excreted by individual person under ordinary

dietary condition in 24 hours or per day. Normal diuresis: for men – 1500 ml/day;

for women – 1200 ml/day.

Polyuria state is indicated in patients when urine output is much higher than

normal (more then 3 L/day). It is considered at patients with chronic nephritis,

diabetes insipidus, chronic pyelonephritis.

Oligouria state is the diminished excretion of daily urine, and it is observed at

patients with febrile state, toxicosis, diarrhea, vomiting, and acute nephritis.

Anuria (nearly complete suppression of urinary excretion) is observed at patients:

1) under nervous shock; 2) at acute diffuse nephritis caused by poisoning with

lead, mercury or arsenic compounds.

173

2. The pH of urine

All the acids, ammonia salts and urea make special pH of urine; the average value

of it is about 5,3-6,5. The pH of urine depends on the diet of patient. Strong

vegetarians` urine in pH is higher then 6,5. After animal food intake the pH of

urine changes to value lower then 5, 3. The pH of urine may be decreased at

diabetes mellitus associated with ketoacidosis, at diseases accompanied with

extensive excretion of amino acids (aminoaciduria state). The alkaline urine is

observed in cystitis and pyelitis, at intake of some drugs, also as sequent to strong

vomiting. The pH of the urine is inversely proportional to the acidity of the urine .

3. The specific gravity of urine

The normal value must be in region 1,012 – 1,020 g/ml. The specific gravity of

urine may be very low (about 1,001-1,004 g/ml) in patients with diabetes insipidus.

At oligouria state it may be lower then normal, too. Polyuria state at patients with

diabetes mellitus may be accompanied with the increase of this index (higher then

1,020 g/ml) at the expense of glucose present in the urine.

4. The color of the urine

The urine of healthy humans is transparent, straw yellow or amber liquid. The

presence of pigments such as stercobilin, urochrome, uroerythrin gives those

colour for urine. Abnormal pigments observed in the urine at pathologic states can

change it to colour:

1) dark (urobilin formed from excess urobilinogen that is not transformed in

the liver at liver parenchyma damage);

2) green or blue (intensive putrefaction of proteins in the intestine causes the

accumulation of indoxyl sulpharic acid in the urine); uroglaucin at scarlatina state;

3) dark brown like beer (there is the conjugated bilirubin presence in the urine);

4) black (due to presence of homogentisic acid oxidation product at

Alkaptonuria state);

5) red shade at presence of blood pigments, they include red blood cells

(hematuria state) and hemoglobin (hemoglobinuria state).These pigments are

observed at the damage of urinary tracts by kidney stones or at acute cystitis.

174

The urine may be not transparent when sediments are present in it. It may be at

pathologies associated with the damage of urinary tracts by kidney stones, with the

accumulation in the urine some salts (calcium oxalates), with the appearance of

epithelial cells in the urine and with excretes from vagina of women.

5. Special smell of the urine

The urine slight smell is associated with the presence of ammonia salts and urea in

it usually. But it may be changed at:

1) Maple syrup urine disease (like maple syrup odor);

2) Phenylketonuria (like mouse odor);

3) Intensive putrefaction of proteins in the intestine (the smell of rotten meat);

4) Glucosuria state at diabetes mellitus (special fruity odor);

5) The appearance of excretes from vagina of diseased women at pathologies

such as syphilis and gonorrhoea can also change the smell of the urine (like

the smell of rotten meat).

THE PATHOLOGICAL COMPONENTS OF THE URINE

Proteins of the urine

Proteins are found in minimal amounts (less 150 mg/day) in the urine of healthy

people and they can’t be detected by color reactions used for proteins. If the color

reaction for proteins is positive in the urine, this component is considered as

pathological and proves proteinuria state. This state is determined at patients with

acute glomerular nephritis; extra renal reasons: inflammation of urinary tracts,

affected prostate gland, at the burns and fever, at the trauma of urinary tract

(hemoglobinuria). Proteinuria state is accompanied with the change of

physicochemical properties of the urine: 1) hemoglobinuria is associated with

pink-red color of the urine; 2) the density of urine becomes higher then normal at

proteinuria state at the expense of proteins; 3) the urine has the big foam after

shaking.

175

Ketone bodies

In minimal amounts they are at healthy people but not detected by color reactions

in the urine. If the color reaction for ketone bodies in the urine is positive they are

considered as pathologic components. The high levels of them are accompanied

with long time starvation or with diabetes mellitus (severe form). The pH of urine

becomes lower then normal in this case.

Glucose

It is practically absent in the urine of healthy people, but is observed at patients

with diabetes mellitus (all types) when the levels of glucose in the blood are higher

then 9,5 mmole/L. Glucosuria state is observed in patients in this case. Polyuria

state in this case accompanied with the increase of urinary density (higher then

1,020 g/ml) at the expense of glucose present in the urine.

Bile pigments

Urobilin formed from excess urobilinogen and conjugated bilirubin are

pathological components and must be absent in the urine of healthy persons. Their

appearance in the urine first of all is the signal for the problems with liver function,

and is accompanied with the jaundice development in patient. The color of the

urine is changed at their presence (see above).

Creatine

It is practically absent in the urine of healthy adults, and is considered as

pathologic component. Creatine is determined in the urine at developed muscular

dystrophy in patients and at old people with the deficiency of motor function for

skeletal muscles in person.

176

EXERCISES FOR INDEPENDENT WORK. In the table with test tasks

emphasize keywords, choose the correct answer and justify it:

№ Test tasks: Explanations:

1. A 34-year-old patient was diagnosed

with chronic glomerulonephritis 3

years ago. Edema has developed

within the last 6 months. What

caused the edema?

A. Liver dysfunction of protein

formation

B. Hyperosmolarity of plasma

C. Proteinuria

D. Hyperproduction of vasopressin

E. Hyperaldosteronism

2. Examination of a 43 y.o. anephric

patient revealed anemia symptoms.

What is the cause of these

symptoms?

A. Folic acid deficit

B. Vitamin B12 deficit

C. Reduced synthesis of erythro-

poietin

D. Enhanced destruction of

erythrocytes

E. Iron deficit

3. A biochemical urine analysis has

been performed for a patient with

progressive muscular dystrophy. In

the given case muscle disease can be

177

№ Test tasks: Explanations:

confirmed by the high content of the

following substance in urine:

A. Urea

B. Porphyrin

C. Hippuric acid

D. Creatine

E. Creatinine

4. Kidney insufficiency in patient is

accompanied with:

A. Excess levels of urea in the blood

plasma

B. Excess levels of potassium ions

in the blood plasma

C. Disturbed clearance

D. Disturbed filtration and

reabsorption processes

E. All that is placed above

5. Point out the most important

compensatory mechanism in

metabolic acidosis:

A. Hyperventilation

B. Increased NH3 excretion by

kidneys

C. Increased filtration of phosphates

D. Increased HCO3- production

E. Urea production in the liver

6. Point out main source of ammonia

in kidney tissue:

178

№ Test tasks: Explanations:

A. Urea

B. Aspartate

C. Glutamine

D. Glutamate

E. Uric acid

7. Choose normal amount of proteins

excreted in urine/24 hours:

A. Less than 150 mg

B. 200 mg - 225 mg

C. 450 mg – 500 mg

D. More than 800 mg

E. 150 mg – 250 mg

8. Name organic compound which is

terminal for humans and not

reabsorbed in renal tubules:

A. Globulins

B. Glucose

C. Albumin

D. Creatinine

E. Bilirubin

9. Choose the specific gravity region

(g/ml) for urine of healthy person:

A. 1.005-1.015

B. 1.030-1.040

C. 1.015-1.020

D. 1.030-1.040

E. Less then 1.010

179

№ Test tasks: Explanations:

10. Creatinine levels in the urine and

blood are used to test kidney

function. Creatinine is useful for this

test because it is not significantly

reabsorbed nor secreted by kidney,

and metabolically it is:

A. Produced at a constant rate

B. Produced only in kidney

C. A storage form of energy

D. An acceptor of protons in renal

tubules

E. A precursor for phosphocreatine

11. Appearance of albumins in the urine

of diseased person may be at:

A. Acute nephritis

B. Chronical nephritis

C. Severe form of diabetes

mellitus

D. Pyelonephritis

E. All that is placed above

12. Choose the main biochemical tests

for diagnostics of kidney diseases:

A. Urea content in the blood

plasma and in the urine

B. Creatinine content in the blood

180

№ Test tasks: Explanations:

and urine

C. Sodium ions content in the

blood and urine

D. N-acetyl-beta-D-glucose-

aminidase activity (blood serum,

urine)

E. All that is placed above

13. What organic compounds

accumulate in final urine at severe

form of diabetes mellitus?

A. Albumins

B. Glucose

C. Ketone bodies

D. Bilirubin conjugated

E. All that is placed in positions A,

B, C

14. Kidney insufficiency development

will cause the infringements in those

processes:

A. Erythropoietin synthesis and

secretion

B. Calcitriol synthesis

C. Mineralization of bone tissue

D. Creatine synthesis

E. All that is placed

181

№ Test tasks: Explanations:

15. The infringement in glomerular

filtration mostly is associated with

appearance in the urine of this class

compounds. Name it:

A. Lipids

B. Proteins

C. Amino acids

D. Keto acids

E. Carbohydrates

16. Renal clearance may be calculated

using this compound concentration

value in the blood serum and in

urine of patient. Name it:

A. Inulin

B. Creatine

C. Free ammonia

D. Ammonia salt

E. Indican

17. Utilization of excess protons in renal

tubule lumen may be due to:

A. Aspartic acid

B. Creatinine

C. Uric acid

D. Ammonia

E. Water

182

№ Test tasks: Explanations:

18. This compound impossible to find

out in the urine of healthy person:

A. Globulin

B. Alanine

C. Pyruvate

D. Oxaloacetate

E. Carbonic acid

19. Acute tubular necrosis is associated

with increase of this index in the

blood serum of patient. Name it:

A. Creatine

B. Free amino acids

C. Pyruvate

D. Cholesterol total

E. Urea

20. This vitamin derivative is produced

in renal tubules mainly to control

calcium ad phosphate ions levels in

the blood. Name it:

A. FAD

B. NAD+

C. Calcitonin

D. Calcitriol

E. Angiotensin II

183

LITERATURE

Basic:

1. Berezov T. T. Biochemistry : translated from the Russian / T. T. Berezov, B. E.

Korovkin. - М. : Mir, 1992. - 514 p.

2. Ferrier D. R. Biochemistry : Lippincott illustrated reviews / D. R. Ferrier ; ed.

by.: R. A. Harvey. - 6th ed. - India : Lippincott Williams & Wilkins, 2015. -

560 p.

3. Murray R. K. Harper's Illustrated Biochemistry / R. K. Murray, D. K. Granner,

V. W. Rodwell. - 27th ed. - Boston [etc.] : McGraw Hill, 2006. - 692 p.

4. Satyanarayana U. Biochemistry : with clinical concepts & case studies / U.

Satyanarayana, U. Chakra Pani. - 4th ed. - India : Elsevier, 2015. - 812 p.

Additional:

1. Deitmer J. W. Strategies for metabolic exchange between glial cells and

neurons / J. W. Deitmer // Respir. Physiol. – 2001. – N 129. – P. 71-81.

2. Hames D. Instant Notes in Biochemistry / D. Hames, N. Hooper. – [2nd ed.].

– UK : BIOS Scientific Publishers Ltd, 2000. – 422 p.

3. Hertz L. Intercellular metabolic compartmentation in the brain: past, present

and future / L. Hertz // Neurochemistry Int. – 2004. – N 2-3. – P. 285-296.

4. Jurcovicova J. Glucose transport in brain - effect of inflammation / J.

Jurcovicova // Endocr. Regul. – 2014. – Vol. 1. – N. 48. P. 35-48.

5. Koolman J. Color Atlas of Biochemistry: textbook / J. Koolman, K.-H.

Roehm. – 2nd ed. – Stuttgart-New York : Thieme, 2005. – 467 p.

6. Kutsky R. J. Handbook of vitamins, minerals, and hormones / R. J. Kutsky.-

[2nd ed.]. - New York : Van Nostrand Reinhold, 1981. – 492 p.

7. Lieberman M. Medical Biochemistry: textbook / M. Lieberman; A. Marks, C.

Smith. - 2nd ed. - New York : Lippincott Williams & Wilkins, 2007. – 540 p.

8. Marks D. B. Biochemistry: The Chemical Reactions of Living Cells / D. B.

Marks, D. Metzler - [2nd ed., vol. 1,2] - USA : Elsevier Academic Press,

1994.- 1974 p.

184

9. Marshall J. W. Clinical Chemistry : textbook / J. W. Marshall, S. K. Bangert.-

Fifth edition. – China : Mosby, 2004. – 422 p.

10. Molecular Biology of the Cell / B. Alberts [et al]. – [6th ed.]. – NY : Garland

Science, 2015. – 1465 p.

11. Newsholme E. A. Functional Biochemistry in Health and Disease / E. A.

Newsholme, T. R. Leech. - UK : John Wiley & Sons Ltd, 2010.-543 p.

12. Smith C. Basic Medical Biochemistry: A Clinical Approach: textbook / C.

Smith, A. Marks, M. Lieberman. - 2nd ed. - New York : Lippincott Williams

& Wilkins, 2009. - 920 p.

185

CONTENT

Introduction…………………………………………………………………... 3

The role of water-soluble and fat-soluble vitamins in the metabolism of

humans. Vitamin similar substances. Antivitamins ...................................

4

Biochemistry of muscular and connective tissues……………………...... 28

Biochemistry of nervous tissue................................................................. 61

Biochemical functions of the liver at healthy and diseased people.................. 83

Xenobiotic transformation in humans. Microsomal oxidation…………….… 108

Biochemistry of blood tissue. Proteins of blood plasma. Non-Protein

components of blood plasma at healthy and diseased people.....................

131

The role of kidneys in the regulation of water and salts metabolism. The

normal and pathological components of urine..................................................

162

Literature.......................................................................................................... 183